ANDROLOGIE

ANDROLOGIE
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273
SOCIETE D’ANDROLOGIE DE LANGUE FRANCAISE
Année 2006
Dominique DELAVIERRE
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TABLE DES MATIÈRES
ANDROLOGIE - VOLUME 16, NUMÉRO 4, DÉCEMBRE 2006
Abstracts of the Fourth European Congress of Andrology and 23rd Congress of the
French Speaking Society of Andrology, Toulouse, France 7 – 10 December 2006
Programme Organizing Committee
F. Wu (United Kingdom), chairman
European Academy of Andrology
E. Baldi (Italy), T. Cooper (Germany), A. Grootegoed (The Netherlands), E. Jannini (Italy),
N. Joergensen (Denmark), G. Verhoeven (Belgium), B. Zorn (Slovenia)
French-speaking Society of Andrology
D. Delavierre (France), M. Bailly (France), D. Royère (France), P. Thonneau (France)
Local Organizing Committee
P. Thonneau, chairman
T. Almont , M. Bailly, L. Bujan, D. Delavierre, J. Drevet, R. Mieusset, J. Parinaud, P. Plante,
D. Royère, M. Soulié
• Editorial
281
• Plenary Lectures PL 01 to PL 06
283
• Symposia S 01 to S 23
287
• Oral Communications OR 01 to OR 24
301
• Posters PO 001 to PO 116
319
• Authors Index
395
279
280
Editorial
A propos du présent numéro d’Andrologie
Ce numéro d’Andrologie est très différent des précédents.
D’une part, il ne contient aucun article mais est constitué des résumés des lectures plénières, des
conférenciers invités, des présentations orales sélectionnées et des posters présentés lors du 4ème
Congrès Européen d’Andrologie, manifestation de l’Académie Européenne d’Andrologie, des 7
au 10 décembre 2006 à Toulouse.
D’autre part, il est en langue anglaise, langue utilisée (écrite et orale) lors de ce congrès.
Nous avons ainsi fait de ce numéro d’Andrologie un ‘Livre des résumés’ (Abstract book) qui sera
emporté par tous les participants à ce congrès et qui proposera aux lecteurs habituels d’Andrologie
une vision de l’andrologie quelque peu différente.
En vous souhaitant une lecture fructueuse,
Roger Mieusset
281
282
Abstracts of the Fourth European Congress of Andrology and
23rd Congress of the French Speaking Society of Andrology
Andrologie 2006, 16, N°4 283-286
Plenary Lectures
(PL 01 to PL 06)
PL 01
The history of the testis and testosterone
treatment
E. NIESCHLAG
Institute of Reproductive Medicine of the University
D-48129 Münster, Germany
Email: [email protected]
The biological effects of the testes and testosterone (T) are
known since antiquity. Aristotle (384-322 BC) knew the effects
of castration and his hypothesis on fertilization is one of the
first scientific encounters in reproductive biology. Over
centuries castration has been performed as punishment,
but also to preserve the soprano voices of prepubertal boys.
The Chinese imperial (and other oriental) courts employed
castrates as overseers of harems; the last of the Chinese
eunuchs died in 1996.
The era of testis transplantation and organotherapy was
initiated by John Hunter in London who transplanted testes
into capons in 1786. The intention of his experiments was
to prove the “vital principle” as the basis for modern
transplantation medicine, but Hunter did not consider
endocrine aspects. Adolph Berthold postulated internal
secretion from his testicular transplantation experiments in
1849 in Göttingen and is thus considered the father of
endocrinology. Following his observations, testicular
preparations were used for therapy, popularised by selfexperiments by Charles-Édouard Brown-Séquard in Paris
(1889), which can at best have had placebo effects.
Nevertheless, testis preparations were consumed until quite
recently for the enhancement of virility. In the 1920s Sergio
Voronoff transplanted testes from animals to men, but their
effectiveness was disproved by the Royal Society of Medicine
in 1927. Experimentation with testicular transplantation
continued to be popular in Russia. Today testicular
transplantation is being refined by stem cell research and
germ cell transplantation.
Modern androgen therapy started in 1935 when Ernest
Lacquer isolated T from bull testes in Amsterdam. In the
same year T was then chemically synthesized independently
by Adolf Butenandt in Göttingen and Leopold Ruzicka in
Basel. Since T was ineffective orally it was either compressed
into subcutaneous pellets or was used orally as 17_-methyl
T, now obsolete because of liver toxicicity. In the 1950s
longer-acting injectable T enanthate became the preferred
therapeutic modality. In the 1950s and 1960s research
concentrated on the chemical modification of androgens in
order to emphasise their anabolic effects. Although anabolic
steroids have largely disappeared from clinical medicine,
they continue to live an illegal life for doping in athletics. In
the 1970s the orally effective T undecanoate was added to
the spectrum of preparations. In 1992 WHO, NIH and FDA
postulated preparations of natural T mimicking physiological
serum levels, a demand first met by a transdermal scrotal
film. Non-scrotal skin patches followed and finally in 2000
transdermal T gels became available. The most recent
additions to T substitution therapy, the short-acting buccal T
and the long-acting injectable T undecanoate, also fulfil the
demand for physiological serum levels.
PL 02
Reproductive epigenetics and transgenerational
toxicity
J. TRASLER
McGill University-Montreal Children’s Hospital Research
Institute, Departments of Pediatrics, Human Genetics and
Pharmacology & Therapeutics, McGill University,
Montreal, QC, Canada.
E-mail : [email protected]
The term ‘epigenetics’ refers to heritable non-sequence
based mechanisms that regulate gene activity. Three main
283
types of mechanisms, including DNA methylation, RNAassociated silencing and histone modifications have been
associated with the epigenetic silencing of genes. To date, the
most well studied
DNA modification associated with the modulation of gene
activity is methylation of cytosine residues within CpG
dinucleotides occurring at about 25 million sites throughout
the genome. DNA methylation plays a role in regulating genes
during development and has been implicated in gene
regulation, genomic imprinting (variation in the expression of
a gene according to its maternal or paternal origin), and X
inactivation. Abnormalities in DNA methylation have been
linked to cancer as well as growth and behavioral defects. DNA
methylation is catalyzed by DNA (cytosine-5)-methyltransferases (DNMTs), is initiated in the germ line and then further
modified during early embryo development. In the field of
male-mediated developmental toxicity, adverse effects on
the offspring in animal studies often occur at levels too high
to be accounted for by mutagenesis, leading to the suggestion
that alternative mechanisms, including epigenetic processes,
may be affected.
Recent studies have reported that male-mediated effects on
the progeny can be passed across generations and have
implicated epigenetic mechanisms. In addition, a number of
recent studies have linked the use of assisted reproductive
technologies with growth and genomic imprinting disorders
in children; epigenetic processes appear to be involved as the
imprinting disorders found were associated with DNA
methylation abnormalities. However, in the latter studies it
was unclear whether the birth defects were related to the
underlying infertility or the treatments (i.e. ICSI, superovulation,
culture conditions) being used.
Since DNA methylation events and enzymes are well
conserved across mammals, the rat and mouse have served
as excellent models relevant to human studies. Using the
rodent model, our results and those of others indicate that DNA
methylation is highly regulated in the male germ line and
implicate alterations in the enzymes involved in DNA
methylation, the DNMTs, and DNA methylation, with
abnormalities in germ cell as well as embryo development.
The presentation will describe recent studies on the timing and
mechanisms underlying the acquisition and mainteneance
of DNA methylation patterns in gametes and early embryos
as well as the consequences of altering these patterns. Three
models relevant to male-mediated effects will be reviewed,
DNMT deficiency, effects of cytosine analogues and defects
in folate (methyl donor) pathways.
PL 03
Health of children conceived through
intracytoplasmic sperm injection, in vitro
fertilization, and natural conception
A.G. SUTCLIFFE
Institute of Child Health, Royal Free and University College
Medical School, University College London, Department of
Community Child Health, 250 Euston Road,
London, NW1 2PQ
In vitro fertilisation has been carried out for nearly 30 years
and in developed countries 1% or more of births are from
assisted reproductive therapies (ART). These children now
represent a significant proportion of the population but until
recently little has been known about their health.
Some of the morbidity associated with assisted reproductive
therapies does not result from the techniques as such but result
from the underlying health risks of being sub fertile.
Much of the increased risk associated with ART is related to
higher birth order. However, there are apparent specific
increased risks of intrauterine and subsequent perinatal
problems, and urogenital malformations in boys, even in
singleton ART infants. There is no apparent increase in
problems within families.
Long term follow-up of ART children to reproductive age and
beyond is necessary.
Keywords : childhood, assisted reproduction, outcome
PL 04
Gene regulation in spermatogenesis and
spermiogenesis - insights and implications for
human infertility
P. SASSONE-CORSI
Department of Pharmacology, Gillespie Neuroscience,
Univeristy of California, Irvine, California 92697-4625,
USA E-mail : [email protected]
No abstract provided.
284
PL 05
Stem cells in spermatogenesis
M. STEFANINI, E. VICINI
Dept of Histology and Medical Embryology, University of
Rome “La Sapienza”, Via Antonio Scarpa 14 00161 Rome,
Italy E-mail : [email protected] or,
[email protected]
The highly efficient process of sperm production is dependent
on proper hormone balance, local cellular interactions and,
more importantly, on the biological activity of spermatogonial
stem cells (SSCs), the stem cells of germline. Because of the
ability to transmit genes from one generation to the next,
SSCs represent the only replicating, potentially totipotent,
population of cells in the adult body. A major breakthrough in
understanding SSC biology was represented by the successful
demonstration of spermatogonial transplantation and
subsequent proliferation and differentiation of the transplanted
cells in the recipient animal (1). Germ cell transplantation
represents, to date, the only bioassy for SSCs and appears
to have relevant potential applications. Manipulations of these
cells in vitro, before transfer to a recipient subject, represents
an opportunity to modify germline for generating transgenic
experimental animals and introduction of new genes directly
into the male germline could be of great interest for agricultural,
zoological and clinical purposes. By FACS analysis and
transplantation technology, surface antigenic profile of SSCs
has been partially defined, however none of the markers
identified is specific for SSCs, since they are expressed also
by other germ cells and/or by somatic cells in the testis (2).
Using Hoechst staining of isolated seminiferous tubule cells
and FACS analysis, we were able to identify a testis “side
population” (T-SP) population that, by transplantation
technology, we demonstrated to be enriched in stem cell
activity, evaluated 2 months after transplantation (3). Further
characterization of T-SP SSCs by double vital staining with
Hoechst and rhodamine, allowed us to understand that T-SP
SSCs represent only a subset of SSCs and that a variety a
SSC subpopulations, possibly endowed with different functional
properties, could exist (4). It has been demonstrated that a
combination of growth factors, such as GDNF, bFGF and
EGF, induces the proliferation of spermatogonia with stem cell
potential in vitro, thus providing the possibility to manipulate
SSC genome. Transgenic mice and rats have been produced
by retroviral infection of SSC (5) and, more recently,
homologous recombination has been demonstrated to be
feasible in mouse SSC (6). On this direction, we analyzed the
feasibility of the in vitro Cre delivery to germ cells in order to
obtain gene inactivation in stem cell germline (7).
Conditional mutagenesis represents a powerful approach to
analyze gene functions in mammalian cells. Site-specific
recombinases such as the bacteriophage P1 recombinase Cre
have been used to induce gene modification in a spatial
and/or temporal manner (8). However, the use of site-specific
285
recombination in genetic studies is limited by difficulties to
express the recombinase in cells at the desired time and
place. Recently a new tool for the in vitro deletion of loxP
flanked genes has been described. In this approach
enzymatically active cell-permeable Cre recombinase is
delivered to cultured target cells (9). The HTNC (HIS-TAT-NLSCRE) recombinase has been shown to induce deletion of
lox-flanked gene in about 90% of ES cells or primary
splenocytes (9). Genetic analysis in spermatogonial stem
cells (SSC) is highly warranted but, presently, no Cre transgenic
mice are available to perform conditional gene inactivation in
postnatal SSC.
To test if HTNC induces conditional gene inactivation in germ
cells, we thought to treat germ cells isolated from ROSA26R
mice and to detect recombination events by means of germ
cell transplantation. Genetic analysis has demonstrated that
after transplantation, donor-derived spermatogenic colonies
originate from a single SSC thus germ cell progeny inherit the
founder SSC genotype (1). We anticipated that recombination
events occurring in HTNC-transduced SSC will generate
colonies of donor-derived spermatogenesis where germ cell
progeny bear the same recombination events. Recombinant
colonies would therefore be easily recognizable as blue
colonies in the recipient testis after X-gal staining owing to the
activation of the ROSA 26R locus.
Germ cells were isolated from adult ROSA26R mice and
enriched in SSC by Percoll purification (10). Germ cell aliquots
were treated in vitro with HTNC and transplanted in the testis
of busulfan-treated recipient mice. Two months after
transplantation, animals were sacrificed and testis analyzed
to detect donor-derived blue colonies generated from modified
SSC. Donor-derived blue colonies were found in 6 out of 11
tranplanted testis. We have demonstrated, for the first time,
the feasability of in vitro conditional gene inactivation in stem
cell germline. This experimental strategy may be used to
perform conditional gene inactivation in order to analyze gene
functions in germ cells during spermatogenesis. The possibility
to perform postnatal gene inactivation may be relevant for the
study of lox-flanked genes whose inactivation is either
embryonically lethal or interferes with prenatal germ cell
development. This strategy may also be used to introduce
stable genome modification in mice germline.
Bibliography
1. Brinster RL and Zimmermann JW : Proc. Natl. Acad. Sci.,
1994, 91 : 11298.
3. Falciatori I et al. : FASEB J., 2004, 18 : 376-378.
4. Vicini in preparation
5. Kanatsu-Shinohara M et al. : Biol. Reprod., 2005, 72 : 236.
6. Kanatsu-Shinohara M et al. : Proc. Natl. Acad. Sci., 2006,
103 : 8018.
7. Corallini S et al. : 14th European Workshop on Mol and Cell
End of the Testis, Miniposter Book, 2006, II25.
8. Nagy A. : Genetics, 2000, 26 : 99.
9. Peitz M et al. : Proc. Natl Acad. Sci., 2002, 99 : 4489.
10. Kubota H et al. : Biol. Reprod., 2004, 71: 722.
PL 06
Pathophysiology of erectile dysfunction
M. MAGGI
Department of Clinical Physiopathology, Andrology Unit,
University of Florence, Florence, Italy
In unicellular organism genetic information to the offspring is
just duplicated, and therefore, subsequent generations are
identical to the preceding ones (parthogenetic reproduction).
It is, overall, a monotonic, asexual reproduction. The individual
diversity of the more complex organisms derives from the
development of sexual reproduction, which requires two
distinct partners (the couple) having distinct phenotypic and
behavioural futures (maleness and femaleness).
Couple reproduction allows billiards of possibility to exchange
genetic material and is of great evolutionary advantage and
very favourable for species evolution. However, sexual
reproduction between heterogeneous individuals carries
patent risks. Although the couple reproduction geometrically
increased genetic benefits, threats and problems are similarly
amplified. In fact, due to partner interdependence, sexual
reproduction can be problematic either for reproductive or
for sexual reasons, resulting in one hand in couple infertility
and in the other one in couple sexual dysfunction. In fact,
couple sexual function should be viewed as the net result of
the sexual fitness of both partners.
Only few decades ago it was generally thought that the majority
of male sexual dysfunctions (including erectile dysfunction)
were mostly related to psychological problems, and to anxiety
in particular. Hence, at that time, the only recognized treatment
was psychotherapeutic, i.e psychoanalysis and behavioral
therapy. Nowadays, it is well recognized that this picture is
limitative, as biological and relational domains, beside
psychological components, have a relevant impact on male
sexual response. Indeed, male sexual response can be seen
as an integrated feed-forward interaction among an intrapsychic system (the individual’s sexual identity and sense of
well-being), a biological system (cardiovascular, hormonal,
neuronal), and a relational system (the context for a sexual
relationship).
Understanding the relative contribution of each of these
components is essential for a correct diagnostic and/or
therapeutic approach to male sexual dysfunction and, in
particular, to erectile dysfunction (ED). From a biological point
of view, male sexual activity is essentially characterized by a
T-driven synchronization of sexual desire, arising in the brain,
and its transmission to the periphery, allowing penile erection.
The most important pathway underlying the penile erection
is the nonadrenergic/noncholinergic signalling, which through
the release of nitric oxide (NO), leads to an intracellular
286
increase of cyclic GMP (cGMP), the main secondary
messenger mediating tumescence in the penis. Interestingly,
both cGMP formation and degradation are affected by
testosterone (T). Because T positively controls both the
initiation (NOS) and the end (PDE5) of the erectile process,
its net effect on erection might result even null. Hence,
erections are still possible in hypogonadal conditions where
a decreased cGMP formation, due to impaired NO production,
is counterbalanced by a reduced cGMP hydrolysis.
Abstracts of the Fourth European Congress of Andrology and
23rd Congress of the French Speaking Society of Andrology
Andrologie 2006, 16, N°4 287-298
Symposia
(S 01 to S 23)
S 01
Early Diagnosis and Management of Prostate
Cancer
F.C. HAMDY
Academic Urology Unit. School of Medicine & Biomedical
Sciences University of Sheffield, Sheffield, UK
Prostate cancer is a significant cause of morbidity and mortality
in the United States and Europe. It is the second most common
cancer in men in the European Union, with 85,000 new cases
and approximately 40,000 deaths each year. The natural
ageing of the population, combined with the continued and
widespread use of improved diagnostic tests such as serum
prostate specific antigen (PSA), are resulting in an increase
in the numbers of men diagnosed with localised prostate
cancer. Screening to identify organ-confined disease has
provoked much public and scientific attention and there is
intense debate about its role in improving men’s health. While
there are strong advocates of screening, the findings from most
reviews of the scientific evidence conclude that it is insufficient,
at present to recommend routine population screening because
of the lack of evidence that this would improve survival and
the quality of men’s lives. Particular concerns in these reviews
relate to the lack of knowledge about the natural history of
screen-detected disease, and the lack of evidence about the
effectiveness of treatments.
In particular, to date, whilst the recent Scandinavian SPCGIV randomized controlled trial of watchful waiting versus
radical prostatectomy has demonstrated a survival benefit
as well as reduced progression in men receiving surgery
versus no treatment for clinically localized prostate cancer, no
survival advantage has been shown to date in screen-detected
cases for any of the major treatments (radical prostatectomy,
radical radiotherapy including brachytherapy and “watchful
waiting” otherwise known as “Active Monitoring” or “Active
287
Surveillance with delayed intervention”). Each can result in
damaging iatrogenic complications and outcomes, including
various levels of incontinence and impotence for radical
interventions and anxiety relating to the presence of cancer
in untreated patients. Furthermore, recent observations from
the Prostate Cancer Prevention Trial in the US have revealed
that the incidence of the disease is considerably higher than
expected at low PSA levels, with a significant risk of overdetection and over-treatment in the majority of screen-detected
cases.
Despite these uncertainties, opportunistic screening for
prostate cancer is widespread in many countries.Two
randomised trials of screening are ongoing ; the Prostate
Lung and Colorectal and Ovarian Trial (PLCO) in the USA and
the European Randomised Screening for Prostate Cancer
(ERSPC). There are two current trials of localised prostate
cancer treatments: the Prostate Testing for Cancer and
Treatment study (ProtecT) in the UK and the Prostate Cancer
Intervention Versus Observation Trial (PIVOT) in the USA.
Whilst controversies persist in the management of localized
disease, many advances have been made in the treatment
of locally advanced, as well as metastatic prostate cancer.
Improved technology and better understanding of the biology
of prostate cancer have allowed new combination treatment
strategies to be developed. Sophisticated radiotherapy
combined with androgen suppression appear to improve
survival in locally advanced disease, and fine tuning of
interventions allow protection of the skeleton and improved
palliation in metastatic and hormone refractory disease, using
novel chemotherapeutic agents and bone resorption inhibitors
such as bisphosphonates.
There is a considerable paradigm shift in the general
management strategy for prostate cancer, from the
development of active monitoring programmes for low-risk
tumours, to the conversion of previously debilitating advanced
disease into a chronic, manageable condition with improved
outcomes and quality of life for our patients.
S 02
Microarray-based molecular profiling of advanced
and hormone-refractory prostate cancer
S 03
Role of estrogen receptor β in the pathogenesis
and progression of prostate cancer
D. DONDI1, D. SAU1, M. PICCOLELLA1,
M. TORTORETO3, G. PRATESI3, P. CIANA2, A. MAGGI2,
V. GUERINI1, M. MOTTA1, A. POLETTI1*.
M. WOLF, H. EDGREN, I. MILLS, A. CARLES, O. POCH,
S. KILPINEN, M. PELTOLA, R. AUTIO, D. NEAL, B.
WASYLYK, J. SCHALKEN, O. KALLIONIEMI
EU FP6 PRIMA (Prostate Cancer Integral Management
Approach) VTT Technical Research Centre of Finland and
University of Turku, Finland ; University of Cambridge, UK;
Institut de Génétique et de Biologie Moléculaire et
Cellulaire, Illkirch, France ; Tampere University of
Technology, Tampere, Finland; Radboud University
Nijmegen Medical Centre, The Netherlands
There are few treatment options available for hormonerefractory and metastatic prostate cancer, largely as a result
of incomplete understanding of the molecular mechanisms
underlying the disease progression. In order to characterize
clonal selection mechanisms leading to the failure of androgen
deprivation therapy, we have analyzed a unique sample set
of 19 advanced prostate cancers, consisting of hormonerefractory prostate cancers (HR-PRCA) and untreated
advanced tumors using oligo-CGH and gene expression
microarrays. In addition, publicly available existing gene
expression data from normal prostate tissues and untreated
prostate cancers were used as a reference to validate the
relevance of the gene targets identified here.
The genetic changes identified by oligo-CGH in the two groups
were partly overlapping, but HR-PRCA also displayed many
unique alterations (such as gains/amplifications at Xq12,
1q21-q32, 2q31-q33, 3q21-q28 and deletions at 15q14-q22
and 15q24-25). A novel data integration method, DNA/RNA
Outlier Analysis, was used to identify the targets genes for the
genetic changes. Several outlier genes were identified as
targets for genomic amplifications and deletions in both sample
groups, such as AR as a target gene for Xq12 amplification
in HR-PRCA, and PTEN as a target for 10q23 loss, as well
as a several other novel genes at these and other loci. Based
on all the identified outlier hits, Wnt signaling pathway showed
the most significant enrichment in both sample groups
indicating that deregulation of this critical pathway in prostate
cancer occurs through genome-level alterations.
In summary, we have applied here an integrative microarray
based analysis on gene copy number and expression of
advanced prostate cancers, which has led to the identification
of several novel targets with putative relevance in hormonerefractory prostate cancers. Ongoing functional studies will
further clarify their therapeutic potential.
1 Institute of Endocrinology ; 2 Department of
Pharmacological Sciences, University of Milan, Milano,
Italy, via Balzaretti 9, 20133 Milano, Italy . 3 Istituto
Nazionale Tumori, Via Venezian, 1. 20133 - Milano. *E-mail
: [email protected]
In earlier stages, prostate cancer (PC) depends on androgens
and its growth can be usually controlled by androgen blockade.
However, androgen-ablation therapy often induces androgenindependent PC, which is generally characterized by an
increased invasiveness. Recently, we found that the
testosterone derivative, dihydrotestosterone (DHT) inhibits
PC cell migration through an androgen receptor (AR)independent mechanism. This effect has been linked to the
DHT metabolite 5alpha-androstane-3beta,17beta-diol (3betaAdiol), which is unable to bind AR, but interacts with the
estrogen receptor beta (ERbeta). 3beta-Adiol inhibits cell
migration of PC cells through the activation of the ERbeta
signaling, while, surprisingly, estradiol is not active, suggesting
the existence of different pathways for ERbeta activation in
PC cells. The inhibitory effects of 3beta-Adiol on PC cell
migration appear to be mediated by an overexpression of Ecadherin, a protein known to be capable to reduce metastasis
formation of breast cancer and PC cells. In fact, 3beta-Adiol
is not active on PC cells in which E-cadherin expression has
been silenced using a selective siRNA.
To further investigate the growth properties of PC cells in
vivo, we implanted DU145 cells stably transfected with AR
(DU145-AR) and DU145-mock s.c. into nude male mice to
characterize their receptor state. Tumor growth was followed
up to 25 days. The DU145-mock xenografts kept an
exponential growth in such time frame ; castration only
marginally influenced tumor growth. Interestingly, testosterone
seemed to be able to reduce the growth of the tumors
generated by DU145-mock suggesting that an androgenic
metabolite (3beta-Adiol ?) may be responsible for this effect.
On the other hand, tumor growth of DU145-AR xenografts was
significantly lower and further inhibited by androgen deprivation.
After testosterone replacement, the growth rate was restored
to that of DU145-AR xenografts in intact animals, suggesting
that the molecular changes present in the stably transfected
DU145-AR cells influence their growth properties in in vivo
systems.
The present data demonstrated that 1) testosterone may
exert an estrogenic effects downstream in the catabolic
process present in the prostate; 2) the estrogenic effect of
288
testosterone derivatives (ERbeta-dependent) inhibits cell
migration, although it is apparently different from that linked
to estradiol on the same receptor and may be protective
against PC invasion and metastasis. These results correlate
with the clinical data reporting that alterations in gene coding
for 3beta-HSDs (the enzymes responsible for 3beta-Adiol
formation) are strongly correlated with hereditary PC.
S 04
- detection of first genes that are allow to distinguish very
early seminomas from nonseminomas ;
- discovery of new gene expression markers of rare germ
cell tumours that do not originate from CIS (infantile tumours
or spermatocytic seminomas of elderly men) ;
- discovery of new markers applicable for clinical diagnosis
of CIS, including CIS cells found in semen samples, e.g.
TFAP2C (AP2 gamma).
Some of these findings are currently tested in clinical studies
and begin to benefit patients.
Benign prostatic hyperplasia – current
management options and future prospects
S 06
C. SCHULMAN
Dept. Urology, Erasme Hospital, University Clinics of
Brussels, route de Lennik 808, B-1070 Brussels, Belgium
Email : [email protected]
Fetal androgen disruption and chronic adult germ
cell apoptosis - a model for the testicular
dysgenesis syndrome
No abstract provided.
M. BENAHMED
S 05
Germ cell cancer : clinical applications of gene
expression profiles
Inserm U-407, Faculte de Medecine Lyon Sud, BP 12,
69921 Oullins Cedex, France Email :
[email protected]
No abstract provided.
E. RAJPERT-DE MEYTS
S 07
Department of Growth & Reproduction, Copenhagen
University Hospital (Rigshospitalet), Denmark
Fertility after testis cancer
Recent years have seen an explosive increase in gene
expression studies because of the availability of highthroughput methods, in particular microarrays (cDNA- or
oligonucleotide- arrays). DNA chips - as the microarrays are
popularly called – enable the investigator to evaluate relative
expression of thousands of genes simultaneously in one
experiment. This ingenious technology has provided
researchers with an ocean of new information concerning
global gene expression in many types of normal cells or
tissues, and changes of their gene expression profiles in
diseases, including cancer. The germ cell cancer research field
has also benefited from these studies. Among the most
important findings, to name just a few, are :
E. HUYGHE
Human Fertility Research Group, Hôpital Paule de Viguier,
330 Avenue de Grande Bretagne, 31059 TOULOUSE
cedex 9, France
- high expression of pluripotency genes and stem-cell related
factors in the pre-invasive carcinoma in situ (CIS) and
embryonal carcinoma (EC) and EC-derived cell lines ;
Testicular cancer (TC) is the most common cancer in men 20
to 35 years of age. Its prognosis is excellent and TC represents
a model of curable cancer (cure rate exceeding 95%). For longterm survivors, often in the reproductive age, fertility
preservation is a major preoccupation.
- construction of new expression-based classifiers allowing
to predict the predominant histological component of
nonseminomatous tumours (nonseminomas) ;
Epidemiological, histological and clinical data point out a
common aetiology between testicular germ cell cancer, male
genital abnormalities (such as cryptorchidism) and conditions
289
related to male reproductive health (such as infertility). More
than half of the patients with TC already have testicular
impairment before orchiectomy. The degree of spermatogenic
dysfunction is higher than what can be explained by local
tumour effect and by a general cancer effect. Histological
abnormalities are frequently observed in the controlateral
testis.
Treatments such as cytotoxic chemotherapies and
radiotherapy are associated with significant gonadal damage
in men. Cisplatin-based chemotherapy for TC results in
temporary azoospermia in most men, with a recovery of
spermatogenesis in about 50% of the patients after 2 years
and 80% after 5 years. A cumulative dose of cisplatin at 400
mg/m2 is predictive of the occurrence of long-term effects
on sperm production. Second line chemotherapies may contain
alkylating agents that are damaging agents for
spermatogenesis.
The germinal epithelium is very sensitive to radiation-induced
damage: Testicular doses of less than 0.2 Gy have no
significant effect on FSH levels and sperm counts. Doses
above 0.2 lead to a dose-dependent increase in FSH and
reduction in sperm concentration. The time to recovery, if it
occurs, is likely to be dose dependent. It is considered that
doses of 1.2 Gy and above are at high risk of definitive
impairment of spermatogenesis. Limitation of the radiation field
to the para-aortic field, total radiation dose less than 30 Gy
and testicular shielding are ways to limit radiation-induced
impairment of fertility.
Information and counselling about semen cryopreservation,
chance of recovery of spermatogenesis and current available
methods of ART are mandatory. Cryopreservation of
spermatozoa is a simple and practical approach to preserve
fertilizing potential, and its proposal is part of good clinical
practice. Wait-and-see strategies and ways to decrease
morbidity of treatments should be strongly promoted and
prospective trials comparing patients with and without cytotoxic
treatment should be developed to ascertain the extent to
which treatment affects long-term fertility. Cooperation between
oncologists, urologists and andrologists on this topic remains
essential.
S 08
Cryptorchidism – relaxins and other factors
abnormalities with recorded frequency of 3-4% among the
newborn boys. Before sex determination both female and
male embryonic gonads are located in the same high
abdominal position. The developing testes are migrating
through a multiphase process of testicular descent (TD), first
into low abdominal position and then into developing scrotum.
Critical role in TD belongs to the proper differentiation and
growth of gubernacular ligaments. Analysis of the mouse
mutants revealed a number of genes involved in this process.
INSL3 (insulin-like 3) is a peptide hormone of relaxin-insulin
family, expressed in Leydig cells in testis. INSL3 signals
through its cognate G protein-coupled receptor LGR8, which
is expressed in gubernacular cells and controls the
transabdominal phase of TD. The inguinoscrotal stage of TD
is believed to be mediated mainly by androgens. Several
transcription factors, such as Hoxa10, Hoxa11, and Desrt,
caused cryptorchidism in mice, suggesting an involvement of
additional signaling pathways in TD. Using different mouse
transgenic lines we have analyzed the regulation and
interactions between these genes. The mutation analysis of
INSL3 and LGR8 genes in human patients with testicular
maldescent revealed mutant alleles present exclusively in
affected patients. A unique missense mutation (T222P) in the
ectodomain of the LGR8 receptor has been identified
exclusively in patient group in some populations. The
expression analysis of the mutant protein revealed that the
mutation severely compromised receptor cell membrane
expression. Consecutively, we have also shown compromised
physiological properties of the mutant INSL3 variants identified
previously in cryptorchid patients. Thus, mutations in the
INSL3 and LGR8 genes might be responsible for the etiology
of some cases of cryptorchidism in humans.
S 09
The role of chromatin in the establishment of
paternal epigenetic information during
spermatogenesis
S. ROUSSEAUX, A.K. FAURE, J. THEVENON,
E. ESCOFFIER, C. LESTRAT, J. GOVIN, S. HENNEBICQ,
B. SELE, C. CARON, S. KHOCHBIN
Unité INSERM-UJF U309, 38706 La Tronche Cedex,
France E-mail : [email protected]
I. A. AGOULNIK
Department of Obstetrics and Gynecology, Baylor College
of Medicine, Houston, Texas, 77030, USA
Cryptorchidism is a one of the most frequent congenital
290
It is now admitted that, in addition to genetic information, the
spermatozoon conveys information, named “epigenetic”,
which is not encoded by the DNA sequence. Epigenetic
information is known to be crucial for the development of the
embryo.
In somatic cells, the epigenome conveys the epigenetic
information, which is supported by the DNA methylation as
well as by modifications of the genome packaging structures,
or epigenome, which consists of the nucleosomal chromatin
(DNA wrapped around somatic histones).
spermatogonia. Germline inactivation of Rara results in a
testicular degeneration comprising some, but not all, abnormal
features observed upon dietary vitamin A deficiency, whereas
germline inactivation of either Rarb or Rarg do not cause
primary testicular defects.
During post-meiotic maturation of the male germinal cells, or
spermiogenesis, the epigenome is completely re-organised
since most somatic histones are replaced by sperm-specific
basic proteins, the protamines, which are responsible for the
tight compaction of the haploid genome within the sperm
nucleus.
A selective ablation of the RARα gene in mouse Sertoli cells
(RaraSer–/– mutation) yields testis defects identical to those
generated upon inactivation of RARα in the whole organism,
indicating that all RARα functions in male reproduction are
Sertoli cell-autonomous. During postnatal testis development,
the RaraSer–/– mutation abolishes cyclical gene expression
in Sertoli cells. It also induces testicular degeneration features,
as well as a delay in spermatogonial Stra8 expression, two
hallmarks of RA deficiency. Thus, our data demonstrate that
RARα is a master regulator of the Sertoli cell cycle and plays
important paracrine functions in spermatogonia differentiation
and survival of germ cells. However, ablation of all three
RARs in Sertoli cells does not induce an arrest in
spermatogonia differentiation and does not abolish Stra8
expression, as does vitamin A deficiency. These phenotypic
differences point to a cooperation between distinct RA
signalling pathways mediated by RARα in Sertoli cells and by
RARγ in spermatogonia during spermatogenesis.
Although this process has long been described, very little is
known about the molecular events and factors involved. We
have identified several chromatin modifiers, which could have
a crucial role during post-meiotic chromatin re-organisation.
Moreover, it is now known that the sperm genome is not
uniformly packed with protamines, and that some histones
remain, as well as some other non-histones, non-protamines
basic proteins. Our recent data suggest that specific regions
of the genome could be associated with specific structures
within the sperm nucleus.
The heterogeneity and specificity of this structure organising
the sperm genome could convey specific paternal epigenetic
information to the zygote, which could play a key role during
development.
Finally, the implications of these data in the field of male
infertility will be discussed.
Additionally, ablation of all three RXR (α, β and γ isotypes) in
Sertoli cells does not recapitulate the RaraSer–/– mutant
phenotype, providing thereby the first evidence that RARα
exerts functions in vivo independently of its heterodimerization
with a RXR.
S 10
S 11
Retinoic acid signalling in the testis
Connexins – relevance to rodent and human
spermatogenesis
M. MARK, N. VERNET, C. DENNEFELD, F. GUILLOU, P.
CHAMBON, N.B. GHYSELINCK
Institut de Génétique et de Biologie Moléculaire et
Cellulaire (IGBMC), 67404 Illkirch Cedex, Communauté
Urbaine de Strasbourg, France.
M. BERGMANN
Institute of Veterinary Anatomy, Histology & Embryology,
Justus-Liebig-University, Gießen, Germany
Development and functioning of the mammalian seminiferous
epithelium, require a complex assortment of hormones and
cytokines. Amongst these, retinoic acid (RA), the active
metabolite of vitamin A (retinol) regulates spermatogonia
differentiation and spermatid adhesion properties. RA acts
through binding to nuclear receptors, the Retinoic Acid
Receptors (RARα, β and γ isotypes), which are liganddependent transcriptional regulators. In general, RAR
transduce the RA signal in the form of heterodimers with
Rexinoid Receptors, RXRα, β and γ. Each RAR is detected
in a specific cell-type of the seminiferous epithelium: RARα
in Sertoli cells, RARβ in round spermatids and RARγ in A
291
Gap junctions contain channels that connect neighbouring cells
and constitute the basis for direct intercellular communication
via exchange of ions, second messengers and metabolites.
Each channel consists of two hemichannels, called connexon,
which are formed by the hexameric assembly of protein
subunits, connexins (Cx), a familiy of highly related proteins,
that consist of at least 21 members in human and 19 in
rodents. In the testis, gap junctional intercellular communication
is well known between interstitial Leydig cells and endothelia
of blood vessels, and within the seminiferous epithelium
between Sertoli cells, and between Sertoli cells and germ
cells.
In rodent testis, six isoforms have been demonstrated so far.
Cx37 was solely found in blood vessel endothelia, while Sertoli
cells express Cx 26, Cx32, Cx33, and Cx43. Germ cells are
positive for Cx31 and Cx 43. In the testis, Cx 43 seems to be
the most important and predominant connexon. In the
seminiferous epithelium, it is first expressed together with
the pubertal terminal differentiation of Sertoli cells and it`s
expression depends on the seminiferous epithelial cycle. The
importance of Cx 43 to gametogenesis in indicated by severe
depletion of germ cells in prenatal male and female mice
lacking the Cx43 gene. Postnatal spermatogonial proliferation
is impaired in Cx43-null mutants, and this defect can not be
restored by insertion of Cx32 or Cx 40 coding regions into
the respective region of Cx43-/- mice. It is impossible to
evaluate the physiological role of Cx 43 or Cx 26 by knockout
mouse models, because Cx43 knockout leads to altered
cardiac morphology and perinatal death, and Cx 26 deficient
mice die on embryonic day 11 because of placental deficiency.
In order to circumvent perinatal lethality and pleiotropic effects,
we generated a mouse line that carries the floxed connexin
43 coding region flanked by loxP recognition sites for the Cre
recombinase. Sertoli cell specific deletion was achieved by
crossing these floxed mice with mice harbouring the transgene
under control of Sertoli cell specific AMH gene. First data
show, that these Sertoli cell specific Cx 43 knockout mice
show a prepubertal arrest of spermatogenesis at the level of
spermatogonia indicating, that Cx43 expression plays the
key role in the initiation of spermatogenesis, which is not
replaced by the up-regulation of other Cx´s. Cx26 seems to
be restricted in the apical region of rodent seminiferous
epithelium, i.e. in spermatids and/or Sertoli cells. Cx 33 is a
member of the gap junction showing several specialities. In
contrast to most other Cx`s which are also expressed in other
tissues, Cx 33 expression seems to be restricted only to the
testis. It was shown to inhibit the expression of Cx33 and to
reduce Cx43. Its functional role is believed to limit the capacity
of cells to create functional channels by enabling formation
of heterotypic channels with other cells. The Cx33 gene,
which is mapped to the X chromosome in the rat and mouse
genome has no orthologs in the genome of humans or any
other mammals, such as dog, cattle, pig, horse, and marmoset
monkey, which have been investigated so far.
In the human Cx43 and Cx26 expression was intensely
investigated. As in rodents, Cx43 is firstly found during pubertal
Sertoli cell terminal differentiation and shows stage dependent
expression in adult normal seminiferous epithelium. Cx26 is
weekly expressed and located at inter Sertoli cell junctional
sites. Both Cx`s have been found to be functional Sertoli cell
markers, indicating the state of Sertoli cell differentiation in
testes of infertile patients showing spermatogenic impairment.
Sertoli cells in seminiferous tubules showing an arrest of the
seminiferous epithelium reveal a persistant AMH expression
together with an abberant diffuse cytoplasmic Cx26 staining,
and absence of Cx43 expression indicating a prepubertal
state of Sertoli cell population without functional intercellular
communication. This is also true for Sertoli cells associated
292
with preinvasive testicular intraepithelial neoplasia (TIN), syn.
: Carcinoma in situ (CIS) which is known to be the precursor
of germ cell tumours such as seminomas and non seminomas
with the exception of spermatocytic seminoma. In TIN tubules
Sertoli cells show a progressive down-regulation of Cx43
indication the loss of intercellar communication between
Sertoli cells and between Sertoli cells and preinvasive tumor
cells. Aberrant gap junctional intercellular communication
and/or loss of connexin expression has been demonstrated
to correlate with neoplastic transformation in several human
tissues such as prostate, breast, lung, and brain. Thus the loss
of gap junctional intercellular communication might lead to
uncontrolled mitotic division of CIS cells and finally to solid
invasive neoplasia.
References
Brehm R., Steger K. : Regulation of Sertoli Cell and germ cell
differentiation. Adv. Anat. Embryol. Cell. Biol., 2005, 181 : 193.
S 12
Databases and data mining in studies on
spermatogenesis
M. PRIMIG
Biozentrum & Swiss Institute of Bioinformatics,
Klingelbergstrasse 50 -70, 4056 Basel, Switzerland
Email : [email protected]
No abstract provided.
S 13
Human epididymal proteins: regulation and
interactions with spermatozoa
R. SULLIVAN
Centre de Recherche en Biologie de la Reproduction and
Dépt Obstetrique-Gynécologie, Université Laval, Canada
[email protected]
Vasectomy is the third most common contraceptive method
and the demand for surgical vasectomy reversal
(vasovasostomy) is increasing. Some consequences of
vasectomy on human epididymal physiology will be presented.
Vasectomy disturbs gene expression in the human epididymis.
Vasectomy modifies the pattern of expression of P34H, a
protein expressed in the human corpus epididymidis, added
to the sperm surface during maturation and involved in zona
pellucida binding. While P34H is expressed in the corpus
epididymidis in normal men, its expression is restricted to the
proximal caput epididymidis. This may affect the fertilizing
potential of ejaculated spermatozoa of some
vasovasostomized men.
In fact, an important proportion of vasovasostomized men
are characterized by spermatozoa lacking the P34H protein.
Although the expression of P34H in the epididymis is relocated
under vasectomy, HE1/NPC2, a gene expressed all along
the human epididymis, is down-regulated under vasectomy.
Westerm blot analyses show that many vasovasostomized
men are characterized by high HE1/NPC2 levels on
spermatozoa when compared to fertile donors. HE1/NPC2
association with sperm from vasovasostomized men is not
correlated to low motility per se as spermatozoa from
asthenospermic men have similar levels of HE1/NPC2 as in
normal fertile semen samples. Spermatozoa from
vasovasostomized men with high amount of HE1/NPC2 are
characterized by higher concentration of cholesterol and more
lipid raft domains. Knowing the effect of cholesterol on sperm
physiology, this may affect the fertilizing ability of the male
gametes. HE1/NPC2 is secreted in different glycoforms by
different tissues of human male reproductive tract. These
forms are due to variation in N-glycosylation and only the
deglycosylated form is associated with spermatozoa from
some vasovasostomized men.
Compared to normal men, seminal plasma of vasectomized
men is characterized by a major decrease in immunodetectable
HE1/NPC2 without change in the glycosylation pattern.
Following surgical vasectomy reversal, seminal plasma
HE1/NPC2 is found in similar amount to the ones
characterizing normal men. Considering the potential role of
HE1/NPC2 in cholesterol transport during sperm maturation,
unusual high levels of this protein associated with spermatozoa
of vasovasostomized men may reflect epididymal sequelae
occurring when the vas deferens is obstructed. Taken together,
our results show that vasectomy affects gene expression
along the human epididymis. These changes have
consequences on some biochemical characteristics of
ejaculated spermatozoa of vasovasostomized men. Thus,
sequelae to the human epididymis under vasectomy may not
be reversible in all men following vasovasostomy. This may
explain the discrepancy between surgical success of vas
deferens reanastomosis and fertility recovery.
This work was supported by CIHR (Canada).
S 14
Human epididymal proteins - secretion,
absorption, and luminal fluid composition
J-L. DACHEUX
Equipe "Gamètes Mâles et Fertilité", UMR 6175 INRACNRS-Université de Tours-Haras Nationaux, Physiologie
de la Reproduction et des Comportements, 37380 Nouzilly,
France Email : [email protected]
No abstract provided.
S 15
Sperm membrane dynamics : structure and
function
R. JONES
The Babraham Institute, Cambridge CB2 4AT, UK
[email protected]
Sperm plasma membranes are unusual for their high content
of unsaturated phospholipids and their compartmentalisation
into at least 5 recognizable domains, each with specific and
overlapping lipids and glycoproteins. Elucidating how these
domains are created and maintained over relatively large
distances against the randomising forces of diffusion is central
to understanding membrane remodelling events during sperm
maturation and capacitation. Foremost amongst remodelling
processes is repositioning of specific components from areas
of the spermatozoon where they are ‘inactive’ to regions
where they become ‘active’, either as a result of processing
(e.g. endoproteolysis) or because they form complexes (e.g.
lipid rafts) with new membrane components.
To understand the supramolecular structure of the sperm’s
plasma membrane we have applied a range of quantitative,
high resolution microscopy techniques to investigate the
dynamics of lipid and protein diffusion in live spermatozoa
(boar, ram, bull and mouse) at different stages of development.
The methods include atomic force microscopy (AFM), scanning
ion conductance microscopy (SICM), fluorescence recovery
293
after photobleaching (FRAP), fluorescence loss in
photobleaching (FLIP), single particle fluorescence imaging
(SPFI) and single molecule tracking (SMT). AFM revealed
significant differences in membrane topography between the
acrosome, postacrosome and identified a new region within
the equatorial segment, designated the subsegment (EqSS).
The EqSS is enriched in Hsp70 and constitutively
phosphorylated proteins and is assembled during epididymal
maturation.
FRAP analysis using lipid reporter probes demonstrated
significant differences between the sperm head and tail
domains that, surprisingly, were largely unaffected by lipid
peroxidation from oxygen free radicals. Combined FLIP and
SPFI analysis suggested the presence of a molecular ‘filter’
rather than an absolute diffusion barrier between the acrosomal
and postacrosmal domains. This has been substantiated by
SMT although the nature of the probe and target molecule has
to be taken into account. Thus, sperm membranes can respond
rapidly to agonists in external fluids, be they in the epididymis
or female reproductive tract or artificial media, by remodelling
the composition of specific membrane domains and by
assembly/disassembly of multimolecular complexes that
transduce signals across the membrane. This hierarchy of
responses at each stage of maturation and capacitation
ensures that only fully competent spermatozoa reach the site
of fertilization in vivo.
of Ca2+-fluxes at the plasma membrane is essential for
activation of acrosome reaction upon binding of the zona
pellucida and for chemotactic responses mediated through the
olfactory receptors that have recently been found in human
sperm.
In contrast, progesterone apparently regulates motility by
controlling mobilisation of Ca2+ stored inside the cell. It has
become clear that sperm show surprising sophistication in their
ability to generate complex spatio-temporal [Ca2+]i signals
and use them discretely to regulate the different activities of
the cell, control their ‘behaviour’ and maximise their chance
of success.
Significant progress in understanding the functioning of sperm
and the way in which their activities are regulated opens up
new possibilities for studying fertilisation, for treating male
sub-fertility and for development of new contraceptive
strategies.
S 17
Definition and epidemiology of premature
ejaculation
M. WALDINGER
This work was supported by the BBSRC, UK.
Department of Psychiatry and Neurosexology, Leyenburg
Haga Hospital, Leyweg 275, 2545 Centre Hospitalier, The
Hague, The Netherlands Email : [email protected]
S 16
No abstract provided.
Sperm calcium signalling - nature and function
S 18
S. PUBLICOVER
School of Biosciences, University of Birmingham, UK
Pathogenesis and diagnosis of premature
ejaculation
Prior to fertilisation a human sperm must complete a series
of tasks in order both to reach the egg and to be competent
to fertilise when it arrives. These functions must be activated
discretely and must be timed appropriately. They are
dependent entirely upon 2nd messenger signalling, particularly
[Ca2+]i. Compared to somatic cells, spermatozoa appear
extremely simple, reduced to only those components
necessary to achieve their single, specific purpose.
However, recent studies have transformed our understanding,
revealing the spermatozoon to be a complex, highly-organised
and tightly-regulated cell. Sperm express their own, apparently
unique, ‘toolkit’ of Ca2+ homeostatic mechanisms. Regulation
294
F. LOMBARDO
Department of Medical Pathophysiology, University of
Rome “La Sapienza”, Rome, Italy
Premature ejaculation (PE) is a symptom rather than a disease.
In fact, it can be caused by short frenulum of the prepuce,
penile hypersensitivity and reflex hyperexcitability, prostatitis
and thyroid hyperfunction. Some researchers believe that PE
is not a psychological disorder but a neurobiological
phenomenon. This is a very interesting pathophysiological
explanation, based on the role of serotoninergic system in the
control of ejaculation. As a matter of fact, the effectiveness
of serotoninergic antidepressants demonstrates that central
neurotransmission is involved in ejaculatory control, but not
that serotonin hypoactivity is the cause of PE, as suggested.
In fact, many psychological disturbances provoke a
neuroendocrine imbalance.
On the other hand, the classic psycho-sexological approach
affirms apodictically that PE is a psychosexual disorder, which
is ‘all in the mind’ with a psychogenic aetiology and
pathogenesis that must be treated with psychotherapy.
S 19
Management of premature ejaculation
S. DROUPY
Dept of Urology, Academic Hospital of Bicetre, 78 Rue du
General Leclerc, 94270 Le Kremlin-Bicetre, France
Email : [email protected]
No abstract provided.
The possibility that PE may involve marital problems should
always be considered, even though it may be difficult to
discern whether the couple’s troubles are the cause or the
effect of PE. Whenever possible, sexual symptoms should be
assessed in the context of the couple. For this reason, it is
essential to compare the male’s description of the symptom
with that of his partner. Perception of penetration time is in
fact very subjective.
The physical examination of the patient with PE is usually
normal: pathological findings are unlikely to be associated
with this condition. However, penile biothesiometric
measurement of the penile shaft, the glans penis and the
mid scrotum has been proposed as a useful method to
evaluate and qualify penis sensitivity, but denied by others.
The biothesiometer is a vibrating device with a fixed frequency
of 50 Hz and variable amplitude which is placed on these
genital areas. The patient is asked to inform the examiner of
the first sensation of vibration as the amplitude is slowly
increased.
A possible flow chart of PE diagnosis is proposed. It should
include prostate evaluation by transrectal ultrasonography
and standardized Meares & Stamey protocol (1968). Urethral
and midstream bladder urine, expressed prostate secretions
by prostate massage, and post-massage urine samples are
collected, examined microscopically, and cultured
bacteriologically. Prostate inflammation is diagnosed if 10 or
more white blood cells per high power field are present in the
expressed prostate secretions. Non-bacterial prostatitis is
defined by evidence of prostate inflammation together with
negative urine and prostate fluid cultures. Prostate infection
is defined by a colony count 10 times greater in the expressed
prostate secretion or post-massage urine sample than in the
urethral urine sample. The presence of Chlamydia
Trachomatis, Trichomonas vaginalis, Mycoplasma hominis,
Candida species, and Ureaplasma urealyticum should be
carefully checked.
Negative subjects should be further studied for the possible
presence of hyperthyroidism and, in selected cases,
neurological diseases.
295
S 20
Antiviral defences of the testis
N. DEJUCQ-RAINSFORD
GERHM-INSERM U. 625 Université de Rennes 1, Campus de Beaulieu, 35042 RENNES cedex, Bretagne,
France
Viral infection of the testis can have damaging consequences
for the fertility and general health of the individual : it may
disrupt the reproductive and endocrine functions, as illustrated
by mumps virus and HIV infection in human, and potentially
lead to testicular cancer (although the latter remains to be
formally proven). It may also participate to the spreading of
viruses from host to host through contaminated semen.
Moreover, viral infection of the germ cells may result in the
transmission of virus-induced mutations to subsequent
generations.
Acquired immune reactions are normally suppressed in the
testis in order to prevent germ cells destruction. We seek
whether the testis possesses innate defense mechanisms to
counteract viral attack and limit harmful infiltrations of
specialized immune cells. Our research was initiated in the
rat since there are no documented viral infections of the testis
in this animal. Interferons (IFNs), discovered through their
ability to “interfere” with viral replication, represent the main
component of the cell antiviral response. These cytokines
act in an autocrine/paracrine manner by inducing the
expression of various proteins (the so-called IFN-induced
proteins), the three best characterized in term of antiviral
activity being the 2’5’ oligoadenylate synthetase (25OAS),
the double strand RNA activated protein kinase PKR and the
Mx proteins. IFNs and IFN-induced proteins expression was
investigated in different rat testicular cell types in both basal
conditions and following in vitro viral stimulation (Sendai virus,
an RNA virus of the paramyxovirus family). The protection of
the germ cells in the seminiferous tubules is of prime
importance because their infection may result in the total
destruction of the germ line or the dissemination of the virus
or viral DNA to all germ cell classes, including the spermatozoa
themselves. Spermatogonia are one of the most important
potential targets because unlike the meiotic and postmeiotic
germ cells, they are not isolated from the molecules and
agents produced by the interstitial compartment.
The basal tubular position of these germ line precursors, a
priori, exposes them to the risk of contamination in cases of
viral infection. Despite the high potential risk of infection of
spermatogonia, these cells do not constitutively express
biologically active IFNs and they respond only weakly to
stimulation by the Sendai virus in vitro. Moreover, they only
weakly express the antiviral protein Mx and do not express
2’5’AS or PKR. Similarly, early spermatids and pachytene
spermatocytes, although expressing low levels of IFNs in
basal conditions, respond very weakly if at all to exposure to
the Sendai virus and do not produce any of the 3 IFN-induced
antiviral proteins tested (2’5’AS, PKR and Mx).
It was therefore suggested that the germ cell antiviral protection
system may involve mainly the somatic peritubular and Sertoli
cells within the seminiferous tubules and the somatic Leydig
cells and macrophages within the interstitium, all of which
have subsequently been shown to express both high levels
of IFNs and IFN-induced proteins in response to viral infection.
Of all the testicular cell types tested, Leydig cells and Sertoli
cells have by far the strongest antiviral potential. Thus, in the
rat testis, a first line of defense against viruses arriving in the
bloodstream is represented by Leydig cells and testicular
macrophages, whilst a second line of defense involves the
myoid cells lining the seminiferous tubules and Sertoli cells.
However, the situation may vary in between species as the
first results obtained in human Leydig cells show lack of IFN
production when exposed to a range of viral stimuli, including
mumps virus and the RNA virus-like, synthetic double strand
RNA poly IC. Despite this, virally stimulated Leydig cells overexpress two antiviral proteins, the 25OAS and MxA protein.
To fully apprehend the innate immune status of the human
testis, the antiviral capabilities of all testicular cell types will
need to be tested.
296
S 21
Viruses and semen
C. PASQUIER
Laboratoire de Virologie, Institut Fédératif de Biologie, 330
avenue de Grande Bretagne, TSA40031, 31059 Toulouse
Cedex 9, France Email : [email protected]
Most sexually transmitted viruses can also be found in male
or female genital secretions and detected by cell culture or
genomics. HBV, HSV, CMV, HHV-8, HPV, HTLV and HIV
have been studied in human semen. Some other viruses can
be detected in semen using new improved molecular biology
techniques, but they are poorly transmitted during sexual
intercourse. This is particularly true for HCV. Many other
viruses that are probably non-pathogenic may be detected in
semen. All viruses involved in viremia or associated with white
blood cells can be, at least in theory, present in semen.
The viruses in semen, can be present as free virus particles
in the semen plasma, as infected cells (with productive or
latent infections), and in the spermatozoa themselves. The
spermatozoa are unlikely to harbour productive infections,
but they may have latent infections and virus reactivation
cannot be excluded. This has been shown for human
endogenous retroviruses (HERV) and remains a discussed
possibility for HBV. The infected cells are usually lymphocytes,
monocytes or polymorphonuclear cells. An increase risk of virus
shedding is associated with the presence of these cells in
semen and when there is genital inflammation or infection.
Free virus particles in the semen can come from the blood
by passive diffusion during the viremia phase of infection or
from local virus replication. The characteristics (genome
sequence) of virus produced by local replication in semen
are often different from those of viruses in the blood, reflecting
compartmentation.
Semen is screened to detect viruses with pathogenic potential
so as to limit sexual transmission and the risk of transmission
during medically assisted procreation (MAP). Screening is
based on virus acquisition risk factors, clinical signs, systematic
or oriented biological screening. Vaccines, when available, are
then used to avoid transmission to the partner. Antiviral
treatments are becoming more and more efficient and can cure
a patient by eradicating virus from his/her body in some cases
(HCV for example). But they usually only limit virus replication
or keep it below the level needed to control clinical evolution.
These antiviral treatments can reduce the virus concentration
in the semen and thus the risk of transmission, depending on
their ability to diffuse into the male genital tract. The assays
used to detect virus in semen have improved greatly in recent
years and are now very sensitive and less influenced by
semen PCR inhibitors. However, a negative result does not
mean that virus is absent, since there is always a detection
threshold in all assays of virus genomes.
Sperm processing techniques can further decrease the risk
of transmission from patients that may have residual virus in
their semen (eg. HIV) by eliminating semen plasma and nonspermatozoa cells and by keeping only spermatozoa for MAP.
These methods have proved to be effective for treating semen
containing HIV-1 and HCV and should be just as effective for
removing other viruses.
Nevertheless, the way many viruses are shed into the semen
has not yet been characterized, especially during incubation
phase or when the infection is asymptomatic or acute. This
should be taken into account in the future management of the
risk of transmission of viral diseases.
S 22
Assisted reproductive technology in couples with
HIV-infected male partners
C. GILLLING-SMITH
Chelsea & Westminster Hospital, 369 Fulham Road,
London SW10 9NH Email : [email protected]
The development of highly active antiretroviral therapy
(HAART) over the last 10 years has transformed the prognosis
of patients infected with human immunodeficiency virus type1 (HIV) living in the developed world. HIV has been reclassified
as a chronic disease with good life expectancy and quality and
many heterosexual couples, where one or both partners are
infected, are now considering parenting as a realistic option
In a heterosexual couple where the male partner is infected,
unprotected intercourse caries a risk of transmitting HIV to the
uninfected female partner and potential child of 0.1% per act
of intercourse. Unfortunately viral load in semen correlates
poorly with that in serum and men on antiretroviral medication
with undetectable viral loads can still have detectable virus
present in semen.
Sperm washing is a specific risk-reduction option available to
HIV serodiscordant couples where the male is infected trying
to conceive. Semen is centrifuged in a density gradient to
separate live sperm, which does not carry HIV, from seminal
plasma and non-germinal cells which may carry virus and
then inseminated into the female partner at the time of
ovulation. If a couple have additional fertility issues, sperm
washing can be combined with ovulation induction, in vitro
fertilisation (IVF) or intracytoplasmic sperm injection (ICSI).
In order to ensure the final preparation used in clinical practice
is free of HIV and safe to use, an aliquot of washed sperm is
tested for detectable HIV RNA prior to the sample being used
for treatment. A nucleic acid-based sequence amplification
(NASBA) or similar commercial PCR assay can be used.
297
The clinical program for sperm washing at The Chelsea and
Westminster Hospital was established in 1999 and has
performed the largest series on treatments in the UK. 106
couples have undergone 293 cycles of IUI with a live
birth/ongoing pregnancy rate per cycle of 10.2% and clinical
pregnancy rate of 14.3%. These figures are similar to national
statistics for IUI using donor sperm and compare favourably
to results reported from other centres in Europe with similar
programs for serodiscordant couples. In terms of safety, there
have been no seroconversions in either female partner or
child on rigorous follow-up (HIV testing of female partner for
up to 6 months after the last treatment with washed sperm)
and 27 healthy children have been born to date through the
IUI program alone (45 through the sperm washing program
including IVF and ICSI cases). In our series, the cancellation
rate due to a positive NASBA post wash was 3.3%.
Semen samples from 106 HIV-positive men undergoing sperm
washing IUI weer compared with those from a control group
of 234 HIV-negative men undergoing IUI (Nicopoullos et al.,
2004). Markers of HIV were assessed and although the
majority of HIV-positive men had sperm parameters within the
defined WHO ‘normal’ range, ejaculate volume, sperm
concentration, total count, progressive motility and normal
morphology were all significantly lower in the HIV-positive
group compared to the HIV-negative controls (p < 0.05). There
was a significant positive correlation between CD4 count and
sperm concentration, total count and total and progressive
motility and a significant negative correlation with normal
sperm morphology of both raw and post preparation samples.
There was no correlation between viral load, years since
diagnosis, use of or duration of use of HAART with any semen
parameter. This is the largest analysis of semen parameters
in HIV-positive men to date.
We also analysed the factors which had an impact on IUI
outcome in our first 140 cycles of IUI with sperm washing.
Clinical pregnancy rate was significantly higher in cycles
where the man had a low viral load (<1000copies/ml; 29%
versus 11%, p=0.05) and in cycles where the man was on
HAART (27% versus 9%, p=0.02).CD4 count had no impact
on IUI outcome. Although these data clarify the factors that
influence IUI outcome, they do not shed light on the precise
mechanism by which HIV and markers of HIV infection alter
semen parameters and IUI outcome which should be the
subject of further investigation. Our present advice is that the
decision to start or stop medication should be not be based
on concern for fertility but rest primarily on viral load and CD4
count parameters and health of the individual.
Further reading
Gilling-Smith C., Nicopoullos J.D., Semprini A.E.,
Frodsham L.C. : HIV and reproductive care - a review of
current practice. BJOG. 2006, 113(8) : 869-878.
Frodsham L.C.G., Boag F., Barton S., Gilling-Smith C. :
Human immunodeficiency virus infection and fertility care
in the United Kingdom – demand and supply. Fert. Steril.,
2006, 85 : 285-289.
Nicopoullos J.D.M., Ramsay J.W.A., Almeida P.A., GillingSmith C. : The effect of HIV on sperm parameters and the
outcome of IUI following sperm-washing. Hum. Reprod.,
2004, 19 : 2289-2297.
S 23
Bacterial male genital tract infection and sperm
quality
E. VICARI, S. LA VIGNERA, F. GARRONE,
A.E. CALOGERO
Section of Endocrinology, Andrology and Internal Medicine,
Department of Biomedical Sciences, University of Catania,
Catania
The field of bacterial infection and sperm quality, stagnant for
at least three decades, has changed from the nineties years.
Two different panel of scientists evaluated this research area
ending up with two independent consensus conferences. The
first, of mainly urological background (NIH Chronic Prostatitis
Collaborative Research Network), focused on prostatitis
classification and only two categories (II and IV) were
associated with male infertility (Krieger et al., 1999; Nickel et
al., 2001). Sperm analysis was included among the optional
tests and therefore it does not play a pivotal role. This justifies
the contradictory data available on the influence of urogenital
tract infection on sperm quality with a full array of effects
reported which range from a negative impact on all main
sperm parameters (density, motility and morphology) or on
sperm motility only, to no sperm parameter alteration at all (see
Weidner et al., 1999, for review).
The second task force, of uro-andrologic background (WHO
Task Force on the Diagnosis and Treatment of Infertility (WHO,
1993), identified a diagnostic category affecting male
reproductive function and fertility which was named male
accessory gland infection (MAGI). In this classification, sperm
analysis is viewed as a fundamental step of the diagnostic
workup. In fact, MAGI is diagnosed in presence of one or
more sperm parameter abnormalities (oligo- or astheno or
teratozoospermia) associated with a combination of two or
more of the following factors :
- Factor A : history positive for urinary tract infections and/or
sexually-transmitted diseases and/or physical urogenital
examination ;
- Factor B : expressed prostate signs of infection and/or
inflammation ;
- Factor C : ejaculate signs of infection.
According to the NIH classification, seminal bacterial infection,
corresponding to chronic bacterial prostatitis (category II, NIH
classification), has a marginal epidemiologic role since bacterial
prostatitis (acute or chronic) accounts for a low number of
patients with prostate symptoms (5-10%) (see Weidner et
al., 1999, for review). Recently, Li and colleagues reported a
higher rate of bacterial infection (24.2%) in patients with
prostatitis type IV (Li et al., 2004). On the contrary, MAGI has
been reported to account for a wide range of frequency (1.6-
298
39.1%) of infertile patients according to the various infertility
clinic settings (Comhaire et al., 1986; Andreeßen et al., 1993;
Vicari, 2000; Diemer et al., 2003; Li et al., 2004).
In the course of urogenital bacterial infection, sperm quality
is the final product of germ intrinsic properties (degree of
virulence, bacterial load), time of interaction the microorganism
and the involvement of one or more sex accessory glands.
Some Gram Enterobacteriaceae, such as Escherichia coli,
Klebsiella sp., Proteus, Serratia, Pseudomonas sp, etc., have
been recognized as known prostate pathogens (category II,
NIH classification) having a strong association with a clear
positive clinical history (prior and/or recurrent urinary tract
infection, sexually transmitted disease, congenital uro-genital
abnormalities) and some uro-genital abnormalities at the
physical examination. On the other hand, the only presence
of some microrganisms is interpreted by some investigators
as "probable" (when Gram-positive pathogens, such as
Enterococcus sp, and Staphylococcus aureus, are present)
or "possible" (when coagulase negative pathogens, such as
Staphylococcus, Chlamydia, Ureaplasma, anaerobes are
present) prostate infection. The major difficulty in interpreting
microbiological findings is the presence of contaminating,
indigenous microbiota, or of inhibitory substances known to
be present in the prostatic secretions, as well as previous
courses of antibiotics. Thus, the diagnosis of bacterial prostatitis
may be confirmed by quantitative bacteriological cultures in
the semen (growth of >103 pathogenic bacteria or >104 nonpathogenic bacteria in seminal plasma diluted 1:2 with saline
solution) (Comhaire et al., 1980) or segmented cultures, i.e.
four (Meares and Stamey, 1968) and/or two (Nickel, 1997)
glass test.
In addition, underlying prostate and systemic chronic
comorbids have to be taken into account. Thus a negative
impact on sperm quality may arise from one (or more) of the
following mechanisms: 1) secretory dysfunction of one or
more male accessory glands; 2) deterioration of
spermatogenesis; and 3) (unilateral or bilateral) organic or
functional sub-obstruction of the seminal tract. Gland secretory
dysfunction represents the most relevant cause of negative
impact of sperm quality. It induces an inflammatory response
(leukocytospermia, reactive oxygen species and/or cytokines
release, such as IL-1β, IL-6, IL-8, TNFα; and autoimmune
processes) (Ochsendorf, 1999; Vicari, 2000; Vicari & Calogero,
2001; Weidner et al., 2002; Vicari et al., 2002; Diemer et al.,
2003). These bioactive substances may persist even following
successful treatment with antimicrobials, since the initial
antioxidant capacity (mainly based on epididymal biological
micronutrients of the seminal plasma) is progressively
exhausted, thereby impairing sperm function by inducing
DNA damage and/or apoptosis (Agarwal et al., 2003; Sanocka
et al., 2003). Gland structural abnormalities may, in addition,
play a negative role. In fact, infertile patients with MAGI and
elevated bacteriospermia (>105 CFU/ml) or with Clamydia or
Ureaplasma urealitycum infection (at urethral swabs after
prostate massage) have a higher number of ultrasonographyc
abnormalities involving more glands (prostato-vesiculoepididymitis, epididymo-orchitis) (Vicari, 1999; Vicari et al.,
2006). These patients show also to have an increased
inflammatory response and an impaired semen quality directly
related to MAGI extension (prostatitis < prostato-vesiculitis <
prostato-vesiculo-epididymitis) (Vicari, 1999) or a strong
association with sperm abnormality (Bayasgalan et al., 2004).
In conclusion, sperm parameters represent the "end-target"
of many possible pathophysiological mechanisms which may
contribute to the onset of infertility related to urogenital
infections. Therefore, despite an open debate with pros and
cons on the role of MAGI in male infertility is going on, the
andrologist should at least consider MAGI as a risk factor of
male infertility (Bayasgalan et al., 2004), different from one
country to another. Thus, MAGI may be or may become an
effective cause of male infertility throu the above-mentioned
multiple pathophysiological mechanisms which in turn impair
sperm function.
Support :
1. Agarwal A, Saleh RA, Bedaiwy MA : Role of reactive oxygen
species in the pathophysiology of human reproduction.
Fertil. Steril., 2003, 79 : 829.
2. Andreeβen R, Sudhoff F, Borgmann V, Nagel R : Results
of ofloxacin therapy in andrologic patients suffering from
therapy-requiring asymptomatic infections. Andrologia,
1993, 25 : 377.
3. Bayasgalan G, Naranbat D, Radnaabazar J, Lhagvasuren
T, Rowe PJ : Male infertility : risk factors in Mongolian men.
Asian J. Androl., 2004, 6 : 305.
4. Comhaire F, Verschraegen G, Vermeulen L : Diagnosis of
accessory gland infection and its possible role in male
infertility. Int. J. Androl., 1980, 3 : 32.
5. Comhaire FH, Rowe PJ, Farley TMM : The effect of
doxycicline in infertile couples with male accessory gland
infection: a double blind prospective study. Int. J. Androl.,
1986, 9 : 91.
6. Diemer T, Hales DB, Weidner W. Immune-endocrine
interactions and Leydig cell function: the role of cytokines.
Andrologia, 2003, 35 : 55.
7. Krieger JN, Nyberg L Jr, Nickel JC : NIH consensus definition
and classification of prostatitis. J.A.M.A., 1999, 282 : 236.
8. Li HJ, Xu P, Liu JS, Xing GW, Pan TM, Yang BL, Soing YH,
Huang YF : Prevalence of chronic prostatiti and its effect
on male infertility. Zhanghma Yi Xue Zazhi, 2004, 84 : 369.
9. Meares EM, Stamey TA : Bacteriological localization patterns
in bacterial prostatitis and urethritis. Invest. Urol., 1968, 5
: 492.
10 Nickel JC, Downey J, Hunter D, Clark J. Prevalence of
prostatitis-like symptoms in a population based study using
the National Institute of Health chronic prostatitis symptom
index. J. Urol., 2001, 165 : 842.
11. Nickel JC : The Pre and Post Massage Test (PPMT) : a
simple screen for prostatitis. Tech. Urol., 1997, 3 : 38.
12. Ochsendorf FR : Infections in the male genital tract and
reactive oxygen species. Hum. Reprod. Update, 1999, 5 :
299
399.
13. Sanocka D, Jedrzejczak P, Szumala-Kakol A, Fraczek
M, Kurpisz M : Male genital tract inflammation: the role od
selected interleukins in regulation of pro-oxidant and
antioxidant enzymatic substances in seminal plasma. J.
Androl., 2003, 24 : 448.
14. Vicari E. : Seminal leukocyte concentration and related
specific radical oxygen species production in different
categories of patients with male accessory gland infection.
Hum. Reprod., 1999, 14 : 2025.
15. Vicari E. : Effectiveness and limits of antimicrobial treatment
on seminal leukocyte concentration and related specific
radical oxygen species production in patients with male
accessory gland infection. Hum. Reprod., 2000, 15 : 2536.
16. Vicari E, Calogero AE. : Effect of treatment with carnitines
in patients with prostato-vesciculo-epididymitis. Hum.
Reprod., 2001, 16 : 2338.
17. Vicari E, La Vignera S, Calogero AE. : Antioxidant treatment
with carnitines is effective in infertile patients with prostatovesciculo-epididymitis and elevated seminal leukocyte
concentration after treatment with non-steroidal antiinflammatory compounds. Fertil. Steril., 2002, 78 : 1203.
18. Vicari E, La Vignera S, Castiglione R, Calogero AE. :
Sperm parameters abnormalities, low seminal fructose and
reactive oxygen species overproduction do not discriminate
patients with unilateral or bilateral post-infectious
inflammatory prostato-vesiculo-epididymitis. J. Endocrinol.
Invest., 2006, 29 :18.
19. Weidner W., Colpi GM, Hargreave TB, Papp GK, Pomerol
JM, Ghosh C : EAU Working Group on Male Infertility. EAU
Guidelines on male infertility. Eur. Urol., 2002, 42 : 313.
20. Weidner W, Krause W, Ludwig M : Relevance of male
accessory gland infection for subsequent fertility with special
focus on prostatitis. Hum. Reprod. Update, 1999, 5 : 421.
21. World Health Organization WHO manual for the
standardized investigation and diagnosis of the infertile
couple. (Rowe P, Comhaire F, Hargreave TB and Mellows
HJ, eds.), Cambridge University Press, 1993.
300
Andrologie 2006, 16, N°4 301-317
Abstracts of the Fourth European Congress of Andrology and
23rd Congress of the French Speaking Society of Andrology
Oral Communications
(OR 01 to OR 24)
OR 01
Anti-prostasome autoantibodies in serum from
prostate cancer patients
K.G. RONQUIST, L. CARLSSON, G. RONQUIST,
L. LARSSON
Department of Medical Sciences, Sweden
Email : [email protected]
Objectives : Prostasomes are secretory granules
synthesized, stored and released by normal and neoplastic
human prostate epithelial cells. The average size is 150 nm
in diameter (range 50-500 nm) and the bilayered membrane
has a very high cholesterol/phospholipid ratio making up an
interesting new biological environment. Prostasomes seem
to have several important functions in the reproduction
process. Prostasomes escaping from the urogenital tract
mount an immune response against themselves. Other cell
types have also been proved to produce extracellular vesicles
in a similar way called exosomes. Each vesicle type has a
unique membrane protein composition reflecting the cell
from which it originates. The aim with this study was to
identify the prostasome-derived proteins that were
immunogenic in serum from prostate cancer patients.
Materials and Methods : The aim was to identify the
prostasome-derived proteins that were immunogenic in
prostate cancer patients. Prostate cancer patients' sera (n
= 44) with high enzyme-linked immunosorbent assay (ELISA)
titers against prostasomes were selected for immunoblotting
against purified seminal prostasomes separated on 2D gels.
Twenty-five of the recognized proteins were isolated and
analyzed by means of mass spectrometry.
Results : Out of 44 patients' sera, 31 (70%) demonstrated
in immunoblotting experiments reactivity against several
prostasomal protein bands in the molecular weight range of
10-200 kDa. Some of the bands (55, 70 and 170 kDa) were
more frequently recognized by the patients' sera.
Concomitantly run control sera generated only very weak or
no bands at all. The most frequently occurring prostasomal
proteins were identified as heat shock proteins (HSP 70,
71) and clusterin.
Conclusion : Prostate cancer consistently remains a difficult
clinical enigma. Therefore, the development of novel
strategies for diagnosis and treatment (e.g. immunotherapy)
of prostate cancer is essential. This study identified the most
important molecular targets of autoantibodies against
prostasomes generated in connection with the development
of prostate cancer in man. These immunogenic prostasomal
proteins could be appropriate target molecules for specific
immunotherapy of prostate cancer patients.
OR 02
Prospective multicenter study on safety and
efficacy of intramuscular injections of 1000 mg
testosterone undecanoate in hypogonadal men
under conditions resembling real-life
H.M. BEHRE1, J. ELLIESEN2, AND THE
TESTOSTERONE UNDECANOATE STUDY GROUP
1 Andrology Unit, Dept. of Urology, Martin-LutherUniversity, Halle, Germany ; 2 Schering AG, Berlin,
Germany
Objective : The study was performed to evaluate
intramuscular testosterone undecanoate (TU) for treatment
301
of hypogonadal men under conditions which resemble reallife situations as closely as possible (e.g., variable injection
intervals, minimum selection of patients, i.e., including
hypogonadal patients with co-morbidities). It was intended to
test the safety profile of TU under long-term treatment with
respect to serum levels of prostate-specific antigen (PSA)
[primary study variable], prostate size measured by transrectal
ultrasonography, physical examinations and standard safety
laboratory. A further aim was to demonstrate that sustained
serum testosterone (T) levels in the physiological range can
be retained under real-life conditions, preserving satisfaction
with the treatment.
Design : Open-label, prospective, multicenter (16 centers in
Germany), one-arm study.
Material and Methods : Men with hypogonadism, aged 18
– 75 years, with serum total T levels < 10 nmol/l and no
contraindications for T therapy were included. 1000 mg TU
(Nebido®) were given at individually adjustable loading
intervals of 6 – 10 weeks between the first 2 injections and
maintaining intervals of 10 – 14 weeks (total: 6 TU injections).
Results : Of 161 patients screened, 96 could be included (age,
48.6 ± 12.7 years [mean ± SD]; height, 178.85 ± 7.84 cm; body
weight, 93.33 ± 15.71 kg; body mass index, 29.22 ± 4.80
kg/m2). Trough concentrations of total T (considered as an
indicator of the adequacy of substitution) increased from 5.60
± 3.03 nmol/l at baseline to levels between 9.79 ± 3.96 and
14.02 ± 6.06 nmol/l in the course of treatment phase. Median
values of trough T after the 2nd TU injection were 12 – 13.25
nmol/l. Mean PSA serum concentration of 0.81 ± 0.80 ng/ml
at screening moderately increased to 1.23 ± 1.33 ng/ml after
the 6th TU injection. At the time of the 5th TU injection, 75%
of the patients had PSA values ≤ 1.40 ng/ml (3rd quartile) ;
at the time of the final examination the 3rd quartile was
1.5 ng/ml. There were 7 patients with elevated PSA values
above the age-specific normal range at the final examination
compared to 1 patient at screening; however, only 2 of them
had values within the age-specific alert ranges. Malignant
changes were excluded by prostate biopsies in 4 patients
with a PSA level above 4 ng/ml. Mean prostate volume
increased from 20.66 ± 11.44 ml at screening to 22.54 ± 13.11
ml at the time of the 5th injection and 22.63 ± 12.84 ml at the
final examination. Physical examinations and standard safety
laboratory confirmed the safety of TU. 92.5% of the patients
assessed the treatment with TU as satisfying. Of them 40.4%
were “very much satisfied” and 43.6% “much satisfied”.
Conclusions : Under conditions resembling real-life situations,
intramuscular TU (Nebido®) appears to be a safe and efficient
drug for treatment of male hypogonadism. The majority of
patients express high satisfaction with the treatment.
Support : The study was supported by Schering AG, Germany.
302
OR 03
Androgens modulate the availability of active
glucocorticoids by regulating 11-SS-HSD1 activity
in murine adipocytes in vitro
A.M. ISIDORI, S. PIEROTTI, D. GIANFRILLI,
E. GIANNETTA, V. BONIFACIO AND A. LENZI
Cattedra di Endocrinologia, Dipt. Di Fisiopatologia Medica,
Univ. “La Sapienza” di Roma.
Objective : In humans, testosterone treatment affects body
fat distribution and metabolism. The mechanisms involved
in these processes are not yet entirely understood. We
hypothesized that sex hormones modulate the activity of [type
1] 11-ß-hydroxy-steroid-dehydrogenase (11-HSD1) -the
enzyme involved in the intracellular activation of
glucocorticoids.
Design : We measured intracellular fat accumulation, leptin
production (Lep) and 11-HSD1 activity in differentiating or
fully mature 3T3-L1 cells under the conditioning effects of
sex hormones.
Materials and Methods : 11-HSD1 activity was estimated
calculating the molar conversion of [H3]-dehydrocorticosterone
(DHC) into [H3]-corticosterone. Steroids were extracted using
solvents, separated by TLC and measured by ß-scanning of
the TLC plate. Testosterone (T), DHT and estradiol (E2), in
the presence/absence of androgen receptor antagonist
(flutamide) and aromatase inhibitor (formestane) were tested
at various time-points (2 to 6 days) and doses (1 nmol to 1
µmol/L) using carchoal-stripped serum and/or minimum serumfree cell cultures. Various treatment condition using a synthetic
Liver X Receptor (LXR) Agonist were also tested.
Results : We found that 3T3-L1 cells acquire 11-HSD1 activity
at early stages of differentiation and that the activity increases
steadily during differentiation in parallel with fat accumulation.
We demonstrated that androgens (T and DHT) enhance 11HSD1 activity dose-dependently either in mature and
differentiating cells (form 10% when treated with 1 nM T, to
40% with 1 µM T ; conversion measured at 90 minutes
incubation with 15 nM [H3]-DHC + 15 nM cold-DHC). We
also showed that androgen modulation of enzymatic activity
was similar to that achieved by insulin (5 ng/ml) -a known
regulator of 11-HSD1. Conversely, LXR agonist inhibited 11HSD1 activity dose-dependently. We also found that the effect
of androgens, but not insulin, were partially blunted by pretreatment with flutamide (200 nM). In addition, we showed that
T treatment on differentiating cells, inhibited leptin production
and fat accumulation/release while increasing 11-HSD1 activity.
The latter finding suggests that the effects of androgens on
the enzyme were independent of their effects on differentiation
and fat storage.
Conclusions : In summary, we showed that in an experimental
murine model androgens may regulate adipocytes metabolism
by affecting the availability of intracellular glucocorticoids.
The implication of these findings on testosterone replacement
therapy remains to be established with human models and
in vivo studies.
OR 04
Sex steroid regulation of kisseptin/GPR54
system, a new regulator of the neuroendocrine
reproductive axis, in human fetal GnRH secreting
neurons
0.01-100nM) stimulated gene expression of both KISS-1 and
GPR54. In addition, we found that leptin (1nM), an adypocytederived hormone known to act on the hypothalamus to
influence reproduction, increased the expression of KISS-1
and GPR54 mRNAs.
Conclusions : Overall, our results indicate that FNCB4 cells
represent a valuable tool to study in vitro the regulation of KISS1/GPR54 system in order to identify those permissive signals
that determine the precise timing of puberty onset and related
disorders.
OR 05
Inhibition of Phosphodiesterase type 5 is
associated to an in vivo and in vitro improved
activity of circulating angiogenic cells in men
with erectile dysfunction
A. MORELLI1, R. MANCINA1, L. VIGNOZZI1,
M. LUCONI2, G. FORTI1, M. MAGGI1
1 Andrology and 2 Endocrinology Units, Department of
Clinical Physiopathology ; 3 Department of Anatomy,
Histology and Forensic Medicine, University of Florence,
Florence, Italy ([email protected])
S. FRANCAVILLA1, M. BOCCHIO1, F. PELLICCIONE1,
G. PASSAQUALE2, S. NECOZIONE3, C. FERRI2
Objective : The hypothalamic GnRH neurons, arising from
the olfactory placode region, remain immature until puberty.
The molecular mechanisms underlying their reawakening at
puberty remain to be completely elucidated. Recently, the G
protein-coupled receptor 54 (GPR54) and its endogenous
ligand kisspeptin, encoded by the KISS-1 gene, have been
crucially involved in the hypothalamic mechanisms that time
puberty. We used the previously characterized human fetal
olfactory GnRH-secreting neurons, FNC-B4, in order to study
the KISS-1/GPR54 system.
Design and Methods : FNC-B4 are immature neurons that
can differentiate in vitro and express both neural and olfactory
markers. Moreover, these cells express and release GnRH
in response to sex steroids and odorants, as well as migrate
in response to GnRH and endothelin-1. By real-time RT-PCR
and western blot analysis we studied the kisspeptin and
GPR54 expression in FNC-B4 in response to sex steroids
stimulation.
Results : We report that these human neuronal precursor cells
express both gene and protein of kisspeptin and GPR54.
With immunohistochemistry we localized both kisspeptin and
GPR54 in sections of fetal olfactory mucosa, as well as in FNCB4 cells. Moreover, we observed that sex steroids regulated
both gene and protein expression of KISS-1/GPR54 system.
In particular, we found that increasing concentrations (0.01100nM) of 17beta-estradiol significantly decreased
(IC50=0.3nM) in a dose-dependent manner the amounts of
KISS-1 and GPR54 mRNA. Conversely, androgens (DHT,
303
Chairs of 1 Andrology, 2 Internal Medicine, 3 Epidemiology,
University of L’Aquila, Italy. Presenting author e-mail :
HYPERLINK "mailto : [email protected]"
[email protected]
Objective : Circulating angiogenic cells (CACs) contribute to
repair of the vessel wall and dysfunctional CACs are associated
to endothelial dysfunction in men with vascular risk factors
(VRFs).
Design : We investigated whether inhibition of
phosphodiesterase type 5 (PDE5) in men with erectile
dysfunction (ED) and VRFs is accompanied to changes of
activity of CACs.
Materials and Methods : Thirty six men with ED and VRFs
were randomised to 4 weeks of tadalafil (20 mg/other day) or
placebo treatment. The number of ex vivo expanded functional
CACs, identified by uptake of 1,1’-dioctadecyl-3, 3,3’, 3’tetramethylindocarbocyanine-labelled acetylated low-density
lipoprotein (DiLDL) and concomitant Ulex europaeus agglutinin
I (UEA-1) binding, was determined at baseline and after
treatment.
Results : The number of cells double-positive for DiLDL and
for UEA-1, per high-power field fluorescence microscopy was
significantly reduced in patients compared to 10 controls
(26.88±6.3 and 74.41±17.1 respectively; P<0.0001) and was
markedly increased after tadalafil treatment (40.69±13.07
versus 25.82±6.49; P<0.0001) but not after placebo. The
percentage variation of the number of CACs was correlated
with the percent change of Flow-mediated dilation in the
brachial artery by ultrasound, after treatment (R=0.59,
P=0.0004). The migratory activity of ex vivo expanded CACs
determined in a Boyden chamber system, was significantly
increased after incubation for 3 hours with tadalafil (10-6M)
(number of migrated cells:1073±363 versus 702± 204;
P=0.0007). The effect on number of cells after tadalafil
incubation was not due to an increased proliferation of CACs.
CACs proliferation was assessed by the incorporation into
cellular DNA of 5-bromo-2’-deoxyuridine (BrdU) and
subsequent staining with a TRITC-labelled mouse monoclonal
antibody anti-BrdU. The number of BrdU-positive cells
evaluated with an inverted fluorescence microscope did not
change after 3 and 24 hours of tadalafil incubation (10-6M).
Conclusion : In conclusion PDE5 inhibition has a beneficial
in vivo and in vitro effect on vascular homeostasis by improving
circulating angiogenic cell function.
OR 06
rabbit) and “in vivo” as erectile response elicited by electrical
stimulation of cavernous nerve (diabetic rats). T-substitution
(30 mg/kg, weekly) completely reverted hypogonadism and
diabetes-induced penile hypersensitivity to Y27632. To test
whether this effect was due to a T-dependent regulation of
RhoA/ROK gene expression, we measured RhoA/ROK
mRNA. Both isoforms of ROK (ROK1 and ROK2) were
analyzed by real time RT-PCR in penile samples of each rat
group. We found that ROK1 mRNA was significant increased
(p<0.05) in penile tissues from diabetic animals and restored
to the control values by T, as also confirmed by semiquantitative
RT-PCR in rabbit. Conversely, expression of RhoA and ROK2
mRNAs was not influenced neither by diabetic condition and
by T administration. Accordingly, ROK1 protein expression,
as evaluated by western blot and immunohistochemistry
analysis, resulted increased in penile samples from diabetic
animals and normalized by T.
Conclusions : Our data further support the hypothesis that
the activation of RhoA/ROK signalling contributes to diabetesrelated erectile dysfunction. Moreover, treating hypogonadism
in course of diabetes, may restore erectile function by
maintaining a correct balance between pro-erectile NO/cGMP
and anti-erectile RhoA/ ROK pathways.
Testosterone regulates RhoA/Rho-kinase
signalling in two distinct animal models of
chemical diabetes
OR 07
Cryptorchidism and in utero exposure to
endocrine disruptors in Nice area : absence of
correlation between neonatal examination and
concentrations of selected xenobiotics in cord
blood and maternal milk
L. VIGNOZZI1, A. MORELLI1, S. FILIPPI2, M. LUCONI3,
G. FORTI1, M. MAGGI1
1 Andrology and 3 Endocrinology Units, Dept. of Clinical
Physiopathology ; 2 Interdep. Lab of Functional and
Cellular Pharmacology of Reproduction, Depts. of
Pharmacology and Clinical Physiopathology; University of
Florence, Florence, Italy ([email protected])
Objectives : The RhoA/Rho-kinase (ROK) signalling pathway
is up-regulated in erectile tissue of diabetes animal models
and may contribute to diabetes-related erectile dysfunction
(ED). In previous studies we demonstrated that testosterone
(T) restores diabetes-induced ED by influencing the
NO/cGMP/PDE5 pathway. The aim of our study was to
investigate the effect of T on RhoA/ROK signalling in course
of diabetes.
Design and Methods : Two distinct animal models of chemical
diabetes: alloxan-induced in the rabbit and streptozotocininduced in the rat were used.
Results : In both models, hypogonadism was observed,
characterized by reduced T and atrophy of androgendependent accessory glands. Diabetic animals showed a
significant increase in responsiveness to increasing
concentrations of Y27632, a highly selective ROK inhibitor,
as evaluated either by “in vitro” contractility study (diabetic
304
F. BRUCKER-DAVIS1-2, A. BONGAIN 3, B. DUCOT4,
P. THONNEAU5, K. WAGNER-MAHLER6,
P. FÉNICHEL1-2
1 Endocrinology Department, CHU Nice, 06200 Nice,
France. 2 Unité INSERM, Nice, France. 3 Gynaecology
Department, CHU Nice, 06200 Nice, France. 4 Unité
INSERM, Paris, France. 5 CHU Toulouse, France. 6
Pediatry Department, CHU Nice, Nice France
Corresponding author : [email protected]
The etiology of cryptorchidism, a frequent congenital
malformation, is not completely elucidated, involving genetic
and acquired factors.
Objectives : We decided to test the hypothesis that
environmental xenobiotics with hormonal action (endocrine
disruptors) are involved. Data on in utero exposure are scarce
in France. Thus, we realized a snapshot of in utero exposure
to selected xenobiotics in our area, looking for correlations with
the testicular location at birth.
Design : We have studied over 3 years (2002-2005) newborn
boys born at 2 maternity wards (CHU Nice, and CHG Grasse),
screening them for cryptorchidism and for exposure (cord
blood and maternal milk) to selected xenobiotics known for
their anti androgenic and/or estrogenic effects. In addition,
parents were asked to fill confidential questionnaires about
their reproductive history, their origin, occupation, hobbies
and other potential exposure. This study was funded as a
PHRC 2001 and apporved by the Ethical board of our
institutions.
Material and methods : Of 6246 boys born at or after 34
weeks, 101 were cryptorchid (incidence of 1.6% in Nice and
Grasse). 95 were included in this study (48 from Nice and 47
from Grasse), as well as 2 controls matched for gestationnal
age, date and place of birth, and when possible ethnic origin.
151 serum (67 cryptorchid and 84 controls) and 125 milk (56
cryptorchid and 69 controls) were tested for the presence of
7 non planar PCBs, DDE, dibutylphtalate and its metabolite
monobutylphtalate, hexachlorobenzene, linuron, procymidone
and vinclozolin. Xenobiotic measurements were performed by
gaz chromatography coupled to mass spectometry at
accredited laboratory (Laboratoire de l’Environnement de
l’Agglomération Niçoise).
Results : All samples were positive for one or more xenobiotics
confirming the ubiquitous exposure of foetuses/infants in our
area. Concentrations were generally higher in milk, as expected
for lipophilic compounds. Concentrations, however, were
often lower than in other studies. We established scores of
exposure (high, low, negligible) using composite scores
including ÂPCBs, DDE and phthalate. We have found no
significant differences between cryptorchid and controls. As
predictive factor of high exposure, we found maternal age, and
for DDE, the geographical origin of the mother (subsaharian
and north Africa).
Conclusion : The incidence of cryptorchidism in our area is
1.6% at birth. The exposure to endocrine disruptors is
widespread, but at a lower level than in other reported series.
Our current data does not support a direct link between
exposure to a given xenobiotic and cryptorchidism in our
population.
OR 08
Does histopathological tumor type or vascular
invasion influence spermatogenesis in testicular
cancer ?
J.E. LACKNERa, A. KOLLERb, G. SCHATZLa,
M. MARBERGERa, C. KRATZIK a
Medical University of Vienna, Vienna, Austria
a Department of Urology
b Department of Pathology
Objectives : To assess quality and activity of spermatogenesis
in the contralateral testicle at the time of orchiectomy and to
assess whether any tumor related factor such as tumor type
or vascular invasion is a risk factor for impaired
spermatogenesis. An additional aim was to investigate if
areas with normal spermatogenesis for a possible harvesting
of sperms for artificial reproductive technologies could be
found in the contralateral testicle.
Methods : Contralateral biopsy specimens at the time of
unilateral orchiectomy from patients with testicular cancer
were evaluated using the Johnsen score. Median and highest
detectable Johnsen score were counted. The Johnsen score
was correlated to histopathological tumor type such as
seminoma and non seminomatous germ cell tumor (NSGCT)
and to TNM stage.
Results : From overall 76 patients 36 had a seminoma, while
40 patients had a NSGCT. In all patients a stage I disease was
diagnosed: in the seminoma group 13 pT1 and 23 pT2 tumors
and in the NSGCT group 9 pT1, 29 pT2 and 2 pT3 tumors.
The median Johnsen score of 8.9 in seminomas did not differ
significant to that in NSGCT 8.6 (p=0.348). According to the
presence of vascular invasion similar results were seen with
a median Johnsen score of 8.8 (8.1-9.2) in pT1 and 8.8 (8.29.5) in pT2 or higher stages (p=0.389). Areas with a highest
Johnsen score above 8 were found in 88.9% in seminomas
and 92.5% in NSGCT.
Conclusion : Testicular cancer is associated with impaired
spermatogenesis. Neither the histopathological tumor type nor
the presence of vascular invasion was correlated to significant
reduced spermatogenesis. When testicular sperm extraction
from contralateral testis at the time of orchiectomy is planned,
there is a high chance to find areas with normal
spermatogenesis.
305
Risk factors for development of hypogonadism in
men treated for testicular cancer :
a longitudinal study
and T60 (OR 4.4, 95% CI 1.2-16). Those hypogonadal at T0
were at higher risk of hypogonadism at T6 (OR 53, 95% CI
19-145), T12 (OR 125, 95% CI 37-430), T24 (OR 88, 95% CI
26-300) and T36 (OR 121, 95% CI 32-460). We did not find
any predictive value of age, stage of disease, AR
polymorphisms, volume or consistency of contralateral testicle.
J. EBERHARD2, O. STÅHL1-2, Y. GIWERCMAN2,
E.C. SALMONSON3, A. RIGNELL-HYDBOM4,
A. GIWERCMAN2.
Conclusion : Hypogonadism at T0 together with type of
treatment and microlithiasis of the contralateral testis seem
to be predictive factors for post-treatment hypogonadism in
men treated for TGCC. These risk factors should be taken into
consideration in the clinical follow-up of men cured for testicular
cancer.
OR 09
Department of Oncology, Lund University Hospital, Lund
University, Lund, Sweden.
Fertility Centre, Scanian Andrology Centre, Malmö
University Hospital, Lund University, Malmö, Sweden.
Department of Radiology, Lund University Hospital, Lund
University, Lund, Sweden.
Department of Environmental and Occupational Medicine,
Lund University Hospital, Lund University, Lund, Sweden.
OR 10
Increase in testicular cancer incidence in the
Midi-Pyrenees region, France
Background : TGCC (Testicular Germ Cell Cancer) patients
have been suggested to be at risk of developing reduced
Leydig cell function and hypogonadism because of both the
disease per se and its treatment. The aim of current study was
to define clinical and laboratory risk factors for developing
hypogonadism in TGCC patients.
Patients and Methods : During the period 2001-2005, 143
consecutive TGCC patients followed at the oncological
outpatient clinic, Lund University Hospital, were enrolled in the
study. Blood samples were collected at fixed time points (after
orchidectomy, prior to further therapy [T0] and 6, 12, 24, 36
and 60 months [T6, T12, T24, T36, T60] after completion of
therapy). Hypogonadism was defined as serum testosterone
<10 nmol/L and/or LH>10 IU/L. Patients having increased
hCG levels post-orchidectomy were not included. As possible
predictive factors the following were tested: hypogonadism at
T0 (yes/no); age at diagnosis (<30/>30); type of treatment
(adjuvant chemotherapy [ACT]/ 3-4 cycles chemotherapy
[HCT]/adjuvant radiotherapy [RT]); stage of disease (I/II/III
or IV); contralateral testicular microlithiasis on ultrasound
(yes/no); testicular consistency (normal/abnormal); testicular
volume (<15ml/>15ml); androgen receptor CAG and GGN
repeat length. For each post-treatment time point, odds ratios
for hypogonadism were calculated by use of binary logistic
regression. As controls 104 Swedish fishermen aged 24-49
years were used (median: 40 years).
Results : As compared to controls, significantly increased
odds ratios (OR) for hypogonadism was found at T6 (OR:
5.3, 95% confidence interval [CI] 1.6-17) and T12 (OR 3.2,
95% CI 1.1-9.3) in HCT treated men. Patients treated with ACT
or RT did not have any higher risk of becoming hypogonadal
than controls. Microlithiasis was predictive for hypogonadism
at T0 (OR 11, 95% CI 1.2-112), T12 (OR 3.9, 95% CI 1.1-13),
T24 (OR 3.0, 95% CI 1.0-8.8), T36 (OR 5.4, 95% CI 1.7-17)
306
M. WALSCHAERTS1-4, E. HUYGHE1-2, R. MIEUSSET1,
M. DAUDIN1-3, P. PLANTE1-2, P. THONNEAU1-2
1 Equipe de Recherche en Fertilité Humaine (EA 3694),
Hôpital Paule de Viguier, 31059 Toulouse
2 Service d’Urologie et d’Andrologie, Hôpital Paule de
Viguier, 31059 Toulouse
3 CECOS (Centre de Conservation du Sperme), Hôpital
Paule de Viguier, 31059 Toulouse
4 Corresponding author : Hôpital Paule de Viguier, Groupe
de Recherche en Fertilité Humaine (EA 3694), 330 av de
Grande Bretagne, TSA 70034, 31059 Toulouse Cedex 9,
France. [email protected]
Numerous international studies have observed constant and
recent progression in the incidence rate of testicular cancer
since the middle of the 20th century in industrialised countries.
Objective : To find a predictive mathematical model of the time
trend of testicular cancer incidence in the Midi-Pyrenees
region of France.
Design - Materials and Methods : Study of a consecutive
series of 506 cases of testicular cancer occurring over a 20year period (1980-1999) in the Midi-Pyrenees region. Patients
were recruited in a standardised manner by the CECOS and
the principal anti-cancer centres of the region, which made
it possible to obtain a quasi-representative study population
of testicular cancers in this area. Date of birth, year of tumour
diagnosis and histological type were available for all patients.
Incidence rates were standardised to the world population. An
age-period-cohort model was adjusted using Poisson
regression.
G. TOFTe, J.P. BONDEe, C. GIWERCMANa, T. TIIDOa,
Y.L. GIWERCMANa AND INUENDOl
Age standardised (word population) incidence rates of testicular
germ cell tumors (TGCTs) from 1980-1984 to 1995-1999 in
Southern France.
Results : Over the whole of the study period, we observed
a marked increase in the incidence of testicular cancer (cf.
figure), for both seminomas and non-seminatous germ-cell
tumours (mean annual increase 5% and 8%, respectively).
The incidence rate rose from 1.27 to 3.04 for 100 000 men
between 1980 and 1999. The age-cohort submodel appeared
to be predictive of the time trend of testicular cancer incidence
and revealed the existence of a birth-cohort effect.
Conclusion : The increased incidence of testicular cancer
recorded in the Midi-Pyrenees region over the last 20 years
reveals a birth-cohort effect similar to that observed in a
number of European countries. This birth cohort effect is an
argument in favour of the hypothesis now supported by
numerous epidemiologists, that of environmental and/or
occupational exposures to risk factors occurring early in life
or even during the embryonic period.
OR 11
Androgen receptor gene CAG repeat length as a
modifier of the association between persistent
organohalogen pollutant exposure and semen
characteristics
a Molecular Reproductive Medicine Research Unit,
Department of Clinical Sciences, Fertility Centre, Malmö
University Hospital, Lund University, Malmö ;
b Division of Occupational and Environmental Medicine
and Psychiatric Epidemiology, Lund University, Lund
University Hospital, Lund, Sweden ;
c Centre for Arctic Environmental Medicine, Nuuk,
Greenland ;
d Institute of Public Health, Department of Occupational
and Environmental Medicine, University of Aarhus, Aarhus,
Denmark ;
e Department of Occupational Medicine, Aarhus University
Hospital, Aarhus, Denmark ;
f Department of Environmental Toxicology, National
Institute of Hygiene, Warsaw, Poland ;
g Regional Clinical Center of Urology and Nephrology,
Kharkiv State Medical University, Kharkiv, Ukraine ;
h Laboratory of Human Reproduction, Kharkiv State
Medical University, Kharkiv, Ukraine ;
i Section of Toxicology and Biomedical Sciences, BIOTECMED, ENEA Casaccia Research Centre, Rome, Italy ;
j University of Modena and Reggio Emilia, Reggio Emilia,
Italy ;
k Istituto di Biologia e Genetica Universita` Politecnica
delle Marche, Ancona, Italy ; l www.inuendo.dk.
Objectives : Exposure to Persistent Organohalogen Pollutants
(POP) was suggested to impair male reproductive function.
A gene-environment interaction has been proposed. No genes
modifying the effect of POP on reproductive organs have yet
been identified. We aimed to investigate whether the CAG and
GGN polymorphisms in the androgen receptor (AR) gene
modify the effect of POP exposure on human sperm
characteristics.
Design : Multi-centre prospective collection of biological
material from partners of pregnant women (Greenland ;
Kharkiv, Ukraine and Warsaw, Poland) and men from general
population (Swedish fishermen).
Materials and Methods : Semen and blood from 680 men
(mean (SD) age 34 (10) years) from Greenland, Sweden,
Warsaw (Poland) and Kharkiv (Ukraine) were collected. POP
exposure was assessed by measuring serum levels of 2,2’,
4,4’5,5’-hexachlorobiphenyl (CB-153) and dichlorodiphenyl
dichloroethene ( p,p´-DDE). Semen characteristics (volume,
sperm concentration, total count, proportion of progressively
motile, morphology) and DNA Fragmentation Index (DFI).
CAG and GGN repeat lengths were determined by direct
sequencing of leukocyte DNA.
A. GIWERCMANa, L. RYLANDERb,
A. RIGNELL-HYDBOMb, B.A.G. JÖNSSONb,
H.S. PEDERSENc, J.K. LUDWICKIf, V. LESOVOYg,
Results : Taking into consideration the inter-centre differences
in exposure levels and genotype distribution and including age
and time of abstinence as confounders, a statistically significant
V. ZVYEZDAYh, M. SPANOi, G.C. MANICARDIj,
D. BIZZAROk, E.C. BONEFELD-JØRGENSENd,
307
interaction was found between the CB-153 group and CAG
repeat category in relation to sperm concentration and total
sperm count (p=0.03 and p=0.01, respectively). For p,p’DDE, in the European cohorts a significant interaction was
found in relation to DFI (p=0.01). For CAG<20 sperm
concentration and total sperm count were 35% and 42%
lower, respectively, when the group with CB-153 exposure
above median was compared to that below the median. DFI
was 40% higher in high p,p´-DDE exposure group for CAG≤21.
Conclusions : The observed effect of POP exposure on
sperm number was not seen when the genotype was not
taken into consideration. This is the first study indicating geneenvironment interaction in relation to semen parameters. AR
CAG repeat length might modify the susceptibility of an
individual to the adverse effects of POP exposure on semen
quality. These findings need to be confirmed by other studies.
Support : The study was supported by The European
Commission to the 5th Framework Programme Quality of
Life and Management of living resources, Key action four on
environment and health, (Contract no. QLK4-CT-2001-00202),
running 01.01.02-30.06.05. www.inuendo.dk. The Ukrainian
part of the study was supported by a grant from INTAS
(Contract no. 2001 2205). The Swedish part of the study has
also been funded by the Swedish Medical Research Council
(Grant No : 521-2004-6072) and the Swedish Research
Council for Environment, Agricultural sciences and Spatial
Planning.
population have become priority targets for healthcare
providers. A multi-centre longitudinal population cohort study
was undertaken in Europe to investigate the relationships
between age-related changes in hormones, their potential
causes and the common clinical features of ageing in men.
The present work aims to describe the changes in the
reproductive endocrine axis in ageing men, and to identify the
underlying mechanisms and associated aetiological factors.
A total of 3369 men aged 40 to 79 years were randomly
recruited from the general population of eight cities
(Manchester, Leuven, Tartu, Malmo, Szeged, Lodz, Florence
and Santiago de Compostela). Total testosterone (TT), sex
hormone binding globulin (SHBG), Luteinising hormone (LH)
were measures in a central laboratory (Roche, Elecys E170)
on a single fasting blood sample obtained before 11.00h.
Free T (FT) was calculated from TT and SHBG.
There was a significant age-related decline in TT and FT and
an increase in LH and SHBG.
Multiple linear regression, linear spline fitting and multinomial
regression analyses were used to investigate the combined
effects of age and other predictors. We identified four distinct
loci of dysregulation in the feedforward/feedback mechanism
in the reproductive endocrine axis, which simultaneously
contributed to the overall pattern of circulating hormone
changes with increasing age. Each of these mechanisms
was associated with a specific factor/aetiology.
Age : with increasing age, the testicular response to LH was
impaired and compensated by increased LH The testicular
dysfunction was amplified by the age-related increase in
SHBG which resulted in a steeper decline in FT compared to
TT.
OR 12
Mechanisms underlying the age related fall in
testosterone in men : results from the european
male ageing study (EMAS)
Obesity : (general and central) – high BMI and waist
circumference were associated with low TT and FT. This was
not accompanied by a compensatory rise in LH indicating
the presence of hypothalamus/pituitary dysfunction.
Co-morbidity : was associated with a more exaggerated
rise in LH in men over the age of 70 indicating that testosterone
feedback at hypothalamus/pituitary level was impaired or
attenuated.
A. TAJAR1, F.C.W. WU1, T. O’NEILL1, J.D. FINN1,
G. BARTFAI2, F. CASANUEVA3, G. FORTI4,
A. GIWERCMAN5, I. HUHTANEIMI6, K. KULA7,
M. PUNAB8, A.J.S. SILMAN1, D. VANDERSCHUEREN9
AND THE EMAS GROUP
Smoking : increased SHBG, total testosterone and LH with
only a minimal change in free T. This was compatible with a
primary effect on the liver which triggers the initial rise in
SHBG followed by a resetting of the feedback at a higher
level of LH to maintain normal free testosterone.
1 University of Manchester (UK), 2 Szeged University
(Hungary), 3 Universidade de Santiago de Compostela
(Spain), 4 University of Florence (Italy), 5 Lund University
(Sweden), 6 University of Turku (Finland), 7 Medical
University of Lodz (Poland), 8 Medical University of Tartu
(Estonia), 9 Katholieke Universiteit Leuven (Belgium)
We conclude that the age-related decline in circulating
testosterone embraces a combination of specific
dysregulations of hypothalamic-pituitary-testicular axis
functions, each of which is associated with a distinct risk
predictor. These mechanisms suggest that ageing is
associated with compensated primary hypogonadism which
develops into overt primary hypogonadism in the presence
of comorbidity. Obesity on the other hand is associated with
secondary (hypogonadotrophic) hypogonadism. Smoking is
associated with increased TT due to elevated SHBG.
With the continuing rise in average life expectancy, care and
prevention of disease/disability for the burgeoning elderly
308
email : [email protected]
OR 13
Analysis of X-Y interactions in developing
spermatids and characterisation of novel Y
transcripts
Other deregulated transcripts indicate downstream responses
to the deletions. The array data highlighted the spermatidspecific up-regulation of X-linked genes in these deleted
models (2).
Figure 2 : shows the X chromosome bias of up-regulated
genes in the 100% Yq deletion model.
Southern blotting and BLAST searches were used to determine
autosomal, X or Y linkage for novel unmapped transcripts, and
to confirm the mapping of selected deregulated transcripts.
Intriguingly, we discovered that an up-regulated transcript,
the RIKEN cDNA AK006152, had a male specific locus (figure
3a). This transcript was further mapped to Yp by PCR on
genomic DNA from three mouse lines with deletions on Yp,
indicating this gene is located either on telomeric Yp or within
the centromeric Rbmy cluster (figure 3b). Transcription of the
male specific locus AK006152 was shown by RT-PCR (figure
3c) to be testis specific.
L. FERGUSON1, P.J.I. ELLIS1, P.S. BURGOYNE2,
N.A. AFFARA1
1 University of Cambridge Department of Pathology,
England
2 National Institute for Medical Research, England
[email protected]
Objective : A series of mouse lines with deletions on the Y
chromosome display phenotypes ranging from
teratozoospermia to complete infertility and grossly abnormal
sperm heads, depending on the extent and location of the
deletion. Some of the sub-fertile models display a sex ratio
distortion towards females. We have extended our
transcriptional analysis of these models to a comprehensive
gene set and mapped a selection of genes whose expression
is altered by the deletions. Our goal is to identify novel Ylinked genes contained within the deletions and elucidate
downstream genetic mechanisms linking genotype to
phenotype.
Design / Materials and Methods : Array and quantitative RTPCR data (1,2) was extended to wider gene-sets (a 36,000
probe oligonucleotide set and the MmcDNAv1 chip.) Labelled
testis cDNA from each of four Y deletion models was cohybridised with labelled testis cDNA from wild-type mice of the
same strain as a common control. Lowess normalisation and
Z-score analysis was used to select significantly regulated
genes. Expression profiles were confirmed using RT-PCR. The
chromosomal location and copy number of novel (previously
uncharacterised) transcripts of interest was determined via
Southern blotting and confirmed by PCR analysis of genomic
DNA from the deleted models.
Figures : 3a : Southern blot identifying male specific band. M
= male, F = female.
3b : Genomic PCR identifying the presence of the loci in all
3 deletion models indicating a distal or proximal location on
Yp common to all 3 models.
3c : RT-PCR confirming expression of the transcript is testis
specific.
Southern blotting has also identified Y chromosome
homologues of other up-regulated X-linked and autosomal
genes (data not shown).
Characterisation of deleted Yq linked genes may help in
understanding the mechanism linking these deletions to the
up-regulation of X-linked and Yp-linked genes and may be
important in defining the phenotypes of these Y deletion
models. (3)
Conclusions : We can conclude that while there is an overall
deregulation across the genome in the mouse Y chromosome
deletion models, the number of genes up regulated on the X
chromosome is significantly above average. This indicates a
de-repression of X-linked genes (and some Yp-linked) as a
consequence of these MSYq chromosomal deletions.
Figure 1 : MA plots of oligonucleotide arrays for each Y
deletion model. Red plots are significantly up or down regulated
transcripts with a Z score >1.28 (p = 0.1).
We also conclude that several significantly down-regulated,
uncharacterised transcripts in the Yq deletion models have Yq
genomic loci and may have a role in the phenotypes of these
models.
Figure 2 : Genomic distribution of significantly up-regulated
genes in the 100% Yq deletion model with respect to
chromosomal gene density (ensembl) is biased towards the
X chromosome.
Further studies of these novel Yq transcripts and the upregulated X and Yq linked genes may provide insight into the
mechanism of this proposed competitive interaction of the X
and Y chromosomes in murine spermatogenesis.
Results : Figure 1 : shows the significant differentially
expressed transcripts (red) in the deletion models. Downregulated Y-linked transcripts indicate loci deleted from the Y
chromosome in the various models. Two down-regulated
transcripts with genomic loci within the Sly (Sycp3 like Ylinked) gene family were identified, one of which is transcribed
in anti-sense with respect to Sly.
Support :
1. Ellis P.J.I. et al. Hum. Mol. Gen., 2005, 14, 2705-2715.
2. Ellis P.J.I. et al. Mol. Hum. Reprod., 2004, 10, 271-281.
3. Toure A., E.J. Clemente et al. Genome Biol., 2005, 6, 102.0102.15
309
OR 14
Germline hMLH3 variants in maturation arrest
C. FERRAS1, X. ZHOU2, S. FERNANDES1,
M. SOUSA1-3, A. LINDBLOM2, A. BARROS1-4
patients. T2896C was detected in one secondary MA. C2531T
combined with IVS9+66G->A were identified only in primary
MA cases (3 of 4 patients, 75%; 95% CI 21.9-98.7%; p=0.003
to secondary MA cases. Secondary MA cases revealed none
of these changes (0 of 9, 0%; 95% CI 0-37.1%). The intronic
variant IVS9+66G->A was identified in 5 of secondary MA
cases (55.6%, 95% CI 22.7%-84.7%): two with past history
of spermiogenesis, two with DAZ1/DAZ2 microdeletions and
one with panhypopituitarism.
1 Genetics Department, Faculty of Medicine, University of
Porto, Portugal ; 2 Department of Molecular Medicine,
Karolinska Institutet, Stockolm, Sweden ; 3 Lab Cell
Biology, ICBAS, University of Porto, Portugal ; 4 Centre for
Reproductive Genetics A Barros, Portugal.
C. Ferras (HYPERLINK "mailto:[email protected])1"
[email protected])
Conclusions : Our results suggest that primary spematogenic
arrest is associated with two simultaneous (C2531T and
IVS9+66G/A) hMLH3 variants. As isolated C2531T and
IVS9+66G/A variants were previously described in several
families with colorectal cancer as well as in controls, the
presence of two simultaneous hMLH3 variants in patients
with primary MA might represent an additive predisposing
risk and be the cause of spermatogenic arrest.
Objective : To investigate whether hMLH3 gene mutations
could be associated with meiosis impairment and consequently
with human maturation arrest.
Support :
1. Lipkin et al. (2002),
2. Liu et al. (2003),
3. Loukola et al. (2000),
4. Sousa et al. (2002),
5. Ferrás et al. (2004).
Design : MutL homologue 3 is a member of a family of
conserved proteins involved in DNA mismatch repair during
replication and meiotic recombination. MLH3-deficient mice
do not show microsatellite instability (MSI) and are sterile
due to meiotic arrest (1). Hereditary nonpolyposis colorectal
cancer (HNPCC) is commonly associated with defects of
DNA MMR genes and MSI, but involvement of the human
MLH3 gene in DNA MMR is currently under debate, as
mutations have either been associated or found to be absent
in HNPCC. However, there is no experimental evidence for
a similar hMLH3 involvement in human meiosis.
Material and Methods : Thirteen spermatogenic arrest
patients studied for AZF/DAZ deletions were used for MLH3
mutation analysis. Genomic DNA was isolated from the
testicular biopsies using a DNeasy Tissue Kit (Qiagen). MSI
status was determined using the HNPCC MIS test (Roche)
and the BAT34 mononucleotide marker. The 12 coding exons
and exon-intron boundaries of the hMLH3 gene were studied
by DHPLC and sequencing (2,3). The primary spermatocyte
stage arrest on the testicular biopsies was confirmed by FISH
analysis of chromosomes 7/18/X/Y in isolated germ cells (4).
Primary MA was diagnosed when no evident cause for the
meiotic arrest was found. Secondary MA was used as a
control group and was established with patients with conserved
spermatogenesis in their past history, AZFc and DAZ1/DAZ2
microdeletions (5) or hormonal disturbs like hypogonadotrophic
hypogonadism.
Statistics analyses were performed by using the difference
between two proportions test (Statistica, vs. 5.1) with a
probability value of 0.05 being regarded as significant.
Results : All MA patients had a normal karyotype (46,XY) and
none presented a personal or family history of HNPCC. Four
out of 13 MA patients were considered primary MA and the
remaining 9 cases secondary MA. MSI was negative for all
310
OR 15
Administration of estradiol stimulates
proliferation of premieiotic germ cells and
facilitates precocious maturation of Sertoli cells,
promoting germ cell survival
R. WALCZAK-JEDRZEJOWSKA,
J. SLOWIKOWKA-HILCZER, K. MARCHLEWSKA,
K. KULA
Department of Andrology & Reproductive Endocrinology,
Medical University of Lodz, Poland.
E-mail:[email protected]
Estradiol may participate in spermatogenic onset but its roles
in Sertoli and germ cell multiplication and apoptosis are not
clear.
Methods : Newborn male rats were daily injected between
the 5th and 15th day of life with: 12.5 µg of estradiol benzoate
(EB) or 7.5 IU of human purified FSH (hFSH) or EB+hFSH
or solvents (control–C). On the 16th day blood was taken for
hormone measurements and one testis for histology.
Proliferation of germ and Sertoli cells were quantitatively
evaluated by incidence of PCNA positive (+) and apoptosis
by TUNEL+ cells.
Results : I) Blood level of testosterone was reduced to 25%
of C (p<0.01) after EB, to 49% of C (p<0.01) after EB+hFSH
and increased to 186% of C (p<0.05) after hFSH. Blood
concentrations of prolactin increased to 1425% or 1265% of
C after EB or EB+hFSH. (p<0.001). Blood triiodothyronine was
slightly reduced by EB (84% of C, p<0.05) and by hFSH
(90% of C, p<0.05) and not changed after EB+hFSH. EB did
not influence blood levels of FSH or LH.
II) In all groups first spermatogenesis progressed up to
pachytene spermatocytes. Testes weight and tubule length
were reduced by half of C after EB (p<0.001) and were
doubled after hFSH or EB+hFSH (p<0.001).
Seminal tubule diameter was reduced by EB (69.5±2.6 µm
vs. 79.0±2.8 in C, p<0.001), was stimulated by EB+hFSH
(94.1±1.9 µm, p<0.001) and not changed by hFSH alone.
The same concerned mean area of Sertoli cell nucleus.
Similarly, while the formation of seminal tubules lumen
increased by 3150% of C (p<0.01) after EB+hFSH, it was
not affected by hFSH or EB alone, indicating that precocious
completion of Sertoli cell maturation appeared after
combination of estradiol and FSH with a possible involvement
of prolactin.
III) Incidence of PCNA+ germ cells was increased by all
treatments: 90.8±3.7% of cells for EB; 92.5±2.1 for hFSH
and 91.2±1.6 for EB+hFSH vs. 77.7±2.2 for C (p<0.001).
Incidence of TUNEL+ germ cells was stimulated by EB to
196% of C (p<0.05), not affected by hFSH, and inhibited to
32% of C (p<0.05) after EB+hFSH.
IV) Incidence of Sertoli PCNA+ cells was increased after
hFSH to 420% of C (p<0.001) and reduced after EB or
EB+hFSH to 25% of C (p<0.01). Incidence of Sertoli TUNEL+
cells was increased after EB to 967% of C (p<0.05) and not
affected by hFSH or EB+hFSH.
Conclusions :
1) Administration of estradiol decreases testis maturation and
secretion of testosterone but increases secretion of prolactin.
2) Estradiol, FSH and possibly prolactin increase proliferation
of germ cells and hence all hormones may be important for
triggering spermatogenesis.
3) Estradiol in synergism with FSH and possibly prolactin
(but neither one of the hormone alone) induce acceleration
of testis growth and Sertoli cell maturation, what in turn
protects germ cells from apoptosis.
4) While FSH stimulates, administration of estradiol inhibits
proliferation of Sertoli cells facilitating the shift into
nonproliferative functional phase of Sertoli cell development.
OR 16
Transition protein (TP) expression in spermatids
detected in ejaculates from Infertile men
Y. SOFFER, S. BECKER, L. YOGEV*, L. SHOCHAT, L.
LEWIN, R. GOLAN
Department of Human Molecular Genetics and
Biochemistry Sackler School of Medicine, and *Institute for
the Study of Fertility, Tel Aviv Sourasky Medical Center.
Tel-Aviv University.
Objective : TPs are DNA binding nuclear proteins found in
spermatids during their differentiation. In rats, mice and
humans TP2 appears first, shortly followed by TP1. Then
protamine appears. The objective was to detect TP expression
in seminal spermatids from infertile men .
Design : Out of 60 human semen samples from infertile men
submitted to flow cytometry (FC) using propidium iodide
staining, 23 samples demonstrated immature haploid cells on
FC histograms, with noncondensed immature 1c DNA peaks.
These samples were submitted to immunofluoresent staining
of TP1 and TP2 and examined under confocal microscopy.
Materials and Methods : Samples were smeared on slides
covered with polylysine and incubated in citrate buffer (0.1
M) at 900C for 10 minutes. Antibodies against transition
proteins TP1 and TP2 (provided by Dr. W. S. Kistler) were
added and incubated overnight, slides washed (3 times in
Triton x-100 0.1%) and secondary antibody conjugated with
FITC) was added for fluorescence and confocal microscopy.
Sequential optical sections were prepared to demonstrate
that TP expression was found inside the nuclei.
Results : TP expression was observed in 12 samples out of
23. In 9 samples, TP1 & TP2 were both expressed and in 3
other samples, one of them only. Sequential optical sections
using confocal microscopy demonstrated TP expression inside
the nuclei. Flow cytometry performed on the semen samples
revealed 4 peaks of haploid immature cells with various
degrees of condensation. No clear relationship was found
between degree of condensation and appearance of TP in the
spermatids.
Conclusion : TP expression was demonstrated inside the
nuclei of spermatids in about half of samples of infertile human
semen samples containing immature haploid cells indicating
disturbance of differentiation in spermatids. Similarities between
sperm defects found in TP’s mutants animals and infertile
patients (Shirley et al., 2006) highlight the importance of TP’s
expression research in infertility. In this study, the heterogeneity
of our infertile patients group and its multifactorial etiology didn’t
allow us to correlate TP’s expression with clinical data.
Support : In part, by a grant of Israel Ministry of Health Chief
Scientist Office.
311
OR 17
β in subfertile men
The diagnostic role of Inhibin-β
and its correlation with testicular cytology
β as independent parameters and FNA biopsy result as the
dependent parameter revealed that only Inh-β was a significant
predictor of the FNA result (r2 = 0.289, p < 0,001).
Conclusions : Basal serum Inh-β seems to constitute an
important diagnostic parameter in male subfertility, as it reflects
both Sertoli cell reserve and testicular cytology. On the other
hand, Sertoli cell reserve estimation, as estimated by EFSERT,
does not seem to provide additional information for the
diagnostic or the therapeutic approach of the subfertile male.
P. POLICHRONOU, TH. MIKOS, D.G. GOULIS,
G. GRIMBIZIS, A. PAPANICOLAOU, S. GEROU,
J. BONTIS, J. PAPADIMAS
Presenting author : Prof. J. Papadimas, 1st Department of
Obstetrics & Gynaecology, “Papageorgiou” General Hospital,
Periferiaki Odos, Nea Efkarpia, 564 03, Thessaloniki, Greece.
Unit of Reproductive Endocrinology, 1st Department of
Obstetrics & Gynaecology, Aristotle University of
Thessaloniki, Greece
OR 18
Objective : Inhibin-β (Inh-β) is produced almost exclusively
by Sertoli cells and controls FSH secretion through a negative
feedback mechanism. The role of Inh-β as a diagnostic tool
in male infertility is not fully elucidated. The aim of this study
was to determine basal and stimulated Inh-β serum levels in
fertile and infertile men and to correlate them with testicular
cytology.
Design : Prospective, cross-sectional, clinical study.
Materials and Methods : Sixty-seven subfertile and 29 fertile
men underwent clinical, sperm, basal hormonal and dynamic
Sertoli-cell evaluation (EFSER Test: serum Inh-β levels before
and 24 h and 48 h after administration of 300 IU rhFSH
subcutaneously). Genetic studies were applied as appropriate.
Fifty-four of the subfertile men underwent testicular Fine
Needle Aspiration biopsy (FNA). The final clinical diagnoses
were: Idiopathic Non-Obstructive Azoospermia (INOA – n =
31, 46%, 5 of them with Late-Onset Hypogonadism - LOH),
varicocele (n = 14, 21%), cryptorchidism (n = 10, 15%) and
other causes (n=12, 18%).
Results : There was significant difference between subfertile
and fertile men regarding the basal (55.8 ± 51.2 vs 115.7 ±
54.0 pg/ml, respectively, p < 0,001) but not the stimulated
Inh-β levels (64.6 ± 71.8 vs 69.7 ± 23.6 pg/ml, p = NS at 24
h and 63.3 ± 79.2 vs 87.9 ± 25.1 pg/ml, p = NS at 48 h).
Patients with INOA and LOH, INOA only and cryptorchidism
had the most severe Sertoli cell damage according to basal
Inh-β levels followed by varicocele. There was significant
correlation between basal Inh-β levels and FNA findings (r =
0.488, p < 0.01) with lower Inh-β levels corresponding to
more severe cytology diagnoses (Sertoli Cell-Only Syndrome
–SCOS and maturation arrest). We applied a Receiver
Operative Characteristics (ROC) analysis for sperm retrieval
in testicular FNA (i.e. cytological diagnoses of normal
spermatogenesis and hypospermatogenesis). The area under
the ROC curve was 0.663 for FSH and 0.725 for Inh-β, with
a threshold level of 54 pg/ml having a sensitivity of 59% and
a specificity of 86% for sperm retrieval. Multivariate stepwise
logistic regression analysis having age, mean testicular
volume, serum FSH, serum testosterone and basal serum Inh-
312
Testosterone undecanoate is detected in seminal
plasma and significantly increases serum
dihydrotestosterone
G. MITIOS, N. KAPOLLA, H. LASS*, E. KOUKKOU,
S.C. NICOPOULOU, D.A. ADAMOPOULOS
Endocrine Department, Elena Venizelou Hospital,
115 21 Athens
* Bioanalytics, NV, Organon Oss and Organon
Development GmbH, Waltrop, Germany
Objective : Testosterone undecanoate (TU) has been used
as a complimentary treatment to tamoxifen citrate in men
with idiopathic oligozoospermia (I.O.) with satisfactory results.
It has been postulated that TU through a dihydrotestosterone
(DHT) promoting effect improves mainly epididymal
environment and consequently the quality of spermatozoa.
Design : To examine this possibility, TU has been administered
(40 ng t.i.d.) to 18 healthy, normozoospermic volunteers for
a week, with measurements of TU, total testosterone (TT),
dihydrotestosterone (DHT), E2, SHBG, FSH, LH and PRL.
Steroid hormones were measured in peripheral blood and
on some occasions, in seminal plasma before and after TU
administration. Moreover, steroid hormones were also
measured in a small number of testicular tissue samples from
5 unrelated men who also received TU before biopsy.
Materials-Methods : A home made assay was employed for
TU estimations and AutoDelphia assays for the remaining
hormones. Measurements in seminal fluid and testicular tissue
were made following standard extraction techniques. Analysis
of the results was made following standard procedures.
Results : TU had no effect on serum FSH (pre-: 6.0±0.7,
post-: 5.1±2.5 U/mL), LH (pre-: 5.0±2.0, post-: 4.0±2.0 U/mL),
PRL (pre-: 5.1±3.7, post-: 4.0±2.0 U/mL), SHBG (pre-:
30.0±13.8, post-: 26.0±12.2 nmol/L), TT (pre-: 4.88±1.63,
post-: 4.24±1.69 ng/mL) and E2 (pre-: 27.6±8.0, post-: 26.9±5.4
pg/mL). Following TU treatment, a marked increase of DHT
was observed in peripheral blood (pre-: 0.46±0.20, post-:
1.14±0.74 ng/mL, P<0.001, ratio: 0.41±0.15, increase: 248%)
whereas no marked differences were noted for the remaining
hormones. In seminal plasma, E2 and TT did not change
after TU (E2: 73.4±19.3 vs 70.2±24.3, TT: 0.6±0.1 vs 0.7±0.2)
whereas DHT was not measured (by mistake). In the same
fluid, TU was detectable in 6 of the 12 the samples assayed
(11.1±8.0 ng/mL) and serum levels were comparable at
12.7±7.6 ng/mL. The blood to seminal plasma ratio was 1.14
signifying that the average seminal TU concentration was
87.4% of that of peripheral blood.
In testicular tissue extracts, high E2 and T values (E2:
92.5±54.3 pg/mL, T: 48.8±16.3 ng/mL of extract dilution) were
seen but TU was undetectable.
Correlation analysis among the parameters examined showed
that E2 and TU in serum and seminal plasma were significantly
correlated both before and after treatment (pre : P<0.01, 0.02
post: 0.04, 0.02). Moreover, correlations were noted before
and on TU between E2 and DHT in serum (P<0.02, 0.01)
and seminal plasma (P<0.001 for both).
Conclusions : By and large, it appears that TU administration
increased markedly serum DHT levels, making a beneficial
effect on epididymal and accessory gland function a distinct
possibility. TU was measurable in a number of seminal plasma
samples in a concentration close to that of peripheral blood,
indicating a clear presence of this lipophylic agent in the
relevant compartments of the male genital tract. These two
findings may contribute to the understanding of TU’s beneficial
effect in sperm dynamics in I.O. patients.
Support : T and DHT hormone assays were performed at
Department of Bioanalytics, NV Organon, Oss The
Netherlands, whereas TU and other hormones were analyzed
at the Department of Bioanalytics, Organon Waltrop GmbH,
Waltrop, Germany. The study was sponsored by NV Organon,
Oss, The Netherlands.
de Medicina de Lisboa ; 3 Instituto de Patologia e
Imunologia Molecular da Universidade do Porto ; 4
Unidade Pluridisciplinar de Reprodução Humana, Hospital
de Santa Maria, Lisboa, Portugal
([email protected])
Objective : The highly repetitive structure of the AZFc locus
is a putative generator of Y diversity. We hypothesize that such
genetic variability could account for the variable phenotypes
detected in patients with partial AZFc deletions. To test this,
we characterized with novel amplicon-specific markers the
exact structure of the AZFc locus of men with partial AZFc
deletions, in order to identify sequence rearrangements that
could be associated to specific infertility phenotypes.
Design : Gene and transcription unit copies previously mapped
to AZFc amplicons were compared in silico in order to find
amplicon specific nucleotide markers. Thus 7 novel ampliconspecific sequence family variants (SFVs) were designed and
characterized in patients with partial AZFc deletions. These
patients were selected after a previous STS screening of
fertile and infertile men. Results were correlated to Yhaplogroup and (in)fertility phenotype.
Materials and Methods : The available reference sequence
copies of the PRY gene, TTY4 transcript and the grey
amplicons were aligned and compared (ClustalW software,
EMBL-EBI). Significant amplicon-specific nucleotide variants
associated to differential endonuclease restriction patterns
were selected, resulting in 7 new amplicon-specific SFVs:
b2, g1, gr, b3, g2, g3 and b4.
An initial screening for partial AZFc deletions was performed
using a set of STSs (sY142, sY1197, sY1192, sY1291, sY1206
and sY1201) in 299 idiopathic infertile men and 220 fertile men.
Deletions were confirmed by Eco-RV DNA blotting using the
49f probe.
The AZFc ampliconic composition of the selected patients
was characterized by sequencing all novel SFVs and by
digestion profiling of previously described SFVs (DAZ-SNV
I, DAZ-SNV II, sY586 and GOLY-SFV) and STS (Y-DAZ3).
Y-haplogroup analysis of the patients having deletions was
performed by typing 35 Y-SNPs. Statistical analysis was
performed using SPSS v13.0 software.
OR 19
Characterizing the ampliconic composition of
men with partial AZFc deletions – insights into
how sequence rearrangement dynamics shape
the AZFc locus
P. NAVARRO1,2, L. PEREIRA3, L. GUSMÃO3,
J. CALHAZ4, C.E. PLANCHA2, J.GONÇALVES1
1 Centro de Genética Humana, Instituto Nacional de
Saúde Dr Ricardo Jorge ; 2 Unidade de Biologia da
Reprodução, Instituto de Medicina Molecular, Faculdade
Results : Partial AZFc deletions were significantly more
frequent in infertile men than in fertile controls : 16/299 (5.4%)
vs. 3/220 (1.4%; P<0.05). Although by STS typing all of the
19 deletions fit into the gr/gr profile, AZFc amplicon content
in these patients varied significantly (14 different AZFc
structures). Interestingly, the previously described gr/gr deletion
mechanism could only be resolved in patients belonging to
the R haplogroup (12/19). In this haplogroup, 7 r1/r3, 3 g1/g3
and 2 r1/r4 deletions were characterized, as well as 4 gene
conversions and 3 amplicon inversions. No correlation between
AZFc structure and fertility was detected since the ampliconic
compositions recorded for fertile men were also observed in
oligo and azoospermic patients.
Conclusions : The sequence rearrangement events operating
313
in the AZFc locus result in high sequence diversity. This is
observed on a large scale in different haplogroups, and on a
smaller scale inside each haplogroup. This diversity results
in different substrates for deletion mechanisms, which are
reflected in the diverse products of the gr/gr deletion resolved
by the new markers. Our data indicate that partial AZFc
deletions reflect the evolutionary history of that particular
AZFc locus and that, although a fertility risk, correlation
between a specific AZFc composition and infertility cannot be
established, suggesting that AZFc genes are part of a
multifactorial network.
Support : Partially funded by Fundação para a Ciência e
Tecnologia: #SFRH/BD/16662/2004, and POCTI/
SAU/97/2001.
OR 20
SNV (Single Nucleotide Variants) or STS
(Sequence Tagged Sites) : Which choice for
partial AZFc deletions and DAZ copies
detection ?
I. AKNIN-SEIFER1,2, R.L. TOURAINE2, A.K. FAURE3,
K. MCELREAVEY4 , J. CHOUTEAU5, R. LEVY1
1 Laboratoire de Biologie de la Reproduction, Hôpital Nord,
CHU de Saint-Etienne. 2 Laboratoire de Génétique de
l'hôpital Nord, Saint-Etienne. 3 Laboratoire de Biologie de
la Procréation, CHU de Grenoble. 4 Reproduction, Fertilité
et Populations, Institut Pasteur, Paris. 5 Clinilab, SaintMartin d’Hères. Tel. : 04-77-82-83-07
Fax : 04-77-8287-84 e-mail : [email protected]
Aim of study : Partial deletions of the AZFc region
(AZoospermia Factor) of the Y chromosome have been
reported to be a significant risk factor for oligo-/azoospermia.
Within the AZFc region, the DAZ (Deleted in AZoospermia)
gene family is organized into two clusters with a variable
number of copies with 99.9% homology. DAZ1/DAZ2 deletions
have been found only in infertile men, as opposed to
DAZ3/DAZ4 deletions found also in fertile men. The aim of
the present study is to compare two methods for partial AZFc
deletions detection: one based on STS (Sequence Tagged
Sites) and the other one based on SNV (Single Nucleotide
Variant) which allows to distinguish one copy from the 3
others.
Design and Location : In this retrospective study, first, we
used previously published STS to screen infertile patients
and controls in order to detect partial deletions of AZFc region.
Secondly, we designed a method to detect SNV using DHPLC
314
(Denaturing High Performance Liquid Chromatography).
Materials and Methods : Patients : 850 infertile patients
exhibiting a non-obstructive oligozoospermia or azoospermia
and 72 normozoospermic fertile men have been analyzed.
SNV: Detection of SNV-I, SNV-II, SNV-III (for DAZ4, DAZ1 and
DAZ2 respectively) using DHPLC. DAZ3 is identified using a
gene copy specific STS amplification. STS : Screening of 6
STS: sY1161, sY1197, sY1191, sY1291, sY1206 and sY1201,
in biplex.
Results : STS : gr/gr deletions (corresponding to the absence
of sY1291), b1/b3 deletions and b2/b3 deletions were found
respectively in 38 men (4.8%), 2 men (0.3%) and 2 men
(0.3%) with oligozoospermia or azoospermia. They were not
detected in fertile normozoospermic men. SNV: 2.9% of
patients and 0.5% of controls exhibited a DAZ1 deletion.
4.9% of patients (38) but none normospermic exhibited a
DAZ4 deletion. Surprisingly, DAZ2 and DAZ 3 "deletions"
could be found both in controls and in patients (23% versus
14.8%, and 10% versus 16%, respectively).
Concordant results between the two methods were observed
for 33 gr/gr deletions: 17 were DAZ1/2 deletions (57%) and
16 were DAZ3/4 deletions (43%). The 5 others cases were
gr/gr deletions according to STS with unexpected results with
SNV analysis: one DAZ1/DAZ2/DAZ4 deletion, two with an
isolated DAZ2 deletion, and two without any DAZ copy deletion.
We also confirmed the two b1/b3 and the two b2/b3 deletions
using SNV analysis. We consider that the SNV-III and YDAZ3 used for DAZ2 and DAZ3 are not useful, since they are
absent in control patients (either these markers are not always
present, or the absence of the corresponding copy has no
consequence on fertility). Conversely, the absence of DAZ1
and DAZ4, detected using SNV-II and SNV-I respectively, is
predictive of infertility and correlates well to the STS detection
results (exact Fisher's test p<10-3). No correlation with semen
or clinical parameters could be found for infertile patients,
neither with STS or SNV results.
Conclusions : Study of SNV II and I, present in DAZ1 and
DAZ4 respectively, by DHPLC, seems to be an easy method,
completing the STS analysis. SNV analysis can bring additional
information on the size and type of the partial AZFc deletions.
Together with the Y haplogroups determination (in progress),
STS and SNV combined analysis is needed to have a more
accurate prognostic tool for these small rearrangements of the
Y chromosome.
References :
Fernandes A.T., Fernandes S., Goncalves R. et al. : DAZ
gene copies : evidence of Y chromosome evolution. Mol.
Hum. Reprod., 2006 Jun 15.
Repping S., Van Daalen S.K., Brown L.G. et al. : High mutation
rates have driven extensive structural polymorphism among
human Y chromosomes. Nat. Genet., 2006, 38 : 463-467.
Lynch M., Cram D.S., Reilly A. et al. : The Y chromosome gr/gr
subdeletion is associated with male infertility. Mol. Hum.
Reprod., 2005, 11 : 507-512.
Lin Y.W., Thi D.A., Kuo P.L. et al. : Polymorphisms associated
with the DAZ genes on the human Y chromosome. Genomics,
2005, 86 : 431-438.
Ferlin A., Tessari A., Ganz F. et al. : Association of partial
AZFc region deletions with spermatogenic impairment and
male infertility. J. Med. Genet., 2005, 42 : 209-213.
Giachini C., Guarducci E., Longepied G. et al. : The gr/gr
deletion(s) : a new genetic test in male infertility ? J. Med.
Genet., 2005, 42 : 497-502.
OR 21
Natural transmission of USP9Y gene mutations :
a new perspective on the role of AZFa genes in
male fertility
C. KRAUSZ, S. DEGL’INNOCENTI, F. NUTI, A. MORELLI,
E. GUARDUCCI, G. FORTI
Department of Clinical Physiopathology, Andrology Unit,
University of Florence, Italy Corresponding
author : [email protected]
Objective : Deletions of the AZoospermia Factor Regions
(AZF) of the Y chromosome are associated with severe
spermatogenic failure and represent the most frequent
molecular genetic cause of azoospermia and severe
oligozoospermia. The exact role of the candidate AZF genes
is largely unknown due to both the extreme rarity of naturally
occurring AZF gene specific mutations and the lack of
functional assays. Only two cases of finely characterized and
confirmed isolated AZF gene mutations have been reported
to date. In both cases the mutations removed the USP9Y
gene in the AZFa region.The associated phenotype in both
cases was infertility due to severe impairment of the
spermatogenesis.
Here, we report the fine characterization of two different
deletions in the USP9Y gene (one of the two candidate genes
in the AZFa region), which have been transmitted through
natural conception in two unrelated families.
Materials and Methods : A total of 995 men affected by
infertility have been screened for AZFa (USP9Y and DBY) and
AZFb (eIF1AY and HSFY) gene specific deletions in our
laboratory. In addition, 350 normospermic men were screened
for gene specific deletions and resulted not to be deleted for
any of the two USP9Y markers.
Since USP9Y is composed of 46 exons, we use two primer
pairs, one mapping to exon 22 (GeneBank G54725), and
another to exon 46 (GeneBank G34983). We used a number
of additional gene specific markers to localize the breakpoints
315
in patient two patients. The deletion junction has been then
amplified using long range PCR, and the amplified products
was sequenced by automatic sequencer (ABI PRISM 310,
Applied Biosystems).
Results : After the routine and gene specific screening we
found two patients (1115 and A333) with partial USP9Y deletion
(one of the two candidate genes in the AZFa region). The
deletion in patient A333 removed the first 28 exons, whereas
in patient 1115 the last 13 exons of the USP9Y gene. In patient
1115, RT-PCR revealed the presence of the 5’ end of USP9Y
transcript and, as expected, the absence of its 3’ end,
suggesting the translation of a truncated protein. In patient
A333 it is highly unlikely that the undeleted, 3’ portion, of
USP9Y was transcribed, as the deletion removed 28 kb of
sequence 5’ of the normal position of the first exon. The
screening of the male relatives of both subjects enabled us
to detect the transmission of deletion through natural
conception in both unrelated families. The associated mild
testicular phenotype (moderate oligoasthenoteratozoospermia), in both cases, is in sharp contrast with that of
the two previously reported infertile patients bearing a mutation
of the same gene.
Conclusions : In conclusion, to date the USP9Y gene has
been considered as one of the major Y-linked spermatogenesis
genes, based on both its position within the AZFa region and
previous reports that correlated USP9Y mutation to severe
spermatogenic failure and infertility. This view is now
substantially changed since our findings clearly demonstrate
that during human spermatogenesis, USP9Y is more likely a
fine tuner which improves efficiency rather than a provider of
an essential function. More importantly, the observed natural
conceptions suggest that the protein is not required for the
final sperm maturation process or for the acquisition of sperm
fertilizing ability providing a new perspective on the role played
by the USP9Y gene in male fertility.
OR 22
Effect of prostasomes on human spermatozoa.
An in vitro study
S. BECHOUA, I. RIEU, B. SION, G. GRIZARD
Laboratoire de Biologie de la Reproduction, Université
d’Auvergne, EA975, 63000 Clermont-Ferrand, France
Objective : Prostate gland secretions are rich in prostasomes,
vesicles known to play an important role in male fertility.
Prostasomes have an unusually high concentration of
cholesterol relative to phospholipids (2:1 a compared with
1:1 found in plasma membranes). However, their mechanisms
of action are still poorly understood.
In the laboratory, Picherit et al. have reported that human
spermatozoa incubated for 3 hours with cyclodextrine (an
acceptor of cholesterol) exhibited a strong correlation between
the degree of tyrosine phosphorylation of a 107 KDa protein
and ionophore A23187-induced acrosome reactions.
It is known that loss of cholesterol from sperm membranes
occurs during capacitation. Since evidence indicates that
prostasomal molecules, especially cholesterol, can be
transferred to sperm under certain conditions, we hypothesized
that transfer of cholesterol to sperm membranes could affect
some spermatozoa properties. Therefore, we investigated
the effect of prostasomes on 1) the tyrosine phosphorylation
of a 107KDa protein band, 2) sperm lipid profile.
Materials and Methods : Human spermatozoa were selected
by Percoll density centrifugation and incubated for 3 hours at
37oC under an atmosphere of 5% CO2 in air, in the absence
or presence of increasing concentrations of prostasomes.
Quantities of prostasomes used for experiments were
expressed in terms of cholesterol (0.3 nmol to 45 nmol of
cholesterol / 100 µl of incubation medium). After incubation
either Western blotting analysis or HPTLC were performed.
Results : The cell viability measured after 3 hours incubation
in the absence or presence of prostasomes was not modified
compared with the sperm population viability measured before
incubation.
We observed that tyrosine phosphorylation of the 107KDa
protein band was inhibited when sperm were incubated with
increasing concentrations of prostasomes; the differences
relative to untreated controls were significant for 30 and 45
nmol of cholesterol per 100 µl of incubation medium. We also
noted a significant increase in the amount of cholesterol and
the main classes of phospholipids. These enrichments were
dose dependent.
Conclusion : We concluded that prostasomes in vitro are
capable of inhibiting events (tyrosine phosphorylation) known
to be activated during the capacitation process. The changes
in the lipid composition after 3 hours incubation, and more
specifically enrichment in cholesterol could be responsible of
this inhibitory effect. These results suggest that prostasomes
could in vitro maintain human sperm in a non capacitated
state and partly explain the anti-capacitating properties of
seminal plasma. Further in vitro studies are required to
complement this work.
References : Picherit-Marchenay C., Bréchard S., Boucher
D., Grizard G. : Correlation between tyrosine phosphorylation
intensity of a 107KDa protein and A23187-induced acrosome
reaction in human spermatozoa. Andrologia, 2004, 36 : 370377.
316
OR 23
Regulation of mitochondrial DNA content in
human sperm: implications for male (in)fertility
A. AMARAL1,2, J. RAMALHO-SANTOS1, J. ST JOHN2
1 Center for Neuroscience and Cell Biology, University of
Coimbra, Portugal ; 2 The Mitochondrial and Reproductive
Genetics Group, University of Birmingham, UK
Amaral A. ([email protected])
Human mitochondrial DNA (mtDNA) encodes 13 polypeptides
of the electron transfer chain, the apparatus that is responsible
for the generation of ATP through the process of oxidative
phosphorylation. The integrity of the mitochondrial genome
is particularly important in those cells with high energy
demands, such as sperm. Its replication is dependent on
nuclear-encoded factors, including DNA Polymerase Gamma
(POLG) and mitochondrial transcription factor A (TFAM).
POLG is the DNA polymerase specific to mtDNA replication.
TFAM activates mtDNA transcription and thus replication,
and is thought to be a regulator of mtDNA copy number.
Down-regulation of TFAM was proposed to cause the 10-fold
reduction in mtDNA copy number during spermiogenesis.
Furthermore, low levels of expression of either of these two
proteins have been associated with mtDNA-depletion
syndromes.
The aim of this study was to investigate the significance of
the expression of POLG and TFAM in the quality of human
sperm and to determine whether these two factors influence
sperm mtDNA content. We have analysed 93 sperm samples
from men attending a Portuguese infertility clinic and
categorized them into three groups, as evaluated according
to the World Health Organization Guidelines (1999) : i)
normozoospermic ; ii) samples with one or two abnormal
semen parameters; and iii) oligoasthenoteratozoospermic
(OAT). POLG and TFAM expression was evaluated using
immunocytochemistry and Western Blotting. MtDNA content
was determined in 42 samples by the ratio of mtDNA copies
to the nuclear-encoded Beta-Globin gene, through real-time
quantitative PCR. POLG and TFAM expression was localised
to the midpiece of mature sperm.
The percentage of those sperm expressing these proteins
was significantly lower in OAT than in normozoospermic
samples (P < 0.01 for POLG, P < 0.05 for TFAM). The mean
mtDNA copy number for human sperm was determined at 21.6
± 5.8. However, in contrast to POLG and TFAM levels, the
mtDNA content was significantly higher in OAT samples (46.7
±15.0) than in normal samples (6.8 ± 1.8, P < 0.01) and in
samples with 1 or 2 defects (11.3 ± 4.5, P < 0.05). Indeed and
paradoxically, a negative correlation was found between
mtDNA copy number and the percentage of sperm expressing
POLG (R = -0.582, P <0.001).
We propose that the reduction in mtDNA content observed
in normal samples reflects normal spermiogenesis. The high
number of mtDNA copies associated with OAT samples may
reflect one of many outcomes arising from defective
spermiogenesis. We further propose that, in normal samples,
mature sperm increase their levels of POLG and TFAM
expression in an attempt to compensate for the low mtDNA
content. However, this mechanism appears to be ineffective
due to the persistence of low copy number.
In conclusion, tight regulation of nucleo-mitochondrial
interactions might be essential for the effective completion of
spermiogenesis and subsequent sperm function.
A.A is supported by FCT Portugal (SFRH/BD/12665/2003).
This work was supported by FCT (POCTI/ESP/38049/2001),
by Instituto Investigação Interdisciplinar, University of Coimbra
(III/BIO/50/2005) and by British Heart Foundation (PG/04/117).
OR 24
Tyrosine kinases and protein kinase A play
different roles in capacitation and motility in
human spermatozoa
M. LUCONI, G. VARANO, A. LOMBARDI, G. FORTI,
E. BALDI
Dept. Clinical Physiopathology Andrology Unit, University
of Florence. Italy ([email protected])
Although the role of protein phosphorylation/dephosphorylation
in regulation of sperm functions is well known, the precise
timing of activation and reciprocal function of protein kinase
A (PKA) and tyrosine kinases (TKs) in the development and
maintenance of sperm motility and capacitation is still unclear.
By western blot analysis of human sperm using PY20 antibody
directed against tyrosine phosphorylated protein motif, we
demonstrated that during sperm capacitation tyrosine
phosphorylation is the first signal to be activated which is
responsible of the time-dependent increase in sperm motility.
Blocking TKs from the beginning of capacitation results in a
significant inhibition of sperm motility parameters evaluated
by CASA, associated by a reduction of PKA activity, evaluated
using an antibody against PKA activated substrates. On the
contrary, inhibition of PKA by H89 slightly affects sperm motility
and tyrosine phosphorylation but only at the very first step of
capacitation (30 min), whereas such processes are unaffected
at longer time (2h-3h), suggesting a precise timing in regulation
of sperm motility. In the model we propose, in fact, TKs are
immediately activated when capacitation starts and act
upstream of PKA, probably through tyrosine phosphorylation
317
of AKAP3 which results in PKA recruitment and activation in
sperm tails (Luconi et al, J Cell Sci, 2004). Among TKs
activated during capacitation, p60src has been identified for
the first time. Interestingly, PKA contributes to stimulate TKs,
thus a positive loop is established between tyrosine
phosphorylation and PKA activity. At longer time, the relative
importance of PKA declines and motility seems to be mainly
maintained by TKs, as demonstrated by the absence of effect
of H89 at 2 hour capacitation.
318
Andrologie 2006, 16, N°4 319-393
Abstracts of the Fourth European Congress of Andrology and
23rd Congress of the French Speaking Society of Andrology
Posters
(PO 001 to PO 116)
PO 001
Obesity and male reproduction function
S. PFLIEGER-BRUSS1, F. WEMBER1, R.H.
BÖDEKER2, W.B. SCHILL1, H.C. SCHUPPE1
1 Centre of Dermatology and Andrology, and 2 Institute for
Medical Informatics, Justus Liebig University, Giessen,
Germany ([email protected])
Objective : Reproductive function may be affected by
environmental and occupational exposures as well as
changing lifestyle. However, whether or not these factors
contribute to an increasing risk of male reproductive health
problems including poor semen quality is stirring an ongoing
debate. On the other hand, obesity is becoming increasingly
prevalent worldwide and is now considered to be one of the
most important health concerns in many countries. Notably,
the impact of the woman´s body mass index (BMI) on
fecundity is well established, whereas only few reports
addressed the potential reproductive hazards of obesity in
males. Therefore, this study aimed at investigating the
relationship between BMI and parameters of reproductive
function in men attending an infertility clinic.
Patients and Methods : The retrospective study included
a total of 496 men undergoing andrological examination for
infertility work-up. Apart from infertility, inclusion criteria
comprised age (18-50 years), BMI >18.5 kg/m2, uneventful
andrological history, and normal genital examination. Patients
with genetic or congenital abnormalities, genital disorders,
systemic disease, therapeutic use or abuse of drugs, as well
as seminal signs of inflammation or obstruction were
excluded. Semen analysis had been performed according to
WHO guidelines extended by biochemical markers and
microbiology. For statistical analysis, patients were
categorized according to BMI considering cigarette smoking
and positive seminal bacteriology as confounders.
Results : A normal BMI (>18.5 to <25 kg/m2) was found in
209 men (42.1%), 287 patients (57.9%) showed a BMI >25
kg/m2. Compared to the general population in Germany, the
prevalence of obesity was markedly increased, with the
highest difference among the subgroups of men aged 25-29
years and 30-34 years (39.7 vs 61.2% and 48.3 vs 62.3%,
respectively). Semen analysis revealed normozoospermia in
35.4% of men with normal weight, but only in 25.4% of those
with a BMI >25 kg/m2. However, this trend was not significant
and also the confounding factors showed no marked effect
on semen quality. A significant negative correlation could be
observed between BMI and serum testosterone, whereas
gonadotrophin levels remained unaffected.
Conclusions : The results confirm previous reports describing
a decrease of serum testosterone with increasing BMI. The
high prevalence of obesity among men attending an infertility
clinic suggests that this factor may contribute to male
reproductive health problems, although the trend towards
deterioration of semen quality with increasing BMI is not
significant. With regard to the alarming prevalence of obesity
among adolescents, this issue might become increasingly
important in clinical andrology in order to prevent at least some
cases of male subfertility.
PO 002
Incidence of dysspermia categories in a
contemporary diagnostic setting
D.A. ADAMOPOULOS, S. NICOPOULOU,
C. MICHALAKIS, A. PAPPA, E. KOUKKOU, E. VENAKI
Andrology Clinic, Endocrine Dept., Elena Venizelou
Hosp., 11521 Athens, Greece
Objective : To classify sperm disturbances in relation to
possible aetiological factors using the standard clinical
approach, employing all available diagnostic tools and
319
following WHO’s guidelines for categorization (W.H.O., 2000).
Design : Analysis of the clinical material of an Endocrinology
Department embedded Andrology Clinic of a busy inner-city
hospital in Athens for classification of sperm disturbances
was made using the diagnostic categories proposed by W.H.O.
but expanded to include new classes not previously reported
(e.g. epididymopathies, multifactorial causes, etc.).
Materials-Methods : A total of 774 cases investigated for
couple subfertility and found to be dysspermic have been
selected from a large database from which normozoospermic
men have been excluded. The diagnostic classification was
based on meticulous history taking, physical examination,
semen analysis (W.H.O., 1999 criteria,) imaging techniques,
endocrine tests (including inhibin-B), and when needed,
testicular biopsy and chromosome and molecular analysis.
Results : On the basis of the factors identified these cases
were groupped into 3 major categories: (a) single-factor group
(37.3%), (b) two-factor group (34.0%) and (c) three or more
factors group (28.27%).
In the single-factor group, the incidence recorded was in the
following order: 1. Idiopathic OTA (40.6%), 2. varicocele
(18.7%), 3. epididymopathy (12.8%), 4. environmental (8.0%),
5. infections (5.3%), 6. acquired testicular damage (4.8%), 7.
congenital anomalies (3.2%), 8. systemic causes (2.1%), 9.
endocrine causes (1.6%), 10. sexual-ejaculatory dysfunction
(1.3%) and four other diagnostic groups with less than 1% or
no representation. In the two-factors group, epididymopathy
(31.3%), varicocele (26.5%) and environmental (20.6%) factors
were the most frequently encountered components of the
various combinations. Finally, in the three or more factors
group, the main components were environmental (24.8%),
varicocele (19.2%) and epididymopathy (19.0%).
As it is obvious, in single factor aetiology idiopathic OTA was
the principal category whereas in all multifactorial combinations
varicocele, environmental factors and epididymopathy were
dominating the field.
Conclusions : The diagnostic categories formulated in this
analysis differ significantly from the relevant data presented
from the much larger series from other centers (Nieschlag,
2001). In our material, multiple factors have been recognized
in the great majority of the cases and the single-factor category
was clearly in a minority. It appears that the detailed diagnostic
work-up with the introduction of new investigational parameters
was instrumental in explaining, at least in part, the marked
differences between the two series. On the other hand, one
should also count the differences between the relevant
populations in terms of environment, prevailing health
conditions, socioeconomic status, etc. As it becomes obvious,
studies charting each population’s patterns of dysspermia
should be available in each different population as a
prerequisite for a sound reproductive policy for the male.
2. World Health Organization WHO : Manual for the
standardized investigation, diagnosis and management of
the infertile male. Rowe P.J. et al. eds. Cambridge, CUP,
2000.
3. Nieschlag, E. : Classification of andrological disorders. In:
Nieschlag E., Behre H.M. eds. Andrology, Male Reproductive
Health and Dysfunction. Berlin, Springer, 2001.
PO 003
Interests of the post coital test in the
investigation of the infertile couples
N. ABID, N. BEN JAMAA, M. AJINA, A. SAAD
Service of Cytogenetic and Biology of Reproduction,
university Hospital F Hached Sousse, Tunisia. Email :
mounir.ajina @rns.tn
Objective : to establish the profile of the cervical mucus and
the sperm to infertile couples of the Tunisian centre.
Design and place : Service of Cytogenetic and Biology of
Reproduction, University Hospital F Hached Sousse, Tunisia.
Methods : Our study concerns 49 infertile couples which
benefited of at least: a postcoital test (PCT) and a spermogram.
Test was realized in meadow ovulatory period among the 11th and the 12-th day of the cycle after an average sexual
intercourse of 8 hours and a 3 days average sexual abstinence.
The exo cervical mucus is taken with an a bit long crowbar,
we measures the filance between the two branches of the
crowbar; on the other hand the endocervical mucus is taken
by means of an aspiglaire. The endo and exo cervical mucus
are examined under microscope between blade and small strip.
Results : The majority of the couples (63%) benefited from
a hormonal treatment before the taking of the cervical mucus.
Only 18% of the patients benefited during the PCT of a double
taking endo and exocervical. Post coïtal test was positive in
89% of cases and negative in 11% of cases. Only 12% of the
patients have cervical infertility and a single patient has an
azoospermia.
Conclusion : post-coïtal test is a test of reliable diagnosis to
infertile couples because according to our results the majority
of the patients who benefited from PCT are porters of cervical
infertility.
References :
References :
1. World Health Organization WHO : Laboratory Manual for
the Examination of Human Semen and Sperm-Cervical
Mucus Interaction. 4th ed., Cambridge, CUP, 1999.
1. J.-R. Zorn; Place actuelle du test de Hühner dans
l'exploration de la stérilité conjugale; Gynécologie
Obstétrique & Fertilité, 34, 2, 2006 : 142-14.
320
2. S. Hammamah et al. ; Médecine et biologie de la
reproduction, 2ème édition Masson, 2004, p 152.
3. Clinical Guideline 11: Fertility: assessment ant treatment
for people with fertility problems. 1.4.6 Post-coital testing
of cervical mucus.
correlated with the grade of the venous reflux. Doppler
ultrasonography is an essential diagnostic tool before surgery
and a valuable measure of postoperative results.
PO 005
PO 004
Epidemiologic features of varicocele in a large
cohort of Greek men with infertility
The place of Doppler ultrasonography in
evaluating male infertility
P.D. KANTARTZI, D.C. GOULIS, P.K. ILIADOU, C.
TSAMETIS, D.G. GOULIS, J. BONTIS, J. PAPADIMAS
V. CAUNI1, D. DINU2, C. PERSU1, P. GEAVLETE1
Unit of Reproductive Endocrinology, 1st Department of
Obstetrics & Gynaecology, Aristotle University of
Thessaloniki, Greece
1 Department of Urology, Saint John Emergency Clinical
Hospital, Bucharest, Romania 2 “C.I. Parhon” Institute of
Endocrinology, Bucharest, Romania
([email protected])
Introduction and Objectives : Male infertility may be the
cause of several disorders, so that identifying a correct
diagnosis and an appropriate treatment may represent a
challenge for the clinician. Our goal was to evaluate in a
retrospective study the correlations between spermatic
parameters, scrotal abnormalities and the resistive index in
infertile males, as determined by Doppler ultrasonography.
Patients and methods : Our study group consists of 56
infertile males, aged between 21 and 46 years old, and 40
fertile males, as a control group. The evaluation protocol
included andrological examination, hormonal profile, and
determination of antispermatic antibodies, prostatic and
testicular Doppler ultrasonography.
Results : Testicle hypotrophy was diagnosed in 40.7% cases,
26% patients presented cysts or calcifications of the
epydidimus, 32% had chronic prostatitis, 12% had
spermatocystitis, 10% had testicular tumors. 29.64% of the
infertile patients had olygospermia. Varicocele was present
in 71.42% of cases, and in 31.57% cases the varicocele was
bilateral – stage I varicocele in 26.6%, stage II in 40% and
stage III in 33.4%. Doppler ultrasound combined with the
Valsalva maneuver identified three grades of reflux : grade I
in 22.2% cases, grade II in 35.5% cases and grade III in
42.3% cases. The mean value of the resistive index in the
control group was 0.56, in patients with varicocele the mean
value was 0.46, and in patients with azoospermia the mean
value was 0.90. The severity of the spermatogenetic
dysfunction was better correlated with the grade of reflux
than with the clinical stage of varicocele.
Conclusions : Varicocele is the cause of infertility in about
50% of the patients studied. The severity of olygospermia is
321
Objective : Varicocele is a common cause of male infertility
and one of the most controversial issues in the field of
Andrology. The main aim of this study was to analyze the
epidemiologic, clinical, hormonal and sperm parameters in a
large cohort of infertile men with varicocele in a northern
Greek population. A secondary aim was to detect changes of
these parameters in men who underwent surgical repair of
varicocele.
Design : Retrospective, epidemiologic, descriptive, clinical
study.
Materials and Methods : We accessed medical records of
925 infertile men that were examined in our outpatient clinics
between 1991 and 2005 ; 429 (46%) of them having either a
clinical varicocele or a surgically repaired varicocele were
included in the study. Studied parameters included age of
male and female partners, type and duration of infertility,
testicular volume, side and grade of varicocele, FSH, LH,
prolactin, testosterone and sperm parameters before and
after the surgical repair where available. Of the 429 men, in
272 (64%) varicocele was the only cause of infertility, whereas
other additional causes included infection (n=77 ; 18%),
idiopathic non-obstructive azoospermia (INOA) (n=40 ; 9%),
cryptorchidism (n=16 ; 4%), obstruction (n=7 ; 2%) and other
causes (n=17 ; 3%).
Results : Studied parameters are illustrated in the table.
Results are given as mean ± standard deviation or percentage.
In the subgroup of men (n=87) who underwent surgical repair
of varicocele, no statistically significant changes were found
in sperm and hormonal parameters after the operation. The
lack of statistical significance remained when we analyzed
separately men with varicocele only and men with varicocele
plus INOA.
Material and Methods : 22 infertile men, aged 20 - 45 years
old, with varicocele and normal hormone levels, were included.
The evaluation protocol included an andrological examination,
the evaluation of hormonal status, two spermograms in a
three months interval, antispermatic antibody measurement,
sperm cultures, cytogenetic exam and testicular pulse Doppler
ultrasound. Venous reflux associated with the Valsalva
maneuver was also determined. Patients were reassessed 2,
4 and 6 months after surgery for varicocele, by seminal liquid
analysis, antispermatic antibodies measurement. Also,
ultrasonography was routinely performed.
Conclusions : Varicocele is a very common finding in infertile
men, although an etiologic relationship between varicocele and
male infertility is difficult to be established. According to our
findings, surgical repair does not seem to be generally effective,
thus it should be applied only in a meticulously selected group
of men.
Results : Antispermatic antibodies were present in 77.8% of
the infertile patients, significantly correlated with the grade of
the venous reflux and less dependant on the clinical stage of
the varicocele. The spermograms showed a wide range of
abnormalities, from astenospermia to severe oligoasthenoteratozoospermia. These abnormalities were more
severe in patients with intratesticular varicocele and high
grade venous reflux. After surgery, the testicle hypotrophy
stopped its progression in all patients. Ultrasound evaluation
2 months after surgery showed the disappearance of the
venous reflux at the Valsalva maneuver in 90% of cases. All
patients who induced spontaneous pregnancies were under
35 years old.
Presenting author: Prof. J. Papadimas, 1st Department of
Obstetrics & Gynaecology, “Papageorgiou” General Hospital,
Periferiaki Odos, Nea Efkarpia, 56403, Thessaloniki, Greece.
e-mail: [email protected]
Conclusions : Varicocele induces autoimmune processes,
with an increase in spermatic antibodies. Surgery for varicocele
significantly increased the fertility potential in men under 35
years old. Future research is needed in order to reveal the
interdependent mechanisms involved in spermatogenesis
abnormalities and decreased spermatic motility noted in males
with varicocele.
PO 006
PO 007
The varicocele - a frequent cause of male
infertility
Effect of varicocele on the formation of antisperm
antibodies "ASA"
V. CAUNI1, D. DINU2, C. PERSU1, C. DUMITRACHE2,
P. GEAVLETE1
ABD ALLAH M. ATTIA, ALAA H. MARAEE, KHALED A.
ALI, HAYAM M. HANOUT, AZZA G.A. FARAG* AND
EMAN N. EL SHAFEY
1 Department of Urology, Saint John Emergency Clinical
Hospital, Bucharest, Romania 2 “C.I. Parhon” Institute of
Endocrinology, Bucharest, Romania
([email protected])
Dermatology, Andrology & S.T.Ds and Clinical Pathology
Depts*, Minoufiya University Corresponding author :
Hyperlink "mailto:[email protected]"
[email protected]
Introduction : Although the pathophisiology of the varicocele
is a well known matter, its correlation with male infertility and
with the optimal timing of surgery in men with associated
spermatogenesis abnormalities and decreased spermatic
motility is still controversial. Our objective is to asses the
consequences of the varicocele on the reproductive function.
322
The exact etiopathology through which varicocele can affect
fertility potential is still unknown.
This work aims to study the effect of varicocele on ASA
formation and its correlation with the stress pattern detected
in cases of varicocele.
The study included two groups: thirty evident varicocele
(grades II and III) infertile patient (group I) and fifteen
varicocele-free fertile, age matched volunteers as a control
group (group II).
Both patients and controls were subjected to; standard semen
analysis, expressed prostatic secretion examination; excluding
those having chronic prostatitis and detection of both serum
and seminal plasma ASA by ELISA.
The results showed that; the varicocele patients have highly
significant low sperm density and percent of actively motile
sperm (p<0.001) and significantly higher level of abnormal
forms (p < 0.05) compared to controls.
The results also showed that; in varicocele group; both serum
and seminal plasma ASA are negatively correlated with sperm
density (r=-0.732 and -0.66 - p < 0.001) respectively, and
percent of actively motile sperms (r=-0.739 and -0.771 - p <
0.001) respectively, whereas serum and seminal plasma ASA
are positively correlated (r=0.685, p<0.001), denoting that
seminal plasma ASA are derived from the systemic circulation.
Results : The semen analysis found an improvement of all
the parameters for all the patients without normalization except
for the morphology. The post operative outcome was
statistically significant for the density. The majority of the
patients (76% to 92%) had abnormal preoperative values of
semen except for the morphology (28%). Semen analysis
parameters were improved for 48% to 64% of patients
depending on the parameter. For patients with abnormal
semen, the lower the mean preoperative value of a parameter,
the higher it becomes postoperatively. The post operative
pregnancy rate was 31,42%. The fertile patients were those
with a younger age, a shorter duration of infertility, constantly
improved semen analysis parameters compared to infertile
patients.
Conclusion : Values and improvement of semen analysis
parameters are more favourable in patients with young age
and those with bilateral varicocele and a secondary infertility.
Keywords: Fertility, Semen analysis, Varicocele repair.
In conclusion; varicocele can precipitate to the formation of
ASA. The latter can be considered as one of the etiopathogenic
mechanisms through which varicocele can affect the fertility
potential.
PO 009
PO 008
Primary varicocele and fecundity : Post surgical
assessment of sperm and fecundity parameters
Varicocelectomy and scrotal temperatures in
infertile men with varicocele
T. ALMONT1, E. HUYGHE1,2, P. PLANTE1,2,
P. THONNEAU1, L. BUJAN1, R. MIEUSSET1
1 EA 3694 " Recherche en fertilité humaine – Santé de la
reproduction dans les PVD ", Hôpital Paule de Viguier, 330
av de Grande-Bretagne, TSA 70034, 31059 Toulouse
Cedex, France 2 Service d’Urologie - Andrologie, Hôpital
Rangueil, Toulouse, France
L. NIANG, I. LABOU, O. ALI, M. JALLOH, R.KANE,
M. NDOYE, S.M.GUEYE
Service d’Urologie et d’Andrologie Hôpital Général Grand
Yoff, Dakar – Etoile [email protected]
Objectives : To assess the post operative evolution of male
infertility (semen analysis parameters) and the fertility
outcomes.
Materials and Methods : We underwent a retrospective
study including 50 patients with a varicocele operated
according to Palomo procedure in the department of Urology
of Hôpital Général de Grand Yoff. The parameters studied were
related to semen analysis (density, mobility in the first hour
and the vitality) and the spermocytogramme (count of normal
spermatozoa). This analysis was done once before the
operation and twice after the operation (between the 3rd and
8th month and from the 9th month).
323
Objectives : Increased scrotal temperature is one of the
various factors either associated with or considered as a
cause of impaired spermatogenesis in infertile men with
varicocele. Some studies have reported a reduced scrotal
temperature after varicocelectomy. We investigated whether
this finding was true, and if such a reduction in temperature
persisted over time after surgery.
Materials and Methods : This retrospective study included
2 groups of infertile patients with a left varicocele: an operated
group of 116 patients, and a non-operated group of 40 patients
included in an intrauterine insemination programme (IUI).
Varicocele was diagnosed by clinical examination (grade I:
Valsalva positive ; grade II: palpable; grade III: visible). In
both groups clinical examination was performed before surgery
or before inclusion in the IUI programme (T0) and again at 3
months (T3) in both groups, and at 6 months (T6) after surgery
in the operated group. Scrotal temperatures were measured
on each side with a special thermometer with the patient
naked and in a supine position for at least 10 minutes.
Results : Comparison of scrotal temperature values at T0 and
T3 revealed no significant change in left and right mean
temperatures in the non-operated group, while both left and
right mean temperatures decreased significantly in the
operated group.
In the operated group, comparison of scrotal temperature
values at T0, T3 and T6 indicated that left and right mean
temperatures were significantly lower at T3 and T6, with no
difference between T3 and T6 values.
Considering a value <35.1°C as the upper limit of normal
scrotal temperature, an abnormal temperature (>35.1°C) was
observed in 51% (59/116) of the operated group before
surgery. In this subgroup of patients with abnormal temperature
before surgery, temperature was normal in 86% (51/59) 3
months after surgery. In the subgroup of patients with normal
temperature before surgery, scrotal temperature was still
normal in 91% (52/57) but abnormal in 9% (5/57).
Conclusions : Surgery induced a reduction in mean scrotal
temperature in a group of infertile patients with a left varicocele.
This reduction was indeed the result of surgery, as mean
scrotal temperature was unchanged in the non-operated
group of infertile patients with a left varicocele. Moreover,
this reduction seems to be durable as temperature values
were still decreased at 3 and 6 months after surgery. However,
considering individual scrotal temperatures, only 51% of the
patients in the operated group had an abnormal value before
surgery.
Support : None.
PO 010
Prevalence of hypospermia and hyperspermia
and their relationship with genital tract infection
in tunisian infertile men
N. ABID1, N. CHAKROUN1, A. SELLAMI1, A.
BAHLOUL2, T. REBAI1, L. AMMAR-KESKES1
1 Laboratory of Histology-Embryology, Faculty of
Médecine, Sfax-Tunisia. 2 Research Unit "male infertility",
Habib Bourguiba hospital, Sfax-Tunisia. Correspondance
to Pr. Ammar-Keskes Leila, e-mail: lkeskes@ yahoo.fr
Objective : The objective of our work was to determine the
frequencies of hypospermia and hyperspermia and to establish
324
their possible associations to other spermatic abnormalities
and their possible relationship with the genital tract infection.
Design and Setting : Our retrospective study concerned
2332 spermiograms performed between 1995 and 2005 into
the laboratory of Histology of Medicine University of Sfax,
among patients consulting for couple infertility.
Materials and Methods : The spermiograms were carried out
according to the standardized method of WHO. We
distinguished three great groups according to the semen
volume : Gn (normospermia), GH (hyperspermia) and Gh
(hypospermia) ; from Gh we individualized one subgroup
called Gh1 (severe hypospermie : volume<1ml). For all these
groups, we determined the average values of semen
parameters and compared them using the Student test; the
frequencies of semen abnormalities (azoospermia,
asthenospermia,
necrospermia,
oligospermia,
leucocytospermia and teratospermia) were also determined
and compared, using the Chi-2 test. The threshold of
significance of p value was<0.05.
Results : The total prevalence of the hypospermia was 18.3%;
severe hypospermia was found in 3.7% of cases. The
frequency of the hyperspermia was only 6.8%. Many significant
differences were found between Gh1, Gn and GH, mainly
concerning motility, total spermatozoa count and vitality, which
were lower in Gh and Gh1 groups, comparatively with Gn
and GH groups, as well as the average rate of coiled tails and
the frequencies of semen abnormalities; in fact the frequencies
of asthenospermia, necrospermia, leucocytospermia,
oligospermia and azoospermia were higher in Gh and Gh1,
comparatively with Gn and GH. In addition, in Gh and Gh1,
azoospermia was more frequently associated to abnormal
pH than in Gn and GH ; thus basic pH (>8.5) was associated
to azoospermia, respectively in 32.5% and 46.15% of Gh
and Gh1 patients, versus 19.44% and 16.6% of Gn and GH
patients; in the same way, azoospermia was associated to an
acid pH in 4.6% and 15.38% of Gh and Gh1 patients, versus
0% of Gn and GH patients.
Conclusion : It seems that hypospermia is associated to an
active genital infection responsible for many spermatic
disturbances and for the appearance of biological inflammation
signs (leucocytospermia). But, our results led to suggest that
hyperspermia is not related to an evolutive genital tract
infection, since the average values of the principal semen
parameters, as well as the frequencies of semen abnormalities
were comparable with those found in the normospermic group.
We could also suggest in the light of these results that
hyperspermia would be associated to a recent infection that
did not yet induced semen quality disruption. In addition, we
suggest that among azoospermic patients, hypospermia is
related to a prostatic pathlogy (prostatitis with high pH in
seminal plasma) than to a seminal vesicule deficiency (low
pH in seminal plasma).
PO 010 bis
PO 011
Prevalence of asymptomatic inflammatory
prostatitis in young healthy men in Estonia
Genital tract infectious and inflammatory
pathology and male infertility
P. KORROVITS1,2, K. AUSMEES2, R. MÄNDAR1
M. PUNAB2
A. SELLAMI-BEN HAMIDA1,2, L. AMMAR-KESKES1,2,
N. ABID2, T. REBAI2, N.M. MHIRI1, A. BAHLOUL1
1 Department of Microbiology, University of Tartu, Estonia
2 Andrology Centre, Tartu University Hospital, Estonia
Hyperlink "mailto:[email protected]"
1 Research Unit “male infertility”, Habib Bourguiba
Hospital, Sfax Tunisia 2 Laboratory of Histology and
Embryology ; Faculty of Medicine, Sfax Tunisia
Correspondance to Pr. Ammar-Keskes Leila, e-mail :
lkeskes@ yahoo.fr
Objective : The aim of our study was to determine the
prevalence of asymptomatic inflammatory (NIH category IV)
prostatitis in young healthy men in Estonia.
Materials and Methods : The study group consisted of 562
men (291 Estonians, 271 Russians) aged 16-25 years (mean
age 18.8 years). Cytologic examination of their ejaculate
(using Bryan-Leishman stained slides) was performed. In
addition, all subjects were clinically examined for possible
pathologies in genital region and basic semen parameters
(volume, concentration and motility). Subjects with any clinical
symptoms of inflammation were excluded.
Results : The prevalence of asymptomatic inflammatory
prostatitis (>1 million WBC (white blood cells) per ml in sperm,
according to WHO guidelines) was 6.0%, but when we used
lower threshold suggested by our previous studies (>0.2
million WBC/ml), the prevalence was 19.0%. No difference
between the two ethnic groups were found when seminal
parameters and inflammatory markers were compared. In
this study the preliminary analysis did not show any significant
effect of leukocytospermia on sperm quality. We did not detect
any seasonal differences in the prevalence of asymptomatic
inflammatory prostatitis.
Conclusions : Asymptomatic inflammatory prostatitis is
common among healthy young males, suggesting the need
for further studies in order to investigate pathogenetic
mechanisms of the disease. Our study also suggests that
more attention should be paid to evaluation and treatment of
asymptomatic inflammatory prostatitis in young men.
Prevalence data we found should be taken into account when
estimating the total prevalence of all forms of chronic prostatitis,
both symptomatic and asymptomatic.
Support : The study was supported by Estonian Science
Foundation (grant no 5701) and EU 6th FP project QLRT-20010291.
325
Objective : To assess the prevalence of genital tract infection
and inflammation on male infertility and to elucidate the
importance of both clinical, biological and ultrasonographic
investigations in the diagnosis of the male chronic genital
tract infection.
Design : Retrospective study.
Setting : Research Unit “male infertility”, Habib Bourguiba
Hospital, Sfax Tunisia and Laboratory of Histology and
Embryology; Faculty of Medicine, Sfax Tunisia.
Materials and Methods : A total of 220 male partners of
infertile couples were evaluated by the study of their medical
file, biological investigations (semen analysis and culture)
and ultrasonographic examination. Our cohort was subdivided
into two groups : (G1, n= 49) included patients with
genitourinary tract infection/inflammation; and (G2: n=171)
included
patients
without
genitourinary
tract
infection/inflammation. The presence of genital tract
infection/inflammation was attested by the presence of
bacteriospermia or leukocytospermia or by the existence of
several clinical and/or ultrasonographic and/or other biological
abnormal parameters. Statistical analysis was performed
using Statview software. Frequencies were compared by the
chi-square test and means values were compared by Student’s
unpaired t test. Level of significance was fixed at p<0.05.
Results : Genital infection and/or inflammation was detected
in 22.3% in our patients (49/220). In history taking, epididymoorchitis and urethral discharge were found with significantly
higher prevalence in G1 (24.4% and 18.3%, respectively)
than in G2 (5.8% and 5.8%, respectively) ; p values were
0.004 and 0.01, respectively. Urinary tract infection was
detected in 16.3% of patients in G1 and 1.1% in G2 (p<0.001).
Semen culture was negative in 38.8% of patients in G1.
Semen parameters were altered in G1 with significant lower
mean values of motility (total and rapid progressive
spermatozoa), of vitality and of morphology (essentially tail
defects) in G1 than in G2. Hypospermia was significantly
more frequent in G1 than in G2 (32.5% vs 15.4%, p=0.02).
The prevalence of leukocyospermia (>0,5millions/ml) was
also significantly higher in G1 compared with G2 (24.1% vs
2.6%, p =0.01). By ultrasonography examination, genital male
tract abnormalitites (cysts, nodules) and accessory gland
calcifications were founded more frequently in G1 (21.8%
and 11.9%, respectively) than in G2 (2.8% and 0.7%,
respectively), with respective p values : <0,001 and 0.003.
Conclusions : Our results show that genital male tract infection
and/or inflammation occurs frequently in infertile patients.
Negative semen culture has low sensitivity for discrimination
between patients with and without infection. To establish the
diagnosis of infection or silent genital tract inflammation, it
would be necessary to confront the clinical, biological and
ultrasonographic investigation results.
PO 012
Bacteria trigger production of reactive oxygen
intermediates and lipid sperm membrane
peroxidation in in vitro model
M. FRACZEK1, A. SZUMALA-KAKOL2,
P. JEDRZEJCZAK3, M. KAMIENICZNA1, M. KURPISZ1
1 Institute of Human Genetics, Polish Academy of
Sciences, Poznan, Poland ; 2 Unit of Microbiology, Hospital
Medical College, Poznan, Poland ; 3 Clinic of Infertility and
Reproductive Endocrinology, University of Medical
Sciences, Poznan, Poland ([email protected])
Objective : The relationship between the presence of
infectious factor in semen and sperm fertilizing potential
remains to be actively studied. Microorganisms invasion
results in the development of inflammatory process and is
usually accompanied by the oxidative stress. As the role of
particular groups and species of microbes invading, colonizing
or infecting the male reproductive tract is still poorly understood,
we aimed to assess an in vitro effect of the five most often
isolated aerobic, anaerobic and atypic bacterial strains from
semen of our infertile patients. These are : Escherichia coli,
Staphylococcus haemolyticus, Streptococcus oralis,
Bacteroides ureolyticus and Ureaplasma urealyticum. We
have studied their influence on the level of reactive oxygen
intermediates (ROI) generated in co-incubated suspensions
of white blood cells (WBCs) and spermatozoa as well as
quantitating sperm membrane malondialdehyde (MDA).
Design : An in vitro model of semen infection.
Materials and Methods : The venous blood samples were
obtained from 10 healthy adults and WBCs were isolated
using a density gradient centrifugation. Spermatozoa were
isolated by swim-up technique (‘swim-up’ fraction) and
326
discontinuous Percoll gradient (90% and 47% Percoll fractions)
from 10 healthy, normozoospermic volunteers. The
measurement of ROI secretion by WBCs previously incubated
with bacterial strains before and after addition of spermatozoal
fractions was determined by chemiluminescence in the
presence of luminol. MDA concentrations were studied by
using a high-performance liquid chromatography (HPLC) in
sperm lysates after incubation of sperm with bacteria and/or
WBCs.
Results : The presence of bacteria in co-incubated
suspensions of sperm and WBCs was connected with reduced
ROI scavenging potential of sperm, especially of spermatozoa
with the best seminological parameters selected by swim-up
procedure. An increase of detected MDA after incubation of
sperm cells with bacterial strains was observed (which was
a natural consequence of non neutralized ROI in co-incubated
mixture of WBCs and spermatozoa) although no statistic
difference was found. The presence of leukocytes generally
was associated with elevated levels of MDA levels, in ‘swimup’ fraction in particular, and the greatest effect was exerted
by B. ureolyticus and S. haemolyticus (p<0.01 and p<0.05,
respectively, in comparison to spermatozoa incubated with
leukocytes only). Comparisons between MDA content in
spermatozoal fractions with no leukocytes and after leukocytes
incubation with selected bacterial strains exhibited significant
differences when Str. oralis, S. haemolyticus and U.
urealyticum were applied (p<0.05 and p<0.01).
Conclusions : The results obtained in this study constitute
another evidence indicating that bacteria which invade,
colonize or infect the male reproductive tract are important
inducers of the oxidative stress in semen which may play a
crucial role in male gamete dysfunction through the
peroxidative damage of sperm membrane lipids. Our data
support the hypothesis about cooperation between bacteria
and WBCs in triggering both structural and functional defects
in human germ cells. Possibly extended imbalance between
pro-and antioxidative activities in semen, primarly caused by
an infectious factor may lead to limited fertilizing ability of
spermatozoa as a consequence of enhanced ROI reactivity
with cell components.
Support : Committee for Scientific Research of Poland.
PO 013
Costimulatory molecules, chemokine receptors
and proinflammatory cytokines in dendritic cell
population in normal and chronically inflamed rat
testis
(auto)antigens and are in a status to migrate to the local
nymph nodes for T cell activation. They are in a ready migratory
state, but functionally immature.
Our data provide the first firm evidence for the existence of
DC in the testis and in conjunction with the previous
characterization of autoantigens a new tool for the investigation
of the underlying causes of male factor immunological infertility.
M. FIJAK1, L. LUSTIG2, W. VON WULFFEN3,
R. IOSUB1, V.A. GUAZZONE2, E. SCHNEIDER1,
A. MEINHARDT1, C. RIVAL2
PO 014
1 Department of Anatomy and Cell Biology, Justus-LiebigUniversity of Giessen, Aulweg 123, 35392 Giessen,
Germany 2 Center for Research in Reproduction,
University of Buenos Aires, Argentina 3 Department of
Pulmonary and Critical Care Medicine, University of
Giessen Lung Center (UGLC), Germany Hyperling
"mailto:[email protected]"
[email protected]
Uropathogenic E.coli but not commensale E.coli
infection activates Toll-like receptor-4 dependent
signaling pathways in rat testicular cells
S. BHUSHAN1, S. TCHATALBACHEV2, J. KLUG1,
T. CHAKRABORTY1, C. PINEAU3, A. MEINHARDT1#
Dendritic cells (DC) are potent antigen presenting cells and
presentation of self antigens by DC is likely to play an important
role in the initiation of autoimmunity and its progression. Our
recent characterization of testicular autoantigens in a model
of chronic testicular inflammation, i.e. experimental
autoimmune orchitis (EAO) prompted us to investigate the
presence the DC in normal and EAO rat testis.
DC in the testes were quantified by immunohistochemistry
using the DC specific antibodies Ox-62 and CD11c followed
by stereological analysis. The number of DC in EAO testes
(ca. 7x105/testis) increased significantly compared to adjuvant
and untreated control rats (ca. 1x105/testis).
The activation state of the DC is crucial in determining the
outcome of antigenic challenge viz the development of either
T cell immunity or tolerance. To better understand the role of
DC in testicular inflammation, we performed a detailed analysis
of different maturation markers such as costimulatory
molecules, chemokine receptors and cytokines.
We analyzed the expression of CD80, CD86 and MHC class
II molecules on DC by flow cytometry in testicular single cell
suspensions. Moreover, we have isolated testicular DC from
adjuvant control and EAO adult rats by magnetic beads
separation followed by FACS sorting and determined the
expression of mRNAs for chemokine receptors (CCR2 and
CCR7), IL10 and IL12.
Our preliminary results showed no significant differences in
the expression of CD80, CD86 and MHC II between the
investigated groups, but a significantly upregulated expression
of CCR7 and a strong decrease of IL12 mRNA in the EAO
group. The CCR2 mRNA level in EAO animals was not
significantly changed comparing to adjuvant controls. These
data suggest that the DC in EAO testis have already processed
327
Dept. of Anatomy and Cell Biology1, Dept of Microbiology2,
Justus-Liebig-University of Giessen, Germany. INSERM
U.625, Rennes, France3. #Email :
[email protected]
Immunological infertility such as infection, inflammation and
autoimmunity account for at least 12-13% of all cases of male
infertility. Uropathogenic Escherichia coli (UPEC) are the
most common cause of urogenital tract infection, ultimately
leading to infertility by germ cell loss. Toll-like receptor (TLR113) family recognize conserved microbial structures such as
bacterial lipopolysaccharide, peptidoglycan and viral doublestranded RNA, and activate MyD dependent /independent
signaling pathways that result in innate immune responses
against microbial infections. All TLRs activate a common
signaling pathway that culminates in the activation of NFkappa B transcription factors as well as the mitogen-activated
protein kinases (MAPKs) ERK, p38 and JNK.
The so-called “immunological privilege” of the testis is believed
to arise from the need to prevent immune responses against
the autoantigens of the meiotic and haploid germ cells, which
first appear at the time of puberty, long after the establishment
of self-tolerance. Paradoxically, the testis has an active defense
mechanism which is illustrated by the obvious capacity for
inflammatory responses to local and systemic infection.
However, the testicular defence to infection, particularly to
bacteria, are poorly defined on the molecular level.
In a first step, we investigated the mRNA expression pattern
of TLRs in isolated testicular cells by RT-PCR. Most somatic
and germ cell types expressed at least one TLR, with Sertoli
cells (SC) synthesizing some, peritubular cells (PTC) most,
and testicular macrophages (TM) all TLRs. Isolated rat TM,
SC, and PTC were infected with human uropathogenic E.coli
(UPEC) and non-pathogenic commensale E.coli (NPEC).
Following Western blot analysis TLR-4 protein levels increased
2h after UPEC infection in TM and SC. In contrast, TLR-4 levels
in PTC increased only after 6h. Furthermore, we have also
investigated the activation of the MAP Kinases (p38, JNK
and ERK1/2) following UPEC infection in cultured testicular
cells. Phospho-p38 levels were strongly enhanced after 30
min in TM, after 60 min in SC and after 120 min in PTC.
ERK1/2 was phosphorylated transiently after 30 min in TM and
at 60 min in SC, whereas PTC showed no activation. Only in
SC JNK was activated following UPEC infection.
The transcription factor NF-kappa B plays a central role in
immunological processes and several diseases. A primary
level of control for NF-kappa B is through interactions with an
inhibitor protein called I kappa B. Activation of NF-kappa B
to move into the nucleus is controlled by the subsequent
degradation of I kappa B, a process known to prevent
apoptosis and induce proinflammatory cytokine production.
TM and SC displayed no degradation of I kappa B alpha after
UPEC infection, whereas in PTC I kappa B alpha degradation
starts after 1h. This indicates that TM and SC are driven to
apoptosis after UPEC infection, which is supported by
preliminary results from apoptosis assays. Addition of NPEC
to testicular cells showed no effect in any of the above
mentioned experiments, but peritoneal macrophages as
control were sensitive to both UPEC and NPEC. These results
suggest that testicular cells are much more sensitive to UPEC
infection than to other E. coli strains. Moreover, SC and TM
are significantly more sensitive to UPEC infection than PTC
and that apoptosis of SC can be an underlying cause for the
observed loss of spermatogenesis during bacterial infection
of the testis and excurrent ducts.
PO 015
Increasement of apoptotic spermatozoa in
infertile men with inflammatory changes in
seminal plasma
comparison to infertile men with no signs of inflammatory
changes by flowcytometric staining of Annexin-V.
Thereby we could demonstrate that spermatozooas from
infertile men with inflammatory changes showed increased
Annexin-V binding. In the subsequent ejaculate (1 hour after
first ejaculation) we could demonstrate a further significant
increase of apoptotic spermatozooas. Interestingly, we could
detect a high number of apoptotic spermatozooas after swimup. The rate of apoptotic spermatozooas was again higher
within the inflammatory group of infertile men. Furthermore,
we could demonstrate that apoptosis of spermatozooas was
stable over period of up to three hour and did not change
significantly in both group. This suggests that apoptotic
spermatozooas in the swim-up fraction were motile and
initialized the apoptotic process before the procedere.
Taken together our data demonstrate that infertile men with
inflammatory changes in the seminal plasma show an increase
rate of apoptotic spermatozooas which might hamper
fertilisation. Furthermore our data might open a rational basis
for antiinflammatory therapy to reduce apoptosis.
PO 016
Bacteriospermia and in vitro fertilization
B. GODOUET-GETTI1, Y. JASATIS1,
N. MOUSSET-SIMEON1, B. CLAVIER2,
B. MACE1, N. RIVES3
1 Reproductive Biology Laboratory - CECOS, Rouen
University Hospital, Rouen, France 2 Department of
Obstetrics and Gynecology, Rouen University Hospital,
Rouen, France. 3 Reproductive Biology Laboratory CECOS, Centre d'Investigation Clinique Inserm 0204,
Rouen University Hospital, Rouen, France Tél. : 02 32 88
82 25 Fax : 02 35 98 20 07 e-mail : HYPERLINK
"mailto:[email protected]" [email protected]
J.P. ALLAM, F. FROHNHOFFS, I. OLTERMANN,
G.HAIDL
University Hospital Bonn, Department of Dermatology
Objective : It is more than likely that apoptotic sspermatozooas
may hamper successful fertilisation. It is well known that
inflammatory cytokines such as TNF-alpha may induce
apoptosis.
So we investigated apoptotic spermatozooas in infertile men
with inflammatory changes in the seminal plasma in
328
Objective and design : Bacteriospermia is frequent in the
general male population like within men consulting for infertility.
The purpose of in vitro fertilization, classical (IVF) or by
intracytoplasmic sperm injection (ICSI), is to support the
process of fertilization and early embryonic development
within sterile culture media. The presence of bacteria in the
seminal fluid could be a harmful factor in this context. In our
work, we evaluated the effects of a significant bacteriospermia
(<103 bacteria/mL), detected on the semen sample the day
of oocyte puncture, on (i) sperm parameters, (ii) oocyte
fertilization rate, (ii) number and quality of the embryos obtained
as well as (iv) pregnancy rate and evolution.
Materials and Methods : Our study was performed on 514
consecutive cycles of IVF or ICSI. A spermoculture was carried
out on each semen sample collected the day of oocyte
puncture. Volume, pH, sperm count as well as motility (a+b)
constituted sperm parameters studied and were evaluated after
liquefaction according to the World Health Organization. The
number of oocytes collected, inseminated and/or injected
and fertilized as well as the number and the quality of the
embryos represented oocyte and embryonic parameters. The
follow-up of the pregnancies thus obtained was also evaluated.
Sperm preparation and the techniques of IVF and ICSI were
standardized. Statistical analysis was carried out using
software STAVIEW for WINDOWS (Abaccus Concept).
Results : Data collection relates to 514 cycles including 262
IVF (50.97%) and 252 ICSI (49.03%). Bacteriospermia was
detected in 21.21% semen samples. Bacteria most frequently
found were Ureaplasma urealyticum (33.9% of the positive
cultures), followed by Chlamydia trachomatis (11%),
Streptococci other than B (10.1%) and yeasts (1.8%). Among
the various studied sperm parameters, only motility (a+b)
appeared significantly decreased (p<0,0125) in men presenting
a positive culture. No visible sign of infection was observed
in IVF or ICSI culture dishes during this period. No significant
difference was observed by comparing (i) the rates of
fertilization in the infected group (49.68%) versus not infected
(50.41%) as well as (ii) the number and the quality of the
embryos obtained. On 125 pregnancies obtained, 25 (20%)
were in the infected group but without finding any statistically
significant difference in the rate of pregnancy between the two
groups (infected 22.9% versus not infected 24.94%). The
same applies to the frequency of pregnancy complications as
well as fetal and neonatal pathologies.
Conclusion : Bacteriospermia does not constitute a priori a
major factor susceptible to disturb the principal parameters
of IVF or ICSI. The systematic practice of a spermoculture
before Assisted Reproductive Technique (ART) procedure
can be called in question. On the other hand, taking into
account the probable role of certain bacteria in sperm motility
alteration, it appears essential to carry out a spermoculture
in any assessment of male infertility and this before proposing
ART.
329
PO 017
Simian Immunodeficiency Virus in the male
macaque genital tract: target organs and cells
during primary and chronic infection and impact
of early-initiated antiretroviral treatment
A. LE TORTOREC1, A.P. SATIE1, H. DENIS1, B.
JEGOU1, R. LE GRAND2, N. DEJUCQ-RAINSFORD1
1 Inserm, U625, GERHM, univ. Rennes I, Rennes, F35042 France ; 2 CEA, Service de Neurovirologie,
Fontenay-aux-roses, F-92265 France
Objectives : Despite semen being the main vector of HIV
dissemination, the sources of production of the viral particles
contaminating this fluid remain unclear. Numerous studies
have demonstrated compartmentalization of HIV strains
between blood and semen, strongly suggesting viral production
within the male genital tract (MGT). Moreover, men under
antiretroviral treatment may still shed HIV in semen despite
undetectable blood viremia (reviewed in (1, 2)). In this context,
determining the susceptibility of the MGT to HIV infection
and the impact of antiretroviral treatments within this body site
is of prime importance. Our study used SIV-infected macaques
as an animal model to research this issue.
Methods : Macaques inoculated intravenously with
SIVmac251 were sacrificed either during primo-infection at 14
days post-inoculation (n=4) or during the chronic phase (n=7).
In addition, some animals were submitted to antiretroviral
treatment initiated either 4h (n=4) or 7 days post-infection
(n=3) for a 2 week period before sacrifice. Testes, prostates,
epididymis and seminal vesicles were recovered and the
presence of SIV RNA and DNA determined in nested RTPCR and PCR, respectively. Immunohistochemistry and in situ
hybridization were performed to appreciate the distribution of
immune and SIV infected cells.
Results : In chronic and primary-infected untreated monkeys,
viral RNA and DNA were detected in all the genital organs,
with a frequency positively correlated with plasma viral load.
Notably, elevated levels of detection of SIV nucleic acids
were observed during primo-infection. Infection was
consistently more frequent in the accessory glands than in the
testes. The accessory glands often presented varying degrees
of HLA-DR+ inflammatory infiltrates, observed as early as
14 days post-infection, and mainly composed of cytotoxic T
lymphocytes (CD3+TIA-1+). Using combined in situ
hybridization for SIV gag RNA and immunohistochemistry for
specific cell markers, we demonstrated the presence of SIV
RNA co-localizing with CD68+ myeloid cell in the prostate of
chronic and primary infected macaques. Two out of four
animals treated as early as 4h post-infection and four out of
four animals treated 7 days post-infection still displayed SIV
DNA in the accessory glands, in spite of a drastic decrease
of the plasma viral load.
Conclusions : SIV infection of the male genital tract occurs
early, the virus being detected as soon as 14 days postinfection. Infection of the MGT is associated with high blood
viremia and triggers an inflammatory response. Early initiated
treatment applied for a short duration does not prevent the
viral spread to the male genital organs.
References :
1. Dejucq-Rainsford N., Jégou B. : Viruses in semen and
male genital tissues : consequences for the reproductive
system and therapeutic perspectives. Current Pharm.
Design, 2004, 10 : 557-575.
2. Dejucq N., Jegou B. : Viruses in the mammalian male
genital tract and their effects on the reproductive system.
Microbiol. Mol. Biol. Rev., 2001, 65 : 208-231.
Fundings : Inserm, ANRS, Région Bretagne, Sidaction,
Organon.
PO 018
glucosidase threshold level derived from its frequency
distribution in the control group. All men underwent routine
andrological workup. Testicular biopsies were available from
32 patients.
Results : The 2.5-percentile (16 mU/ejaculate) of αglucosidase levels in the control group was chosen as the cutoff value to categorise the study group. 33 patients had αglucosidase levels < 16 mU/ejac. (suspected OA) and 46
were above this threshold (suspected NOA). The suspected
OA group was indistinguishable from the group with proven
OA in all relevant parameters evaluated. In contrast, the group
with suspected NOA differed significantly from the proven
OA group in regard to semen volume, fructose and αglucosidase, which were higher, while testicular volume and
age were lower. No differences in hormone levels were found
except that the control group had higher LH and lower FSH
levels. All patients of the suspected OA group with available
histology had elongated spermatids in their biopsy (16/16) while
in the suspected NOA group several patients with
spermatogenic arrest were identified (6/16 ; 10 had elongated
spermatids) giving a specificity of 100% (pos. predictive value
: 100%) and sensitivity of 62% (neg. predictive value : 38%)
for an α-glucosidase level of 16 mU/ejac.
Conclusions : The α-glucosidase level of 16 mU/ejac., which
is slightly lower than the WHO cut-off level of 20 mU/ejac.
derived from proven fathers, provides a discriminator to help
counselling patients with azoospermia and normal FSH levels
with regard to the chance of finding sperm in a biopsy
(specificity : 100%). However, the sensitivity of 62% is rather
poor. Fructose and zinc do not improve the diagnosis.
Azoospermia and normal follicle stimulating
hormone (FSH) levels - role of α-glucosidase as
discriminator between patients with or without
obstruction
Support : No external support.
References :
1. WHO Laboratory Manual for the Examination of Human
Semen and Semen-Cervical Mucus Interaction. 4th edition,
Cambridge, Cambridge University Press, 1999.
F. TÜTTELMANN, F.M. WERNY, T.G. COOPER,
M. SIMONI, E. NIESCHLAG
Institute of Reproductive Medicine of the University of
Münster, Germany ([email protected])
2. Krause W., Bohring C. : Why do we determine alphaglucosidase activity in human semen during infertility workup ? Andrologia, 1999, 31 : 289-294.
Objective : Although α-glucosidase determination is included
in the WHO Handbook for Semen Analysis (1), its value in the
infertility workup is still under debate (2). We reinvestigated
the diagnostic power of α-glucosidase to distinguish between
obstructive (OA) and non-obstructive azoospermia (NOA).
Design : Retrospective analysis of patients’ data retrieved from
the institute’s database Androbase© (3).
Materials and Methods : 164 healthy volunteers with normal
sperm concentration (> 20 x 106/ml) formed the control group.
The group with proven OA consisted of 86 patients with either
diagnosis of vasectomy (n = 55) or CBAVD (n = 31). 79
patients with azoospermia and normal FSH levels (≤ 7 U/l)
and no apparent clinical reason for their azoospermia (such
as Klinefelter syndrome, post-chemotherapy, tumours, etc.)
were subdivided into two study groups according to an ·α−
330
3. Tüttelmann F., Luetjens C.M., Nieschlag E. : Optimising
workflow in andrology: A new electronic patient record and
database. Asian J. Androl., 2006, 8 : 235-241.
PO 019
Measurement of steroid hormones concentration
in peripheral and spermatic blood in infertile
patients with non-obstructive azoospermia : A
prospective comparative study
PO 020
A long term quality control for a
serum inhibin B assay
A. MAHMOUD, F. COMHAIRE, KAUFMAN J.M.
University Hospital Ghent - Andrology Laboratory
Ghent, Belgium
G. PASQUIER1, L. SIBERT1, Y. JASATIS2, N.
MOUSSET-SIMEON2, B. MACE2, N. RIVES3
1 Urology, Rouen University Hospital, Rouen, France
2 Reproductive Biology Laboratory - CECOS, Rouen
University Hospital, Rouen, France
3 Reproductive Biology Laboratory - CECOS - Centre
d'Investigation Clinique Inserm 0204, Rouen University
Hospital, Rouen, France
Objective : Inhibin B in serum is a well-established marker
for Sertoli cell function and spermatogenesis. The present
study evaluates the reproducibility of an enzyme linked
immunosorbant assay (ELISA) for inhibin B.
Objective and design : The serum levels of steroid hormones
(testosterone, estradiol) were compared between spermatic
vein blood and peripheral blood, in patients with non obstructive
azoospermia (NOA), according to the results of surgical sperm
retrieval. The aim of our study was to identify predictive factors
of successful testicular sperm recovery in patients with non
obstructive azoospermia.
Materials and Methods : A Prospective and comparative
study (supported by a Rouen Hospital Grant and approved
by the local ethical committee) was performed in a population
of of 30 patients with NOA. For each subject who underwent
testicular sperm extraction, serum levels of testosterone (T)
and estradiol (E2) were determined in spermatic vein blood
(Ts and E2s) and in peripheral blood (Tp and E2p). Differences
in hormone levels between the two groups of patients with
either successful or unsuccessful sperm recovery were
analysed using non parametric variance analysis and MannWhitney U-test.
Materials and Methods : Two pooled blood sera with low (PL)
and high activities of inhibin B, were included in each of 57
assays for inhibin B (Serotec, Oxford, UK) and the assay was
performed at our laboratory according to manufacturer’s
instructions.
Pooled serum samples were processed in duplo allowing for
the calculation of inter assay co-efficient of variation (CV)
and the averages of the two readings in the different assays
was used to calculate the inter assay CV. In total data on 3
different pairs of pools used in 57 runs for the period 19982002 was available for analysis.
Results : The average intra-assay coefficient of variation
(CV) for all the inhibin B runs performed at our laboratory
was 6.9 % for the serum pools with low inhibin B levels (PL)
and 4.4 % for those pools with high inhibin B activities (PH).
The inter-assay CV ranged from 19 to 34 % (see table 1).
Table 1 : Intra- and inter-assay coefficient of variation (CV)
for inhibin B measurement.
Results : Data are presented as the mean values with ranges.
Conclusion : The ratio E2s/Ts appears significantly increased
in the group with failure of testicular sperm retrieval (p=0,018).
These data suggest that aromatase activity is higher when
haploid germ cells are absent of the testis. The mechanisms
of action of estrogens in the spermatogenesis remain to be
clarified, especially with studies using another marker like
tissue marker (cytochrome P450 aromatase activity, aromatase
gene (CYP 19) expression).
331
Conclusions : The high interassay variability might be
explained by the use of different batches of inhibin B kits over
the period of 5 years. The within batch interassay CVs is
much lower (data not shown). The high inter-assay CVs over
a long period of time indicates, however, that comparing
results from different studies where different batches of inhibin
B kits were used is not recommended. Support : none
PO 021
Inhibin B as a predictor before testicular sperm
extraction – valuable completion or cost factor ?
No differences in the serum Inhibin B levels were observed
in TESE-positive and TESE-negative pats.. But TESE-positive
and TESE-negative pats. had significant lower serum Inhibin
B levels compared to the control group, p<0.05.
In pats. with normal serum FSH 82% of sperm retrieval
attempts were successful, 27/6 vs 22/23, p<0.05.
In TESE-positive pats. the serum-FSH level was with 14.6±1.9
IE/l significantly lower compared toTESE-negative pats.
20.3±1.9 IE/l (p<0.05).
F. REIHER1, O. RAU1, T. LINDENMEIR1, I. NICKEL2,
J. KLEINSTEIN2, E. ALLHOFF1
1 Klinik und Poliklinik für Urologie, Otto-von-GuerickeUniversität Magdeburg AöR, Deutschland,
2 Klinik für Reproduktionsmedizin und gynäkologische
Endokrinologie, Otto-von-Guericke-Universität Magdeburg
AöR, Deutschland
Email : [email protected]
PO 022
Introduction : Inhibin B as a predictive marker for a successful
testicular sperm extraction (TESE) is discussed controversely
in the literature. The aim of this study was to evaluate the
predictive value of Inhibin B as a reliable marker for
spermatogenesis in patients (pats.) with nonobstructive
azoospermia (NOA).
Materials and Methods : In 78 pats. (33±4.8 years)
undergoing TESE the following parameters were evaluated:
(a) testicular volume; (b) serum follicle hormone (FSH) and
(c) serum Inhibin B level. These parameters were correlated
to the TESE-results (recovery of spermatozoa).
Results : In 49 of the 78 pats. (62%) spermatozoa were
successfully retrieved (TESE-positive). The Serum inhibin B
level was 103,5±12,5 pg/ml in the TESE-positive group.
Compared to a control group (healthy probands) there was
a significant difference regarding serum Inhibin-B levels
between the TESE positive group and control group (103.5
vs. 183.6 pg/ml; p<0.05). We observed also a statistically
significant difference of the serum Inhibin-B levels between
controls and pats. with no evidence of spermatozoa in TESE
(92.8 vs. 183.6 pg/ml; p<0.05). No statistically significant
difference exists for the Serum Inhibin B levels in TESEpositive and TESE-negative pats. (p>0.05).
In contrast, a significant difference in serum FSH was evident.
In TESE-positive pats. the serum-FSH level was 14.6±1.9
IE/l, in TESE-negative pats. 20.3±1.9 IE/l (p<0.05).
In pats. with normal serum FSH 82% of sperm retrieval
attempts were successful, whereas in the group with pathologic
serum FSH only in 42% of attempts sperms were retrieved
(p<0.05). Additionally, spermatozoa recovery correlated also
with testicular volume (p<0.05).
Conclusions : Serum FSH and testicular volume are valuable
marker to estimate the expecting results of a testicular sperm
extraction. In our hands Serum inhibin-B wasn`t able to give
further information predicting the possible evidence of
spermatozoa.
332
Testicular Fine Needle Aspiration Biopsy in the
investigation of the subfertile male with
azoospermia and severe
oligo-terato-asthenospermia
TH. MIKOS, P. POLICHRONOU, G. GRIMBIZIS, A.
PAPANICOLAOU, E. ATHANASIOU, P. SEVASTIADOU,
D.G. GOULIS, B.C. TARLATZIS, J. BONTIS,
J. PAPADIMAS
Unit of Reproductive Endocrinology, 1st Department of
Obstetrics & Gynecology, Aristotle University of
Thessaloniki, Greece
Objective : To describe our experience with testicular Fine
Needle Aspiration Biopsy (FNA) in the investigation of subfertile
men with azoospermia and severe oligo-astheno-teratospermia
(OAT).
Patients and Methods : From 1999 to 2005, 1087 subfertile
men were studied at the outpatient clinics of a referral
Andrology center in Northern Greece. All men underwent
clinical, sperm, basal and dynamic hormonal evaluation.
Genetic studies were applied as appropriate. Azoospermia and
severe OAT were found in 78 (7.9%) and 27 (2.7%) men,
respectively. Testicular FNA underwent 99 (94.2%) of these
men. In addition, 15 men underwent Testicular Sperm
Extraction (TESE) at a time later than FNA.
Results : Etiological diagnosis of the studied men were:
Idiopathic Non-Obstructive Azoospermia (INOA) with or without
Late-Onset Hypogonadism (LOH) (n = 51, 52%),
crytporchidism (n = 14, 14%), congenital agenesis of vasa
deferentia (n = 8, 8%), obstructive azoospermia (n = 7, 7%),
varicocele (n = 13, 13%), Klinefelter syndrome (n = 2, 2%),
and other causes (n = 4, 4%). Overall, the cytological
diagnoses were : normal spermatogenesis (n = 18, 19%),
moderate hypospermatogenesis (n = 16, 17%), severe
hypospermatogenesis (n = 24, 24%), incomplete maturation
arrest (n = 2, 2%), complete maturation arrest (n = 14, 14%),
complete Sertoli-Cell only Syndrome (SCOS) (n = 22, 22%),
and insufficient sample (n = 3, 2%). The cytological diagnoses
in the subgroup of 51 men with INOA were: normal
spermatogenesis (n = 1, 2%), moderate hypospermatogenesis
(n = 7, 14%), severe hypospermatogenesis (n = 18, 35%),
incomplete maturation arrest (n = 2, 4%), complete maturation
arrest (n = 9, 18%), complete Sertoli-Cell only Syndrome
(SCOS) (n = 13, 25%), and insufficient sample (n = 1, 2%).
Finally, during the TESE procedure, sperm was found in 7 men
whereas not found in the remaining 8 men ; of them, the
positive result was predicted during the FNA procedure in 6
men (Positive Predictive Value – PPV: 86%) and the negative
result was predicted in 6 men (Negative Predictive Value –
NPV: 75%).
Conclusion : FNA plays an important role in both the
diagnostic and therapeutic approach of subfertile man. In the
diagnostic approach, a single testicular FNA according to our
results has 51% possibility of detecting sperm in subfertile men
with INOA. Aspirated spermatozoa can then be used for
assisted reproduction techniques, such as Intra-Cytoplasmic
Sperm Injection (ICSI). In the therapeutic approach, FNA has
86% PPV and 75% NPV of predicting TESE results, further
facilitating the ICSI procedure.
Presenting author : Prof. J. Papadimas, 1st Department of
Obstetrics & Gynaecology, “Papageorgiou” General Hospital,
Periferiaki Odos, Nea Efkarpia, 56403, Thessaloniki, Greece.
e-mail : [email protected]
PO 023
Presence and quality of testicular spermatozoa in
azoospermic men according to region of their
residence in Slovenia
I. VIRANT-KLUN, S. DROBNIC, L. BACERKERMAVNER, J. MIVSEK, T. TOMAZEVIC
Department of Obstetrics and Gynaecology, University
Medical Centre Ljubljana, Slovenia
E-mail : Hyperlink "mailto:[email protected]"
[email protected]
to 8 Slovenian geographical regions of their residence.
Additionally, the incidence of testicular cancer in their medical
history was compared among the regions.
Material and Methods : In this study 192 diagnostic testicular
biopsies from 192 infertile men with azoospermia attending
our Department were included. Each biopsy specimen was
checked for the presence of spermatozoa. In patients with
spermatozoa sperm motilility was evaluated (motile and nonmotile spermatozoa). The testicular tissue was cryopreserved
for later in vitro fertilization. The men were divided in 8 groups
by the geographical region of their residence: I. Capital
Ljubljana (n =71), II. Region of _tajerska (n = 30), III. Region
of Gorenjska (n = 25), IV. Region of Primorska (n = 20), V.
Region of Dolenjska (n = 13), VI. Region of Koro_ka (n =
12), VII. Region of Severna Primorska (n = 9), VIII. others –
Idrijsko-Cerkljansko, Posavje, Zasavje (n = 12). All groups of
men were statistically compared according to the presence
and motility of testicular spermatozoa, ICSI outcome, and
the incidence of testicular cancer in their medical history.
Results : The mean male age was 36 years (min. 25 – max.
56 years) with no statistically significant difference among
regions. There was no statistically significant difference among
regions in the proportion of men with testicular sperms (I.
65%, II. 57%, III. 72%, V. 77%, VI. 67%, VIII. 70%), except
for the Primorska region (IV) : a significantly lower proportion
of men (35% ; P < 0.05) due to a higher incidence of Sertoli
Cell Only Syndrome and maturation arrest. Also a higher
incidence of testicular cancer in the medical history was
registered in the men from the region of Primorska than from
other regions (15% vs 4%, P < 0.05). We found no significant
differences in the proportion of men with motile sperms. In
researched group of men 68 ICSI cycles were performed and
16 pregnancies (33.5% pregnancy rate per cycle) resulted (I.
23%, II. 27%, III. 37.5%, V. 25%, VI. 20%, VIII. 25%), none
to a couple from the Primorska region (0%). Ten children
were born (7 girls and 3 boys), 2 pregnancies are still on-going,
and 4 pregnancies ended in a spontaneous abortion.
Conclusions : In this preliminary study, performed in a small
group of azoospermic men, we found that the Primorska
region (towns Koper, Piran, Portoro_, Izola, Ankaran) situated
at the coast of the Northern Adriatic Sea (Gulf of Trieste, Gulf
of Koper, Gulf of Piran) shows negative effects on
spermatogenesis and on incidence of testicular cancer. This
study will be extended to a bigger group of azoospermic men
to exclude possible bias (like genetic abnormalities and
cryptorchidism), and to evaluate the possible environmental
effects of the Primorska region.
Support : We would like to thank to Mr. Matjaz Klun, B. Sc.,
lawyer, Ministry of the Environment and Spatial Planning of
Republic of Slovenia and to Ms. Mojca Pirc, B. Sc.
Objective : Little is known about the effect of geographical
factors on the appearance and type of azoospermia, and
manifestation of testicular cancer.
Design : The aim of this preliminary study was to compare
the presence and quality (motility) of testicular spermatozoa,
and outcome of ICSI in patients with azoospermia according
333
PO 025
PO 024
Recombinant FSH for the treatment of idiopathic
infertility
Fertility in men with renal insufficiency before
and after transplantation
V. CAUNI1, D. DINU2, C. PERSU1, P. GEAVLETE1
J. NOHRA, A. ZAIRI, B. BENGOUDIFA, N. KAMAR,
L. ROSTAING, E. HUYGHE
1 Department of Urology, Saint John Emergency Clinical
Hospital, Bucharest, Romania
2 “C.I. Parhon” Institute of Endocrinology, Bucharest,
Romania. [email protected]
Service d’Urologie et d’Andrologie et *Service de
Néphrologie et de transplantation d’Organe, Hôpital
Rangueil, Toulouse ([email protected])
Introduction : Several experimental studies have
demonstrated the key role played by FSH in normal
spermatogenesis. Our objective was to analyze the results of
the treatment with recombinant FSH in normogonadotropic
patients with severe idiopathic olygospermia.
Patients and Methods : 50 males with idiopathic infertility,
aged between 20 and 43 years old where included, based on
the following criteria : severe olygospermia, infertility diagnosed
at least 3 years ago, normal cariotype, normal ultrasonographic
aspect of the testis, no associated pathology. Hormones
involved in the reproductive function were measured by
immunometry and only patients with normal hormone levels
were included. Circadian patterns of urinary gonadotropes were
determined in controls and infertile patients. In all patients, the
free androgen index and the ratio FSH/LH were determined.
10 men with normal spermograms which conceived in the
last 12 months represented the controls. All patients where
treated for 20 weeks with, recombinant FSH, 50IU, 3 times
a week. In those with low FAI testosterone, undecanoate
40mg/day was added. After 10 weeks of treatment, patients
were reevaluated, using a spermogram and a hormonal profile.
Results : In all cases, circadian patterns of gonadotropes
are very close, characterized by a synchronization of the
secretion of the 2 hormones. After the treatment with
recombinant FSH, the mean concentration of spermatozoids
increased four times. 42% of the patients have normal
spermatic parameters, and a mean FSH/LH ratio of 1 at 20
weeks after the treatment. 52% of the patients induced
pregnancy after the treatment. In 8% cases, pregnancies
terminated with first trimester spontaneous abortions.
Conclusions : Our study proves the efficacy of the treatment
with recombinant FSH in males suffering from idiopathic
infertility. It also proves the necessity of synchronous secretion
of gonadotropic hormones for a normal spermatogenesis.
334
Objective : To determine the status of fertility in men with renal
insufficiency and to analyze the effect of transplantation on
fertility.
Design : self-administrated questionnaire.
Materials and Methods : A series of 239 renal transplanted
men were evaluated through a self-administrated questionnaire
focusing on fertility. Questions concerned the period before
hemodialysis, after hemodialysis, and after transplantation.
Results : Before hemodialysis, 173 men tried to have children,
163 succeeded (94.2%) and 10 patients did not succeed
(5.8%).
After the beginning of hemodialysis, 14 patients tried to have
children. Among them 4 patients succeeded (29%) and 10
patients did not succeed (71%).
After transplantation, 51 patients tried to father and 27
succeeded (53%). Andrological evaluation of the 24 infertile
men revealed erectile dysfunction in 7 cases and ejaculation
disorders in 6 cases. Time elapsed between transplantation
and the onset of pregnancy was 4 +/- 3 years (1-15). Among
them, 8 were hemodialysed due to graft function deterioration.
Among the 43 remaining patients, the fertility rate was 63%.
Conclusion : fertility decrease dramatically in hemodialysed
patients from 94% to 29%. Renal transplantation is a way to
recover fertility up to 63%.
PO 027
PO 026
Modeling of the upper range of scrotal
temperature in a driving situation
Complex molecular analysis of genetic factors in
Russian infertile men
B. BENGOUDIFA, R. MIEUSSET
V.B. CHERNYKH, A.L. CHUHROVA, T.S.
BESKOROVAINAYA, S.V. GUDZENKO, A.A.
STEPANOVA, E.V. IL’YNA, F.S. MYACHINA, L.F.
KURILO, A.V. POLYAKOV
EA 3694 " Recherche en fertilité humaine – Santé de la
reproduction dans les PVD ", Hôpital Paule de Viguier, 330
av de Grande-Bretagne, TSA 70034, 31059 Toulouse
Cedex, France
Objectives : Elevation of testicular or scrotal temperature,
either isolated or associated with a temperature rise of the
whole body, leads to functional disturbances reflected in
decreased quality and quantity of the gametes produced.
This has been demonstrated in experimental studies in fertile
men and observed in studies of infertile populations. However,
determination of maximum scrotal temperature, which we
will call the "upper range" is still an open question. We sought
the upper range by recording the maximum reached during
the temporal gradient of right and left scrotal temperature
and rectal temperature under the particular environmental
conditions of car driving.
Materials and Methods : Eight fertile male volunteers (20 to
48 years old) were studied after a continuous uninterrupted
period of 180 minutes of driving a motor vehicle in a sitting
position. History taking and clinical examination showed that
all men were free of any medical, surgical or occupational
histories, with no clinical varicocele or testicular hypotrophy
or atrophy. Testicular volumes were measured with callipers.
Temperatures were measured every 2 minutes with an AM7001 precision skin thermometer (reading accuracy 0.1%,
precision 0.03°C and sensor type K, Anritsu Meter Co., Tokyo,
Japan).
Mathematical and statistical analysis :
1) Modeling of the physiological parameters which play a part
in determining the upper range of temperature:
Adaptive ability of the scrotum: calculation of immediate and
mean speeds of temperature change.
Mechanisms of scrotal thermoregulation: time to initiation of
thermoregulation.
Scrotal sweating : computing of a periodogram.
2) Stepwise regression method : model selection and Akaike
criterion.
Results and Conclusion : Under the same conditions, the
upper range of scrotal temperature differs from one individual
to another. This upper range is essentially explained by two
factors: the initial values of scrotal temperature (physiological
values with no major thermal constraint), and the ability of the
scrotum to adapt in abnormal conditions such as driving.
Support : None.
335
Research Centre for Medical Genetics of Russian
Academy of Medical Sciences, Moscow, Russian
Federation.
Objective : To evaluate the frequency of common genetic
infertile factors in the cohort of Russian men with azoospermia
or severe oligozoospermia.
Design : Prospective study.
Materials and Methods : We have examined a cohort of
281 Russian infertile men. In all patients azoospermia or
severe oligozoospermia has been diagnosed by sperm
analysis (WHO, 1999). The individuals with chromosome
abnormalities were excluded from study. Molecular
investigation was carried out on leucocytes DNA. All DNA
samples were examined for complete and partial AZF
deletions. Detection of Y-chromosome microdeletions was
carried out according to Laboratory guidelines (Simoni et al.,
1999) by analyzing of recommended STSs plus sY615 in
multiplex PCR. The DNA samples with no deletions have
been tested on partial AZFc deletions in multiplex PCR, which
included following STSs: sY142, sY1197, sY1192, sY1291,
sY1206, and sY1125. Furthermore, eleven common CFTR
gene mutations (del21kb, delF508, delI507, 1677delTA,
2143delT, 2184insA, 394delTT, 3821delT, G542X, W1282X,
N1303K), IVS8 poly (T) variants, and androgen receptor (AR)
CAG-repeat polymorphisms were analyzed in 98 and 68
patients, respectively.
Results : Complete AZF deletions (AZFa – 2; AZFb+c – 2,
AZFc/ b2/b4 – 24) have been found in about 10% of all
examined chromosomes. Also thirty-three partial AZFc
deletions (gr/gr deletions – 7, b2/b3 deletions – 22, del sY1197
– 3, del sY1197 and sY142 – 1) have been revealed in 11.7%
patients (13% men with no complete AZF-deletions). Nine
heterozygous CFTR mutations (∆F508/- – 3, W1282X/- – 2,
2143delT/- – 2, del21kb/- – 1, and 2184insA/- – 1) have been
found in 9 of 98 (9.2%) examined. 5T allele polymorphisms
(7T/5T – 10, 9T/5T – 2) were found in 12.3% infertile men,
including one patient with heterozygous ∆F508 mutation. In
one case we have found determined revealed complete AZFc
deletion (b2/b4) and IVS8-5T variant in heterozygous state
simultaneously.
CAG repeat length in infertile men varied from 15 to 33
(median 22, mean 22.99), moreover 19% examined had
increased CAG-repeat numbers (n≥26).
Conclusions : In Russian azoospermic or severe
oligozoospermic men the frequencies of complete and partial
AZF-deletions are nearly equal. The results of this study
displayed that investigated genetic factors have high
prevalence in severe male infertility. In this situation complex
genetic examination is more preferred.
Support : none.
PO 028
Role of the X-chromosome linked, testis-specific
TAF7L gene in gonadal function and
spermatogenic failure
in the nucleotides sequence of exon 9, 10, 13, and two
changes in the flanking introns of exon 8 and 13, with
concomitant changes in amino acid sequence were observed
in 6 patients. Most of these alterations were also found in 8
controls with the exception of changes in exon 13. Though
none of these changes were previously described in NCBI
database, some are described in a recent publication (1).
There was no association or relationship observed with
reproductive hormones.
Conclusions : The alterations in the cDNA sequence observed
are probably polymorphisms. However, the haplotype observed
in exon 10 and the changes in the intron of exon 8 appears
novel. We report for the first time that these alterations in
nucleotide sequence of TAF7L gene are not associated with
gonadal dysfunction.
Support : Dr O. Akinloye is a visiting research fellow from the
Department of Clinical Biochemistry, College of Health
Sciences, Ladoke Akintola University of Technology, Nigeria
and is supported by the Alexander von Humboldt Foundation.
1) Stuoffs K., Willems A., Lissens W., Tournaye H., Van
Streirteghem A., Liebaers I. : The role of the testis-specific gene
hTAFL in the aetiology of male infertility. Mol. Hum. Reprod.,
2006, 12 : 263-267.
O. AKINLOYE, M. SIMONI, C. CALLIES, J. GROMOLL,
E. NIESCHLAG
Institute of Reproductive Medicine, of the University of
Muenster, Muenster, Germany. Hyperlink
"mailto:[email protected]"
[email protected]
Objective : The precise temporal and partial expressions of
specific transcription regulation factors (TRF) have long been
considered essential for proper execution of spermatogenesis.
Recently, mammals have been speculated to have evolved
more specialised TRF genes. In human the X-linked, testisspecific gene TAF7L gene may be obligatory for maintenance
of spermatogenesis. In this study, we attempted to investigate
the possible role of TAF7L gene in testicular function and
spermatogenic failure.
Design : The study is a case-control retrospective study.
Materials and Methods : Sixteen carefully selected infertile
males with consistent, non-obstructive azoospermia without
elongated spermatids in testis biopsy (available in some) and
with normal serum follicle stimulating hormone levels were
selected for this study. Twenty age-matched men with normal
spermatogenesis with the same ethnic background
(Caucasian) were recruited as controls. DNA extracted from
EDTA whole blood was screened for sequence changes in the
coding regions and part of the flanking introns of the TAF7L
gene by direct sequencing. Amino acid sequence was
compared to the NCBI standard sequence.
(Hyprelink"http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=N
ucleotide&dopt=GenBank&val=BC043391"BC043391).
Semen analysis and hormone evaluation were performed.
Results : Five alterations, consisting of three exonic changes
336
PO 029
DAZ (Delected in AZoospermia) gene: an
evidence of Y chromosome evolution
S. FERNANDES1, A.T. FERNANDES2, R. GONÇALVES2,
M. SOUSA1-3, A. BREHM2, A. BARROS1-4
1 Genetics Department, Faculty of Medicine, University of
Porto ; 2 Lab of Human Genetic, University of Madeira,
Funchal ; 3 Lab Cell Biology, ICBAS, University of Porto ; 4
Centre for Reproductive Genetics A Barros, Porto; Portugal
S. Fernandes, ([email protected])
Objective : Better understanding the heterogeneity and
instability of the AZF loci on the human Y chromosome from
fertile and infertile individuals, to determine which cases could
present higher predisposition for a quick evolution since
oligozoospermia to azoospermia. Check the existence of an
association between the Y-haplogroups and the DAZ gene
partial deletions.
Design : The human Y chromosome is strictly paternally
inherited and, in most of its length, does not engage in pairing
and crossing over during meiosis. The DAZ gene, candidate
to fertility factor, lies within human Y chromosome’s AZFc
region (Azoospermic Factor) whose deletion is a common
cause of spermatogenic failure. The Y single-nucleotide
polymorphisms (Y-SNPs) on the non-recombining Y region
(NRY) are well characterized and permit the construction of
a unique phylogeny of haplogroups. DAZ haplotypes were
defined using Single Nucleotide Variants (SNVs)/Sequence
Tagged-Site (STSs) markers to distinguish the four copies of
the gene (1). The variation of ten Y-chromosome Short Tandem
Repeat (STRs) was used to determine the coalescence age
of DAZ haplotypes in a comparable time frame similar to that
of SNP haplogroups.
Materials and Methods : DNA samples from 97 azoospermic
men ranging from Sertoli-cell-only syndrome (SO) to maturation
arrest (MA) and hipospermatogenesis (HP) were analysed.
A control group of 91 fertile men was also studied. All males
involved in this study gave an informed consent, following
local ethical guidelines. The Y-chromosome typing (24 SNPs
and 10 STRs) was assayed and the haplogroup nomenclature
and phylogeny used was the one proposed by the Ychromosome Consortium (2002). DAZ haplotypes were
determined by analysis of 6 DAZ-SNVs (I-VI) and 2 DAZSTSs (DAZ-RRM3 and Y-DAZ3) (1). The coalescence time
within each DAZ haplotype was defined using 10 STR (2).
Results : An association between DAZ haplotypes and Y
chromosome haplogroups was found and our data shows
that the DAZ gene is not under selective constraints and its
evolution depends only on the mutation rate. The same
variants were common to fertile and infertile men, although
particular DAZ partial deletions occurred only in infertile ones,
suggesting that infertility is due to gene conversion
mechanisms or other chromosomal rearrangements (3).
Conclusion : As DAZ deletions might be a polymorphic event
associated to a specific haplogroup or an individual cause of
infertility, DAZ partial deletions should only be used as a tool
for infertility diagnosis when analysed in combination with Yhaplogroup determinations. The possibility that a mutation
defining a haplogroup could be something more than a single
mutation - for instance, being associated with Y chromosome
rearrangements - remains open for debate.
Support : (1) Fernandes et al. 2002 ; (2) Zhivotovsky et al.
2004 ; (3) Fernandes et al. 2006.
PO 030
Lack of association of Y chromosome gr/gr
deletions with spermatogenic failure
C. RAVEL1,2, S. CHANTOT-BASTARAUD1,2 A.
DUMAINE A1, D. LOURENÇO1, J. MANDELBAUM2, J.P.
SIFFROI2, K. MCELREAVEY1
1 Reproduction, Fertility and Populations. Institut Pasteur,
Paris, France 2 Université Pierre et Marie Curie Paris-6,
EA1533, AP-HP, Hôpital Tenon, Paris, France.
Corresponding author : [email protected]
Objective : Complete deletions of the AZFc region are
associated with spermatogenic failure and occur with a
frequency of 0.00025% in the general male population.
Recently, partial deletions of AZFc have been reported to be
a risk factor for developing infertility.
Design : Here we report our latest data on the investigation
of partial AZFc deletions (gr/gr) in both control
(fertile/normospermic men n=186) and case (spermatogenic
failure, n=329) cohorts from the Tenon Hospital in Paris.
Materials and Methods : DNA from each patient was tested
for partial AZFc deletions using a series of Sequence Tagged
Sites markers from the region. In addition, each sample was
tested with informative polymorphisms to distinguish between
the DAZ1/2 and DAZ3/4 gene clusters as well as CDY1a and
CDY1b. The haplogroup of each individual was defined using
7 binary markers. Finally the samples were tested for DAZ copy
number using dosage assays.
Results : The incidence of gr/gr deletions was similar in each
group: 11/329 (3.3 %) spermatogenic failure, 6/186 (3.2 %)
control samples. Using polymorphic markers for DAZ1/2,
DAZ3/4 and CDY1a /CDY1b, we did not observe a correlation
between absence/presence of these genes and the fertility
status. Y chromosome haplogroup distribution was similar in
each group. Of the 11 cases with the gr/gr deletion, 10 showed
the presence of 2 copies of DAZ, whilst one (control male) had
4 copies of DAZ.
Conclusions : Taken together our data indicate that partial
AZFc deletions are relatively common compared to AZF
deletion. Using available markers and gene dosage assays
we could not distinguish between the case and control groups.
These data are consistent with the hypothesis that the gr/gr
deletion is an inconsequential polymorphism, at least in an
ethnically heterogeneous Parisian population. Further studies
are underway using fiber-FISH technology to understand the
molecular nature of these deletions in both study groups.
337
PO 031
Fine molecular characterization of a new genetic
risk factor for oligo/azoospermia
C. GIACHINI*, F. NUTI*, E. GUARDUCCI*, G.
BALERCIA§, G. FORTI*, C. KRAUSZ*
*Department of Clinical Physiopathology, Andrology Unit,
University of Florence, Firenze, Italy §Division of
Endocrinology, Institute of Internal Medicine Polytechnic
University of Marche, Ancona, Italy
Hyperlink "mailto : [email protected]"
[email protected]
reduced sperm count in the Italian population. The role of
gr/gr deletions as risk factor for reduced sperm count is
currently debated and it maybe related to different factors:
ethnic origin (Y background), lack of confirmation of results
by dosage analysis, analysis of control groups with unknown
sperm counts. We propose that different Y chromosome
backgrounds and subtypes may confer protection against
the deleterious effect of gr/gr deletion and thus a simple STS
based method is insufficient to provide clinically relevant
information.
PO 032
Objective : “gr/gr” deletion, a newly identified partial deletion
of the AZFc region of the Y chromosome, has been reported
as a new risk factor for male infertility. Subsequent studies
questioned the association of this genetic anomaly with
infertility.
Materials and Methods : A part from our study and the
original report in which all deletions were confirmed by
quantitative assays, the detection of gr/gr deletions is currently
based on a sequence tagged site (STS) +/- PCR method.
The confirmation of the deletions PCR results is fundamental
since rearrangements in this region may modify the primer
annealing sequences leading to false deletions. Moreover,
duplications identifiable only by dosage analysis may restore
normal gene dosage and thus be compensate for a partial
deletion. Using a combined method based on STS +/- followed
by quantitative gene dosage and qualitative assays – to
confirm the deletion and to define the number and the type
of deleted DAZ and CDY genes – we identified three different
gr/gr deletion patterns. The heterogeneity of the phenotypes
associated to gr/gr deletions maybe therefore related to
different gr/gr deletion subtypes.
Results : The aim of this study was to further increase the
size of our study population and to provide a tool for the
distinction between pathogenic and neutral types of gr/gr
deletions by fine molecular characterization of the deletions
and Y haplogroup definition. We analyzed a total of 557
patients and 364 controls. The frequency of gr/gr deletions
in patients was significantly different from the normospermic
controls (4,1% versus 0,2%, respectively p<0,001 ; OR=9,7
with CI 95% 1,4-66,2). Furthermore we have calculated how
many subjects, identified with gr/gr deletion by the first step
PCR +/- method were not confirmed by gene dosage and
sequence family variant analysis. We observed a false deletion
rate of 18% (6/33) indicating that STS +/- method alone does
not provide reliable results.
Conclusions : In conclusion, we confirmed on a large group
(total n=921) of subjects that gr/gr deletion is a risk factor for
338
The impact of androgen receptor polymorphism
and parental ethnicity on semen quality in young
men from Latvia
J. ERENPREISS1,2, I. TSAREV2, A. GIWERCMAN2,
Y. GIWERCMAN2
1 Molecular Reproductive Research Unit, Dept. of Clinical
Siencies, Lund University, Sweden ;
2 Andrology laboratory, Riga Stradins University, Latvia. email : Aleksander.Giwercman.@med.lu.se
Objective : Recent studies of young men from the general
population have demonstrated geographical and ethnical
differences in semen quality with high sperm counts in the
Baltic countries. The aim of this study was to investigate
whether semen quality in Latvian men might be related to
the maternally derived polymorphisms in the androgen receptor
(AR) gene or rather to the ethnicity of the fathers.
Materials and Methods : In total 119 military conscripts from
Latvia were included in the study. One hundred and fourteen
men with available AR gene polymorphism data were divided
into two groups according to their mother’s ethnicity: men
with Latvian mothers (n=83) and those with non-Latvian
mothers (n=31). Regions outside of Latvia from which the
parents originated included Russia (n=33), Ukraine (n=9),
and Byelorussia (n=6). To assess the impact of father’s
ethnicity, men were additionally divided into two groups
according to their father’s ethnicity : men with Latvian fathers
(n=77) and men with non-Latvian fathers (n=37). The influence
of ethnic origin and AR polymorphisms on sperm concentration,
total sperm count, semen volume and proportion of
progressively motile sperms was analyzed in general linear
regression models adjusted for the abstinence time. Influence
of the mother’s ethnicity was additionally adjusted for father’s
ethnicity, and vice versa.
Semen analysis was performed according to WHO
recommendations. DNA was extracted from leukocytes and
the numbers of CAG and GGN repeats obtained by direct
sequencing Material was available for sequencing of the CAG
and GGN tracts in 114/119 (96%) and 99/119 (83%) of the men,
respectively.
Results : Men with Latvian mothers had borderline significantly
shorter CAG repeat length (21.6±2.9) as compared with those
with non-Latvian mothers (22.9±3.2, p=0.05). Sperm
concentration did not differ significantly between these two
groups (76±59 and 70±52, p=0.9, respectively). In contrary,
father’s ethnicity exhibited significance for sperm concentration
and total sperm count, with higher numbers in men with
Latvian fathers (n=77) as compared to men with non-Latvian
fathers (n=37) (80±61 vs 62±48, p=0.035, for sperm
concentration; 225.7±209 vs 158.4±134.4, p=0.002, for total
sperm count, respectively). CAG repeat length was associated
with sperm concentration in the whole study population: men
with high sperm concentration (>50 x 106/mL, n=70) had
shorter CAG repeat length (21.4±2.9) than those with low
sperm concentrations (<50 x 106/mL, n=44): 22.8±3.1, p=0.03.
GGN repeat length, in turn, correlated only with semen volume:
men with GGN>23 had higher semen volume (3.2±2.1) as
compared with those with GGN=23 (2.6±1.3, p=0.04) and
GGN<23 (2.0±1.2, p=0.006).
Conclusions : In Latvian men the origin of the father seems
to play a more important role for sperm concentration than the
maternally derived AR polymorphisms.
PO 033
Infertility and reproduction failure in autosomal
translocations carriers
A. ABDELMOULA1, A. AMOURI2, M. MEDDEB3,
A. SALLEMI1, T. REBAI1
1 Laboratory of Histology, University of Medicine, Sfax,
Tunisia 2 Laboratory of Cytogenetics, Pasteur Institute,
Tunis, Tunisia 3 Laboratory of Genetics, Tunis, Tunisia
Auteur correspondant : nouha_abdelmoulabouayedahoo.fr
/ Fax : 00216 74 239 826
Objective : Assessment of the incidence of autosomal
translocation carriers in Tunisian subjects with reproduction
failure and report of their treatment attempts results.
Design and Location : South of Tunisia during 5 years at the
laboratory of Histology of the University of Medicine at SFAX.
339
Materials and Methods : Cytogenetic investigations were
performed in 260 infertile men because of severe male infertility
with low sperm count and in 47 couples because of recurrent
miscarriages.
Results : Out of the 146 oligospermic and 114 azoospermic
men, 35 had an abnormal karyotypes (13.5% : 21% in
azoospermic and 7,5% in oligospermic men).
Chromosome aberrations observed were 24 sex chromosomal
aberrations [comprising 46,XX (n=1), 47,XXY(n=20),
47,XYY(n=1) and 46,X,del(Yq) (n=2)] and 11 autosomal
aberrations including 5 reciprocal translocations in oligospermic
men [t(4;9)(p15.3;p21), t(11;22)(q24;q11),t(2;3)(p24;q26)
t(16;22)(q13;q12) and t(11 ;21)(q13 ;p11)] and 6 robertsonian
translocations [t(13;14) in 2 oligospermic and 2 azoospermic
men and t(14;21) in an oligospermic and an azoospermic
brothers]. The incidence of autosomal balanced structural
abnormalities in oligospermic men was higher than in
azoospermic men especially for reciprocal translocations
(45,5% v/s 0%).
For robertsonian translocations carriers, a maternal meiosis
transmission is recorded in two cases while for reciprocal
translocations, a familial history is noted for 3 cases. In the
other hand, chromosomal abnormalities were recorded twice
out the 47 couples, in a female partner and in a
normozoospermic male partner, who carried reciprocal
translocations [46,XX,t(1;4)(q31;q26) and 46,XY,t
(4;17)(p16;p12)]. None of theses translocations carriers come
to conceive naturally or with assisted conception. In fact,
fertilization failures in previous ICSI attempts (one to 5
attempts) were recorded for 6 couples.
Conclusion : Although ICSI provide a way of treating infertile
men, it seems that translocations carriers need to be assisted
with the most recent advances of reproductive technology
which is the preimplantation diagnosis. In fact, only this
technology may improve the implantation rates and protect
couple from termination of pregnancy. Unfortunately, this
practice is not yet available in our country.
PO 034
Preliminary study on the role of the human gene
izumo in oocyte spermatozoa fusion failure
1. IZUMO is implicated in gametes fusion in mouse but not
in human where several proteins could have a redundant
role in this function.
2. IZUMO is indeed implicated in gametes fusion in humans.
Unfortunately, either we did not studied patients with the
correct phenotype, or we missed the mutations (mutations
outside the coding region or large size rearrangements).
Therefore, protein expression needs now to be studied in
infertile patients.
I. AKNIN-SEIFER1, V. GRANADOS1, R.L. TOURAINE2,
J. CHOUTEAU3, J.P. WOLF4, R. LEVY1
1 Laboratoire de Biologie de la Reproduction, Hôpital Nord,
CHU de Saint-Étienne, France ; 2 Laboratoire de
Génétique Moléculaire, Hôpital Nord, CHU de SaintÉtienne, France ; 3 Clinilab, Saint Martin d'Hères, France ;
4 Laboratoire de Biologie de la Reproduction, CHU Jean
Verdier, Bondy, France. e-mail : [email protected]
References : Inoue N., Ikawa M., Isotani A., Okabe M. : The
immunoglobulin superfamily protein Izumo is required for
sperm to fuse with eggs. Nature, 2005, 10 ; 434(7030) : 2348.
PO 035
Aim of study : Oocyte fertilization results from gametes
fusion. Molecular complexes mediate this process. In ART,
fertilization failure occurs in 10% of the attempts and fertilization
is very often achieved by intra cytoplasmic sperm injection.
This technique bypasses the physiological gamete membrane
interaction with a risk of decreased embryo quality and genomic
imprinting anomaly.
Izumo, first sperm membrane protein shown to be essential
for fusion in mice, is hidden under plasma membrane and
accessible only after the acrosome reaction. This novel
immunoglobulin superfamily (IgSF), type I membrane protein
with an extracellular immunoglobulin domain, was also
detected in human and is encoded by a gene composed of
10 exons. We decided to look for mutations of the IZUMO gene
in a group of infertile patients with a phenotype fitting to the
features of the KO mouse model.
Design and Location : Retrospective and multicentric study.
Materials and Methods : Patients : 22 normozoospermic
patients with past of intrauterine insemination failure followed
by unexplained IVF fertilisation failure. 23 " controls "
normozoospermic and fertile volunteers. 20 fertile men from
general population (unknown semen parameters)
PCR : Amplification of the 9 coding exons of the gene (exon
1 is non-coding). Sequencing : mutation screening.
Results : No punctual mutation could be found in any of the
65 studied men. Three different polymorphisms were observed
in exon 4 and intron 4 : c321C>T ; c397+17G>A ;
c397+109G>A (neutral mutations without any predictable
consequence on the protein) and one was observed in exon
10 (c998_999CG>TT) resulting in an alanine to valine
substitution (pA333V). Only two combinations of these
polymorphisms were observed (namely, C-G-G-CG and T-AA-TT). There were no significant differences in the frequency
of these genotypes in our 3 studied populations.
Conclusions : Two hypothesis :
340
Gamete aneuploidy rate and implantation failure
F. VIALARD1,2, I. HAMMOUD1, D. MOLINA-GOMES1,
M. ALBERT1,2, M. BAILLY1, J. SELVA1,2
1 Departement de biologie de la reproduction,
cytogénétique et gynécologie obstétrique, CHI Poissy St
Germain, 78303 Poissy Cedex ; 2 INSERM U407, Faculté
de Médecine Lyon Sud, 69924 Oullins Cedex
Objective : Implantation failure (more than 10 embryos
transfered without pregnancy) is a situation known to be
associated to an increased risk of aneuploidy in embryos and
considered as a PGD indication in many countries. Our aim
was to evaluate the gamete aneuploidy incidence in this
situation in France, a country where PGD is not allowed for
this indication.
Materials : 3 couples groups were considered: group 1 (n=11)
control couples with pregnancy obtained after IVF with fertile
sperm and female infertility, group 2 (n=20) couples with
pregnancy obtained after IVF for male or idiopathic infertility,
group 3 (n=32) couples with implantation failure.
Methods : 1000 spermatozoa per patient were analysed by
FISH (chromosome X, Y et 18 (Abbott) in the 3 groups. In group
3, 1st polar body (PB1) was also analysed by FISH with the
Polar Body™ PGT multicolour (Abbott) kit, in the 22 couples
who accepted the procedure during ICSI.
Results : Sperm aneuploidy rate was increased in group 2
(1,6% ; p<0,0005) and 3 (2,1% ; p<0,001) in comparison to
group 1 (0,6%). In group 3, 2 patients presented a very high
aneuploidy rate (>5%) in spermatozoa (6,5% et 25%). This
did not happen in group 1 and 2. 8/32 patients in group3 and
6/20 patients in group 2 exhibited a moderate but increased
aneuploidy rate in sperm (between 2 and 5%). This did not
happen in group 1.
PB1 aneuploidy rate was high (average:35,4%) in the 127 PB1
analysed in group 3. For 3 patients, this rate was very high,
more than 2/3 abnormal oocytes, analysing only 5
chromosomes!
All together, 22% couples (5/22) had no particular
chromosomal risk in gametes, 68% (15/22) presented an
increased spermatic (> 2%) or oocyte (> 1/3) aneuploidy rate
and 10% of couple (2/22) had both.
Conclusion : Those results confirm that implantation failure
has a heterogeneous origin and that gamete chromosome
abnormality rate is one of the major factors explaining it.
Genetic counselling seems to be necessary after those
analyses and those results may influence the next ART
attempts, and future pregnancy chance. Indeed,
preconceptionnal and when possible preimplantation diagnosis
can be indicated for these couple (78% in our series) with
increased gamete aneuploidy risk and implantation failure.
PO 036
We report a consanguineous family with two siblings, a male
and a female, who have a typical Kartagener's syndrome and
another dead brother who seems to be affected. The diagnosis
was established on the basis of clinical grounds and familial
history of a 33-year old man who was referred for cytogenetic
exploration because of 11 months history of male infertility with
azoospermia in semen analysis.
Clinical history and physical examination revealed presence
of situs inversus totalis, chronic bronchorrhea and
bronchiectasis, chronic nasal symptoms, sinusitis and infertility.
Serum FSH, LH and testosterone were normal and the patient
had a normal 46,XY karyotype. The patient's sister was also
affected and had similar features with the same respiratory
symptoms and situs inversus totalis. After some difficulties to
conceive, she gave birth to a healthy female child and a male
stillborn with multiple congenital abnormalities. Their third
sister had also history of foetal congenital abnormalities.
Diagnosis of Kartagener's syndrome was confirmed by electron
microscopy study of ciliary structure. Epithelial cells were
obtained from nasal and bronchic biopsy specimens. Ciliary
ultrastructure showed qualitative defects in dynein arms with
absence of the outer dynein arm and an abnormal additional
microtubule at the central doublet probably secondary to
radial spokes abnormalities.
Genetic diagnosis consisting of mutation research in DNAI1
gene at 9p13-p21 (20 exons), DNAH5 gene at 5p15-p14 (79
exons) and DNAH11 gene at 7q21 (82 exons) may be helpful
for familial genetic counselling but remains hard because of
the extent of these genes and heterogeneity of the disease.
The infertile patient, who sought an ICSI treatment, was
informed about possible rates of testicular sperm retrieval
after TESA (Testicular sperm aspiration) and all possible
genetic risks through a genetic counselling.
Genetic counselling in azoospermia with primary
ciliary dyskinesia
N. ABDELMOULA1, A. SALLEMI BEN HMIDA1,
R. REKIK5, M. MEDDEB5, A. AMOURI3,
S. KAMMOUN2, H. JAAFOURA4, T. REBAI1
PO 037
1 Faculty of Medicine of Sfax University, Tunisia
2 University Hospital Hedi Chaker of Sfax, Tunisia
3 Institute of Pasteur of Tunis, Tunisia
4 Faculty of Medicine of Tunis University, Tunisia
5 Private sector, Tunisia
Auteur correspondant : nouha_abdelmoulabouayedahoo.fr
/ Fax : 00216 74 239 826
Primary ciliary dyskinesia is a rare etiology of infertility in
man. It is an autosomal recessive disease characterized by
defective cilial ultrastructure in ciliated cells and affects about
1 in 20000 newborns. Regarded as a subgroup of primary
ciliary dyskinesia, Kartagener's syndrome is characterized
by the simultaneous presence of chronic bronchorrhea with
bronchiectasis, chronic sinusitis and situs inversus. Infertility,
occasionally described in males, is caused by ultrastructural
defects of sperm tails with always total asthenozoospermia.
341
Transcriptional study of apoptosis during the first
wave of spermatogenesis in gonadotropin
deficient mice
O. CHAUSIAUX, M. ABEL, R. FURLONG,
H. CHARLTON, N. AFFARA
HMGG group, Department of Pathology, Cambridge
University, UK [email protected]
Objectives : The objective of this study is to investigate the
transcriptional events involved in the infertility phenotypes
seen in luteinizing hormone receptor Knock-out (LuR-KO)
and Hypogonadal (hpg) male mice. The LuR-KO lacks the
receptor for LH causing the failure of Leydig cells to secrete
androgen. Therefore spermatogenesis arrests at the round
spermatid stage and leads to male infertility. Hpg lacks GnRH
preventing the pituitary from secreting either LH or FSH.
These hpg mice are also infertile with spermatogenesis arrest
at the pachytene spermatocyte stage. Here we concentrate
on apoptosis pathways in the testis during the early stages
of spermatogenesis.
Design : Microarray analysis was used to identify biological
processes that are differentially expressed during the
premeiotic and meiotic stages of the first wave in the two
mouse models compared to the wild type. Apoptosis was
found to be one of the mechanisms highlighted by this study.
TUNEL assays were performed to confirm the occurrence of
apoptosis. Transcriptional profiles of some genes were
confirmed by QRT-PCR.
time points. This data is normalised to the overall composition
of the library.
Figure 4 : Expression level of selected genes involved in
apoptotic pathways in the model compared to the age matched
WT. Only genes with a fold change >1.5 are shown. The antiapoptotic genes are shown in green, the pro-apoptotic in
orange and the regulators of apoptosis in grey. Eg: Aven is
1.5 fold down regulated in LuR-KO at day 3 compared to the
age matched control.
Transcript
Localisation
Bcl2l11/Bcl-Rambo
PGC ( Tanaka et al 2002)
Faf1
Spermatogonia,
Spermatocytes , Spermatids,
Somatic cells (Ryu et al, 1999)
Api5
Spermatogonia, Spermatocytes 1 ,
Somatic cells (Clemente et al, in
press)
Btg2
Spermatogonia, Spermatocytes 1 ,
Somatic cells (Clemente et al, in
press)
Bcl2l11/Bim
Spermatogonia, Spermatocytes ,
Spermatids, Somatic cells
(Meehan et al, 2001)
Bcap29
Spermatocytes, Spermatids
(Clemente et al, in press)
Dap
Spermatogonia, Spermatocytes ,
Spermatids (Clemente et al, in
press)
Btg2
Spermatogonia, Spermatocytes 1 ,
Somatic cells (Clemente et al, in
press)
Figure 1 : MA plots for LuRKO at day3, day 8, day 13 and day
19. The number of genes differentially expressed is indicated
for each time point, and they are labelled in red. For hpg, the
number of differentially expressed genes increases at each
time point and is larger than in LuR-KO. The lack of both
gonadotropins has a strong effect on gene expression.
Aven
Spermatogonia, Spermatocytes ,
Spermatids (Ina et al,2003)
Diablo
Spermatocytes (El Chami et al,
2005)
Cflaf/Flip
Spermatocytes
(Giampretri et al,2003)
Figure 2 : MA plots for hpg at day3, day 8 and day 13. The
number of genes differentially expressed is indicated for each
time point, and they are labelled in red.
Fasl
Spermatocytes, Spermatids
(D’Alessio et al, 2001)
Daxx
Spermatocytes,
Spermatids,Somatic cells
(Lopez et al 2002)
Materials and Methods : Microarray experiments were
performed comparing the models to an age matched control
on the MmcDNAv1 library. Each experiment included 4
technical replicates and 2 biological replicates. Technical
reproducibility was assessed using a windowing procedure
described by C.Tseng. The Z score method described by J.
Quackenbush was used to identify differentially expressed
genes. Source was used to categorise genes into different GO
groups (gene ontology). GO data was normalised taking into
account the library composition and the number of genes
differentially expressed in each experiment. TUNEL assays
(Apoptag kit, Chemicon) and immunohistochemistry were
performed on paraffin sections. Apoptotic figures were also
identified on H&E sections.
Results : Differentially expressed genes for each model at
each time point are highlighted in figures 1 and 2. The number
of genes affected by the mutation increases in the last 3 time
points in LuR-KO, possibly due to increasing differences in
cell composition.
GO data indicates that, at the transcriptional level, apoptosis
genes are over represented in the model at all time points
except at day19 in LuR-KO. Most apoptosis related
transcriptional events occur at day 8 in both models. In hpg
at day 8, there is a bias towards apoptosis related genes in
the list of differentially expressed genes (almost 2.5 times
the expected amount). Figure 4 presents a selection of genes
whose expression is altered in the models. Figure 5 indicates
localisation of the transcripts during the WT first wave of
spermatogenesis.
Figure 3 : Proportion (% compared to WT) of genes related
to apoptosis that are differentially expressed at the different
342
Figure 5 : Known localisation in wild type testis of the selected
transcripts involved in apoptosis.
Figure 6 : TUNEL assays. Histology presents TUNEL assay
results. Apoptotic cells are labelled in brown and are mainly
of later stages of spermatogenesis. The histogram presents
the average number of apoptotic cells per tubule, p value
<0.1 is indicated with a star (data is not yet available for hpg
adult).
Conclusions : As confirmed by TUNEL assays (Figure 6),
apoptosis is involved in eliminating the germ cells that stop
differentiating
in
hpg
and
LuR-KO
testis.
Immunohistochemistery results for active Caspase 3 and
Cytochrome-c should be available for the conference. This will
allow a better understanding of the apoptotic pathways induced
during the first wave of spermatogenesis and how they might
contribute to the infertility phenotype in the two models.
Table1 : Serum testosterone levels on day 28.
Support : Our work is funded by the Department of Pathology
at Cambridge University.
Table 2 : Serum testosterone levels on day 65.
PO 038
Table 3 : Percentage of haploid cell population of testis on day 65.
The effects of Human Chorionic Gonadotropin on
germ cells maturation and testosterone secretion
in mouse testis
N.R. AKBARZADEH1, 2, M.M. AKHONDI2, M. JEDDI
TEHRANI3, M.R. SADEGHI2, E. JAVADI1
1 Department of Biochemistry, School of Basic Sciences,
Azad University, Science and Research Campus, Tehran,
Iran. ([email protected]) ; 2 Department of
Reproductive Endocrinology & Embryology, Reproductive
Biotechnology Research Center, Avesina Research
Institute, ACECR, Tehran, Iran ; 3 Department of
Reproductive Immunology, Monoclonal Antibody Research
Center, Avesina Research Institute, ACECR, Tehran, Iran ;
4 Department of Endocrinology, Endocrinology &
Metabolism Research Center, School of Medical Science,
Shariati Hospital, Tehran, Iran
Objectives : Testis function including sperm production and
androgen secretion is controlled by hypophyseal
gonadotrophins. Androgen production is induced by luteinizing
hormone (LH) and promotes germ cell proliferation and
maturation. LH has more than 90% similarity with Human
Chorionic Gonadotropin (hCG) in structure and function,
accordingly hCG is used in research and clinic in spite of LH
presence. The aim of this study is to evaluate the effects of
different dosages of hCG on germ cell maturation and
testosterone secretion in immature testis of neonate mouse.
Design : The present study was designed to examine
testosterone secretion and germ cell proliferation in mouse.
Moreover, the long-term effects of this hormone have been
evaluated on the germ cell proliferation. Because the effects
of hCG could be dose related, varying doses of hCG were
used. Simultaneously, the effects of hCG injection on androgen
status of the animals were evaluated.
Materials and Methods : 24 mice (C57BL/6) aged 15 days
old divided into 4 equal groups that group 1 served as control.
343
The groups 2, 3, and 4 were exposed to 5, 10 and 50 IU hCG
respectively that was injected on days 15 and 25 of the mouse
life. The level of serum testosterone was determined on days
28 and 65 of the mouse life. The different testicular cell
populations were determined according to DNA contents by
Flow Cytometric Analysis. The data thus obtained were
analyzed by Cell Quest software. Statistical analysis was
done using SPSS software program. The Kruskal-Wallis test
was applied for comparison of all groups and Mann-Whitney
test was applied for comparison of any group with the control
group and P values less than 0.05 were considered statistically
significant.
Results : Day 28 testosterone in case groups were shown
to have increased, as compared to the control group by
increase in hCG dose, with the highest values in group 4 as
shown in Table 1. However, only the difference between group
4 (highest hCG dose) and the control group was found to be
significant. In contrast to day 28, different results were found
for day 65. In this regard, the testosterone levels were declined
with increasing dose of hCG in different groups in comparison
to the control group. Accordingly the group 4 had the lowest
level of testosterone (Table 2). There was also no statistically
significant difference detected between the case groups and
the control group. Additionally, DNA Flow Cytometric analysis
revealed that the germ cell numbers had remarkably declined
in case groups in comparison with the control group on day
65 and this reduction was statistically significant for groups
3 and 4 (Table 3).
Conclusions : The present study used a mouse model and
demonstrated that hCG, administrated at levels comparable
to clinical dosages, adversely affects the testicular germ cell
population and androgen production. With maturation, there
is an increase in haploid cell proportion with a simultaneous
decrease in the other cells. Thus, the significantly reduced
haploid cell population seen postpubertally in group 4 in the
present study is consistent with these reports and implies
impaired germ cell maturation. In the present study, only a
profound reduction in testosterone levels, as seen in group
4, was associated with a reduced haploid cell population.
This might imply that mild to moderate androgen withdrawal
may be safely tolerated by the germ cells. It may be that
Leydig cells are also sensitive to androgen withdrawal;
however, additional studies are necessary in this regard.
Support : This study was supported by Avesina Research
Center.
Materials and Methods : The single cell suspensions were
analysed by flow cytometer after staining with Propidium
Iodide (PI). The slides were incubated in citrate buffer (0.1 M)
at 900C for 10 minutes. Antibodies against transition proteins
TP1 and TP2 (generously provided by Dr. W. S. Kistler) were
added and incubated overnight, washed (3 times in Triton x100 0.1%) and secondary antibody (goat anti rabbit IgG
conjugated with FITC) was added for fluorescence microscopy.
In order to correlate between dpp where TP were first
expressed and the step of spermiogenesis, fixed tubuli from
each dpp were stained with PI and analyzed by CLSM. On
each dpp we determine the most advanced spermiogenic
step by focusing on the spermatocyte and spermatid layers.
Results : Using fluorescence microscopy TP2 appeared at
27dpp (Step 6), and peaked at 34 dpp during maturation of
spermatids during spermiogenesis. TP1 appeared later (at
28dpp, step 8) and peaked at 35dpp. Using flow cytometry
we could demonstrate that the chromatin condensation process
started before the appearance of TPs.
PO 039
Conclusion : Confocal microscopy demonstrated that TP2
was first expressed in step 6 and TP1 in step 8 during
spermiogenesis in the hamster. Chromatin condensation in
the hamster started before the expression of TPs.
Transition protein expression in spermatids
during the first spermatogenic wave in golden
hamster
Support : In part, by a grant of Israel Ministry of Health Chief
Scientist Office.
S. BECKER, L.M. LEWIN, L. SHOCHAT, Y. SOFFER,
R. GOLAN
PO 040
Department of Clinical Biochemistry, Sackler School of
Medicine, Tel-Aviv University
Analysis of gonosomic aneuploidy in men with
severe teratozoospermia
Objective : Spermatogenesis is a sequence of events which
starts in germinal cells (spermatogonia) and ends in mature
sperm cells (Spermatozoa). The process consists of
spermatogonial proliferation, meiosis and spermiogenesis
(spermatid differentiation). In spermiogenesis, the round
spermatids, the product of meiosis, are the first haploid cells.
They undergo biological, morphological and cytological
differentiation to produce mature spermatozoa. Among other
changes histones in the chromatin are presumed to be
replaced, first by transition proteins (TPs) and then by
protamines. The objective of the present project was to
determine the age of hamsters and step of spermiogen-esis
where TPs are first expressed.
Objective : The aim of this study is to evaluate the incidence
of spermatic aneuploidies in men with severe teratozoospermia
and to determine an eventual relation between aneuploidies
and a specific morphology of spermatozoa.
Design : Male hamsters undergoing the first spermatogenic
wave, from 26-42 day postpartum (dpp), were sacrificed by
CO2 asphyxiation ; testes were surgically excised and single
cell suspensions were prepared for flow cytometric analysis.
In addition, a portion of each testis was fixed in Bouin solution,
embedded in paraffin and prepared for staining on slides for
confocal laser scanning microscopy (CLSM).
Material and Methods : Fluorescent in situ hybridization
(FISH) with direct label fluorescence DNA probes specific for
chromosome X, Y and 8, was performed on spermatozoa
sampled from 30 patients with severe teratozoospermia
(abnormal forms > 80%). Results were compared with those
of spermatozoa sampled from 12 healthy men with normal
semen profiles.
344
M. MEHDI, H. EL GHEZEL, M. AJINA, A. SAAD
Laboratoire de Cytogénétique et Biologie de la
Reproduction, CHU Farhat Hached, Sousse, Tunisia
Corresponding author : [email protected]
Results : A minimum of 10000 spermatozoa was analysed
per chromosome probe. The mean frequency of
teratozoospermia in patients was 91 ± 6.99%. There was a
statistically significantly increased frequency of XY, XX and
YY disomies in the patients with severe teratozoospermia
compared with the normal donors (1.42 vs 0.31%, 1.13 vs
0.19% and 1.11 vs 0.24%, respectively, p<0.001 in all
comparisons). The rate of total diploidy was significantly
increased in the patients compared with normal donors. The
difference was statistically significant (1.46 vs 0.16%, p<0.001).
The sex-chromosomal anomalies due to the meiosis I (XY)
are less important than the anomalies due to the meiosis II
(XX or XY).
There was a correlation between macrocephalic spermatozoa
and diploidy (r = 0.37, p<0.05) and a correlation between
macrocephalic spermatozoa and nullisomy XY (r = 0.48,
p<0.05).
Conclusion : These data add further evidence that patients
with severe teratozoospermia have an increased sperm
aneuploidy rate and that this is particularly high in
macrocephalic spermatozoa.
PO 041
Design : Cross-sectional case study.
Materials and Methods : We investigated testicular samples
of 110 men with non-obstructive azoospermia in Avesina
Infertility Clinic, Tehran, Iran during 2005-6. Semi-quantitative
nested RT-PCR was performed in order to determine the
expression of SYCP3. We also evaluated the expression
level of gene during spermatogenesis using the
histopathological scoring of all samples.
Results : Testicular expression of SYCP3 mRNA was
observed in 67/110 (60.9%) patients. The expression level
correlated with the level of spermatogenesis (p<0.0001). It was
expressed in patients with hypospermatogenesis and
maturation arrest, while expression was not observed in
spermatogonial arrest, Sertoli cell– only syndrome and
testicular atrophy. Sensitivity, specificity, positive and negative
predictive values of negative SYCP3 expression for detection
of patients with Sertoli cell-only syndrome were 88.5%, 92.3%,
86.1%, 93.7%, respectively.
Conclusion : our findings indicate that SYCP3 is expressed
in human testis and is restricted to germ cells. The gene
expression in a specific level of spermatogenesis can help
histopathological findings in prediction of level of
spermatogenesis. Therefore, SYCP3 is suggested as a
possible molecular marker for spermatogenesis in men with
non-obstructive azoospermia.
Support : This research was supported by Molecular Medicine
Network, Ministry of Health & Medical Education, Iran.
Expression of Synaptonemal Complex Protein 3
(SYCP3) mRNA in Testis : A Possible Molecular
Marker for Spermatogenesis in Patients with
Azoospermia
PO 042
M. AARABI1*, H. SOLTANGHORAEE2, M. AARABI3,
N. AMIRJANNATI2, M.A. AKHONDI1,
M.H. MODARRESSI4-5
Longitudinal study of inhibin B values during
childhood and puberty in a boy with Klinefelter's
syndrome
1 Reproductive Biotechnology Research Center, Avesina
Research Institute, Tehran, Iran.
2 Avesina Infertility Clinic, Avesina Research Institute,
Tehran, Iran.
3 Academic Unit of Clinical Pharmacology, University of
Sheffield, Sheffield, United Kingdom.
4 Nanobiotechnology Research Center, Avesina Research
Institute, Tehran, Iran.
5 Department of Medical Genetics, Tehran University of
Medical Sciences, Tehran, Iran.
*Will be presented by : Dr. Mahmoud Aarabi MD (Email:
[email protected])
A.F. RADICIONI, E. DE MARCO, E. CAMA, A. ANZUINI,
C. PICCHERI, A. LENZI
Department of Medical Pathophysiology , 1st University of
Rome “La Sapienza”, Italy
Objective : To investigate the expression of the Synaptonemal
Complex Protein 3 (SYCP3) gene as a molecular marker for
spermatogenesis in men with non-obstructive azoospermia.
345
Objective : Klinefelter’s Syndrome (KS) is characterised by
degenerative changes in the seminiferous epithelium, resulting
in fibrosis of the seminiferous tubules and Leydig cell
hyperplasia. This degenerative process leads to the typical
small, firm testes seen with this syndrome. It mainly affects
germ and Sertoli cells, although their numbers range from
normal to subnormal during infancy (Muller et al., 1995). In
normal boys Inhibin B (InhB) levels progressively increase
throughout puberty, until they reach normal adult values as
a marker of mature spermatogenesis (Radicioni et al., 2005).
Design : Case presentation, clinical and laboratory evaluation.
Clinical and Laboratory Evaluation : We studied a KS boy
(karyotype 47,XXY, early diagnosis by amniocentesis) from
the age of 4.5 to the end of puberty. At the first visit, his height
was cm 107.5 and weight kg 18.200, with mean testicular
volume (MTV) 1.0 ml (Prader orchidometer) and
parenchymatous consistency. Subsequent visits documented
a regular height growth curve and hormone levels and testicular
volumes as reported in the table. Spontaneous puberty began
aged 12.5 yrs with regular progression to stage G5 (Tanner,
1962).
Conclusions : These data demonstrate childhood and early
puberty InhB levels similar to normal values. Mid-puberty saw
a rapid drop in gonadal hormone. This suggests – in our case
at least – that Sertoli cell function is normal up to stage G3.
Following this, in normal subjects spermatogenesis activity
begins and InhB originates from the communication between
Sertoli and spermatogenesis cells, whereas in our patient
InhB values underwent a rapid decrease.
FSH in childhood and early puberty was also found to be
within the normal range for the age, and began increasing
on entering mid-puberty. Testicular volume increased up to the
beginning of spontaneous puberty and underwent a further
small growth the following year, at which point penis
morphology and pubic hair growth showed a normal
progression.
PO 043
Effect of cowhage (mucuna pureins) on
reproduction in male albino rats
S. PRAKASH, S. SURESH, E. PRITHIVIRAJ
Department of Anatomy, Dr. A.L. Mudaliar Post Graduate
Institute of Basic Medical Sciences, University of Madras,
Chennai 600 113. India
Objective : To test alcoholic seed extract of cowhage (Mucuna
pruriens) for aphrodisiac and spermatogenic potential in male
albino rats.
Design : Albino rats weighing around 180 to 200 gms were
classified into various groups, group I (G1) received saline and
group II (G2), group III (G3) and group IV (G4) received 150,
346
200 and 250 mg/kg of body weight of extract (seed)
respectively. Animals received daily oral feeding of either
extract or saline for 50 days.
Materials and Methods : Following investigations were done
on 15th, 30th and 50th days of survival 1.Mating behavior :
Computer analysis of the video recorded mating behavior
using responsive female rats. Parameters analyzed were
mount latency, intromission latency, ejaculation latency, postejaculatory interval, mounting and intromission frequency. By
60th day, animals were sacrificed by over dose of anesthesia.
2. Sperm analysis : Epididymal sperm were collected and
following parameters were analyzed; sperm concentration,
viability and motility. 3. DNA/ Chromatin integrity test : Sperm
smear was stained with acridine orange (AO), aniline blue (AB)
and methyline blue (MB) to study DNA integrity. 4. Biochemical
and Hormonal analysis : Blood was collected periodically
were used for these analyses. 5. Anti-oxidant, histological
and histomorphometrical study : Testis and epididymis were
carefully collected and weighed individually; tissues were
processed for anti-oxidant (superoxide dismutase, reduced
glutathione, glutathione reductase, glutathione transferase,
glutathione peroxidase and vitamin C and E) and Hematoxylin
and Eosin stained sections were used histological and
histomorphometric analysis.
Results : Biochemical values indicate mild toxic effect in G4.
There was an increase in sperm concentration, in G2, G3, and
G4, when compared with G1, peak was in G3. However,
number of sperm cells with chromatin integrity was slightly
reduced in drug administrated groups, than control. Similarly,
mating behavior scoring indicates an increase in ejaculation
time and number of intromission, which was more pronounced
in G3, than G2 and G4. Testosterone level was increased by
15th and 30th days in G2, G3 and G4. However; there was
no progressive increase with course of dose, but maintained
at a higher level than normal. Anti-oxidant levels were near
normal in G3, whereas slight increase was observed in others
(G2, G3 and G4). Increase in testicular and epididymal weight
was seen in experimental groups. Histological and
histomorphometric study revealed increased spermatogenic
activity in G2, G3 and G4.
Conclusion : Increase in ejaculation time, number of mounting
and sperm concentration signify that seed of this plant is
having the potential to increase sexual activity and sperm
production. Effects seem to be dose dependant as G3 exhibit
excellent result. However, reason for more number less,
sperm with DNA/chromatin integrity, might be due to increased
(could be premature) spermiation or /and less time given for
sperm to mature in the ducts before being ejaculated. Increase
in testosterone level signifies the androgenic property of the
extract. Testicular and epididymal weight increase supported
by histological analysis clearly demonstrate that, the cowhage
having the potential to increase sperm production, as well
as producing aphrodisiac effect in male rats. However, a longterm study is warranted to analyze its beneficial and toxic
side effects of this extract, before testing it on specific fertility
problems.
Support : Work was supported by University Starter fund
from University of Madras.
PO 044
Regional variations in semen quality in France
A. MULLER1,4, J. AUGER2, P. JOUANNET2, L. BUJAN1,
A. SPIRA3, P. THONNEAU1
1 EA 3694 " Recherche en fertilité humaine – Santé de la
reproduction dans les PVD ", Hôpital Paule de Viguier, 330
av de Grande-Bretagne, TSA 70034, 31059 Toulouse
Cedex, France
2 Service de Biologie de la Réproduction-CECOS, Hôpital
Cochin, 75014 Paris, France
3 INSERM (National Institute For Health and Medical
Research) U569, 94276 Le Kremlin-Bicêtre, France
4 Corresponding author: A. Muller, EA 3694 " Recherche
en fertilité humaine – Santé de la reproduction dans les
PVD ", Hôpital Paule de Viguier, 330 av de GrandeBretagne, TSA 70034, 31059 Toulouse Cedex, France. Email : [email protected]
Several studies have suggested that the semen concentration
of fertile men has declined in recent decades in many
industrialised countries. In France, a retrospective study of
sperm donors from 1973 to 1992 found a decrease in sperm
concentration in a northern region (Paris) over the study
period. Nevertheless, no sperm concentration decrease was
observed in south-western France (Toulouse) during the same
period (1977-1992). For the authors, these findings may
suggest a regional difference in semen quality which could
be due to different environmental exposures between the two
French regions. Nevertheless, these conclusions must be
taken with caution due to potential bias linked to the long
inclusion period (starting in 1973 and ending in 1992) with
possible modifications in sperm analysis methods during this
time.
Objective : In order to re-analyse geographical variations in
semen concentration between north and south-western
France, we conducted a prospective study (REPRHOM) of
sperm characteristics, using standardised methodology and
including laboratory quality control.
Design, Materials and Methods : From 2002 to 2003, male
partners of pregnant women were recruited in maternity
departments in Paris and Toulouse, using the same
standardised recruitment protocol. A semen sample was
collected from each volunteer. Quality control showed no
significant inter-laboratory variability in sperm concentration
assessment.
Results : During the study period, sperm characteristics were
examined in 129 subjects living in a northern region of France
(Paris area) and 79 in a south-western area (Toulouse). No
difference was observed in sperm concentration, mobility or
347
other characteristics between men living in Paris and in
Toulouse.
Conclusions : Our results are in contradiction with those of
an earlier study where sperm quality was found to be higher
in men living in Paris compared with those living in Toulouse,
between 1973 and 1992. Although the authors suggest that
the environment may play a role in such regional variation,
recruitment and measurement bias cannot totally be excluded
(recruitment began 30 years ago and continued for 20 years).
Moreover, even if this environmental hypothesis could be
considered to have an impact in the 1970s, we believe that
pollutant exposure has drastically changed 30 years later,
especially in the south-west of France where intensive
prevention has limited environmental pollution, especially for
pesticide exposure. In conclusion, our comparative study did
not confirm regional variations in sperm characteristics between
north and south-western France in the early 2000s.
Acknowledgements : This work was supported by the
European Union QLK4-CT-1999-01422 and grants from the
French Ministry of Health Department (DGS), the French
Research Department, the French Agency for Environmental
Safety (AFSSE), the Total-Fina-Elf Group and the AGRICA
Group.
PO 045
Impact of occupational and environmental
exposures on semen parameters : evaluation of
an investigation methodology.
G. DE FLEURIANa,b,c, J. PERRINa,c,
I. SARI-MINODIERb,c, A. BOTTAb,c, J.M. GRILLOa,c,
M.R. GUICHAOUAa,c
a Laboratoire de Biologie de la Reproduction. AP-HM – La
Conception. 147, bd Baille ;
b Service de Médecine et Santé au Travail Faculté de
Médecine. 27, bd J Moulin ;
c Laboratoire de Biogénotoxicologie et Mutagenèse
Environnementale (EA 1784 - IFR PMSE 112), Faculté de
Médecine, 27, bd J Moulin ; 13385 Marseille Marseille
cedex 5 France
Tel. : +33 (0)491 38 13 76 ; Fax : +33 (0)491 38 38 97 ;
email : Hyperlink
"mailto:[email protected]"
[email protected]
Objective : To study relations between occupational and
environmental exposures and semen parameters.
Design and setting : Retrospective study between novembre
2004 and november 2005, La Conception University Hospital,
Marseille, France.
Materials and Methods : Population: 340 volunteer patients
(aged 18 to 55) consulting for infertility evaluation in
Reproductive Medicine Laboratory. Methods: association of
Reproductive Medicine Laboratory, Occupational Medicine
Unit and Genetic Toxicology Laboratory. Patients exposures
were assessed by an occupational physician before semen
analysis, using a standardized questionnaire. Semen
parameters were assessed according WHO criteria. Statistical
analysis : Chi-squared test, ANOVA means comparisons and
multivariable logistic regression model.
Results : Population : 80% of patients showed abnormal
semen parameters, 20% showed normal semen parameters
(considered as control patients). Main occupational sectors
were: office workers (15% of patients), professional drivers
(14%) and building trade workers (11%). Statistical analysis:
no occupational sector was over-represented in patients with
abnormal semen parameters, but this population was
significantly more exposed than control patients to: solvents,
colouring, gaz and fumes, cement, polycyclic aromatic
hydrocarbons, heat and cannabis (p<0.05). Analysis by logistic
regression showed that the risk for abnormal semen
parameters was associated with exposure to cement (OR =
5.2 ; p = 0.022), cannabis (OR = 4.0 ; p = 0.047) and
environmental pollution (OR = 3.8 ; p = 0.052).
Conclusions : The findings indicated an association between
impaired semen parameters and exposure to some
environmental or occupational toxic agents. This approach
could be systematically used during infertility evalutation, in
order to assess reproductive toxicant exposure of patients.
The perspectives are to complete the investigation by the
use of biomarkers of exposure in semen.
References :
Younglai E.V., Holloway A.C., Foster W.G. : Hum. Reprod.
Update, 2005, 11 : 43-57. Thonneau P., Bujan L., Multigner
L., Mieusset R. : Human. Reprod., 1998, 13 : 2122-2125.
Sram R.J., Binkova B., Rossner P., Rubes J., Topinka J.,
Dejmek J. : Mutat. Res.,1999, 428 : 203-215.
Whan L.B., West M.C., McClure N., Lewis S.E. : Fertil. Steril.,
2006, 85 : 653-660.
PO 046
Estrogens regulate epididymal contractility
through RhoA/Rho-kinase signalling
S. FILIPPI, A. MORELLI, L. VIGNOZZI, R. MANCINA,
G. FORTI, M. MAGGI
Interdep.Lab of Functional and Cellular Pharmacology of
Reproduction, Dep.of Pharmacology and Clinical
Physiopathology, 1Andrology Unit, Dep.of Clinical
Physiopathology, University of Florence, Florence, Italy,
(Hyperlink "mailto:[email protected]"
[email protected]),
Objective : Epididymis is a sex steroid-sensitive duct provided
with spontaneous motility, allowing sperm transport. We
previously demonstrated that human epididymis expresses
an high abundance of mRNA for ER-alpha and ER-beta. We
also demonstrated that in epididymis estrogens up-regulate
either OT responsiveness, acting at the receptor level, and
responsiveness to endothelin-1 (ET-1), another well known
stimulator of epididymal motility. However, we did not find
any significant change either at gene or protein level in ET1 and its cognate receptors. Hence, other molecular effectors
should mediate the increased sensitivity to ET-1. In particular
we hypothesized that estrogens up-regulate some contractile
effectors, such as RhoA/Rho-kinase pathway, downstream to
the ET-1 receptors.
Design and Methods : To investigate the effect of changing
endocrine milieu on RhoA/Rho-kinase pathway, we induced
hypogonadism (hypo) in rabbits with a single administration
of 2.9 mg/Kg of a long-acting GnRH analog, triptorelin, and
we replaced hypo rabbits with different sex steroids
(Testosterone, T 30 mg/Kg weekly or estradiol valerate, E2,
3.3mg/Kg weekly).
Results : After 8 weeks from GnRH analog administration,
T plasma levels were decreased and the relaxant effect of the
Rho-kinase inhibitor,Y27632 on ET-1 pre-contracted
epididymal strips, was significantly decreased. T administration
restored T plasma levels, but not Y27632 sensitivity in the
epididymal strips. E2 not only completely restored Y27632
responsiveness but even amplified it, as indicating that the
RhoA/Rho-kinase calcium sensitizing pathway is up-regulated
by E2. Accordingly, RT-PCR studies indicate that Rho kinase
gene was strongly induced by E2 but not by T. To verify
whether endogenous estradiol is involved in the regulation of
Y27632 responsiveness, we treated intact rabbits with an
aromatase inhibitor, letrozole (1.25 mg/day) for 21 days.
Blocking aromatase activity abolished Y27632 responsiveness
in rabbit epididymis.
Conclusions : Our results support the hypothesis that
epididymis is a male target for E2, which regulates its motility
tuning up contractile hormones and local peptides
responsiveness by increasing RhoA/Rho-kinase signalling
and therefore calcium sensitivity.
348
PO 047
An in vitro model for epididymal sperm
maturation
epididymis with bovine caput spermatozoa is able to trigger
the development of global and progressive motility. In contrast
to unconditioned serum-free medium, the epididymal
environment maintains sperm vitality and motility for at least
72 hours supporting the key role of epididymal cell-cell
interactions for sperm maturation.
C. MEHNERT1, J. FECHNER2, T. STALF1,
H.R. TINNEBERG1, R. HENKEL3, H.C. SCHUPPE2
PO 048
1 Center for In vitro Fertilization (CIF), 2 Center of
Dermatology and Andrology, Justus Liebig University,
Giessen, Germany, 3 Department of Medical Biosciences,
University of the Western Cape, Bellville, South Africa
([email protected])
Sperm phagocytosis by mononuclear cells in the
ejaculate : a marker of epididimydis pathology ?
A quantitative ultrastructural analysis
Objective : The fertilization potential of spermatozoa is
critically dependent on maturation in the epididymis. One of
the key features is progressive motility, which is gradually
acquired during the epididymal transit. Cellular and molecular
mechanisms underlying epididymal sperm maturation,
however, have not yet been elucidated in detail. In order to
investigate the interaction between epididymal epithelium
and spermatozoa during this process, we developed a coculture system of epididymal epithelium and spermatozoa in
a defined, serum-free culture system.
Material and Methods : Primary cell cultures of epithelial
cells obtained from caput, corpus and cauda epididymis were
grown in culture medium (RPMI 1640) supplemented with
5% fetal bovine serum until confluence. On the day of
confluence (usually day 6 after preparation), cell cultures
were washed twice with serum-free medium. The same
medium was used for further cultivation. Spermatozoa freshly
obtained from bovine caput epididymis were added to the
epithelial cell cultures at a concentration of 10 x 106 /ml. As
a negative control, caput spermatozoa were incubated with
fresh serum-free medium alone. Motility was measured with
a CASA system (Microptic, Spain) every 24 hours until 72 hours
of co-culture.
Results : Caput spermatozoa showed a low rate (7%) of
global motility immediately after preparation. Global motility
increased in both, negative control (20%) and co-cultures
with epithelial cells of all three epididymal regions (50%) after
24 hours incubation time. However, changes in motility were
significantly lower in the control group. Global as well as
progressive motility in the co-culture setting was maintained
at high values until termination of the experiment after 72
hours. In contrast, global motility of the negative control was
largely reduced after 24 hours. Furthermore, spermatozoa
cultivated in unconditioned medium showed a significant loss
of vitality associated with a high level of disintegration, i.e.
between head and flagellum.
Conclusions : Our data demonstrate that a co-culture system
of epithelial cells from bovine caput, corpus and cauda
349
F. PELLICCIONE1, G. CORDESCHI1, R. MIHALCA1,
M. BOCCHIO1, S. NECOZIONE2, S. FRANCAVILLA1
Chairs of 1 Andrology and 2 Epidemiology, University of
L’Aquila, Italy Presenting author e-mail : Hyperlink "mailto :
[email protected]" [email protected]
Objective : Experimental epididymidis obstruction is
associated with appearance in the epididymal lumen of
macrophages undergoing sperm phagocytosis. This is the
result of epididymal barrier disruption and exposure of sperm
to the immunosystem. Macrophages undergoing phagocytosis
of sperm and bacteria (Ma-Phago) are observed by
ultrastructural analysis of ejaculate in infertile men.
Design : We analysed in a large number of ejaculates
evaluated by quantitative ultrastructural analysis with
transmission electron microscope (TEM) the relationship
between the presence of Ma-Phago as a marker of sperm
exposure to the immunosystem, and seminal parameters.
Materials and Methods : 430 ejaculates with a
leukocytospermia < 1x106/ml and a negative MAR test, were
analysed by routine semen analysis and by TEM. Specimens
were grouped on the basis of the presence by TEM of MaPhago. The quantitative ultrastructural analysis included the
fraction of heads with normal chromatin and acrosome
organization, the fraction of degenerating sperm with
fragmented membranes, the fraction of sperm with amorphous
heads (altered shape associated with altered chromatin
condensation and a disorganized acrosome).
Results : 26.7% of samples showed Ma-Phago. Compared
with the others, these showed a decreased number of sperm
total count (p<0.0001) and a reduced sperm forward motility
(p<0.04) but no difference for sperm morphology. The
quantitative TEM analysis showed that group with Ma-Phago
showed a significative incresed fraction of degenerating sperm
(p=0.0002), and of amorphous heads (p=0.019). The fraction
of degenerating sperm was correlated negatively with sperm
forward motility (r=0.35, p<0.0001) and sperm total count
(r=0.30; p<0.0001). A multivariate logistic analysis showed that
a low number of total ejaculated sperm and an increased
number of degenerating sperm indipendently predicted the
presence of Ma-Phago (OR 1.72; CI 1.10 to 2.28 and OR 1.85
CI 1.19 to 2.88 respectively).
Conclusion : The presence of macrophages undergoing
phagocytosis of sperm and bacteria is frequently observed in
ejaculates of subfertile men and is associated to a decreased
number of sperm count but normal morphology, suggesting
it may be a marker of an inflammatory sub-obstruction at
epididymal level. Supported by MURST, PRIN 2005
PO 049
Correlation of apoptosis and aneuploidy in
spermatozoa of oligoasthenozoospermic patients
nuclei, and the non-apoptotic nuclei were counterstained with
DAPI (1mg/ml) (Vector Laboratories). At least 500 spermatozoa
per sample were evaluated using a fluorescent microscope.
Statistical analysis was performed with SPSS-PC V11.0
Software. As all continuous variables were symmetrically
distributed (as shown by analysis of variance using the ShapiroWilk test) the parametric Pearson’s r correlation test was
used to examine the relationship between sperm aneuploidy
and TUNEL reactive spermatozoa. A p value of <0.05 was
considered statistically significant.
Results : The mean percent aneuploidy for all five
chromosomes was 1.35, while the percentage of TUNEL
positive spermatozoa was 45.75. No statistically significant
correlation (r=-0.407, p=0.075) was observed between
chromosome aneuploidy (five chromosomes) and TUNEL
reactive spermatozoa.
Conclusions : The data obtained from the 20
oligoasthenozoospermic patients showed that the total sperm
chromosome aneuploidy for five chromosomes (X, Y, 13, 18
and 21) did not correlate with sperm deoxyribonucleic acid
fragmentation (evidenced by TUNEL assay).
Support : The project was funded by the European Social
Fund and National Resources – (EPEAEK II) PYTHAGORAS
(grant “Pythagoras” 70/3/7361).
K. PLASTIRA, R. ANGELOPOULOU, P. MSAOUEL,
K. ZANIOTI, D. MANTAS
Department of Histology and Embryology, Medical School,
Athens University, Greece
PO 050
Objective : To evaluate the cytogenetic constitution of
spermatozoa from twenty oligoasthenozoospermic (OA)
patients by analyzing the rates of aneuploidy for the
chromosomes 13, 18, 21, X and Y and to determine if any
correlation exists between the DNA fragmentation and the
incidence of sperm chromosome aneuploidy.
Design : Basic research laboratory experiment.
Twenty patients (aged 24-50, mean age : 37.3±7) were referred
to the department of Assisted Reproduction with the view of
participating in an ICSI program, after experiencing at least
two years of infertility. Semen analysis was carried out, based
on the WHO 1999 guidelines and a severe male factor
condition was the main cause of infertility due to
oligoasthenozoospermia (n=20).
Materials and Methods : Dual and triple colour FISH
(Fluorescence in situ hybridization) was applied, according to
standard manufacturer procedures (Vysis, Inc.). The probes
13, 18, 21, X and Y were used and the stained samples were
observed using an Axiolab Zeiss microscope. A total of 20.000
spermatozoa were counted.
Nick end labelling of DNA breaks in sperm nuclei have been
performed with a mixture of biotinylated dUTP (Roche, stock
50 nmol) and TdT (Amersham, stock 9 units/µl) in TdT buffer.
After the end of the reaction, the slides were incubated with
Texas Red Streptavidin (1mg/ml), which revealed the apoptotic
350
Ultrastructural nuclear defects and increased
chromosome aneuploidies in spermatozoa with
elongated heads
N. PRISANT1.2*, D. ESCALIER2, J.C. SOUFIR2 ,
M. MORILLON2, M. MISRAHI3, G. TACHDJIAN1.2
1 Department of Genetic and Reproduction, APHP, Antoine
Béclère Hospital, Clamart; France. 2 Department of
Andrology, APHP, Kremlin Bicêtre Hospital, Kremlin
Bicêtre, France. 3 Department of Hormonology and
Molecular Biology, Kremlin Bicêtre Hospital,
Kremlin Bicêtre, France.
*To whom correspondence should be addressed a t: Dr N
Prisant. Service de Génétique et Reproduction. Hôpital
Antoine Béclère- 157, rue de la porte de Trivaux 92140
Clamart, France. e-mail : [email protected]
Objective : Elongated spermatozoa are characterized by a
head length higher than 5µm. Cellular and molecular
mechanisms leading to elongated sperm heads are not known.
An increased nuclear volume has been reported to be
associated with sperm head abnormal elongation. The
incidence of sex chromosome abnormalities is increased
significantly in morphologically abnormal spermatozoa
(macronuclear spermatozoa). Whether anomalies of the
chromosomal content are responsible for the increased nuclear
volume of elongated spermatozoa is questioned.
Design : The nuclear status of elongated spermatozoa was
evaluated by ultrastructural and cytogenetic analyses.
PO 051
Characterization of M540 bodies present in
human semen
Setting : Teaching hospital.
Materials and Methods : Fourteen men with at least 30% of
spermatozoa with an elongated nucleus. Five fertile men
were used as controls. Medical history, family infertility and
personal habit were recorded. Semen parameters, semen
biochemical markers, karyotypes and Y chromosome
microdeletions were assessed in all patients. Sperm
morphology was analysed by a quantitative ultrastructural
analysis. Sperm chromosomal content was assessed by three
colour fluorescence in situ hybridisation (chromosomes X, Y,
18). Sperm aneuploidy rates were compared between patients
and controls with a Mann-Whitney statistical test. Differences
were significant when P<0.05.
Results : Semen parameters (median) of the patient group
as compared to the control group were ranged as follows: total
sperm count (67.1 Mo ± 10.6 vs 219 Mo ± 47), percentage
of progressive sperm cells (25% ± 10 vs 25% ± 15), sperm
viability (66% ± 18 vs 72% ± 22) and normal spermatozoa
(3.5% ± 3.5 vs 69% ± 5). The patients presented a polymorphic
teratozoospermia, where a majority of spermatozoa display
more than one type of abnormality. Elongated sperm head
rates were ranged from 30% to 75% (median : 48.5%) in the
patient group versus 0% to 2% in the control group. No
anomalies of sperm biochemical markers were found in either
group. All the men showed a normal karyotype (46,XY) and
an absence of Y chromosome microdeletion. Quantitative
ultrastructural analysis revealed the presence of nuclei of
various sizes and shapes, including a mean of 13% of
spermatozoa with an increased nuclear volume and a mean
of 10% of binucleated sperm cells. Moreover, the chromatin
was undercondensed in 30% of the spermatozoa. Aneuploidy
rates of gonosomes and chromosome 18 were significantly
higher in patients with elongated sperm heads as compared
to the values of control sperms (1.8 and 4.3 fold increase,
p=0.02 and p=0.006, respectively).
Conclusions : This study demonstrates for the first time that
the chromatin compaction and the numerical chromosome
content can be altered in spermatozoa with an elongated
head and that elongated sperm nuclei could arise from meiotic
non-disjunctions.
Financial support : None
351
S. MARCHIANI, M. MURATORI, G. FORTI, E. BALDI
Department of Clinical Physiopathology, Andrology Unit,
Center of Research, Transfer and High Education
DeNothe, University of Florence, Florence, Italy
([email protected])
In semen of subfertile subjects, membrane-surrounded round
bodies (termed as M540 bodies) are present. M540 bodies
stain promptly with merocyanine 540 (M540), a probe revealing
somatic apoptosis. M540 bodies appear virtually devoid of
chromatin as nuclear dyes fail to stain them. Further, M540
bodies have heterogeneous size and density and result
ubiquitinated. Finally, their amount correlates with poor quality
semen parameters.
In this study we challenged the hypothesis that M540 bodies
are apoptotic bodies, derived from testis apoptosis. By flow
cytometry, we investigated whether M540 bodies exhibited
apoptotic key markers, by excluding from the analysis semen
nucleated cells.
We found that M540 bodies express all the apoptotic markers
investigated : caspases activity, Fas receptor, p53 and TUNEL
positivity (percentages of positive M540 bodies were
respectively: 68.1±20.7% ; n=7 ; 13.7±10%, n=6 ; 42.2±13.5%,
n=4 and 22.2±18.7, n=8, mean±SD). The latter parameter
increased after treatment with exogenous nuclease, suggesting
that M540 bodies do contain fragmented DNA.
In this study we show that M540 bodies resemble closely
apoptotic bodies as they express key apoptotic markers.
Occurring of DNA fragmentation was surprising to us. On the
other hand, if M540 bodies are apoptotic bodies, their DNA
content is expected to be very low but intensively fragmented.
In agreement with this, our data show that DNA bound to
M540 bodies is detectable by TUNEL but not by nuclear
staining. In M540 bodies, caspase activity occurs at a greater
extent than Fas receptor, p53 and DNA fragmentation. The
higher positivity observed for caspases with respect to Fas,
p53 and DNA fragmentation could be explained by the different
localization of caspases (cytoplasm, present in all M540
bodies) and p53 and DNA (nucleus, present only in a fraction
of apoptotic bodies) and by the use of a generic probe for
detecting caspases.
PO 052
PO 053
Reconsidering DNA fragmentation in light of
presence of M540 bodies in human semen
Relationship between DNA fragmentation and
ubiquitination in human sperm
M. MURATORI, S. MARCHIANI, L. TAMBURRINO,
G. FORTI, E. BALDI
M. MURATORI, S. MARCHIANI, C. PUCCI, G. FORTI,
E. BALDI
Department of Clinical Physiopathology, Andrology Unit,
Center of Research, Transfer and High Education
DeNothe, University of Florence, Florence, Italy.
([email protected])
Department of Clinical Physiopathology, Andrology Unit,
Center of Research, Transfer and High Education
DeNothe, University of Florence, Florence, Italy
([email protected])
The origin and impact of sperm DNA fragmentation (DF) has
been not yet understood. Most studies investigated DF by flowcytometry, a technique able to measure large number of
sperm automatically. The recent identification, within semen,
of M540 bodies (round elements virtually lacking of chromatin
and occurring at high extent in Oligoasthenoteratozoospermic
patients) which are included in the FSC/SSC region
characteristic of sperm, raised the question whether they may
be confounding in the analysis.
In human sperm, ubiquitination seems to have both positive
(for instance in the sperm-oocyte interaction) and negative
(marking defective sperm) roles. Indeed, both normal and
abnormal sperm result ubiquitinated. Abnormal sperm are
often DNA fragmented.
We determined percentages of DF in sub-fertile men by
excluding M540 bodies from the analysis by labelling samples
with both TUNEL (to detect DF) and propidium iodide (PI, to
distinguish sperm from M540 bodies).
By flow cytometry, we simultaneously detected DNA
fragmentation and ubiquitination (the latter by two different
antibodies: an IgM against recombinant human and an IgG
against rabbit ubiquitin, from here on indicated respectively
IgM and IgG). In principle, the technique should distinguish
four populations of sperm : A, not ubiquitinated not fragmented;
B, not ubiquitinated fragmented ; C, ubiquitinated fragmented
and D, ubiquitinated not fragmented.
The population formed solely by sperm (population A) resulted
quite more DNA fragmented than population formed by sperm
plus bodies (population B, difference : 8.6 ± 13.9%, mean ±
SE; range: from -5.0 to 64.8%). Although testing only 25
subjects, DF in population A correlated with morphology (r=0.40, p < 0.05), total motility (r=-0.36, p < 0.05) and sperm
number (r=-0.37, p < 0.05), at variance with population B (not
significant correlations with any parameter). After gating out
M540 bodies, we found that PI labelling separated a less
fluorescent sperm population (PIdimmer) from a brighter one
(PIbrighter) and, mostly important, the latter was entirely DNA
fragmented, in all the samples investigated. In addition, the
PIdimmer population dramatically correlated with morphology
(r=-0.46, p < 0.02) and total motility (r=-0.58, p < 0.01) whereas
PIbrighter did not.
We found that when the IgM was used, all the fragmented
sperm resulted ubiquitinated (i.e population B is lacking)
whereas with the IgG no fragmented sperm resulted
ubiquitinated (i.e. population C is lacking). This finding suggests
that the two antibodies recognise epitopes on different groups
of sperm proteins. Such result was confirmed by Western
blot analysis: the two antibodies reveal the same protein
bands within the range of 7-182 KD, except for some proteins
within the MW of 25-49 KD that are not detected by the IgG.
Importantly, these data imply that there are ubiquitinated
proteins (those recognised by both antibodies) present in
sperm only if the cell is not DNA fragmented (population D).
In fact, if such proteins were present also in fragmented
sperm, they would be recognized by the IgG, but this is not
the case.
We conclude that sperm DF can have been underestimated
up to now and associated to seminal parameter more closely
than suspected. Such association is mainly driven by the
incidence of an entirely fragmented subpopulation of sperm.
The impact of DF of the other subpopulation has to be entirely
investigated. The heterogeneity within DNA fragmented sperm
could enlighten the origin of sperm DNA fragmentation.
On the contrary, proteins within 25-49 KD (detected only by
IgM) may be present in fragmented (and ubiquitinated
according to IgM, population C) and in not fragmented sperm
(ubiquitinated according to IgM - population D- but not
ubiquitinated according to IgG, population A). Identification of
ubiquitinated proteins, to clarify the meaning of DNA
fragmentation and the function of bad and good sperm
ubiquitination, is underway.
352
PO 054
Nuclear DNA fragmentation of human
spermatozoa
M. PIASECKA1, D. GACZARZEWICZ2,
M. LASZCZYNSKA1, A. STARCZEWSKI3,
A. BRODOWSKA3
1 Laboratory of Embryology, 3 Department of Reproductive
Medicine and Gynecology, Pomeranian Medical University,
2 Department of Animal Reproduction, University of
Agriculture, Szczecin, Poland.
e-mail : Hyperlink "mailto:[email protected]"
[email protected]
progressive motility (rapid+slow) was found in patients with
>4% TUNEL- positive sperm (71 out of 94) than in patients
with ≤4% TUNEL- positive sperm (23 out of 94 men). A
significant negative correlation was observed between DNA
fragmentation and sperm motility (rs= - 0.38) and between
fragmentation and sperm concentration (rs= - 0.33). In electron
microscope, a large number of conglomerates containing
sperm fragments with cytoplasm and a large number of
immature spermatozoa with chromatin and midpiece defects
and with excess residual cytoplasm was noted more frequently
in subjects with low sperm motility and with >4% TUNELpositive sperm.
Conclusion : A high proportion of sperm with abnormal
genomic integrity may occur in the semen of some men with
normal or with low sperm parameters. Our results suggest that
sperm DNA fragmentation may be associated with the
developmental failure in spermatogenic remodeling process,
particularly in patients with low sperm motility. The used test
may be considered as an additional assay in evaluation of
spermatozoa beside a standard semen analysis and may
help to discriminate between fertile and infertile men.
Objective : The aim of our study was to evaluate the incidence
of ejaculated spermatozoa with nuclear DNA strand breaks
in patients of Assisted Reproductive Technique Laboratory.
Design : The patients were classified according to WHO
standards (1999). Two categories of patients were
distinguished: men with normozoospermia (n=26) and men
with abnormal sperm parameters (n=68). The latter group
included 23 subjects with teratozoospermia, 3 with
astenozoospermia, 28 with asthenoteratozoospermia,
oligozoospermia, 1 with oligoasthenozoospermia, 5 with
oligoterato-zoospermia and 8 cases with oligoasthenoteratozoospermia.
Materials and methods : The sperm DNA strand breaks
were identified with the terminal deoxynucleotidyl transferase
- mediated dUTP nick end-labeling (TUNEL) assay (APOBRDU Kit). The TUNEL-positive cells were evaluated in a
flow cytometer (FACSCalibur, Becton Dickinson) and in a
fluorescence microscope (Axioskop, Carl Zeiss). The semen
was examined in JEM-1200 EX (JEOL Ltd) transmission
electron microscope.
Results : TUNEL-positive spermatozoa revealed bright green
fluorescence in the heads. No significant differences were
found in the percentage of sperm with DNA fragmentation
between the patients with normozoospermia (9.42±7.68% ;
median 6.50%) and with low sperm parameters (14.56±15.51%
; 9.00%).
The proportion of TUNEL-positive sperm was significantly
higher in patients with asthenozoospermia (15.10±10.96% ;
12.00% ; 40 out of 94 subjects) and in patients with
oligozoospermia (24.93±20.31% ; 20.00% ; 14 out of 94) as
compared to normozoospermic men or compared to patients
with normal sperm motility (11.69±15.74% ; 6.50% ; 54 out
of 94) or to men with normal sperm concentration
(11.08±11.50% ; 7.00% ; 80 out of 94) respectively.
A significantly lower percentage of spermatozoa with
353
PO 055
Correlation between semen parameters and DNA
fragmentation in human spermatozoa
L. KHANTOUCHE, M. MEHDI, M. AJINA, A. SAAD
Laboratoire de Cytogénétique et Biologie de la
Reproduction, CHU Farhat Hached, Sousse. Tunisia
Objective : The aim of our study was to detect DNA
fragmentation in human spermatozoa, in order to investigate
a correlation between DNA fragmentation index (DFI) and
semen parameters.
Materials and Methods : The patients were divided into two
groups according to their semen parameters, 30 patients with
asthenozoospermia (group B) and 30 patients with
teratozoospermia (group C). The percentage of sperm DNA
fragmentation was evaluated by the TUNEL (Terminal
deoxynucleotidyl transferase-mediated dUDP nick-end
Labelling) assay. Results were compared with those of
spermatozoa sampled from 30 healthy men with normal
semen profiles (group A).
Results : The difference was not significant between the
percentage of DFI in patients with asthenozoospermia and the
normozoospermic men (9.46% ± 8.68 and 8.19% ± 6.84, p
not significant). In addition, no significant correlation was
found between the motility of spermatozoa and the DFI in
the same group.
The patients with teratozoospermia showed a significantly
higher percentage of DNA fragmentation than the control
group (21.37 ± 17.26% and 8.19% ± 6.84, respectively,
P<0.001). There was a positive correlation between abnormal
sperm forms and the DFI (r = 0.44, P < 0.01) in the same group
(C). The DFI was particularly higher in patients with 91-100%
teratozoospermia (group C2) than the other patients with 8090% teratozoospermia (group C1) (29.55 ± 20.55 versus 14.3
± 11.01, respectively). The highest percentage of DFI was
found among the patients having a high percentage of
microcephalic spermatozoa and acrosome anomalies.
Conclusion : Our study noted that impairments of sperm
parameters were associated with an increase of DNA
fragmentation, this association was strictly related to atypical
forms. This finding suggests that teratozoospermia may be
the critical sperm parameter associated with hypofertility and
when exceeds the proportion of 90 % it would be a prudent
sign for analysis of sperm DNA fragmentation.
PO 056
The production of peroxynitrite and proteins
nitrotyrosine by human spermatozoa : influence
on kinetic sperm features
E. BULDREGHINI, A. VIGNINI*, L. MAZZANTI*,
F. MANTERO§, M. BOSCARO, G. BALERCIA
Division of Endocrinology, Institute of Internal Medicine and
* Institute of Biochimestry, Polytechnic University of
Marche, Italy ; § Department of Medical and Surgical
Sciences, University of Padua, Italy. Corresponding author
: [email protected]
Objective : Like all cells living under aerobic conditions,
spermatozoa product reactive oxygen species, mosty
originating from normal activity. There are many studies to
support a role for (superoxide anion) O2- and NO- (nitric oxide)
in sperm pathophysiology. Under physiological condition these
compounds exist at very low concentration. Peroxynitrite
(ONOO-) formed in vivo from O2- and NO- can mediated
oxidation, nitration reaction (addition of an NO2- group), leading
to an impaired function, toxicity and alterations in signalling
pathways. In fact ONOO- reacts rapidly with proteins, lipids
and DNA. The nitration reaction gives rise to 3-nitrotyrosine
which represents a protein modification specific for ONOO-
354
formation in vivo : tyrosine nitration is a widely used marker
of peroxynitrite. In our study we have determined ONOOproduction in semen and its correlation with kinetic features
in spermatozoa, and we set out to determine whether protein
tyrosine nitration takes place in the same sample in order to
elucidate any pathogenic involvement in sperm cells motility.
Design : Basic study.
Materials and Methods : Semen samples from 29 normal
fertile donors (control group) and 69 infertile patients affected
by idiopathic asthenozoospermia were analyzed according to
WHO 1999 criteria. After liquefaction one aliquot of semen were
diluted to 5x106 spermatozoa/mL with Dulbecco’s PBS
(Phosphate buffer saline) and proxynitrite concentration was
measured through the fluorescence of the DCFDA probe.
Protein tyrosine nitration was determined with Western
ImmunoBlot with an appropriate antibody. Curvilinear velocity
(VCL) and straight line velocity (VSL) of sperm cells were
determined using a Motion Analysis CASA system.
Results : The control exhibited ONOO- production significantly
lower than asthenozoospermic patients (9.11 ± 3.37 vs 27.46
± 5.77 respectively, p< 0.001) ; furthermore, ONOO- exhibited
a significant inverse correlation with the motility parameters.
Moreover, in the western immunoblot there was an increase
in the nitration of the tyrosine residues in the
asthenozoospermic samples compared to controls.
Conclusions : Althought at level of the whole organism, the
reactive chemistry of ONOO- can be considered beneficial,
becaue of its cytotoxicity to bacteria or other invading organism,
the formation of protein 3-nitrotyrosine, in vivo, has been
shown in a number of inflammatory conditions in human and
experimental animals. Our results suggest that higher levels
of peroxynitrite are producer in sperm cells of
asthenozoospermic infertile when compared to normospermic
fertile donors and ONOO- concentration is inversely related
to sperm motility. Tyrosine nitration is enhanced by higher
peroxynitrite levels. Thus, a possible pathogenic role in infertile
men when asthenozoospermia is the main critical problem may
be suggested.
PO 058
PO 057
Oxidative stress biomarkers levels in seminal
plasma from normozoospermic and
asthenozoospermic semen samples
Enzymatic and non-enzymatic measures of
oxidative stress in seminal plasma from
normozoospermic, asthenozoospermic,
asthenoteratozoospermic and
oligoasthenoteratozoospermic males
A. KHOSROWBEYGI1*, N. ZARGHAMI2
1 Department of Biochemistry, Lorestan University of
Medical Sciences, Iran
2 Drug Applied Research Center, Tabriz University of
Medical Sciences, Iran
*E-mail: [email protected]
N. ZARGHAMI1*, A. KHOSROWBEYGI1
1 Drug Applied Research Center, Tabriz University of
Medical Sciences, Iran
2 Department of Biochemistry, Lorestan University of
Medical Sciences, Iran *E-mail : [email protected]
Objective : There is growing evidence that damage to
spermatozoa by reactive oxygen species (ROS) play a key
role in male infertility. The aims of the present study were to
assess seminal plasma levels of total antioxidant capacity
(TAC) and activities of catalase and superoxide dismutase
(SOD) in men with asthenozoospermia, asthenoteratozoospermia and oligoasthenoteratozoospermia compared to
normozoospermic males.
Design : A case-control study with simple random sampling
was designed. The case group consisted of 46 men with
seminal parameters abnormalities that divided into three
categories:
asthenozoospermic
(n=15),
asthenoteratozoospermic (n=16) and oligoasthenoteratozoospermic (n=15), according to WHO criteria. The
control group consisted of 16 males with normozoospermia.
Materials and Methods : Catalase activity was measured by
Aebi spectrophotometeric method. The measurement of SOD
activity and TAC was carried out by commercially available
colorimetric assays. Differences between groups were
assessed using Mann-Whitney U test and Kruskal-Wallis test.
Coefficients of correlation were calculated using Spearman’s
correlation analysis. All hypothesis tests were two-tailed with
statistical significance assessed at the p value <0.05 level with
95% confidence intervals
Results : Both catalase activity and TAC levels in all three
categories of case group were significantly lower than
normozoospermic control males (p<0.05). But SOD activity
did not show a significant difference between these groups.
Both catalase activity and TAC showed a positively significant
correlation with sperm motility and normal sperm morphology.
Conclusions : Decreasing seminal plasma antioxidants levels
especially catalase activity and TAC could have significant role
in etiology of sperm abnormalities.
Support : This research was granted by Drug Applied
Research Center of Tabriz University of Medical Sciences.
355
Objective : Poor sperm-forward motility (asthenozoospermia)
is considered to contribute to the infertility of a significant
number of males, and many cases of decreased sperm motility
are not completely understood. One of factors that potentially
can cause asthenozoospermia is oxidative stress. The aims
of this study were to (i) compare seminal plasma
malondialdehyde (MDA), 8-isoprostane, and total
homocysteine (tHcy) levels in normozoospermic vs.
asthenozoospermic males and to examine their association
with sperm motility and also to (ii) investigate the relationship
between seminal plasma tHcy levels and lipid peroxidation,
as measured by MDA and 8-isoprostane.
Design : We designed a case-control study with a simple
random sampling. The case group consisted of 15
asthenozoospermic males. This group was compared with 15
normozoospermic men.
Materials and Methods : Seminal plasma level of 8isoprostane was measured using enzyme immunoassay (EIA)
method. Absorbance was measured at a wavelength of 405
nm using enzyme-linked immunosorbent assay (ELISA) reader
and data was presented as pg/ml. The intra-assay coefficient
of variation was <10%. The sensitivity and specificity of the
8-isoprostane assay were 5 pg/ml and 100%, respectively.
Levels of tHcy were measured using EIA method. Absorbance
was measured at a wavelength of 450 nm using ELISA reader.
The intra-assay coefficient of variation was <10%. The
sensitivity of the tHcy assay was 2.0 µM. MDA levels were
determined by the thiobarbituric acid (TBA) assay. The
concentration of MDA was expressed as µM. The MannWhitney U test was used to compare two groups. Coefficients
of correlation were calculated using Spearman’s correlation
analysis. All hypothesis tests were two-tailed with statistical
significance assessed at the p value <0.05 level. The data are
expressed as the mean ± SEM.
Results : MDA levels were higher in asthenozoospermic
subjects than in control subjects. No differences were seen
in 8-isoprostane and tHcy levels in asthenozoospermic subjects
and controls. Seminal plasma 8-isoprostane levels showed
an inverse significant correlation with sperm motility and also
with normal sperm morphology. Seminal plasma levels of
MDA showed an indirect correlation with sperm motility. No
relationship was found between MDA and normal sperm
morphology. Finally, the correlation between tHys and lipid
peroxidation, as measured by MDA and 8-isoprostane, was
examined. Seminal plasma levels of tHcy showed no
correlation with lipid peroxidation.
Conclusions : Seminal plasma levels of 8-isoprostane and
tHcy showed no significant differences between
normozoospermic and asthenozoospermic men. Sperm
motility correlated inversely with seminal plasma levels of 8isoprostane and MDA. No relationship was found between tHcy
and lipid peroxidation. We also concluded that homocysteine
metabolism may not be impaired in asthenozoospermic males.
However, a larger sample size is required to confirm these
findings.
Support : This research was granted by Drug Applied
Research Center of Tabriz University of Medical Sciences.
PO 059
An investigation of the relationships between
sperm morphology parameters, reactive oxygen
species production in semen and sperm DNA
status
R. MENKVELD1, R. HENKEL2, S. SCHUELLER3,
I. HOPPE4, W. STARKER4
Materials and methods : Semen samples of 70 men attending
the IVF clinic of the Department of Obstetrics and Gynaecology,
University of Jena, Germany, were analysed according to the
1999 WHO manual guidelines. In addition, the TUNEL and
chromomycin A3 (CMA3) assays were performed and ROS
production of spermatozoa was determined by means of the
use of dihydroethidine (DHE). Semen smears were stained
according to the Papanicolaou method and evaluated
according to strict Tygerberg criteria including the acrosome
index (AI), detailed classification of specific abnormalities
and the calculation of the teratozoospermia index (TZI). The
results were analysed with the MedCalc® statistical program
for basic descriptive statistics of the resultant semen and
functional parameters and rank correlations tests performed.
Results : The mean (±SD) age of the 70 men was 35.1 (±5.8)
years. The mean total motility was 57.9% (±19.1) and sperm
concentration 51.96 (±51.1) X 106/mL semen. The mean
percentage of morphological normal spermatozoa was 3.79%
(±2.5), the mean AI was 5.6% (±3.1) and a mean TZI of 1.71
(±0.26) was found. Positive correlations were found between
the percentage of TUNEL-negative spermatozoa and the AI
(r = 0.277; P = 0.0246), and the percentage of normal sperm
morphology (r = 0.359; P = 0.0036). Negative correlations were
found between the percentage of TUNEL-negative
spermatozoa and the percentages of spermatozoa with head
defects (r = -0.349; P = 0.0046), spermatozoa with neck
defects ( r = -0.255; P = 0.0387), spermatozoa with small
acrosomes (r = -0.330; P = 0.0073), spermatozoa with tail
defects (r = -0.371; P = 0.0026), the TZI (r = -0.288; P =
0.0194) and elongated sperm heads ( r = -0.260; P = 0.0346)
as well as percentage of live (vital) spermatozoa (r = 0.6090;
P <0.0001). A negative correlation was also revealed between
the percentage of spermatozoa with small acrosomes and
sperm viability (r = -0.2927; P = 0.0189) as well as ROS
activity in the ejaculate and sperm viability (r = -0.3102; P =
0.0189). A strong negative correlation was found between
sperm ROS production and CMA3-negative spermatozoa (r
= - 0.320; P = 0.0083).
Conclusions : Based on the results obtained in this study it
is indicated that normal sperm morphology and normal sperm
DNA status (CMA3-negative and TUNEL-negative) are strongly
correlated. This supports the idea that problems in the sperm
DNA remodelling during chromatin condensation might be
one cause of sperm DNA fragmentation. In addition,
spermatozoa with small acrosomes seem to be particularly
susceptible to ROS activity and therefore present with a low
viability, as already indicated in a previous study (Menkveld
et al., 2003).
1 Department of Obstetrics and Gynaecology, Tygerberg
Hospital and University of Stellenbosch, South Africa ; 2
Department of Medical Biosciences, University of the
Western Cape, South Africa ;
3 Department of Urology, University of Jena, Jena,
Germany and 4Department of Obstetrics and
Gynaecology, University of Jena, Jena, Germany.
([email protected])
Reference : Menkveld R., El-Garem Y., Schill W-B., Henkel
R. : J. Assisted Reprod. Genet.,2003, 20 : 432-438.
Objectives : To study the relationship between sperm
morphology parameters, the production of reactive oxygen
species (ROS) within the spermatozoa and sperm DNA
damage.
Design : Prospective analytical study, performed at the
Andrology laboratory of the Department of Obstetrics and
Gynaecology, University of Jena, Germany.
356
Support : Jenapharm, Jena, Germany.
PO 060
Mortimer et al. : Hum. Reprod., 1990, 5 : 99-103.
Rowe et al. : WHO Manual for the Standardized Investigation,
Diagnosis and Management of the Infertile Male. Cambridge,
Cambridge University Press, 2000.
The Teratozoospermia Index : A useful sperm
morphology parameter for inclusion in the new
WHO semen evaluation manual ?
WHO laboratory manual for the examination of human semen
and sperm-cervical mucus interaction. 3rd ed. Cambridge,
Cambridge University Press, Cambridge, 1992.
R. MENKVELD
Andrology Laboratory, Department of Obstetrics and
Gynaecology, Tygerberg Academic Hospital and University
of Stellenbosch, South Africa.
( HYPERLINK "mailto:[email protected]" [email protected]).
Objective : The teratozoospermia Index (TZI) was introduced
in the 1992 WHO manual as an additional sperm morphology
parameter. However, the suggested “normal” reference value
of 1.6 was not applicable as this was the value published by
Jouannet et al. (1988) for the multiple anomaly index (MAI)
based on only three sperm abnormality classes, while the
TZI was bases on four sperm abnormality classes. The
objective of this study was to perform a literature study
investigating the general use of the TZI and find a useable
normal reference value based on appropriate data.
Design : A structured literature review study.
Materials and Methods : A PubMed literature search was
performed with the word TZI in the title or text as well as
available literature for reference to the TZI in their text.
Results : Only three references could be found providing
normal cut-off values for the TZI. The first was in the WHO
manual for the investigation of the infertile male (Rowe et al.,
2000) and the other two by Menkveld et al. (1998, 2001).
Rowe et al. (2000) suggested that with a TZI <1.7 successful
fertilisation may be expected with IVF and that with a TZI
>1.9 ICSI was indicated, but no data or references were
given. Menkveld et al. (2001) found a cut-off value of 1.46 when
comparing a fertile and subfertile population and a value of
1.64 for in vitro fertilisation based on an oocyte fertilisation rate
of >50%. The TZI however, showed the poorest predictive
value when compared with normal morphology and the
acrosome index.
Conclusions : The TZI as a predictor of male fertility potential
is seldom used in the literature and has little predictive value
for in vivo or in vitro fertilisation outcome. Therefore, it can be
regarded as unnecessary parameter which can be deleted from
the new WHO manual.
References :
Jouannet et al. : Int. J. Androl., 1988, 11 : 379-394.
Menkveld et al. : Hum. Reprod., 1998, 13(Abstract Book 1) :
52.
Menkveld et al. : Hum. Reprod., 2001, 16 : 1165-1171.
357
PO 061
Sperm morphology assessment: comparison
between the manual method (David’s criteria) and
a computer assisted analysis (Kruger’s criteria)
B. SION, L. VELEMIR, L. JANNY, G. GRIZARD
Laboratoire de Biologie de la Reproduction,CHU, CECOS,
Hotel Dieu , 63000 Clermont-Ferrand, France
[email protected]
Objective : Manual morphologic analysis with the David’s
Classification is used in most French laboratories. Now
computer assisted analysis is proposed for objective and
reproducible analysis of the spermatozoa morphology. We
compared the results of morphological analysis obtained with
automatised analysis using Kruger’s criteria to those obtained
with manual lecture and David’s classification.
Materials and Methods : Semen samples were selected
from patients referred to our laboratory of reproductive biology
for FIV. Analysis was performed both on spermatozoa from
ejaculate and on spermatozoa selected (fraction 90%) on
Sperm Filter® (cryos, cryo bio system, France).
For the manual analysis, a small drop containing spermatozoa
was placed on a slide and a smear was performed. After
fixating in a solution of 95% alcohol and 10-15 min air-drying,
the slide was stained using the modified Schorr method. The
slides were examined under a microscope (400_) and at least
100 spermatozoa were observed.
For the computer assisted analysis, Hamilton Thorne
research’s system was used. An aliquot of semen was washed
and after centrifugation, the resultant pellet, was resuspended
to obtain a concentration of 100 106/ml. Twenty µl of the
sample was spotted on polylysine coated slide and allowed
to air-dry at room temperature. The concentration and the
droplet size were standardized to produce 10–20 sperm per
high-field magnification. Spermatozoa selected on Sperm
Filter® gradient were directly spotted on slide.
The air-dried slides were stained with Diff-Quik stain (Merck
Diagnostica, Germany). Approximately 100 sperm cells were
evaluated per slide and the percentage of normal sperm
morphology, calculated by the computer, was recorded.
Results : To evaluate the reproducibility of the automatised
method, coefficients of variation (CV) were determined at the
critical steps of the analysis. Measurements were performed
- i) after several readings of the same slide, - ii) after readings
of several smears of the same sample, -iii) after readings of
several preparations of the same semen. No difference in
the CV (about 20%, n=4) were observed in these three cases.
The percentage of normal sperm morphology obtained by
the two methods were closely correlated R2= 0.51, P<0.01
(n = 33) (Figure 1).
Figure 1: percentage of normal sperm morphology in semen
with the manual or automatised methods (n = 33).
A significant correlation was also found in selected
spermatozoa R2= 0.54, P<0.01 (n = 10).
Conclusion : The main factor of variability for this analysis
is linked to the slides reading. The application of computerassisted technology for the evaluation of sperm morphology
may have reached a level of accuracy and precision that
makes it acceptable for routine application and it is less time
consuming. These results indicate that Hamilton Thorne
research’s system might be used as an initial screening test
for the evaluation of sperm morphology in order to make
decisions in planning strategies for the treatment of infertile
couples.
Average height, weight, BMI also as well as average total
testicular volume (26,0±4,3 vs 26,4±4,3) in both groups had
shown significantly no differences. There was no significant
difference among volume (3,7±1,7 vs 3,5±1,4 ml) and pH
(7,7±0,4 vs 7,6±0,9) of the ejaculates. Average of sperm
concentration in men with TZ (78,7±76,2x106/ml) has appeared
considerably smaller than in men with NZ (121,8±81,7x106/ml,
p=0,000). At absence of distinctions in parameters of sperm
motility (a) 25,8±10,5% vs 27,2±10,3%, b) 39,5±9,4% vs
41,0±11,1%, c) 11,0±5.5% vs 10,3±5,7%, d) 24,0±10,8% vs
21,9±9,0%), and their viability (72,7±11,1% vs 75,8±7,0%).
The percentage of spermatozoa with normal morphology was
13,5±8,2% in TZ group and 37,2±8,1% (p=0,000) in NZ group.
There are significant differences in morphologically modified
spermatozoa with a small (7,7±8,6% vs 4,6±4,5%, p=0,000),
cigar-shaped (17,1±12,5% vs 5,2±3,8%, p=0,000), pearshaped (11,5±9,2% vs 4,5±3,9%, p=0,000), double head
(1,0±1,7% vs 0,5±1,0%, p=0,001), defective neck (11,6±6,6%
vs 6,9±3,8%, p=0,000) and tail defects (8,5±5,9% vs 5,3±3,2%,
p=0,000). The average maintenance of leukocytes in the
ejaculate had no significantly differences between the men
with TZ and NZ (0,5±3,0 vs 0,3±1,3x106/ml).
Frequency of occurrence left-side (18,4% vs 14,2%) and
bilateral (16,1% vs 17,5%) varicocele in men with TZ and NZ
has appeared approximately identical, as well as frequency
of occurrence bilateral (47,2% vs 42,0%) and unilateral leftside (13,3% vs 11,7%) testicle hypotrophy.
There also was no significant difference in chronic prostatitis
(22,9% vs 21,0%) while total frequency of detectability sexually
transmitted infections were significantly 1.4 high in comparison
with TZ group.
PO 062
Teratozoospermia : clinico- laboratorial analysis
At the same time TZ group has significantly high frequency
of inflammatory parotitis (20,5% vs 1,7%, p=0,000), infectious
hepatitis A (9,6% vs 0,0%, p=0,001), acute orchitis (3,3% vs
0,8%), surgical operation on account of left-side varicocele
(8,2% vs 0,0%, p=0,002), chronic tonsillitis (3,7% vs 0,8%)
and acute appendicitis (17,8% vs 10,1%, p=0,000. in the
anamnesis.
While in a NZ group were more often observed unilateral
epididymo-orchitis(12,6% vs 3,9%, p=0,000), trauma of
scrotum (16,0% vs 4,6%, p=0,000), infectious hepatitis
B(12,6% vs 3,0%, p=0,000), surgical operation on account of
hemorrhoids (17,7% vs 0,5%, p=0,000), adenoids (10,1% vs
2,5%, p=0,000) and inguinal hernias (12,6% vs 8,6%). Besides,
correlation dependence is found out between sperm
concentration and percentage of spermatozoa with a normal
structure (r=0,29, p=0,000) in men with TZ.
M.V. KORYAKIN1,2, N.P. GONCHAROV1,2,
A.D. DOBRACHEVA1,2, N.S. DALANTAEVA3
1,2 National Centre for Human Reproduction,
Endocrinology Research Centre, Moscow, Russia
3Lomonosov Moscow State University, Russia
([email protected])
One of the most mass manifestations of disturbance of
spermatogenesis is a teratozoospermia (TZ). Its reasons
however are unknown. The purpose of this study became
revealing possible factors leading development TZ. For this
purpose 572 men (mean age 32,3±7,1 years) with TZ and 120
men (mean age 32,4±7,6 years) with normozoospermia (NZ)
as the control are surveyed.
358
Thus, higher maintenance of spermatozoa with a small, cigarshaped, pear-shaped, double head, defective neck and tail
defects comes to light in men with TZ. Secondly, it is possible
to carry a number of the transmitted infection-inflammatory
diseases bacterio-viral etiology as rule extragenital localizations
to the factors potentially leading to TZ. Thirdly, value of prostate
inflammatory diseases, chronic sexually transmitted infections
and varicocele in genesis of TZ is minimal.
PO 063
PO 064
In vitro beneficial effect of date seed oil extract
supplementation on human spermatozoa
acrosome reaction
Relationship between human sperm morphology
and acrosomal function
Y. EL-GAREM1, R. HENKEL2, R. MENKVELD3,
W.B. SCHILL4
F. BEN ABDALLAH2, N. CHAKROUN1, S. BESBES2,
H. ATTIA2, H. BASMA2, L. AMMAR-KESKES1.
1 Department of dermatology,University of Alexandria,
Egypt. 2 Department of Medical Biosciences, South
Africa.3 Reprod. Biol. Unit, Tygerberg Hospital and
University, South Africa.4 Center of dermatology and
andrology, Justus Liebig Universität, Germany. Hyperlink
"mailto:[email protected]" [email protected]
1 Laboratory of histology, Faculty of medicine in Sfax,
Tunisia. 2 Research Unit : Pathology and oxydatif stress,
Superior Institut of biotechnology in Sfax, Tunisia.
Correspondance to Pr. Leila Keskes :[email protected]
Objective : In this study, we investigated the relationship
between functionality of the acrosome and sperm morphology.
Design : Acrosome reaction (AR) was separately determined
in live and dead sperm and in those with normal, small, and
large sized acrosomes by means of the triple stain.
Material and Methods : In 50 patients, triple stain was
performed before and after induction of acrosome reaction.
Sperm samples were washed with human tubal fluid medium
containing 1% serum albumin and split into 2 aliquots for test
and control. IN live and dead spermatozoa the percentage of
acrosome-reacted cells was separately determined in those
with normal sized, small and large acrosomes. Morphology
was analyzed according to strict criteria after Papanicolaou
stain.
Results : AR and morphology correlated regarding detection
of large and small sized acrosomes, but not for normal sized
acrosomes. Spontaneous AR was significantly influenced by
acrosomal size. Sperm with large (11.4%) and normal (9.2%)
acrosomes exhibited a significantly higher percentage of life
spontaneously acrosome-reacted sperm than those with small
acrosomes (4.5%). Sperm with small acrosomes were
associated with a higher percentage of cell death.
Conclusion : The results indicate that sperm with small
acrosomes are more susceptible to cell death and nonphysiological acrosomal loss. Acrosome size reflects the
physiological capability of sperm function and therefore male
fertility potential.
Objective : Date seed oil (DSO) is a natural mixture of
antioxidants containing polyphenols and α tocopherols. The
aim of this study was to test the antioxidants effect of DSO
on human spermatozoa acrosome reaction assessed in vitro,
before and after oxidative stress induced by hydrogen peroxide
(H2O2).
Design : Prospective study.
Setting : Laboratory of histology, Faculty of medicine in Sfax,
Tunisia.
Materials and Methods : The study was carried out on motile
spermatozoa (spz) selected from infertile sperm samples
(n=16) by a discontinuous gradient method, using sill select
(selection medium) and ferticult (culture medium). DSO was
obtained by lipid extraction protocol according to Besbes and
al [1] and dissolved in no toxic Gum Arabic. After selection,
motile spz were incubated in CO2 (5%) at 37°C for 3 hours,
necessary time for spontaneous acrosome reaction; then,
four experimental situations were tested: E1 (motile spz in
ferticult added with 0.01mM H2O2 alone); E2 (spz in ferticult
added with 0.01% DSO alone), E3 (spz in ferticult added
initially with 0.01 % DSO then with 0.01mM H2O2, 30 minutes
later) and us control situation: E4 (spz in ferticult alone). After
that, all the preparations were allowed in CO2 (5%) in 37°C
for 30 min, then used for the determination of reacted acrosome
(RA) spz percentages by an immunofluorescence method,
using flurescein isothiocynate FITC-PSA according to Cross
and al [2].
Results: We found that the percentage of RA spz in the
presence of DSO was higher than in the situation without
DSO (control) and that it was reduced in the presence of
H2O2, but without reaching signification (figure1). However,
we noted that in E3 experimental situation (DSO and H2O2),
the percentage of the RA spz was significantly higher than in
E1 situation (H2O2 alone).
Conclusion : Our results led to suggest that DSO has
beneficial effects on spermatozoa functions, and enhances
their fertilization capacity. So we can propose the use of the
359
purified DSO “in vivo” or “in vitro” treatment essays in male
infertility.
References :
1. Besbes S., Blecker C., Deroanne C. et al. : Date seed oil
: phenolic, tocopherol and sterol profiles. J. Food. Lipids,
2004, 11 : 251-256.
2. Cross N.L., Morales P., Overstreet J.W., Hanson F.W. :
Two simple methods for detecting acrosome reacted human
sperm. Gamete Res., 1986, 15 : 213-226.
PO 065
Tyrosine phosphorylation patterns in human
sperm incubated in HTF media or bound to solid
state hyalutronic acid
Results : We determined the TP pattern in different regions
of sperm. TP occurred in the equatorial segment, acrosomal
area, neck, and principal piece of sperm. The extent of TP
significantly increased at 0 and 4 hours. We observed major
TP changes, related to elapsed time and HA-binding, only in
the neck region and principal piece of sperm. The % sperm
with neck and principal piece phosphorylation in HTF at 0
time and 4 hours were: 14.4±5.3 and 41.5±8.8 (p=0.02,
N=3777 sperm) compared to 21.9%±4.9% and 51.5±5.5%
(p=0.01, N=3555 sperm) in the HA-bound sperm fraction,
respectively.
Conclusions : The capacitation-related pattern of TP in
different regions of the sperm increased in a time-related
fashion. The extent of TP in the sperm neck and principal
piece, a pattern that is characteristic for marker of sperm
activation, increases with time and contacts with HA.
Furthermore, it is of interest that the sperm TP pattern changes
are similar in sperm that are bound to HA or to zona pellucida
of oocytes.
This research was supported by the NIH (OH-04061; HD19505)
S. CAYLI1, L. SATI2, D. SAKKAS3, R. DEMIR3,
G. HUSZAR3.
1 Justus Liebig University, Institute of Anatomy and Cell
Biology, Giessen, Germany ; 2 Akdeniz University,
Department of Histology and Embryology, Antalya, Turkey ;
3 Yale University School of Medicine, Department of
Obstetrics and Gynecology, New Haven, CT,USA
Introduction : During spermiogenesis there is a sperm plasma
membrane remodeling that facilitates the formation of zona
pellucida and hyaluronic acid (HA) binding sites. Upon binding
to zona pellucida, human sperm undergo capacitation-related
changes. Sperm with arrested maturation can fail to bind to
the zona pellucida and HA. Also, the sperm selection attributes
of HA and zona pellucida is similar: HA-bound sperm show
no cytoplasmic retention, persistent histones or DNA
fragmentation. Previous studies have shown that specific
protein tyrosine phosphorylation (TP) changes in spermatozoa
are related to events associated with capacitation and zonabinding. In the present study, we have studied TP patterns in
spermatozoa isolated from media or bound to HA.
Materials and Methods : Immunofluorescence was performed
with antisera for TP. We prepared sperm smears, HA- and
zona-bound sperm from each semen sample suspended in
HTF medium (Irvine Scientific) and using HA-coated slides
(MidAtlantic Diagnostics). The sperm were fixed with 3.7%
formaldehyde after 0 time and 4hr incubations. The slides
were treated with a monoclonal anti-phosphotyrosine antibody
(Sigma). These steps were followed with FITC labeling for
sperm assessment with fluorescence microscopy. Data
analyses were carried out with SigmaStat (Jandel, CA). All
values are mean±SEM.
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PO 066
Toluidine Blue image analysis cytometry for the
prognosis of male fertility potential
I. TSAREV1, T. EBBESEN2, E. ERNST2,
A. GIWERCMAN3, J. ERENPREISS1-3
1 Andrology laboratory, Riga Stradins University, Latvia ; 2
Cryos International Sperm Bank, Denmark; 3Molecular
Reproductive Research Unit, Dept. of Clinical Siencies,
Lund University, Sweden.
e-mail : Aleksander.Giwercman.@med.lu.se
Objective : It is well known that the conventional semen
analysis can be used only for the approximate prediction of
the male fertility potential. Numerous studies have sought a
key factor(s) that would be highly predictive, including also
sperm acrosome status, cell membrane integrity, sperm DNA
integrity, etc. However, to date, no single laboratory test can
precisely assess a man’s fertility potential. Hereby we introduce
the new approach to use the Toluidine Blue (TB) test for
sperm chromatin conformation assessment as a highly
predictive test for the evaluation of the male fertility.
TB test is able to distinguish (a) sperm nuclei with normal
chromatin conformation and normal DNA integrity (light blue
cells), (b) sperms with DNA double-strand breaks and loosely
packaged chromatin (dark purple cells), (c) two “intermediate”
conditions - light purple and intensive blue sperm cell nuclei.
Results of the TB test (proportion of the dark purple cells)
correlates with those from SCSA and TUNEL assays.
Materials and Methods : Sperm samples from 21 sperm
bank donors and 42 infertile men from barren couples with
excluded female factor were subjected to the conventional
semen analysis and TB image analysis cytometry test as
described earlier. ROC curve analysis was used to set the
threshold between fertile and infertile men. Three different data
sets were exploited in this analysis: (a) concentration of TBlight blue cells/mL ejaculate (assuming both TB-test results
and sperm concentration) ; (b) proportion of TB-light blue
cells (sperm concentration not assumed); (c) sperm
concentration only (thresholds of 5, 20 and 40 x106/mL were
tested).
Results : When threshold between fertile and infertile men
was set at 28 millions TB-light blue cells/mL ejaculate, it
possessed high sensitivity, specificity, positive predictive value
(PPV), and negative predictive value (NPV). These values were
considerably lower when only the TB-data or sperm
concentration were considered alone (Table).
PO 067
Evaluation of innovative assays in routine semen
analysis
U. PAASCH, S. GRUNEWALD, M. REINHARDT,
T. BAUMANN, H.J. GLANDER
European Academy of Andrology Training Center,
University of Leipzig, Germany
[email protected]
Objective : Standard semen analysis does not provide
information about subcellular processes in human sperm.
For a better understanding of spermatozoal defects not
monitored by routine semen analyses new fluorescence based
assays were introduced. It was our aim to determine the
stability of the fluorescence signals of these recently developed
tests in the course of time for estimation of practicability.
Design : Prospective study.
Conclusions : Hereby we suggest simple and easy accessible
test with high precision for male fertility potential assessment.
TB test is cheap and easy to perform, and highly correlative
with alternative tests like SCSA and TUNEL, requiring more
expensive equipment and reagents.
Materials and Methods : Semen samples from healthy
donors (n=87) were separated in 4 aliquots to perform the
following assays : 1. detection of active Caspase-3 (FLICA™,
Immunochemistry Technologies), 2. analysis of integrity of
the mitochondrial membrane potential, MMP (Mitosensor™,
Clontech), 3. detection of externalization of phosphatidylserine,
EPS (FITC-labeled, monoclonal mouse anti-human
phosphatidylserine antibody, clone 1H6, Upstate Cell Signaling
Solutions) and 4. detection of CD46 (monoclonal, FITClabeled mouse anti-human CD46, IgG2a, Biomeda).
Afterwards, paraformaldehyd 4% was added to all aliquots.
The fluorescence of each sample was evaluated by flow
cytometry (FACS Calibur, Beckton Dickinson) at day 0, day
3, day 7, day 10 and day 14.
Results : Variances up to ± 5% positive spermatozoa from
the value measured at day 0 were estimated as acceptable
deviation. The Caspase-3 FLICA™ showed mean variances
< 5% at day 3, 7 and 10. At day 14 the mean difference was
7.6%. In contrast, the Mitosensor™ and the EPS detection
showed variances > 5% already at day 3. The CD46-FITC
labeling displayed absolute variances < 5% CD46 positive
spermatozoa at day 3, 7, 10 and 14.
Conclusions : Although immediate analysis of the
fluorescence signals is recommended, it is possible to evaluate
activation of Caspase-3 up to 10 days after the staining in
human spermatozoa. Labeling with CD46-FITC on the
spermatozoal surface gives very stable fluorescence signals
and flow cytometry analysis can be performed at least within
the following 2 weeks. The FACS evaluation of MMP integrity
and EPS detection should be conducted at the same day.
Support : German Research Council GL 199/4-3.
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PO 068
PO 069
Sperm Chromatin Structure Assay (SCSA)
predicts the outcome of ART
SCSA parameters and sperm preparation for IVF
F. GUERIF1, G. ABS1, D. ROYERE1
1 Reproductive Medecine & Biology, Dept of Obs-Gyn &
Human Reproduction, CHRU de Tours, Université François
Rabelais Tours, France e-mail : Hyperlink mailto :
[email protected] [email protected]
Objective : The routine examination of semen does not
identify defects in sperm chromatin structure. Evaluation of
raw sperm DNA damage appears to be a useful tool for
assessing male fertility potential both in vivo and in vitro.
Human semen is heterogeneous in quality within a single
ejaculate. Thus in IVF, it is important to use a sperm processing
technique that enables the recovery of a concentrated and
highly functional sperm population. A study was performed to
examine the effect of semen processing by density gradient
on human sperm DNA integrity.
Design : Descriptive and correlational clinical study.
Setting : This study was performed in the University Hospital
of Tours, France.
Materials and Methods : Semen samples were collected
from 236 patients undergoing IVF in our centre and processed
by density gradient. For each sample (before and after
processing), DNA fragmentation (DFI) was recorded with the
use of Sperm Chromatin Structure Assay (SCSA). Controls
were used to determine intra (n = 20) and inter (n = 63)
variability (both of 5%) of the SCSA in our hands. The difference
between DFI before and after density gradient allowed us to
divide patients in three groups: Group 1 without difference (n
= 37), Group 2 with decreased DFI (n = 181) and Group 3 with
increased DFI (n = 18).
Results : The clinical pregnancy rate was lower (11.1%) in
the Group 3 with an increased DFI after sperm processing by
density gradient compared to Group 1 and 2 (24.3% and
24.9%, respectively). Moreover Group 3 was also associated
with the lowest sperm parameters (numeration and motility)
both before and after density gradient.
Conclusion : Most studies investigating SCSA parameters
used neat semen with conflicting results. Our data show DFI
alteration during sperm processing among IVF patients and
suggest that combining DFI assessment before and after
density gradient might be more accurate. Such results support
the necessity to revalidate SCSA in sperm processed for use
in IVF.
M. BUNGUM1,3, P. HUMAIDAN1, A. AXMON4,
M. SPANO2, L. BUNGUM1, A. GIWERCMAN3
1 Fertility Clinic, Viborg Hospital (Skive), Denmark,
2 Section of Toxicology and Biomedical Sciences,
BIOTEC-MED, ENEA Casaccia Research Center, Rome,
Italy, 3 Fertility Centre, Scanian Andrology Centre, Malmö
University Hospital, Malmö, Sweden, 4 Department of
Occupational and Biomedical Medicine, University of Lund,
Lund, Sweden
Objectives : Since the introduction of assisted reproduction
techniques (ART) there has been a continuous search for
markers with the potential to predict a couple’s chance of
obtaining a pregnancy and to be used for improving the
relatively low baby-take-home rates, which have been held
stable (20-30%) during the last two decades. During recent
years there has been an increased focus on the role of sperm
chromatin integrity in relation to fertility and several sperm DNA
integrity testing methods are now available. One of these
tests, the Sperm Chromatin Structure Assay (SCSA) has
been suggested to be a powerful predictor of fertility in vivo
as well as in vitro. The available data, however, have been
based on limited numbers of treatment cycles. The aim of
this study was to assess the clinical role of SCSA, in IVF,
ICSI and IUI, based on a large study material.
Design : Prospective study
Materials and Methods : 998 consecutive couples undergoing
(ART) were included. Intrauterine insemination with partner’s
sperms (IUI) was performed in 387, IVF in 388 and ICSI in
223 cases. SCSA results were expressed as DNA Fractionation
Index (DFI) and High DNA Stainable cell fractions (HDS).
Reproductive outcome parameters were clinical pregnancy
(CP) and delivery (D).
Results : For IUI, the odds ratios (ORs) for CP and D were
significantly lower in the group with DFI > 30% as compared
to those with DFI> 30% (95% confidence intervals (CI): 0.10
(0.02-0.42) and 0.07 (0.01-0.48), for CP and D respectively)
(Fig 1). No statistical difference between the outcomes of
IVF vs. ICSI was observed in the group with DFI £ 30%. In
the DFI > 30% group, however, the results of ICSI were
significantly better than those of IVF. Comparing ICSI to IVF,
the ORs (95% CI) for CP and D was 3.0 (1.4-6.2) and 2.0 (1.04.5), respectively. Neither sperm concentration nor motility
could add to the prediction of the treatment outcome.
Conclusions : Our study shows that DFI can be used as an
independent predictor of pregnancy and birth in couples
362
undergoing IUI. A high DFI does not exclude successful
treatment, but the OR of pregnancy was 3 times higher by ICSI
than by IVF when the DFI exceeded the level of 30%. When
DFI exceeds 30%, ICSI should be the method of choice, also
in cases where traditional sperm parameters are normal.
Figure1 : Odds ratios (OR) for Biochemical pregnancy (BP)
of intrauterine insemination (IUI) treatment in relation to cut
off level for the DNA Fragmentation Index (DFI).
PO 070
Increased sperm concentration was predictive of the
occurrence of a clinical pregnancy (95.60 ± 17.98 vs 54.47
± 5.48x106 spermatozoa per mL, P=0.04).
In ICSI, a higher MMP predicted a higher fertilization rate
(53.10 ± 3.38 vs 40.50 ± 9.74%, P=0.016). Fewer necrotic cells,
higher MMP and younger males predicted the development
of at least one blastocyst (33.00 ± 3.24 vs 43.50 ± 5.86%,
P=0.039, 57.51 ± 3.46 vs 39.21 ± 5.92%, P=0.039 and 34.1
± 0.7 vs 37.3 ± 1.2 years, P=0.036). Older male age predicted
the arrest of all embryos (40.1 ± 2.1 vs 34.7 ± 0.7 years,
P=0.036). Neither spermiogram nor apoptosis markers could
predict a pregnancy.
Conclusion : Beside male age and sperm concentration,
annexin V and MMP changes, which are early indicators of
apoptosis, may predict the outcome of classical IVF and ICSI.
Sperm annexin V binding, mitochondrial
membrane potential and DNA fragmentation
changes in predicting IVF and ICSI outcome
PO 071
B. ZORN1, A. IHAN2, A. KOPITAR2, M. KOLBEZEN1,
I. VIRANT-KLUN3, H. MEDEN-VRTOVEC3
1 Andrology Centre, Department of Obstetrics and
Gynecology, University Medical Centre, Ljubljana, Slovenia
2 Institute of Microbiology and Immunology, Faculty of
Medicine, University of Ljubljana, Slovenia
3 Reproductive Unit, Department of Obstetrics and
Gynecology, University Medical Centre, Ljubljana, Slovenia
Background : We determined the value of sperm membrane
phospholipid asymmetry, mitochondrial membrane potential
(MMP) and DNA fragmentation changes in predicting the
outcome of classical IVF and ICSI.
Methods : Prospectively we evaluated sperm of men of
infertile couples (n=248) on the basis of spermiogram. After
sperm preparation using gradient centrifugation, apoptosis
markers were assessed by flow cytometric analysis: apoptotic
and necrotic cells by use of PI and annexin V, MMP by means
of DiOC6(3) staining and DNA fragmentation after addition of
acridine orange. One to 6 months later, 45 and 51 couples
were involved in 50 IVF and 64 ICSI cycles, respectively. The
percentage of necrotic cells, the DNA fragmentation index
and the percentage of spermatozoa with high MMP were
correlated with fertilization rate (less or more than 25% of
fertilized oocytes), presence of at least one blastocyst, all
embryos arrested, and occurrence of a clinical pregnancy. Data
were analyzed by logistic regression and analysis of variance.
Results : In IVF, sperm concentration and necrotic cells were
significantly related to fertilization rate (40.53 ± 6.57 vs 74.48
± 7.97x106 spermatozoa per mL, P=0.038 and 30.07 ± 18.77
± 2.39%, P=0.040, respectively). More necrotic cells were
found in couples not obtaining at least one blastocyst or
developing only arrested embryos (27.15 ± 3.73 vs 20.18 ±
2.78%, P=0.03 and 34.83 ± 3.79 vs 18.70 ± 2.40%, P=0.026).
363
Apoptosis signaling in spermatozoa after
standard semen preparation in an IVF clinic
S. GRUNEWALD1, U. PAASCH1, M. REINHARDT1,
V. BLUMENAUE2, F.A. HMEIDAN2, H.J. GLANDER1
1 European Academy of Andrology Training Center,
University of Leipzig, Germany 2 Clinic for Reproductive
Medicine, Gynecology and Endocrinology, Leipzig,
Germany [email protected]
Objective : The inclusion of apoptotic sperm during assisted
reproductive techniques (ART) may be one of the reasons for
suboptimal success rates. The aim of our study was to evaluate
the potential of routine standard semen preparation in the IVF
laboratory to eliminate spermatozoa with activated apoptosis
signaling from patients semen samples prior to ART.
Design : Prospective study.
Material and Methods : Semen samples from 20 infertility
patients scheduled for ART (IVF and ICSI procedures) were
investigated after informed consent. Following density gradient
centrifugation (DGC) and swim up small aliquots were taken
from each sample to analyze the levels of Caspase-3 activation
(CP3) using carboxyfluorescein derivatives (FLICA™) and
the integrity of the mitochondrial membrane potential (MMP)
using a lipophilic cationic dye (MitoSensor™) by flow cytometry.
In addition progressive motility was observed according WHO.
Aliquots from the neat semen served as controls.
Results : Standard semen preparation by DGC and swim up
resulted in improvement of motility (WHO a+b, Mean±SD : from
29.5±10.4 to 73.0±8.8%, p<0.01). Semen samples of patients
contained 46.2±17.7% spermatozoa with intact MMP and
51.8±14.9% spermatozoa with active CP3. After DGC + swim
up the amount of MMP intact sperm was increased to
78.5±11.8% (p<0.01), while the percentage of spermatozoa
containing active CP3 was reduced to 26.1±15.0% (p<0.01).
However, there were inter-individual differences in the
separation effect: Minimal reduction of sperm with disrupted
MMP and active CP3 was 6.0% and 0.7% respectively,
maximum reduction was observed as 65.5% (disrupted MMP)
and 49.3% (CP3).
Conclusion : Semen samples of subfertile patients contain
significantly higher levels of spermatozoa with disrupted MMP
and activated CP3 when compared to results of previous
studies on fertile donors. Although there was a reduction in
the majority of the samples, further reduction might be achieved
by recently developed molecular separation methods
combining the standard semen preparation and Annexin-Vbased elimination of apoptotic spermatozoa.
Support : German Research Council GL 199/4-3.
motile morphologically normal sperm in debris free media.
Results : Total sperm recovery percentage was 33.88±11.37
with DGC and 13.22 ± 4.02 with SU (P < 0.001). Percentage
of increase in progressive motility % was 46.79 ± 24.98 with
DGC and 147.41 ± 58.94 with SU (P < 0.001). Percentage
of increase in total motility % was 36.57 ± 30.88 with DGC
and 84.10 ± 58 with SU (P < 0.001). Total motile sperm
(TMSC) recovery % was 44.92 ± 12.44 with DGC and 25.28
± 5.81 with SU (P < 0.001). Percentage of reduction in round
cell number (RCs)/HPF was 64.51 ± 18.83 with DGC and
90.22 ± 14.87 with SU (P < 0.001). Percentage of increase
in normal morphology % was 27.87 ± 11.30 with DGC and
26.93 ± 11.49 with SU (P> 0.05).
Conclusions : DGC is superior to swim-up in the recovery
of higher number of motile sperm. It can be used in dealing
with low quality semen samples especially in the presence of
oligozoospermia or low native total motile sperm count. SU
is superior to DGC in improving the percentage of motile
sperm. So, It can be used in processing semen samples with
poor motility percentages and relatively good sperm count.
PO 072
PO 073
Comparative study of two semen processing
techniques in idiopathic male infertility
Improvement of sperm characteristics after
sperm preparation by buoyant density gradient
A.A. MOUBASHER1, T.K. AL-HUSSAINI2,
O.M. HASSAN1, E.A. TAHA1
N. ABID, N. BENJAMAA, M. AJINA, A. SAAD
1 Department of Dermatology and Andrology ;
2 Department of Obstetrics and Gynaecology, Assiut
University, Assiut, Egypt
Service of Cytogenetic and Biology of Reproduction,
University Hospital F Hached, Sousse, Tunisia.
mounir.ajina @rns.tn
Objective : To compare two commonly used techniques of
semen processing: swim-up (SU) (Using Ham’s F10, Biochrom
A.G, Berlin, Germany) and density gradient centrifugation
(DGC) (Using Sil-select Plus, Ferti-Pro N.V., BeernemBelgium).
Objective : of our study is to estimate the interests of sperm
preparation technique by swim up on pressure gradient of pure
sperm on sperm characteristics at patients with morphologically
abnormal semen samples within the framework of coverage
of male or mixed infertility.
Design : Prospective experimental study.
Design and place : Service of Cytogenetic and Biology of
Reproduction, University Hospital F Hached Sousse, Tunisia.
Settings : The Department of Dermatology and Andrology,
Assiut University Hospital.
Patients : A group of men diagnosed to have idiopathic male
infertility.
Methods : The study included 44 semen-processing trials for
separate semen samples from men with idiopathic male
infertility using SU in 23 samples, and DGC in the other 21
samples.
Main outcome measure : Comparison between SU and
DGC in the recovery of higher numbers and percentages of
364
Methods : This study included 53 infertile patients presenting
at least to the spermogram a hight percentage of abnormal
forms (> 70%) according to the WHO criteria. The sperm of
the patients was obtained by masturbation after 3 days of
sexual abstinence. The initial examination of the sperm
contains an evaluation of the following parameters: volume,
initial motility, percentage of abnormal forms. Then the sperm
benefits from a centrifugation on 3 layer pure sperm gradient
(90%, 70%, 45%) followed by a wash of the 90% layer in
another environment of culture.
Results : The morphological analysis of spermatozoa after
centrifugation showed a significant increase of the average
percentage of the normal forms which doubles and pass from
18.4% to 36.13%. The indication of multiple abnormalities
(IAM) is improved in a significant way (p < 0,000) passing from
1.6 to 1.38. Also the percentage of sperm cells has
microcephalic, disentangled heads; the abnormalities of the
acrosome double tails, anguled and short spermatozoa are
significantly lowered.
Conclusion : The sperm preparation by centrifugation on
pressure gradient of pure sperm allows a quantitative and
qualitative selection of spermatozoa. This technique allows
us of to improve spermatozoa power fertilizing and obtaining
embryos to be able to evolutionary mattering in case of appeal
to reproduction medical care.
References :
1. Yue Z., Meng F.J., Jorgensen N., Ziebe S., Nyboe Andersen
A. : Sperm morphology using strict criteria after Percoll density
separation : influence on cleavage and pregnancy rates after
in-vitro fertilization. Hum. Reprod., 1995, 10 : 1781-5.
2. Yao Y.Q., Ng V., Yeung W.S., Ho P.C. : Profiles of sperm
morphology and motility after discontinuous multiple-step
Percoll density gradient centrifugation. Andrologia, 1996, 28
:127-31.
3. Hammamah S. et al. ; Médecine et biologie de la
reproduction, 2ème édition Masson, 2004 : 157-161.
PO 074
Prospective cross-over trial comparing the
results of intrauterine insemination with and
without mild ovarian stimulation and timed
natural intercourse in male and unexplained
subfertility
subfertility is controversial. The aim of this study was to
compare the effectiveness of IUI with and without mild ovarian
stimulation in male and unexplained subfertility.
Design : A prospective cross over trial.
Materials and Methods : Seventy-eight couples (duration of
infertility:median=3.9 years; female age: median=35.5 years)
with unexplained or male factor subfertility without ovulatory
dysfunction and other causes of subfertility in the female were
recluted. They were randomly assigned to sequential timed
natural intercourse (NI), IUI and IUI + sequential CC and
human menopausal gonadotrophin (HMG) repeated 3 times,
unless pregnancy occurred.
Results : In this preliminary analysis the results of 326
observed cycles were evaluated. The clinical pregnancy rate
(PR)/cycle was 1.6% in NI, 6.5% in IUI, 8% in IUI+COH (risk
ratio=5; 95% CI: 1.3-18.9 vs NI). A trend toward higher PR
was observed with IUI+COH than in IUI (11.4% vs 6.7%) in
cycles with normal semen, but not in cycles with abnormal
semen (motile sperm count <10x106/mL and/or normal
morphology <15%, and/or MAR test >50%), where PR was
8% in IUI+COH and 7.1% in IUI, excluding cycles where
recovered motile spermatozoa were < 1x10 6.
Teratozoospermia strongly affected the IUI outcome: PR=2/84
cycles (2.4%) vs 13/101 cycles (12.9%; risk ratio=5.4; 95%
CI: 1.5-19.3). In oligo/asthenozoospermia without
teratozoospermia, the PR/cycle was 6/43 (14%).The highest
PR was achieved in the male immunological infertility (4/26
cycles; 15.4%).
Conclusions : This preliminary analysis indicates the
effectiveness of IUI with a mild stimulation with sequential
CC and HMG in infertile couples with unexplained infertility,
while IUI without COH appears to be equally effective in the
male subfertility, thereby representing a proper first-choice
therapy, when >1x106 motile sperm are recovered and
teratozoospermia is excluded.
PO 075
Percutaneous epididymal and testicular sperm
obtaining procedures for intracytoplasmic sperm
injection - 10 years experiences in Fertility Clinic
“Novum” (1996-2005)
A. BARBONETTI, S. SORGENTONE, A. ZUGARO,
R.S. SANTUCCI, F. SCIARRETTA, F. FRANCAVILLA
Andrologic Unit, Department of Internal Medicine,
University of l’Aquila, Italy.
J.K. WOLSKI, K. KOZIOL, P. LEWANDOWSKI, B.
BIARDA, H. MARSZAL, S. TRUBACZ
Objective : Although the effectiveness of intrauterine
insemination (IUI) plus controlled ovarian hyperstimulation
(COH) with gonadotrophins is well established in couples
with unexplained infertility, it carries the risk of ovarian
hyperstimulation and multiple pregnancy. Limited evidence
exists on the effectiveness of IUI with a milder stimulation
with clomiphene citrate (CC). Furthermore, whether the IUI
with COH is more effective than IUI without COH in the male
365
Hyperlink "mailto : [email protected]"jkwolski@op.pl Fertility
Clinic „Novum”, Warsaw, Poland
Objectives : About three years after Palermo’s report
[„Pregnancies after intracytoplasmic injection of single
spermatozoon into an oocyte”, The Lancet 1992;340;17] the
first ICSI (Intracytoplasmic Sperm Injection) procedure in
Poland was done in January 1995, in Fertility Clinic “Novum”,
Warsaw. Next, in the same center, the first ICSI with
percutaneously sperm obtained from epididymis (ICSI-PESA,
Percutaneous Sperm Aspiration) was performed in April 1996
and also the first procedure with testicular sperm (ICSI-TESA,
Testicular Sperm Aspiration) was done in September 1996.
Design : This report presents 10-years experience in
percutaneous sperm obtaining for ICSI-PESA/TESA
procedures in “Novum”.
Materials and Methods : Azoospermic men (mean age 32
y.o.) from infertile couples were qualified for sperm retrieval
procedure as follows: with obstructive azoospermia from
epididymis (PESA) and with non-obstructive azoospermia
from testis (TESA). All procedures were done ambulatory,
under general anesthesia (short IV : Propofol and Fentanyl).
In PESA, both epididymides were punctured percutaneously
using syringe 2 mL with fine needle No 5, then aspirated
liquid was examined under light microscope for sperm
presence for ICSI-PESA. In TESA, testicular tissue from both
testes was obtained using 1.6-mm needle (Hepafix® set,
B.Braun, Melsungen, Germany) and technique tissue
preparations reported by Schoysman [“Modern sperm retrieval
techniques and their usefulness in oocyte fertilization “ BJU
Int. 2001.88.141] were performed for ICSI-TESA. Since 1999,
during regular diagnostic testicular biopsy in each patient,
scheduled cryopreservation and storage of epididymal liquid
and part of testicular tissue were done (the rest of specimen’s
part was fixed in Bouin’s solution for routine histological
evaluation). This procedure made possible to avoid next
testicular biopsies during future ICSI. Mean time of one
procedure was 15-20 minutes. After 2 hours of observation
each patient was verified by anesthesiologist and urologist and
left center. During 2-3 days after procedure commons analgesic
drugs (e.g. Paracetamol) and scrotal ice compresses were
applied; no antibiotics were used as a rule.
Results : These final results were stated by parents’
declarations up to the end 2005 (incomplete unfortunately,
because all childbirths were outside of “Novum”).
In men from both groups no important complications after
sperm obtaining and no prolonged breaks in life activity (work,
study and sex) were reported.
Conclusions : Application sperm obtaining from epididymides
366
and testes in micromanipulation procedures IVF/ICSI-PESA,
TESA makes real possibility to be fathered for men with
obstructive and non-obstructive azoospermia. Percutaneous
sperm obtaining is non-complicated, safe, relatively cheap
minimal invasive procedure.
Support : no declared
PO 076
Evaluation of Motile Sperm Organelle Morphology
Examination in unselected In Vitro Fertilization
with Intra-Cytoplasmic Sperm Injection
N. SERMONDADE1,2, F. VIALARD1, M. BERGÈRE1,
P. CAVELOT1, J. SELVA1, M. ALBERT1
1 Department of Reproductive Biology and Cytogenetics,
Centre Hospitalier Poissy Saint-Germain, 78303 POISSY,
France 2 To whom correspondence should be addressed:
Nathalie Sermondade, nsermondade@hotmail.com
As many teams, we were interested by the results of the
method of high-magnification sperm observation and selection
developed by Bartoov [1] and proposed for ICSI failure specific
indication. We wished to evaluate the technique in unselected
ICSI.
Objective : The aim of the study was to evaluate the relevance
of Motile Sperm Organelle Morphology Examination (MSOME)
compared with usual selection made in ICSI. We determined,
among conventionally selected sperm for ICSI, how many
appeared abnormal when observed with high magnification
and assessed the predictive value of this parameter on
unselected ICSI outcome.
Materials and Methods : The study included 25 successive
unselected ICSI attempts in the IVF Laboratory of the Hospital
of Poissy (France) and 5 controls. ICSI were performed
according to usual protocols used in the laboratory. Twenty
five motile spermatozoa of the migrated fraction, still available
after ICSI, and "injectable" according to conventional
morphology assessment in ICSI ("normal" or "as normal as
possible" with magnification of x200-400) were assessed by
MSOME (superior to x4500) and classified according to criteria
adapted from Bartoov’s work [2] and taking into account
David’s sperm morphology classification [3]. We compared the
results of MSOME, the results of conventional sperm
morphology analysis and ICSI results.
Results : In this small series of ICSI with diverse indications,
we found very high frequencies of abnormalities (over 70 %
vs 46.4% in controls, p<0.0001), particularly nuclear vacuoles.
The rates of anomalies detected with MSOME and
conventional morphology assessment were correlated,
excepted for some of them i.e. nuclear vacuoles. No predictive
value of the morphology of sperm assessed with high
magnification (including vacuoles) was found for fertilization
rate, embryo quality and ICSI outcome. At the opposite of
previous reports, pregnancies were obtained with very
abnormal sperms. In this series of unselected ICSI, nuclear
vacuoles do not seem to have pejorative meaning for
pregnancy outcome.
Conclusions : Our work leads to several perspectives. It
would be interesting to understand the "anatomical" support
of vacuoles observed with MSOME and their meaning. The
question of the phenotype-genotype relation, that is to say the
possible correlation between sperm morphology and genetic
content, could be investigated, for example by studying FISH
and MSOME, Tunel and MSOME. Finally, a prospective
analysis should be performed in clearly defined indications to
validate the potential applications of the method for highmagnification sperm observation and selection.
References :
1. Bartoov B. et al. N. Engl. J. Med., 2001, 345 : 1067-1068.
2. Bartoov B. et al. Fertil. Steril., 2003, 80 : 1413-1419.
3. David G. et al. J. Gynecol. Obstet. Biol. Reprod., 1975,
Suppl.1 : 17-36.
PO 077
The fate of paternal mitochondria in non-human
primate embryos
C.M. LUETJENS, R. WESSELMANN
Design : Prospective animal in vitro study.
Materials and Methods : To study the fate of the paternal
mitochondria we chose a non-human primate model, Callithrix
jacchus. We implemented a hyperstimulation protocol to
increase the number of oocytes collected. Penile stimulation
of the male marmosets was utilized to obtain motile sperm.
The mitochondria of the sperm were stained with different
vital dyes which are integrated into the mitochondrial
membrane. After in vitro fertilization the developing embryos
were cultured and fixed at different cleavage points to track
the sperm-derived mitochondria. Embryos were stained for
markers involved in apoptotic or mitochondrial degeneration
such as cytochrome c, APAF-1; Bcl-2 or ubiquitin. By utilizing
immunofluorescence or laser-scanning microscopy the
paternal mitochondria were located in the blastomeres of the
developing marmoset embryos. Live imaging of the
fluorescence indicator was also used.
Results : We hyperstimulated 39 female marmosets in 81
cycles and obtained 580 oocytes. Paternal mitochondria could
be found in all fertilized embryos up to the 8-cell-stage shortly
before the transition into the morula stage. Although the
cytochrome c remained in the sperm-derived mitochondria up
to this stage, the function of the paternal mitochondria
appeared to be lost. The JC-1 stain indicated that these were
not functional sperm-derived mitochondria. Marmoset sperm
were clearly labeled for ubiquitin, a destruction pathway
marker. However after fertilization ubiquitin did not colocalize
with the sperm-derived mitochondria and had disappeared.
Conclusions : The paternal mitochondria remained functional
up to the 8-cell-stage and there was no indication that the early
embryo has a specific mechanism for dismantling them.
Mitochondrial destruction does not appear to be initiated by
known mechanisms such as the apoptotic or the ubiquitin
pathway. If the developing primate embryo destroys these
mitochondria it must occur after the 8-cell-stage. Paternal
mitochondria are distributed into all blastomeres of the very
early embryo, which may lead to their segregation into
trophoblast cells which do not contribute to the embryo. This
would explain why no paternal mitochondrial DNA is found in
any species analyzed.
Support : Supported by the German Research Foundation
(DFG LU 827/3-1/-2).
Institute of Reproductive Medicine of the University
Münster, Germany E-Mail :
CraigMarc.Luetjens@ukmuenster.de
Objective : Sperm-derived mitochondria are integrated into
the oocyte at fertilization but seem to disappear during the early
cleavage phases of mammalian embryos. The developmental
potential of embryos seems to be closely related to their
ability to destroy these mitochondria, but the mechanisms
underlying their loss of function are not yet understood. By
staining the mitochondria prior to fertilization the fate of the
paternal mitochondria can be followed in the embryo and
distinguished from the much bigger maternal mitochondrial
pool.
367
PO 078
Achievement of twin pregnancy with
spermatozoa from a globozoospermic man after
ICSI treatement
PO 079
Non-steroidal anti-inflammatory drugs and
plasma steroids in male athletes
P. SGRO1, C. ROSSI1, V. FIERRO 1, F. ROMANELLI2,
A. LENZI2 , L. DI LUIGI1
A. SALLEMI1, A. AMOURI2, J. CHERIF1, T. REBAI1,
N. ABDELMOULA1
1 Dept of Health Science, University of Rome IUSM,
Rome; 2 Dept of Medical Pathophysiology, Policlinico
“Umberto I”, University of Rome “La Sapienza”, Rome
paolo.sgro@uniroma1.it
1 Laboratory of Histology, University of Medicine, Sfax,
Tunisia 2 Laboratory of Cytogenetics, Pasteur Institute,
Tunis, Tunisia Corresponding author :
nouha_abdelmoulabouayedahoo.fr / Fax : 00216 74 239
826
Globozoospermia is an uncommon severe form of
teratozoospermia characterized by round-headed sperm with
an absence of acrosomes. Family cases of globozoopermia
suggest that this pathology has genetic origins, but the mode
of inheritance remains unknown. So far, no responsible genes
have been identified. Pregnancies and live birth are rarely
reported even with ICSI treatment.
Objective : We report a successful achievement of a twin
pregnancy in a Tunisian couple in which the male partner
had globozoospermia and referred to us for cytogenetic
evaluation.
Methods and Results : A 35 year old man and his 24 year
old partner, a consanguineous couple sought assisted
conception after 2 years of primary infertility. Semen sample
revealed the following characteristics : volume 4 ml ; sperm
concentration 121.2x106/ml ; 25 percent progressive motility
at 30 min and 65 percent vitality. All the spermatozoa were
round-headed. Hormonal profile was within normal range.
The karyotypes of the two partners were normal. The couple
proceeded, after a first unsuccessful cycle of ICSI, to a second
cycle of ICSI in a Belgium centre. Of 12 oocytes retrieved, 11
were micro-injected and 6 fertilized normally. Four compacting
embryos with eight or more cells were obtained. Two embryos
were transferred 72 h after micro-injection and two others
were cryopreserved for future use. A twin clinical pregnancy
results and culminates in a spontaneous vaginal delivery of
live twin, a female and a male. The infant's birth weight was
respectively 2.3 and 2 kg. The twin show normal development
after 6 months.
Conclusion : Genetic counselling was very hard in this
situation. In fact, if some genetic causes of male infertility
have been identified and are clearly explained to patients, many
others genetic factors of infertility remain largely unexplored.
368
Objective : Arachidonic acid (AA) metabolites modulate the
activity of the hypothalamus-pituitary-gonadal (HPG) and
adrenal (HPA) axis. The aim of the present preliminary
investigation was to evaluate if a short-term treatment with
acetylsalicylic acid (ASA), an inhibitor of prostaglandin (PG)
synthesis, in usual clinical conditions might influence male
athletes' resting plasma steroid hormones concentration.
Design : We evaluated the effects of a ten-day treatment
with ASA on morning plasma free-testosterone, total
testosterone (T), dehydroepiandrosterone sulphate (DHEAS)
and cortisol (F) concentrations in moderately trained males
caucasian athletes affected by minor exercise-related muscleskeletal traumas.
Materials and Methods : Morning plasma free-T, T, F, DHEAS
and their ratios were evaluated immediately before and after
the administration of ASA 800 mg two times a day for ten days,
in twelve subjects with the same baseline characteristics :
chronological age of 19-21 years, height 169.5-193.5 cm,
weight 62.2-85.1 kg, VO2max 51.2±7.9 ml-1min-1 kg-1. As a
control, we evaluated the same volunteers after five/six weeks
of wash-out from ASA administration. For the experimental
control evaluations, the volunteers took placebo twice a day
for ten days and suspended physical exercise for the same
period of time all the volunteers suspended their physical
training during both ASA or placebo treatment.
For therapeutic purposes, the athletes were treated with an
anti-inflammatory therapy and with a ten day break from
physical exercise.
Results : Treatment and time conditions showed a significant
interaction effect on F values, and the comparison between
treatments revealed that morning mean plasma F
concentration was significantly lower after ASA treatment
than after placebo treatment (577.2±98.1 mmol.L-1 vs
588.2±102.6 mmol.L-1, p = 0.023). We showed significant
differences between ASA and placebo treatments, both for ∆F
(-42.5±171.3 and -17.4±156.8, respectively, p=0.045), and
for ∆F% (-0.08±26.5% and 2.83±25.5%, respectively, p=0.04).
It is of clinical interest the marginal interaction effect observed
for T concentrations after ASA treatment, close to statistical
significance (from 24.1±3.9 nmol.L-1 to 28.1±8.2 nmol.L-1, p
= 0.052). The statistical evaluation of T variations suggested
an effect of ASA treatment in increasing plasma T
concentrations. In fact, we showed significant differences
between ASA and placebo treatments, both for ∆T (4.1±5.1
and 0.7±1.2, respectively, p=0.047) and for ∆T% (15.0±20.2%
and 2±5.3%, respectively, p=0.049). It is of great interest that
whereas after placebo plasma T variations ranged from 4.8% to 8.5%, with respect to pre-treatment values, after ASA
treatment this range varied from -7.7% to 44.2%.
Conclusions : In the present study we observed that, in
comparison with placebo, a short-term course of Non-Steroidal
Anti-Inflammatory Drugs (NSAIDs) in moderately trained
athletes is able to influence the resting plasma steroid milieu.
Whereas our previous data suggested the involvement of PGs
in the neuro-endocrine responses to physical stress, the
hypothesis that NSAIDs treatment may act on hormone
pathways secretory activity and/or metabolism also in resting
conditions is quite intriguing, despite the high variability of
the evaluated parameters and the number of volunteers.
PO 080
In men with coronary disease both estradiol and
free testosterone may predispose toward
atherogenic lipid profile but higher blood level of
total testosterone is associated with a fewer
critical coronary stenoses
aimed to assess the correlations between blood levels of sex
steroid hormones or lipid profile and degree of coronary
arteries stenoses in men with CAD.
Methods : 111 men with stable CAD, aged 36-73 yrs,
underwent elective coronary angiography. Dissemination and
degree of coronary stenosis were assessed using 3
angiographic indices. Total cholesterol (T-Ch), high density
lipoproteins cholesterol (HDL-Ch), low density lipoproteins
cholesterol (LDL-Ch) and triglicerydes (TG) were estimated
in a single blood sample. To avoid influence of short-term
fluctuations of the hormones, testosterone, estradiol,
dehydroepiandrosterone sulfate and sex hormone binding
globulin (SHBG) were measured on the basis of two
subsequent blood samples taken with 30-min. interval. These
two samples were mixed together for a single determinations
of each hormone. A free testosterone index (FTI) was
calculated as a quotient of total testosterone (nmol/l) and
SHBG blood levels (nmol/l) multiplied by 100.
Results :
I) Positive, significant correlations were found between blood
level of estradiol and T-Ch (r=0.29, p<0.01), between estradiol
and LDL-Ch (r=0.34, p<0.005) as well as between FTI and
LDL-Ch (r=0.23, p<0.05). Blood level of estradiol negatively
correlated with HDL-Ch/T-Ch ratio (r= -0.21, p<0.05).
II) Blood level of T-Ch positively correlated with an index of
the dissemination of coronary artery stenoses, represented
by sum of all narrowings in 15 coronary artery segments
(r=0.26, p<0.05). In turn, blood level of total testosterone
negatively correlated with an index that represented a number
of critical stenoses (over 50% of artery lumen occlusion) (r=
-0.26, p<0.05).
Conclusions :
1) In men with CAD, blood plasma estradiol level is predictive
for T-Ch and LDL-Ch concentrations and for HDL-Ch/TCh
ratio, while FTI for LDL-Ch, indicating that estradiol and
bioavailable testosterone may predispose toward atherogenic
lipid profile.
2) Higher blood level of total testosterone is predictive for
fewer number of the critcal coronary artery stenoses, indicating
that in men testosterone may have a beneficial effect on the
coronary arteries, irrespectively to the relation between FTI
and LDL-Ch.
K. KULA, J.K. WRANICZ*, I. CYGANKIEWICZ*,
P. KULA*, R. WALCZAK-JEDRZEJOWSKA, J.
SLOWIKOWSKA-HILCZER
Chair of Andrology and Reproductive Endocrinology; *
Chair of Cardiology and Cardiosurgery, Medical University
of Lodz, Poland. E-mail : kkula@csk.umed.lodz.pl
3) Higher blood level of T-Ch is predictive for higher total
number of disseminated coronary artery stenoses supporting
the concept of an involvement of T-Ch in the pathogenesis
of CAD.
Objectives : Estrogen receptors are present in the
cardiovascular system and in men with inherited inactivating
mutations of genes encoding estrogen receptors the
occurrence of premature coronary artery disease (CAD) was
documented, indicating preventive role of estrogen for CAD
in men. However, elevated plasma estrogens have been
found in men surviving myocardial infarction, suggesting that
hyperestrogenemia may be a coronary risk factor. Here we
369
PO 081
Testicular dystenesis syndrome and estrogen
receptor alpha polymorphisms : association with
sperm production and cryptorchidism
with this observation, normospermic subjects with genotype
A had a significantly lower mean sperm concentration (p<0.05)
and a lower total sperm count (p<0.01) with respect to men
bearing genotypes with shorter TA alleles.
AGATA haplotype and SNP12 : We confirmed that SNP12 is
the tag SNP for the AGATA haplotype also in Caucasians. No
association between SNP12 and spermatogenic disturbances
was found in the Italian and Spanish populations. However,
in contrast with the Japanese population, we found a significant
protective effect (OR=0.5) for ESR1 SNP12 on cryptorchidism.
E. GUARDUCCI*, F. NUTI*, J. GALAN§, G. BALERCIA#,
G. FORTI*, C. KRAUSZ*
Conclusions : Our data indicate that specific allelic
combinations of the ER α which confer a stronger estrogen
effect, may influence negatively human spermatogenesis. A
plausible explanation would be that not only deficit of estrogens
but also an exaggerated estrogen action related to this genetic
variant (eventually combined with environmental factors), can
be deleterious. Whether the observed negative effect is the
expression of a disturbance in the early testis development
or in the adult testis remain to be established.
* Andrology Unit, Department of Clinical Physiopathology,
University of Florence, Italy ;
§ Department of Structural Genomics. Neocodex S.L.
Seville, Spain ;
# Division of Endocrinology, Institute of Internal Medicine
Polytechnic University of Marche, Ancona, Italy
Hyperlink "mailto : c.krausz@dfc.unifi.it"
c.krausz@dfc.unifi.it
Objective : Although the importance of estrogens in male
reproduction is indisputable, little attention has been paid to
the role of Estrogen Receptors (ERs) gene mutations in male
infertility. The aim of the present study was to get insights into
the role of ER alpha in spermatogenesis and cryptorchidism
through the analysis of polymorphic sites in the ER alpha
gene : i) (TA)n repeat allelic variant in the promoter region;
ii) five SNPs in distal introns. A previous study on the Italian
population found a significant correlation between the (TA)n
repeat variant and lumbar bone mineral density indicating
that allelic combinations with higher number of (TA)n repeats
are functionally more active. As far as the five distal SNPs are
concerned, a specific haplotype “AGATA” was recently
described as a risk factor for cryptorchidism in Japanese
men. In this ethnic group SNP12 was the tag SNP for the
AGATA haplotype.
Materials and Methods : (TA)n repeats : a large group of
infertile and normospermic men (n=347) was studied by using
an automated sequencer by GeneScan software. Each size
of the PCR products was subjected to direct sequencing on
the autosequencer for the definition of the correct TA repeats
length.
AGATA haplotype and SNP12 : a large group of patients
(n=335) and controls (n=567) of two Caucasian populations
(Italian and Spanish) was analyzed. The genotyping was
performed by direct bidirectional sequencing using an
automated sequencer.
Results : (TA)n repeats : Although the (TA)n polymorphism
failed to show a significant association with male infertility, we
found a significant effect of this polymorphism on sperm count.
In the group of infertile men the mean TA repeats number and
sperm concentration and total sperm number were inversely
correlated, showing an association between higher TA repeat
number (genotype A) and lower sperm production. In line
370
With regard to SNP12 polymorphism, the discrepancy between
our and the Japanese study may be related to genuine ethnic
differences and/or different environmental conditions.
Therefore, the observed associations (although with opposite
effect) with criptorchidism encourage future studies on
independent cases and controls from different ethnic and
geographic origin.
PO 082
Environmental risk factors for testicular cancer
M. WALSCHAERTS1,2, A. MULLER1, L. BUJAN1,
P. THONNEAU1
1 Equipe d’Accueil 3694, " Groupe de Recherche en
Fertilité Humaine ", Institut National de la Santé et de la
Recherche Médicale (INSERM), Hôpital Paule de Viguier,
Université Paul Sabatier, Toulouse, France
2 Corresponding author : Hôpital Paule de Viguier, Groupe
de Recherche en Fertilité Humaine (EA 3694), 330 av de
Grande Bretagne, TSA 70034, 31059 Toulouse Cedex 9,
France walschaerts.m@chu-toulouse.fr
Objective : Numerous research studies have examined the
question of an increased incidence of testicular cancer over
the last 50 years, although its causes are as yet little known.
The environment is a major axis of the search for risk factors
of testicular cancer. Some specific occupational exposures
have been suggested, but their attributable risk is too low to
account for all the cases observed. According to the present
environmental hypothesis, two exposure windows may
intervene in the genesis of testicular cancer : in utero during
the development of male gonads, and later during adult life.
In order to target more closely the risk factors of testicular
cancer, an etiological case-control study was carried out on
these two exposure windows during male life.
Design – Materials and Methods : The data analysed were
taken from the Reprhom study, which involved 229 patients
with testicular cancer and 800 partners of pregnant women.
Results : Concerning male exposure in utero, the fact that
the mother had had a garden (possible exposure to pesticides),
had lived near a factory (possible exposure to pollutants) or
taken treatment during pregnancy (possible exposure to
exogenous hormones), increased the risk of development of
testicular cancer in her son. Concerning exposure during
adult life, certain specific occupational exposures (use of
glue, metallurgy) could be considered as risk factors, but the
latter findings should be interpreted with caution as exposure
was recorded only for the previous three months.
Conclusion : Several previously unknown risk factors for
testicular cancer have emerged, notably intra-uterine factors,
contributing towards the postulate that endocrine disturbances
may partly explain the recent marked increase in testicular
cancer.
Financial support : This work was supported by funding
from the European Union (QLK4-CT-1999-01422) and by
grants from the Direction Générale de la Santé (DGS), the
Département Français de la Recherche, the Agence Française
de Sécurité Sanitaire de l’Environnement (AFSSE), the group
Total-Fina-Elf and the group AGRICA.
Although the cure rate is 95%, a group with bad prognosis
continues to be resistant to treatment. Diagnostic delay is
defined as the time elapsing from the onset of tumour
symptoms to the day of diagnosis. We aimed to assess the
relationship between diagnostic delay, disease stage and
survival rate, and to examine trends in diagnostic delay over
the last 20 years.
Design : Regional population-based study
Materials and Methods : Diagnostic delay was investigated
by mailed questionnaire in 542 patients diagnosed with TC
between 1983 and 2002 in the Midi-Pyrenees region, France.
Information regarding disease, treatments and follow-up was
obtained through their medical records. We analysed
diagnostic delay according to histological type, stage and
survival, and studied trends in diagnostic delay.
Results : Mean diagnostic delay was 3.7 +/- 5.1 months and
was longer in seminoma (4.9 +/- 6.1 months) than in nonseminomatous germ-cell tumour (NSGCT) (2.8 +/- 4.0 months).
Diagnostic delay was significantly linked with disease stage
and the 5-year survival rate in the whole population and in
NSGCT. Duration of diagnostic delay did not change
significantly over the study period.
Conclusion : Diagnostic delay remains highly correlated with
stage and survival, in particular for NSGCT. Programmes to
enhance awareness and knowledge of TC are recommended
in order to diminish the diagnostic delay. Measures that may
be taken to reduce diagnostic delay are notably campaigns
for TC awareness and testicular self-examination.
PO 084
PO 083
Impact on survival of diagnostic delay in testis
cancer
Epidemiology, prognostic and management of
pure teratoma of the testis
P. LABARTHE, E. HUYGHE*, C. MAZEROLLES**, C.
CHEVREAU***, P. PLANTE*, A. HOULGATTE****
Service d’Urologie & Andrologie, *EA 3694 Recherches en
Fertilité Humaine, **Anatomie Pathologique, CHU
Rangueil, Toulouse, ***Institut Claudius Régaud, Toulouse,
****clinique d’urologie de l’HIA du Val de Grâce
E. HUYGHE, M. WALSCHAERTS, J. NOHRA,
C. CHEVREAU, P. PLANTE, P. THONNEAU
EA 3694 Human Fertility Research Group, Service
d’Urologie et d’Andrologie, Hôpital Paule de Viguier &
*Institut Claudius Regaud , Toulouse
(huyghe.e@chu-toulouse.fr)
Objectives : To study prognostic and management of pure
teratoma of the testis.
Design : Multicenter and regional population-based study.
Introduction : Testis cancer (TC) has become a common
cancer, whose incidence is rising especially in young men.
371
Materials and Methods : A retrospective multicenter study
was performed using (1) the data base of the Midi-Pyrenees
region and (2) of the Military Hospital of Val de Grâce. Among
more than one thousand patients diagnosed between 1987
and 2003, 20 had a pure teratoma of the testis. All orchiectomy
specimens were examined by 2 uro-pathologists. All patients
were contacted to perform physical examination and CT scan.
Results : Diagnosis was confirmed in 17 patients, non
teratomatous components were identified in the remaining 3
patients.
Among the 8 patients with stage I pure teratoma, 4 received
adjuvant chemotherapy and 4 were entered in a surveillance
program. With a mean follow up of 105 months, Teratoma
Growing Syndrom occurred in 1 patient and controlateral
seminoma in one patient. In 2005, all the patients were disease
free.
The remaining 9 patients with metastatic teratomas were
managed with chemotherapy followed by resection of residual
tumor masses. With a mean follow-up of 149 months, all the
patients were disease free.
Conclusion : Pure teratoma of the testis are rare tumors
(4% of all non seminomatous germ cell tumors). Their
histological diagnosis is difficult and needs careful examination.
Their prognosis is excellent. Surveillance after orchiectomy
should be proposed in the absence of vascular emboli.
Complete surgical resection of retroperitoneal tumor masses
is mandatory in metastatic forms.
PO 085
Sperm DNA integrity in men treated for childhood
cancer
P. ROMERIUS1,3, O. STÅHL2,3, T. RELANDER2,
M. SPANO4, Y. LUNDBERG GIWERCMAN3,
A. GIWERCMAN3
1 Department of Pediatrics, Lund University Hospital, Lund
University, Sweden ;
2 Department of Oncology, Lund University Hospital, Lund
University, Sweden ;
3 Fertility Centre and Department of Urology, Scanian
Andrology Center, Malmö University Hospital, Lund
University, Sweden ;
4 Section of Toxicology and Biomedical Sciences,
BIOTEC-MED, ENEA Casaccia Research Center,Rome,
Italy.
Objective : The treatment of childhood cancer has greatly
improved, and today approximately 80% of the patients are
cured. The clinical challenge of today is therefore not only to
372
cure, but also to cure at minimal cost. Oncological treatment
of a growing child is associated with several long-term
complications, of which persistent damage to the reproductive
function is one. Men treated for childhood cancer are expected
to suffer from decreased fertility. Furthermore, cancer treatment
implies a potential risk of sperm DNA damage although data
on sperm DNA integrity in childhood cancer survivors are
sparse. The aim of this study was to assess DNA
Fragmentation Index (DFI) as an indicator of sperm DNA
integrity in survivors of childhood cancer. Sperm DNA integrity
is of clinical importance both in terms of fertility and the
potential genetic risk for the offspring of the father treated for
cancer.
Design : The study was based on a patient cohort of 97 male
childhood cancer survivors. They had all received treatment
for a malignant disease in childhood or adolescence during
the period 1970-1989. Patients were asked to deliver a semen
sample, and sperm DNA integrity was assessed with two
methods, Sperm Chromatin Structure Assay (SCSA) and
terminal deoxynucleotidyl transferase dUTP nick end labeling
(TUNEL).
Materials and Methods : In the cohort of 97 men 32 chose
to participate. Due to azoospermia (n=7) or inability to deliver
a sample, semen from 22 men were eligible for DNA
assessment .All samples were analysed with SCSA and 19
were analysed with TUNEL. Mean age at diagnosis of the 22
men was 9 years, and at the time of semen delivery 36 years.
Of the 22 patients 19 were treated with either radiotherapy
or the combination of both radio-and chemotherapy. As controls
served 278 military conscripts.
Sperm DNA integrity was assessed by SCSA, a method
based on the phenomenon that chromatin with abundant
DNA double strand breaks has a tendency to denaturate
when exposed to acid-detergent, whereas normal chromatin
remains stable. The extent of DNA denaturability is expressed
as the DNA Fragmentation Index (SCSADFI). DFI expresses
the proportion of cells containing denaturated DNA. Using
flow cytometry a total of 5000 cells were analysed. The TUNEL
assay quantifies the incorporation of fluorescently labeled
dUTP at breaks of double-stranded DNA. Using flow cytometry
a total of 10000 cells were analysed.
Results : In men treated for childhood cancer, TUNELDFI was
significantly higher than for controls (means: 17.7% vs 12.7%;
p=0.045). SCSADFI was also higher in the cancer group, but
without reaching statistical significance (means: 17.3% vs
13.6%; p=0.072).
Conclusions : We have demonstrated that childhood cancer
treatment induces permanent sperm DNA damage. However,
many questions remain. To what extent is the fertility potential
affected in male survivors of childhood cancer with preserved
spermatogenesis ? Are there any genetic risks for the offspring
of these men, especially considering that these men are
probably prone to an increased need of assisted reproduction
? Finally, in order to evaluate the impact of different treatment
modalities, i.e. radiotherapy and different chemotherapeutic
drugs, a larger material is required.
Support : The study was supported by grants from the
Swedish Childhood Cancer Society, Swedish Cancer Society,
The Gunnar Nilsson Cancer Foundation, Malmö University
Hospital Foundation for Cancer Research, Swedish
Governmental Founding for Clinical Research.
PO 086
Semen cryopreservation and testicular cancer :
analysis of 1158 patients in the French Federation
of CECOS
N. RIVES1, I. BERTHAUT2, M.C. MELUN2,
J. MANDELBAUM2, E. SZERMANN2, A. SAUVALLE2,
N. MOUSSET-SIMÉON2, J. SAIAS2,
C. METZLER-GUILLEMAIN2, C. BARTHÉLÉMY,
M.C CLAVEQUIN2, C. THOMAS2, S. HENNEBICQ2,
M. DAUDIN2, M. CHALET2, G. GRIZARD2, L. BUJAN2,
J.L. BRESSON2
1 Reproductive Biology Laboratory – CECOS - Centre
d'Investigation Clinique Inserm 0204, Rouen University
Hospital, Rouen, France
2 French Federation of CECOS (Besançon, Caen,
Clermont-Ferrand, Grenoble, Marseille, Montpellier, Reims,
Rouen, Tenon, Toulouse, Tours), France
Objective and Design : Testicular cancer is one of the most
common pathologies observed in young males aged between
20 and 35 years (12% of cancers). Sperm quality at the time
of the diagnosis is a major parameter able to modify
spermatozoa resistance to the process of freezing and thawing.
Various studies have reported a decline in semen quality
before therapy in testis cancer depending on the histological
feature of tumour, but not confirmed in all published data.
The objective of our study was to analyse retrospectively,
within 11 CECOS (Centre d’Etude et de Conservation des
Œufs et du Sperme Humain), a population of men who
presented a testis cancer and who consulted for sperm
cryopreservation between January 1, 1999 and December 31,
2003. and to examine (i) their medical history (ii) their fertility
prior to cancer diagnosis (iii) the pre-freeze and post-thaw
semen quality before and after orchidectomy as well as (iv)
the effect of histological features and cancer stage on semen
parameters.
Patients and Methods : A population of 1158 patients was
recruited, before or after orchidectomy but before initiating a
treatment by chemo- and/or radiotherapy. Age, urological
history and fertility at the time of the diagnosis were retained
for each patient. Semen samples were evaluated after
liquefaction according to the World Health Organization.
Sperm parameters taken into consideration were : volume
(mL), sperm concentration (106/mL), total sperm count (106),
373
motility (a+b : %), morphology (% of typical forms), number
of straws per sample, post-thaw motility (a+b : %) and total
number of forward motile spermatozoa per straw (106). The
histological diagnosis as well as the evolutionary stage of
the disease were required for each patient : The statistical
analysis was carried out by Gecem society (Service of
Biometrics, Montrouge, France) with a financial support of
FARO 2005 (Fond d’Aide à la Recherche Organon).
Results : A total of 1158 patients aged between 14 and 56
years (30 years ± 7), presenting a pure seminoma (S: 48%),
a mixed tumour (MT : 33%) or a non seminoma tumour (NST°:
19%) including 13.6% embryonal carcinoma (EC) and 3.8%
teratoma (TE). The majority of the tumours were at stage I
(71%), 19% were at stage II and 10% at stage III of Boden.
A quarter of the patients (26%) presented urogenital history,
cryptorchidism being the most frequent (14%). Patients with
seminoma were oldest (p<0.001) but also most fertile. Patients
with urogenital history (including cryptorchism) were a little less
fertile, moreover, their initial sperm parameters were more
altered. The semen samples were carried out before (28%),
after (58%), before and after (13%) orchidectomy. A decrease
of total sperm count was observed in 43% of the patients as
well a reduced motility in 81% of them. Sperm concentration
and total sperm count were significantly lower before
orchidectomy for S and MT compared to EC (p<0.001),
decreased after orchidetomy for NST (for EC, 17x106 before
vs 87x106 afterwards) and did not vary for S. These sperm
parameters are more altered in the tumours of stage III
(p=0.0008). Initial and post-thaw motility significantly decreased
in patients with cryptorchidism (p<0.001) or non proven fertility
(p=0.007).
Conclusion : This series of testis cancers is the most important
one reported in the literature and calls in question the data
previously published. Indeed, seminoma may alter
spermatogenesis more significantly than the NST (including
EC). Seminoma seems to completely inhibit spermatogenesis
in the testis carrying the tumour.
PO 087
Testicular sperm cryopreservation at the time of
removal of testes and subsequent
intracytoplasmic sperm injection in patients with
testicular cancer
I. VIRANT-KLUN, S. DROBNIC,
L. BAÇER-KERMAVNER, J. MIVSEK, T. TOMAZEVIC,
H. MEDEN-VRTOVEC
Department of Obstetrics and Gynaecology, University
Medical Centre Ljubljana, Slovenia E-mail : HYPERLINK
"mailto:irma.virant@kclj.si" irma.virant@kclj.si
Objective : Testicular cancer is a disease increasing in
incidence in developed countries. It attacks many young men
who do not have a child yet or would like to have more children
after efficient cancer treatment.
Design : The aim of this study was to evaluate, whether the
program of testicular sperm cryopreservation could be set
up at the time of removal of one or both testes in patients who
did not cryopreserve sperm (ejaculate) before starting cancer
treatment and expressed their wish to do so.
Material and Methods : When removal of one or both testes
(orchiectomy) in 9 patients was performed by urologists, a part
of healthy tissue from the removed testis was preserved and
transported in a pre-warmed, sterile Flushing Medium
(MediCult, Denmark) to the IVF laboratory. Approximately 1
hour later it was checked for the presence of spermatozoa
under inverted microscope, frozen in a 20% cryoprotectant
glycerol solution, using a controlled-rate cooling machine,
and stored in liquid nitrogen at -1960C. At the time of use it
was thawed at room temperature and intracytoplasmic sperm
injection (ICSI) was performed using testicular spermatozoa.
Results : The mean age of 9 patients at the time of biopsy
was 30 years. It ranged from 26 to 54 years. Eight patients
were younger than 30 years. All patients had seminoma
tumors in one or in both testes. In 8 patients one testis was
removed because of the tumor; another testis has already been
removed in the past or was abnormal. In 1 patient both testes
were removed. In 8 patients motile spermatozoa were found
in testicular tissue. In one patient the testicular tissue was
thawed 9 years after cryopreservation and 15 oocytes of 32years-old female partner were microinjected with testicular
spermatozoa using ICSI. Seven embryos developed and two,
both at the blastocyst stage after extended culture, were
transferred into the uterus. Female partner conceived and
gave birth to a healthy child. In 8 patients testicular tissue is
still stored after 1 to 10 years after cryopreservation.
Conclusions : In a big proportion of patients with seminoma
testicular cancer motile testicular sperms can be found at the
time of the testes removal which may be cryopreserved for
later use. This offers the testicular cancer patients good
chances to father a child in the future, using ICSI, and means
an enormous psychological support them.
Support : We would like to thank to all urologists, Department
of Urology, to the whole team of gynaecologists, andrologists,
and embryologists of our Department, and to Ms. Mojca Pirc,
B.Sc.
374
PO 088
Cryopreservation of testicular tissue from
prepubertal mice
J.P. MILAZZO1, L. VAUDREUIL1, J.P. VANNIEZ2,
B. MACE1, N. RIVES3
1 Reproductive Biology Laboratory – CECOS, Rouen
University Hospital, Rouen, France
2 Immunologie Hématologie Oncologie Pédiatrique, Rouen
University Hospital, Rouen, France
3 Laboratoire de Biologie de la Reproduction - CECOS,
Centre d'Investigation Clinique Inserm 0204, Rouen
University Hospital, Rouen, France
Tél. : 02 32 88 82 25 Fax : 02 35 98 20 07 e-mail :
nathalie.rives@chu-rouen.fr
Introduction : The increased number of childhood cancer
survivors has focused attention on sequels of treatment, more
specifically on gonad damage and dysfunction.
Cryopreservation of ejaculated spermatozoa should be
proposed for sexually mature boys. However, when boys
failed to collect ejaculated semen samples, or for prepubertal
boys, testicular biopsy and cryopreservation of (i) isolated
testicular spermatozoa when possible or (ii) testicular tissue
should be proposed. However, it is absolutely necessary to
establish an optimal procedure for freezing and thawing
immature testicular tissue before application in humans. In the
present study, we evaluated viability, induction of apoptosis
and morphology of immature testicular tissue after different
cryopreservation protocols in a prepubertal mouse model.
Materials and Methods : Immature testis obtained from sixday-old CD1 mice (Charles River, France) were cryopreserved
using several protocols with either 1,2-propanediol (PrOH) or
dimethyl-sulphoxide (DMSO) as cryoprotectants with a final
concentration of 1,5 M combined with different cooling rate
curves [(i) controlled slow protocol with and (ii) without seeding,
(iii) controlled quick protocol and (iv) uncontrolled protocol].
In the controlled protocol, the cooling rate was regulated by
a Minicool 40 PC (Air Liquide Santé, France). In the
uncontrolled protocol, the samples were placed 24 or 48
hours at -80°C in a freezer before being plunged into liquid
nitrogen. Eight testis were used for each condition and a
minimum of three independent series of experiments was
performed for each condition. Cell viability was determined
by a trypan blue and propidium iodide staining. Apoptosis
markers [Annexin V, terminal deoxynucleotidyl transferase
assay (Tunel)] were evaluated immediately after thawing and
isolation of germ cells using an enzymatic treatment as well
as 24 hours after culture of germ cell suspension at 33°C. We
determined the recovering of testicular cells after thawing
(RTC : 106 per 100mg of testicular tissue) and the rate of
alive and non apoptotic germ cells (RAA : %) in the different
conditions. Morphological evaluation of seminiferous tubules
was also performed, establishing a scoring (ST) of alterations
affecting tubule architecture, cytoplasm and nuclei of germ cells
and somatic cells (Sertoli and Leydig cells). ST varied from
0 (normal morphology) to 10 (major morphological alterations).
Results : For DMSO and PrOH, a controlled and slow cooling
rate curve offered the best condition of cryopreservation.
However, RGC (58 millions per 100mg for DMSO vs 42
millions for PrOH) was the most satisfying with DMSO in a
controlled and slow cooling rate curve without seeding, the
same result was observed for RAA (89% for DMSO vs 62%
for PrOH). Furthermore, this protocol preserved the architecture
of seminiferous tubules (ST = 0.7). Therefore, severe
alterations of seminiferous tubules were observed in protocol
using PrOH with controlled and quick cooling rate curve (ST
= 8.2). DMSO gave the best results in the different cooling rate
curve tested compared to PrOH.
Conclusion : In the different cooling rate curves, DMSO
proved to maintain, after cryopreservation, not solely immature
testicular tissue architecture but also viability of germ cells and
somatic cells, better than PrOH. Substantial modifications in
the cryopreservation procedure should be performed for
PrOH.
PO 089
Subcellular localization and regulation of type 5
phosphodiesterase in coropra cavernosa cells
centrosomes using human myometrial cells (Dolci, S. et al.
Biochem Biophys Res Commun 341:837, 2006). The
centrosomal localization suggested us to investigate the
possibility that PDE5 levels might be related to cell cycle
activity of SMC.
Materials and Methods : In this study we used cultures of
rat corpora cavernosa (CC) cells obtained modifying the
methods of Krall et al. (Krall, J.F. et al. Biol Reprod 39:913,
1988). RNA and protein extracted from CC cells were utilized
for RT-PCR analysis and for western blot using a specific
PDE5 antibody. Immunohistochemistry were performed to
study the localization of PDE5 in adult CC cells.
Results : In rat CC cells we see the same localization of
PDE5 observed in miometrial cells. Thus we studied the
modulation of PDE5 mRNA and protein in conditions in which
the proliferation was inhibited. Indeed, we observed, after 72
hours of serum starvation, a significant increase of PDE5
mRNA levels in adult rat CC (0,027±0,008 v.s. 0,050±0,010;
p>0,05). This result was confirmed by western blot where, in
the same condition, an increase of about 50% of PDE5
proteins levels was found. A similar result was observed when
the proliferation rate of cells was blocked with the MEK inhibitor
U0126 that has been demonstrated to block the proliferation
of SMC. By using an RT-PCR semi-quantitative assay we
observed an increase of PDE5 mRNA levels from 0,034±0,018
within basal to 0,074±0,026 within U0126 treated cells (p>0,05).
We also show that this 100% increase of PDE5 expression
can be reversed by the addiction of serum in 24 hours. In fact,
a dramatic down regulation of PDE5 mRNA from 0,050±0,010
to 0,0154±0,0037 (p>0,02) has been found when serum was
added to cells previously starved for 72 hours. Platelet-derived
growth factor (PDGF) was demonstrated to promote
proliferation of SMC. We observed a significant down regulation
of PDE5 RNA expression in CC60 cells after 24 hours
treatment with PDGF (1,40x10-1±0,018 v.s. 3,98x10-2±0,015
; p>0,02) and this effect was completely reversed by U0126
treatment.
Conclusion : These results suggest that expression of PDE5
is correlated with the progression of cell cycle and
demonstrated that PDE5 is not only the main regulator of
penile erection but can be also considered a marker of
contractile, non-proliferating phenotype of corpora cavernosa
SMC.
E. CAROSA, S. ROSSI, S. DI SANTE, S. DOLCI1,
A. LENZI2, E.A. JANNINI
Endocrinology and Medical Sexology, University of
L’Aquila; 1Anatomy, II University of Rome “Tor Vergata”;
2Endocrinology, University of Rome “La Sapienza”
(jannini@univaq.it).
Objective : In adult animals, smooth muscle cells (SMC) rest
in a quiescent phenotype and different stimuli trigger the shift
from non-proliferating to proliferative phenotype. Proliferation
and contraction are both regulated by the intracellular second
messenger cAMP and cGMP, which are degraded by
phosphodiesterases (PDEs). The most abundant PDE present
in corpora cavernosa is the erectolytic cGMP specific type 5
PDE, the molecular target of sildenafil. We have previously
demonstrate that PDE5 is expressed prevalently in the
cytoplasm and in particular in spots corresponding to
375
PO 090
Effect of sildenafil administration on penile
hypoxia induced by cavernous neurotomy
in the rat
PO 091
Differential effects of two-week treatment with
atorvastatin of elocalcitol, two RHOA/ROK
signalling modulators, on erectile function and
sildenafil responsiveness in spontaneously
hypertensive rats
L. VIGNOZZI1, A. MORELLI1, S. FILIPPI2,
G.B. VANNELLI3, G. FORTI1, M. MAGGI1
1 Andrology Unit, Department of Clinical Physiopathology,
Interdepartmental ; 2 Laboratory of Functional and Cellular
Pharmacology of Reproduction, Departments of
Pharmacology and Clinical Physiopathology ; 3
Department of Anatomy Histology and Forensic Medicine,
University of Florence, Florence, 50139, Italy
(l.vignozzi@dfc.unifi.it)
Objectives : Radical prostatectomy is an effective therapy for
men with clinically localized prostate cancer. A significant
number of men develop erectile dysfunction after radical
prostatectomy (PPED) due to intraoperatory cavernous nerve
injury causing hypoxia and fibrosis of corpus cavernosus.
We established an experimental model of bilateral cavernous
neurotomy (BCN) in the rat in order to investigate whether
sildenafil treatment in PPED patients could prevent penile
tissue damage.
Design and Methods : One, 5 and 10 days after neurotomy,
animals were treated or not with a single dose of sildenafil
(25mg/kg orally) one hour before sacrifice. To analyze penile
oxygenation, rats of each experimental group received (one
hour before sacrifice) an intraperitoneal injection of the bioreductive drug pimonidazole hydrochloride (hypoxyprobeTM1, 60 mg/Kg), which has been recognized as a standard
marker for in vivo imaging and quantification of hypoxia.
Results : With immunohistochemistry for hypoxiprobeTM,
we found that BCN induced massive hypoxia at all times
investigated in corpora cavernosa sections from the
experimental rats, as revealed by computer-assisted
quantitative image analysis. This tissue hypo-oxygenation
was significantly reduced in sections from sildenafil treated
rats at 1 and 5 days after neurotomy, while at 10 days this
reduction was less evident and not significant. In addition,
functional studies indicated that hypoxic corpora cavernosa
tissues were hypersensitive to the relaxant effect of the
endothelin receptor type B (ETB) agonist IRL-1620, due to the
previously described hypoxia-induced overexpression of ETB
receptors. Accordingly, ETB mRNA expression (real time RTPCR) was significantly increased in corpora cavernosa from
BCN rats, and was restored to control levels by sildenafil
administration at all times investigated.
Conclusions : our results indicate that sildenafil treatment
can positively influence penile tissue oxygenation after
cavernous nerve injury, with its effect being more evident the
earlier it is administered.
376
A. MORELLI1, X.H. ZHANG1, S. FILIPPI2, L. VIGNOZZI1,
L. ADORINI3, M. MAGGI1
1 Andrology Unit, Department of Clinical Physiopathology,
2 Interdepartmental Laboratory of Functional and Cellular
Pharmacology of Reproduction, Departments of
Pharmacology and Clinical Physiopathology, University of
Florence, Florence, Italy. 3 Bioxell, Milan, Italy
(a.morelli@dfc.unifi.it)
Objective : Increased RhoA/Rho-kinase (ROK) signalling is
known to impair erectile function. Since it has been recently
described that spontaneously hypertensive rats (SHR) overexpress penile RhoA and are unresponsive to
phosphodiesterase 5 (PDE5) inhibition (Wilkes N et al. Int J
Impot Res, 2004; 16:187), we tested treatments known to
inhibit RhoA activation, on erectile function and sildenafil
responsiveness in SHR.
Design and Methods : SHR (10-weeks old) have been
treated for two weeks with atorvastatin (5 and 30 mg/Kg/day),
or with elocalcitol (30 mg/Kg/day), a non-hypercalcemic vitamin
D receptor (VDR) agonist. The normotensive Wistar Kyoto
(WKY) rats have been used as controls. Erectile function was
evaluated as frequency-dependent intracavernous
pressure/mean arterial pressure (ICP/MAP) ratio after electrical
stimulation (ES) of the cavernous nerve. Gene expression
analysis was performed by real-time RT-PCR.
Results : at the selected concentrations, neither atorvastatin
affected cholesterol, nor elocalcitol affected calcaemia in both
SHR and WKY rats. In WKY, sildenafil (25 mg/Kg by oral
gavage) greatly increased ICP/MAP ratio after ES of the
cavernous nerve, while in SHR, both basal and sildenafilstimulated ICP/MAP ratio were depressed. Atorvastatin did
not affect basal ICP/MAP at any concentration tested. However,
it dose-dependently increased sildenafil effect on ES-induced
penile erection, which was significantly potentiated by 30
mg/Kg dosing. Morever, at this dose, atorvastatin normalized
the over-expression of RhoA mRNA observed in SHR, without
affecting the expression of other genes such as ROK1, ROK2,
PDE5, nNOS, and eNOS. Conversely, elocalcitol, at a dose
known to ameliorate bladder overactivity by inhibiting RhoA
activation (Morelli et al., Prostate, 2006 in press), failed to
restore depressed ICP/MAP ratio, sildenafil responsiveness
and RhoA overexpression in SHR. Gene expression analysis
showed that SHR rats expressed elevated levels of VDR
mRNA in the bladder (almost 5-fold increase over WKY), but
not in the penis.
Conclusions : our data confirm that erectile function and
sildenafil responsiveness are impaired in SHR because of
an increased RhoA signalling. Atorvastatin, at a dose unable
to affect lipids, ameliorates sildenafil effectiveness and downregulates RhoA expression. Conversely, elocalcitol was
ineffective in restoring erectile function in SHR, either alone
or in combination with sildenafil. The differential quantitative
expression of VDR in bladder and corpora cavernosa suggests
a plausible mechanism for the tissue-specific effect of
elocalcitol on RhoA/ROK contractile pathway.
PO 092
Erectile function after brachytherapy for localised
prostate cancer : long term results
Results : Before treatment, 81 (41%) patients had no erectile
dysfunction (ED) (score 22-25); 53 (27%) had mild (score
17-21), 38 (19%) mild to moderate (score 12-16), 11 (6%)
moderate (score 8-11) and 13 (7%) had severe ED (score 17). Overall, 172 (87%) had a score above 12.
After treatment, 21 (13%) patients had no ED ; 26 (16%) had
mild, 46 (27%) mild to moderate, 24 (14%) moderate and 51
(30%) had severe ED. Overall, 93 (55%) had a score above
12.
Among the 172 patients who had a preimplant score above
12, after treatment 19 men (11%) had ceased to be sexually
active, 34 (20%) remained in the same category as before,
43 (25%) deteriorated by one category, 72 (42%) by two or
more, and 4 (2%) patients improved. The median time to the
onset of ED was 8 months. In multivariate analysis, preimplant
erectile function was the best predictor of brachytherapyrelated EF (p<0.0005).
Conclusions : Most of the population treated by brachytherapy
was sexually active before treatment and had no or mild
preimplant ED. The proportion of patients with no or mild ED
decreased from 87% before to 55% after treatment. The
quality of preimplant erectile function is the best predictor of
post-treatment erectile function.
Support : None.
B. DELAUNAY, D. DELAVIERRE*, M. SOULIE, M.
DELANNES‡, J.M. BACHAUD‡, E. HUYGHE
Service d'Urologie et d'Andrologie, Hôpital de Rangueil,
Toulouse, France * Service d'Urologie et d'Andrologie,
Centre Hospitalier d’Orléans, France ‡ Institut Claudius
Regaud, Toulouse, France boris.delaunay@hotmail.fr
PO 093
Objective : To evaluate erectile function before and after
(125)I brachytherapy for localised prostate cancer using a
validated patient-administered questionnaire (IIEF-5).
Design : Prospective non-randomised study with selfadministered questionnaire.
Materials and Methods : A total of 316 patients with preimplant
erectile function (EF) assessed using the International Index
of Erectile Function (IIEF-5) questionnaire underwent
permanent implantation of (125)I seeds between July 2000
and May 2006 for localised prostate cancer. No patient
received supplemental external beam radiation therapy ; 29%
received adjuvant antiandrogen therapy for up to 4 months.
Of the 316 patients, 210 (67%) completed and returned the
questionnaire. Fourteen patients who were not sexually active
were excluded. Mean age was 66 years (43-80). Mean followup was 4 years (2-6). The questionnaire focused on EF without
PDE5 inhibitors. EF was compared before and after
brachytherapy and potential confounding factors (age at
diagnosis, age of partner, diabetes, hormonal treatment, initial
erectile status, dosimetry parameters) were analysed by
univariate and multivariate analysis.
377
Erectile function after short antiandrogen therapy
for localised prostate cancer in sexually active
men
B. DELAUNAY, J. NOHRA, M. SOULIE, M. DELANNES‡,
J.M. BACHAUD‡, E. HUYGHE
Service d'Urologie et d'Andrologie, Hôpital de Rangueil,
Toulouse, France ‡ Institut Claudius Regaud, Toulouse,
France boris.delaunay@hotmail.fr
Objective : To evaluate the impact of short antiandrogen
therapy on erectile function (EF) for localised prostate cancer
using a validated patient-administered questionnaire (IIEF-5).
Design : Prospective non-randomised study with selfadministered questionnaire.
Materials and Methods : A total of 90 patients, mean age
66 years (43-80), presenting with localised prostate cancer
and candidates for prostatic brachytherapy, received
neoadjuvant antiandrogen therapy for up to 4.2 months (26). This consisted of LHRH agonist in 16%, non-steroidal
antiandrogen (NSA) in 9%, cyproterone acetate in 41% and
agonist + NSA in 34%. Erectile function (EF) before and after
treatment was determined using the International Index of
Erectile Function (IIEF-5) questionnaire. We asked the patients
to describe the quality of their erection without PDE5 inhibitors.
We obtained information regarding their erectile function
before and after treatment in 58 of the 90 patients (64%).
Patients who were not sexually active were excluded (n=4).
men were evaluated through a self-administrated questionnaire
focusing on erectile function (IIEF5) regarding the period
before transplantation (last month before) and after
transplantation (at the date of the survey). Mean age was 53
+/- 12 years (23-79). Duration of hemodialysis before
transplantation was 59 +/- 62 months (6 - 300). Duration of
survey after transplantation was of 103 +/- 72 months (7 - 425).
Immunosuppressant therapy consisted of cyclosporine in 120
patients, tacrolimus in 74 patients, sirolimus in 27 patients and
a combination in the remaining cases. Questionnaire focused
on EF without PDE5 inhibitors.
Results : Before treatment, 20 (37%) patients had no erectile
dysfunction (ED) (score 22-25); 14 (26%) had mild (score
17-21), 13 (24%) mild to moderate (score 12-16), 3 (6%)
moderate (score 8-11) and 4 (7%) had severe ED (score 17). Overall, 47 (87%) had a score above 12.
Results : Before transplantation, 125 (43%) patients had no
erectile dysfunction (ED) (score 22-25); 56 (19%) had mild ED
(score 17-21), 58 (20%) had mild to moderate ED (score
12-16), 19 (7%) had moderate ED (score 8-11), 31 (11%)
severe ED (score 1-7) and 3 (1%) had no erection.
After treatment, 5 (12%) patients had no ED; 5 (12%) had mild,
16 (38%) mild to moderate, 5 (12%) moderate and 11 (26%)
had severe ED. Overall, 26 (61%) had a score above 12.
After transplantation, 134 (46%) patients had no erectile
dysfuntion (ED) (score 22-25); 47 (16%) had mild ED (score
17-21), 48 (16%) had mild to moderate ED (score 12-16), 33
(11%) had moderate ED (score 8-11), 27 (9%) severe ED
(score 1-7) and 3 (1%) had no erection.
Among the 47 patients whom initial score was above 12, after
treatment 10 men (22%) had ceased to be sexually active,
15 (32%) deteriorated by two categories or more, 11 (23%)
by one and only 11 men (23%) remained in the same category
as before.
Conclusions : In most men who were previously sexually
active, short antiandrogen treatment for prostate cancer had a
deleterious effect on EF. One man in five ceased to be sexually
active and one in three had a major deterioration of EF.
Support : None.
PO 094
Prognostic factors of erectile dysfunction in renal
transplanted patients
EF was similar in the 2 groups treated by cyclosporine and
tacrolimus (angioneurine inhibitors), and poorer in the sirolimus
group (anti-proliferative agents) (p=0.01): Proportions of men
having severe ED were 9.2%, 9.4% and 40.7% in the
cyclosporine, tacrolimus and sirolimus, respectively.
EF was correlated with the duration of time elapsed from
transplantation : The proportion of men having severe ED
were 33.8% when the time after transplantation was less than
5 years and 9.4% when it was more than 5 years (p=0.02).
EF was correlated with age (p=0.05), but not with the duration
of hemodialysis treatment.
Conclusion : Prognostic factors of ED in renal transplanted
patients are the age at transplantation(<50y), the time after
transplantation(>5y), and the immunosuppressant therapy
(angioneurin inhibitors >> antiproliferative agents).
PO 095
J. NOHRA, N. KAMAR*, B. BENGOUDIFA, A. ZAIRI, L.
ROSTAING*, E. HUYGHE
Erectile dysfunction in patients with myotonic
dystrophy type 1 : correlation with sexual hormones
Service d’Urologie et d’Andrologie et *service de
Néphrologie et de transplantation d’Organe, Hôpital
Rangueil, Toulouse (nohra.joe@yahoo.com)
A.F. RADICIONI1, A. CLEMENZI2, E. DE MARCO1,
E. CAMA1, G. ANTONINI2, A. LENZI1
Objective : To determine the status of erectile function (EF)
in men with renal insufficiency before and after renal
transplantation, and prognostic factors of erectile dysfunction
in this population.
Design : self-administrated questionnaire.
Materials and Methods : A series of 292 renal transplanted
378
1 Department of Medical Pathophysiology ; 2 Department
of Neurological Sciences, 1st University of Rome “La
Sapienza”, Italy
Objective : Myotonic Dystrophy Type 1 (DM1) is an autosomal
dominant disorder caused by a (CTG)n repeat expansion in
the DM1 protein kinase (DMPK) gene. DMPK gene expression
is pleiotropic and includes premature development of agerelated signs, symptoms and metabolic disturbances including
testicular atrophy (63-82% of patients), hormonal dysfunctions
and erectile dysfunction (ED). We evaluated the frequency and
clinical characteristics of ED in DM1 and its relationship with
sex hormone levels.
Design : We studied 32 consecutive DM1 men (age 22-60
yrs, median 41.5) with CTG expansion 60-1570 (median
287.5) and 32 age-matched healthy subjects.
Materials and Methods : All patients and control subjects were
evaluated with an internationally validated 15-item
questionnaire (International Index of Erectile Function-IIEF).
In 20/32 patients (age 22-60, median 47.5) serum follicle
stimulating hormone (FSH), luteinizing hormone (LH), prolactin
(PRL) and testosterone (T) were measured by solid phase
fluoroimmunometric assay (detection limits: 0.05 U/l for FSH;
0.05 U/l for LH; 0.04 ng/ml for PRL and 0.3 nmol/l for T). The
reference ranges, calculated in 50 age-matched healthy
subjects, were 1.6-8.2 U/l for FSH, 1.2-7.7 U/l for LH, 2.3-12.1
ng/ml for PRL and 9.4-33.5 nmol/l for T.
Results : 23/32 patients and 8/32 controls had ED (p<0.001).
ED in DM1 was severe in 9, moderate in 4, and mild in 10
patients. Patients showed lower scores for erectile function
(p<0.001), orgasmic function (p=0.007), satisfaction with
intercourse (p<0.001) and overall satisfaction (p=0.001). In
the control group, ED was moderate in 1 subject and mild in
7. Sexual desire was similar in both groups. CTG expansion
was not correlated with severity of ED. Age, CTG expansion
and ED severity were similar in patients who underwent sexual
hormone assays (n=20) and those who did not (n=12). LH was
increased in 6/20 patients, FSH in 11/20 and PRL in 2/20
patients and T was reduced in 2/20 patients. Patients with ED
(n=15) showed higher levels of both FSH (p=0.01) and LH
(p<0.01) than patients without ED (n=5). All patients with
pathological values of both FSH and LH had ED. CTG
expansion was directly correlated with both FSH and LH
(p<0.01). Age was not correlated with sex hormone levels. 72%
of patients in our DM1 series displayed symptoms of ED,
though sexual desire was unaffected. Blood levels of FSH and
LH were higher than normal in 55% and 30% of DM1 patients
respectively. High levels of these hormones are correlated with
ED.
Conclusion : These results demonstrate a strong correlation
between DM1, hypergonadotropic hypogonadism and ED.
Previous testicular damage may therefore be the main cause
of the onset of ED, although other mechanisms such as
neurological, metabolic and relational factors cannot be
excluded.
PO 096
Sexual rehabilitation with IC PGE1 injections and
oral drug administration in diabetic patients non
responder at oral therapy only
G. PASSAVANTI*, V. PIZZUTI*, R. PAOLINI*, M.
CARLUCCI**, A.M. ALOISI**
* UO Urologia-Andrologia Ospedale Grosseto (Italy)
** Dpt Physiology Univ.Of Siena (Italy)
G. Passavanti email : mpeppina@infinito.it
Objective : Diabetes is an important risk factor in ED, acting
via several mechanisms.
We assessed the efficacy of IC injections (ICI) rehabilitation
and oral systematic therapy in diabetic patients, as well as the
response of controls to oral therapy ‘on demand’.
Materials and Methods : Sixteen diabetic patients with ED
were treated with vasoactive drugs per os when needed
without good results. The patients were then subjected to ICI
rehabilitation with PGE1 20 mcg twice weekly for 4 weeks,
followed by twice weekly administration of oral drugs for 4
weeks. Before and after rehabilitation, the patients completed
a detailed anamnestic protocol to study their libido (always
present) and they responded to questions Q3 and Q4 of the
IIEF. During ICI, a study with ECCD was carried out.
All patients presented Type 2 diabetes: 10 were treated with
oral antidiabetics, 4 were treated with insulin, and in the other
2 patients, treated with insulin, a sensitive neuropathy of the
lower limbs was diagnosed. Fourteen patients were treated
with antihypertensive drugs.
Results : Before rehabilitation, the mean responses to
questions 3 and 4 of the IIEF were 1.6 and 1.5 respectively;
after rehabilitation, the mean responses were 2.68 and 2.5
respectively.
The ECCD test showed an arterial component in 4 cases
and a high EDV in 14 cases.
Four patients (25%) (2 with neuropathy and 2 with advanced
age) did not respond to PGE1 or to oral therapy, 4 patients
(25%) (2 treated with insulin and 2 oral) responded to ICI but
not to oral therapy, while 8 patients (50%) showed a good
response to both injectable and oral therapy, with good Q3
and Q4 scores.
Conclusions : Good endothelial function appears to be
essential for the maintenance of acceptable erectile function.
Diabetes has a negative effect on this function, as does
hypoxia and low perfusion.
Based on the principle that a good erection improves
endothelial function, we tried to determine if oral systematic
379
and intracavernous rehabilitation would improve erectile
function in diabetic patients. The results indicate that diabetes
interferes with erectile function, compromising the effects of
the vasoactive drugs. However, integrated systematic
rehabilitation appears to allow a good erectile response to both
intracavernous and oral therapy in a large number of cases.
Therefore, we suggest a rehabilitative protocol like this one
in the treatment of ED in diabetic patients.
PO 097
Testosterone and metabolic syndrome :
relationship with erectile dysfunction
M. FERNANDEZ, O. RAJMIL, J.A. ARRUS, A.
CARVAJAL, R. MONTAÑES, A. BLASCO
Hyperlink "mailto:mfcamilo@fundacio-puivert.es"
mfcamilo@fundacio-puivert.es Fundacio Puigvert, Spain
Objective : Assessing whether serum concentrations of
testosterone and metabolic syndrome (MS) are related with
erectile dysfunction (ED).
Design : Prospective study in men consulting for ED in whom
penile rigidity was measured.
Material and Methods : 153 men without local penile
alterations consulting for ED were elected for study. Nocturnal
erections with Rigiscan® were measured and rigidity under
60% as cut off point for ED was considered. NCEP ATPIII
criteria was employed for diagnosing MS. Fasting blood
glucose (FBG), glycosylated hemoglobin (HbA1c), total
cholesterol, HDL Cholesterol, triglycerides, LDL Cholesterol.
Total testosterone (TT), sex hormone binding globulin (SHBG),
free testosterone calculated (TT x 100/SHBG) (FT). Blood
pressure, body mass index (BMI) and waist circumference
(WC) were taken. Univariate logistic regression models were
established for each indicator.
Odds Ratios and its 95% Confidence Interval (CI95%) were
displayed. Multivariate logistic regression models adjusted
for age, BMI, Hb A1c, testosterone determinations and MS
were established.
Results : The following tables gather the results of the study.
Conclusions : MS multiplies four times the risk for ED without
taking into account testosterone. The risk of ED is higher with
low FT in the presence of the MS. Elevated levels of
Testosterone and FT combined with MS resist the individual
effect over ED. When comparing groups TT determinations
380
have no significant differences whereas FT seems to
differentiate those with ED. Results in the present population
indicate that ED is related to the metabolic syndrome and
testosterone concentrations.
PO 098
Androtest : a structured interview for the
screening of hypogonadism patients with sexual
dysfunction
G. CORONA1,3, L. PETRONE1, A. D. FISHER1,
G. BALERCIA2, G. FORTI1, M. MAGGI1
1 Andrology Unit, Department of Clinical Physiopathology,
University of Florence, Florence, Italy ;
2 Endocrinology Unit, Polytechnic University of Marche,
Ancona, Italy ; 3 Endocrinology Unit, Maggiore-Bellaria
Hospital, Bologna, Italy. HYPERLINK
"mailto:jocorona@libero.it" jocorona@libero.it
Objectives : Detecting hypogonadism, important in general
population, becomes crucial in patients with sexual
dysfunctions, because hypogonadism can have a causal role
for them and testosterone (T) substitution represents a
milestone for the therapy. At present, three different inventories
have been developed for screening of hypogonadism. All
these instruments demonstrated a good sensitivity but low
specificity. Furthermore, they were all developed for the
screening of hypogonadism in the aging population, and not
specifically designed for individuals with sexual dysfunction.
Moreover, they are self-reported questionnaires (SRQ). This
case-history tool has demonstrated important limits when
compared to structured interviews (SI) in the evaluation of
patients complaining for sexual dysfunction. Until now, there
are no structured interviews available for the screening of
hypogonadism in patients complaining for sexual dysfunction.
Hence, aim of present study is the definition of a brief structured
interview providing scores useful for detecting hypogonadism
defined as low total T (< 10.4 nmol/L, 300 ng/dL) in a
symptomatic population (sexual dysfunction).
Design : A minimum set of items was identified within a larger
structured interview through iterative ROC curve analysis,
with assessment of sensitivity and specificity for hypogonadism
in a sample of 215 patients (Sample A).
Material and Methods : Sensitivity and specificity were
verified in a further sample of 664 patients (Sample B).
Correlation of test scores with PSA, testis volume, and others
clinical and psychological parameters (Middlesex Hospital
Questionnaire modified MHQ) was assessed for concurrent
validity.
Results : The statistical analysis allowed the identification of
a shorter interview format (12 items), including questions
regarding age, history of delayed puberty, presence of pituitary
diseases, history of cryptorchidism, presence of erectile
dysfunction, reduced morning/nocturnal erections, frequency
of autoerotism, feeling with autoerotism, presence of
381
hypoactive sexual desire, reduction of the quantity of the
volume of ejaculate, presence of mild/moderate delayed
ejaculation, body mass index. The ROC curve analysis for the
final version of the interview showed a sensitivity and specificity
for hypogonadism of 76% and 66%, respectively (accuracy
of 0.738±0.05; p<0.0001) when a threshold of > 8 was chosen.
In the validation sample (Sample B), the final 12-item version
of the interview (ANDROTEST) had a sensitivity and specificity
of 68% and 65% (accuracy of 0.700±0.03; p<0.0001), in
detecting hypogonadism and of 71% and 65% (accuracy of
0.716±0.03; p<0.0001), in the screening for low free T (<37
pmol/l). Furthermore, patients with pathological test (i.e score
> 8) showed higher prevalence of hypogonadism related
signs, such as lower testis volume (19.2±4.3 vs 20.1±3.8 ml,
p<0.005) and higher depressive symptoms (MHQ-D score
6.1±3.1 vs 5.3±2.9, p<0.001). Finally, when younger patients
only (<54 years, which represents the median age of the
sample), were considered, Log10 [PSA] levels were
significantly lower in those with ANDROTEST score >8 (0.23±0.04 ng/ml vs –0.14±0.02 ng/ml, respectively ; p<0.05)
Conclusions : ANDROTEST is a quick, and easy-toadminister interview that provides scores for the screening of
male hypogonadism in patients with sexual dysfunction. The
determination of total and free T remains mandatory to confirm
the diagnosis of hypogonadism.
PO 099
The metabolic syndrome and associated sexual
dysfunction : psychobiological correlates
G. CORONA1,4, L. PETRONE1, C. SCHULMANN2,
G. BALERCIA3, G. FORTI1, M. MAGGI1
1 Andrology Unit, Department of Clinical Physiopathology,
University of Florence, Florence, Italy ; 2 University Clinics
of Brussels, Erasme Hospital, Department of Urology,
Brussels, Belgium ; 3 Endocrinology Unit, Polytechnic
University of Marche, Ancona, Italy ; 4 Endocrinology Unit,
Maggiore-Bellaria Hospital, Bologna, Italy. Hyperlink
"mailto:jocorona@libero.it" jocorona@libero.it
Objectives : The aim of present study is to determine
psychobiological characteristics of sexual dysfunction (SD)
associated with metabolic syndrome (MS) as defined by
National Cholesterol Education Program’s Adult Treatment
Panel III, (NCEP-ATPIII) criteria.
Design : A consecutive series of 803 patients attending for
the first time to our University Outpatient Clinic for sexual
dysfunction was studied.
Materials and Methods : Several hormonal, biochemical
and instrumental (penile doppler ultrasound, PDU) parameters
were studied, along with psychopathology scores. The
Structured Interview on Erectile DYsfunction (SIEDY), was also
applied. This is a 13-item interview composed of three scales,
which identify and quantify components concurring to sexual
dysfunctions. Scale 1 deals with organic disorders, Scale 2
with disturbances in relationship with partner, and Scale 3
with psychological traits.
Results : Among subjects studied, 236 patients (29.4%)
were diagnosed as having a MS. Among them 96.5% reported
ED, 39.6% hypoactive sexual desire, 22.7% premature
ejaculation and 4.8% delayed ejaculation. No significant
difference, after adjustment for age, was observed in the
prevalence of ejaculation disorders or in hypoactive sexual
desire in patients with or without MS. Patients with MS were
characterized by greater subjective (as assessed by SIEDY)
and objective (as assessed by PDU) ED and by greater
somatizated anxiety than the rest of the sample. In particular
SIEDY scale 1 score (organic domain of ED), progressively
increased as a function of number of MS components involved
(B= 0.54±0.05; p<0.0001, after adjustment for age).
Furthermore, basal and dynamic (after PGE-1 stimulation)
peak systolic velocity at PDU showed a progressive decline
as the numbers of MS components increased; this trend was
significant even after adjustment for age (B= -0.79±0.23 and
-2.23±0.69 cm/sec, respectively; both p<0.001). The
prevalence of overt hypogonadism (total testosterone <8 nM)
was significantly higher in patients with MS. Circulating total
testosterone decreased as the number of MS components
increased (B=-1.35±0.182 nmol/l ; p<0.0001, after adjustment
for age). Accordingly, the relative risk for hypogonadism was
significantly higher in patients reporting 3 or more risk factors
for MS. Among MS components, waist circumference and
hyperglycaemia were the best predictors of hypogonadism
(OR=2.5[1.3-4.8] and 2.2[1.3-4.4] respectively). Hypogonadal
patients with MS showed higher gonadotropins (5.8[3.5-11.9]
vs 3.9[2.7-5.3] and 12.1[4.3-24.8] vs 5.1[3.4-8.3] U/l for LH
and FSH respectively, both p<0.005) and lower freetestosterone levels, 24.2±11.9 vs 36.9±14 pmol/l, p<0.0001)
suggesting a primary hypogonadism. Among patients with
MS, hypogonadism was present in 11.9% and 3.8% in the rest
of the sample (p<0.0001) and it was associated with typical
hypogonadism-related symptoms, such as hypoactive sexual
desire (66.7% vs 33.3% respectively in hypogonadal and non
hypogonadal patients with MS, p<0.0001), low frequency of
sexual intercourses and depressive symptoms.
Conclusions : Our data suggest that MS is associated with
a more severe ED and induces somatization. Furthermore,
MS is associated with a higher prevalence of hypogonadism
in patients with SD. The presence of hypogonadism can
further exacerbate the MS-associated sexual dysfunction,
adding the typical hypogonadism-related symptoms.
382
PO 100
A comparison of NCEP-ATPIII and IDF metabolic
syndrome definitions with relation to metabolic
syndrome associated sexual dysfunction
G. CORONA1,4, L. PETRONE1, C. SCHULMANN2,
G. BALERCIA3, G. FORTI1, M. MAGGI1
1 Andrology Unit, Department of Clinical Physiopathology,
University of Florence, Florence, Italy ; 2 University Clinics
of Brussels, Erasme Hospital, Department of Urology,
Brussels, Belgium ; 3 Endocrinology Unit, Polytechnic
University of Marche, Ancona, Italy ; 4 Endocrinology Unit,
Maggiore-Bellaria Hospital, Bologna, Italy. Hyperlink
"mailto : jocorona@libero.it" jocorona@libero.it
Objectives : The aim of present study was to verify possible
differences in the prevalence of vasculogenic erectile
dysfunction (ED) and hypogonadism comparing two distinct
new definitions of MetS, as National Cholesterol Education
Program-Third Adult Treatment Panel (NCEP-ATPIII) and
International diabetes Federation (IDF) in patients with sexual
dysfunction.
Design : A consecutive series of 1086 patients attending for
the first time to our University Outpatient Clinic for sexual
dysfunction was studied.
Materials and Methods : Several hormonal, biochemical
and instrumental (penile doppler ultrasound) parameters were
studied. ANDROTEST Structured Interview was also applied.
This a 12-item, recently validated, inventories, which assesses
the degree of androgenization in male.
Results : The prevalence of metabolic syndrome was 32.0%
and 44.7% according to NCEP-ATPIII and IDF criteria,
respectively. Among patients with ED, MetS showed a
significant correlation with PGE-1 stimulated penile flow
(Vpmax) using both definitions (r=-0.187 and -0.123, for
NCEP-ATPIII and IDF criteria, respectively ; all p<0.0001). At
multivariate analysis, after adjustment for age, LDL-cholesterol
and smoking status, only NCEP-ATPIII was significantly
associated with Vpmax (B=-7.7±3.8; p<0.05). At logistic
regression analysis, among NCEP-ATPIII components,
hypertension, elevated glycaemia and hypertriglyceridaemia
were significantly correlated with impaired Vpmax (< 30
cm/sec). Patients with MetS defined according to both criteria
reported lower total (13.6±6.0 vs 17.4±7.2 nmol/l and 14.7±7.4
vs 18.2±6.0 nmol/l,) and free testosterone levels (34.8±14.0
vs 40.8±13.7 pmol/l and 36.2±14.1 vs 42.5±13.5 pmol/l),
higher prevalence of hypogonadism (34.3 vs 11.9% and 25.3
vs 8.7%), and higher ANDROTEST score (9.6±3.0 vs 7.2±3.6
and 9.2±3.2 vs 6.0±3.2) respectively for NCEP-ATPIII and
IDF ; all p<0.0001. To provide further information on possible
differences between definitions, multivariate regression
analysis incorporating the five components of MetS were
used to identify which component from each definition was
independently associated with hypogonadism. These analyses
identified that waist circumference, hyperglycemia and
hypertriglyceridaemia were significantly associated with
hypogonadism for all definitions. Incorporating into the same
regression analysis both MetS criteria as putative predictors
of hypogonadism, after adjustment for age and smoking habit,
both NCEP-ATPIII and IDF were significantly associated with
low testosterone (OR=2.55[1.03-6.33] and 1.62[1.05-2.50]
for NCEP-ATPIII and IDF, respectively ; all p<0.05). In order
to compare NCEP-ATPIII and IDF criteria for MetS, the
prevalence of hypogonadism was assessed in patients fulfilling
either one or both sets of criteria. When IDF, but not NCEPATPIII, criteria were fulfilled, the prevalence of hypogonadism
was significantly lower than that observed in patients fulfilling
both criteria (15.6 vs 34.8% respectively ; p<0.0001).
Conversely, those fulfilling NCEP-ATP-III, but not IDF, criteria
did not show a significant different prevalence of hypogonadism
than those positive for both sets of criteria (30.8 vs 34.8% ;
p=NS).
Conclusions : In patients with ED, NCEP-ATPII criteria seem
to be a better predictor of hypogonadism and impaired penile
blood flow than IDF.
PO 101
Assessment of the relational factor in male
patients consulting for sexual dysfunction : the
concept of couple sexual dysfunction
G. CORONA1,2, L. PETRONE1, F. LOTTI1,
A.D. FISHER1, G. FORTI1, M. MAGGI1
Andrology 1Unit, Department of Clinical Physiopathology,
University of Florence, Florence, Italy; Endocrinology
2Unit, Maggiore-Bellaria Hospital, Bologna, Italy.
HYPERLINK "mailto : jocorona@libero.it"
jocorona@libero.it
Objectives : To date it is not clear to which extent a clinical,
or even a subclinical, sexual dysfunction in the female partner
might associate with erectile dysfunction (ED) in the male
partner. The present study is aimed at the assessment of
clinical features of ED associated with relational disturbances.
Design : A consecutive series of 1140 patients attending for
the first time the Outpatient Clinic for ED sexual problems of
the Andrology Unit of the University of Florence at Careggi
Hospital and reporting a stable (more than 3 months) couple
383
relationship was studied.
Methods : We evaluated the impact of relational factors, as
assessed by SIEDY Scale 2 (exploring, as reported by the
patient, menopausal symptoms, partner’s medical illness
interfering with sexual activity and reduced partner desire
and climax). SIEDY is an easy to administer instrument for
the first screening of ED patient, providing scores for the
relational component (Scale 2) besides those to quantity the
organic (Scale 1) and intrapsychic (Scale 3) components.
Several hormonal, biochemical and instrumental parameters
were also studied, along with psychopathology scores
(Middlesex Hospital Questionnaire modified MHQ).
Results : The presence of a severe ED (inability to obtain an
erection sufficient for intercourse in >75% of occasions) was
significantly (p<0.0001) associated with Scale 1 (r=0.376)
Scale 2 (r=0.200) and Scale 3 (r=0.172) score. The significant
association between SIEDY Scale 2 and ED was confirmed
at logistic multivariate analysis after adjustment for other
SIEDY Scales and patient’s and partner’s age; in fact, the
chance of being affected by severe ED increased by 10 [110] % for each increment of SIEDY Scale 2 score (p<0.05).
Considering other sexual dysfunctions, we found that SIEDY
Scale 2 score was higher in patients with mild to moderate
delayed ejaculation (MMDE) when compared with patients
without DE (2.97±0.51 vs. 1.97±0.07; p<0.01), while no
difference was observed between patient with or without
premature ejaculation. SIEDY Scale 2 scores are associated
with an advanced age of the partner and a long couple
relationship (>10 years), independently from patient’s age. In
addition, an increased relational factor significantly (p<0.0001)
correlates with increased extra-marital affairs (r=0.111),
conflicts in the couple (r=0.279), alcohol abuse (r=0.155) and
presence of depressive symptoms (r=0.182), as assessed
by MHQ questionnaire. The association between depressive
symptoms and SIEDY Scale 2 was confirmed at multivariate
regression analysis after adjustment for psychiatric diseases,
partner’s age, and the sum of MHQ score (Adj. r for MHQD= 0.253; p<0.0001). Furthermore, SIEDY Scale 2 score
increases as function of the reduction in the frequency of
sexual intercourses and in patients reporting a progressively
severe hypoactive sexual desire even after adjustment for
partner’s age and depressive symptoms. Finally, SIEDY Scale
2 did not shown any correlation with pharmacological,
biochemical, hormonal and instrumental parameters assessed.
Conclusions : Our result should encourage the andrologist
to consider the context in which the sexual symptom develops,
analysing the relationship and partner’s behaviour and
diseases. Resolving, or at least ameliorating, the relational
background and the sexual framework might help in treating
male sexual dysfunction.
PO 102
Psycho-biological correlates of free-floating
anxiety symptoms in male patients with sexual
dysfunctions
G. CORONA1,3, L. PETRONE1 , G. BALERCIA2,
F. LOTTI1, G. FORTI1, M. MAGGI1
Andrology Unit1, University of Florence, Florence Italy;
Endocrinology Unit2 Polytechnic University of Marche,
Ancona, Italy; Endocrinology Unit3 Maggiore-Bellaria
Hospital Bologna, Italy. HYPERLINK
"mailto:jocorona@libero.it" jocorona@libero.it
Objectives : Anxiety has a relevant impact on everyday life,
including sexual life, and therefore is considered the final
common pathway by which social, psychological and biological
stressors negatively affect sexual functioning. The aim of this
study is to define the psycho-biological correlates of freefloating anxiety in a large sample of patients complaining
erectile dysfunction (ED) based sexual problems.
Design : A consecutive series of 882 patients attending for
the first time to our University Outpatient Clinic for sexual
dysfunction was studied.
Material and Methods : Patients were interviewed prior to
the beginning of any treatment, and before any specific
diagnostic procedures, using the SIEDY Structured Interview.
This is a 13 items structured interview, composed of three
scales which identify and quantify organic, relational and
intrapsychic domains. Metabolic and hormonal parameters,
nocturnal penile tumescence test and penile doppler ultrasound
(PDU) examination were also performed. MHQ-A scoring
from Middlesex Hospital Questionnaire (MHQ) was used as
putative marker of free-floating anxiety symptoms (AS). All
statistical association among different parameters and MHQA scores were adjusted for total MHQ score, in order to
discriminate the specific effect of free floating anxiety from
generic psychological disturbances.
Results : At univariate analysis, MHQ-A score was significantly
higher in patients complaining difficulties in maintaining erection
in more than 25% of attempts when compared to rest of the
sample (6.5±3.3 vs 5.8±3.3 ; p<0.001). When we considered
other sexual dysfunctions, patients reporting hypoactive sexual
desire and premature ejaculation showed significantly higher
MHQ-A score when compared to the rest of the sample
(6.9±3.3 vs 5.8±3.2 and 6.6±3.3 vs 6.1±3.3 respectively ;
both p<0.05). No correlation was observed between SIEDY
Scale 1 (organic component of ED) and free-floating anxiety
symptoms. Conversely, MHQ-A score was significantly and
positively related to both SIEDY Scale 2 (relational component
of ED ; r= 0.101 ; p<0.05) and SIEDY Scale 3 scores
384
(intrapsychic component of ED ; r= 0.323 ; p<0.0001). In
particular considering relational factors, symptoms of free
floating anxiety were significantly (p<0.005) correlated with
the presence of couple’s (r=0.111) or family’s conflicts
(r=0.141). Furthermore, a higher MHQ-A score was observed
in patients reporting in their partner a low climax or low sexual
desire when compared to the rest of the sample (6.5±3.2 vs
5.9±3.3 and 6.6±3.2 vs 5.9±3.3 respectively ; both p<0.05).
Looking at intrapsychic factors MHQ-A score was higher in
patients reporting low satisfaction and higher stress at work
when compared to the rest of the sample (6.8±3.3 vs 5.4±3.2
and 6.8±3.4 vs 5.5±3.1 respectively ; both p<0.0001).
Furthermore, symptoms of free floating anxiety were
significantly (p<0.05) correlated with cigarette (r=0.108) and
cannabis (r=0.072) smoking. Finally, among physical,
biochemical or instrumental parameters tested, only enddiastolic velocity at PDU was significantly (p<0.05) higher in
the 2nd versus the 1st quartile of MHQ-A (3[0-5] vs 0[-1-3.8]
cm/sec; p<0.05), while the 3rd and 4th quartiles of MHQ-A
did not show any further significant increase over the 2nd
quartile.
Conclusions : In patients with ED based sexual problems,
AS are correlated to many relational and life stressors.
Conversely, organic problems are not necessarily associated
with MHQ-A score.
PO 103
Psychological distress and erectile dysfunction
R. MIHALCA1, M. BOCCHIO1, F. PELLICCIONE1,
S. NECOZIONE2, A. ROSSI3, S. FRANCAVILLA1
Chairs of 1 Andrology, 2 Epidemiology, 3 Psychiatry,
University of L’Aquila, Italy- Presenting author e-mail :
radu_mihalca@msn.com
Objective : The prevalence of mental disorders in men with
erectile dysfunction (ED) is poorly known. Aim of this study
was to investigate psychological distress in men with ED and
to analyse whether mental disorders have a different
prevalence in men with ED associated to atherosclerosis
compared to men with ED of “presumed” non-organic origin.
Materials and Methods : The Symptom Check-List-90 (SCL90), a validated multidimensional self-report questionnaire
that scores nine primary symptom dimensions to identify
psychological distress in outpatient psychiatric and nonpsychiatric care settings, was applied to 250 men with ED (30
to 77 yrs). Intima-media thickness (IMT) of common carotid
arteries, estimated the degree of generalized atherosclerosis
by B-mode ultrasound, and pharmacologically stimulated
peak systolic velocity in cavernous arteries estimated
cavernosal perfusion disorders.
Results : 34% of patients showed psychological distress as
defined as the presence of at least 1 of 9-subscale index
higher than 90th percentile. Depression and anxiety were
respectively observed in 8.8% and in 9.6% of cases. Body
mass index (BMI) positively correlated with each of 9
dimensions, while a negative correlation was found between
the severity of ED (SHIM test) and anxiety (p=0.010) even after
controlling for BMI. No differences were found between men
with or without vascular risk factors in the mean value of the
9 subscale scores although men with atherosclerosis
(IMT≥0.09 mm) showed an increased number of cases with
scores >90th percentiles in all subscales ; in particular
atherosclerosis was associated to an increased risk of
obsessive-compulsive disorders (OR 3.18, CL 1.32 to 7.61)
after controlling for age, BMI, testosterone level.
Conclusion : One third of patients with ED showed
psychological distress. This seemed to be more prevalent in
men with vasculogenic ED thus confirming the association
between clinical atherosclerosis and mental disorders. At
variance with cardiac and cerebral vascular disease,
vasculogenic ED showed an increased prevalence of
obsessive-compulsive disorders, which relevance for the
efficacy of pharmacologic control of ED needs to be
determined.
PO 104
Psycho-biological correlates of delayed
ejaculation in male patients with sexual
dysfunctions
G. CORONA1,3, L. PETRONE1, A.D. FISHER1,
G. BALERCIA2, G. FORTI1, M. MAGGI1
1 Andrology Unit, Department of Clinical Physiopathology,
University of Florence, Italy ; 2 Endocrinology Unit,
Polytechnic University of Marche, Ancona, Italy ; 3
Endocrinology Unit Maggiore-Bellaria Hospital, Bologna,
Italy HYPERLINK "mailto:jocorona@libero.it"
jocorona@libero.it
Objectives : Pathogenesis of delayed ejaculation (DE) is
rather unknown, although the contribution of various
psychological, marital, hormonal and neurological factors has
been advocated. The aim of the present study is to investigate
385
on the psychobiological factors associated with DE in a large
sample of patients referring to an Andrology Clinic for sexual
dysfunction.
Design : We systematically investigated the relative relevance
of the aforementioned factors in 1632 men, seeking medical
help for sexual dysfunction.
Material and Methods : Patients were interviewed prior to
the beginning of any treatment, and before any specific
diagnostic procedures, using the SIEDY Structured Interview.
This is a 13-item interview composed of three scales, which
identify and quantify components concurring to sexual
dysfunctions. Scale 1 deals with organic disorders, Scale 2
with disturbances in relationship with partner, and Scale 3
with psychological traits. The severity of DE was classified
according to Kaplan criteria on a 3-point scale using a standard
question “In the last three months is it difficult to ejaculate during
sexual intercourse?” and rating : 0 (no DE) ; 1 (mild/moderate
DE or MMDE) and 2 (anejaculation/severe DE or ASDE).
MMDE was diagnosed if ejaculation and climax were still
possible, but only with great effort and after prolonged
intercourse (mild DE) or possible only with autoerotism,
although in the presence of the partner, but not during coitus
(moderate DE). ASDE was diagnosed if orgasm and
ejaculation could not be obtained at all (anejaculation) or
could be obtained but only with autoerotism conducted in the
absence of the partner (severe DE).
Results : Among the 1632 patients studied, 82 (5.0%) reported
DE ; of those, 62 reported MMDE and 20 ASDE. Mild and
moderate forms of DE (MMDE) recognized different risk
factors than the most severe ones (anejaculation/severe DE;
ASDE). ASDE was essentially coupled to the presence of
neurological diseases (OR= 10.1[4.1-24.8]) or to the use of
serotoninergic drugs (OR=4.1[1.2-14.7]). Serotoninergic drugs
also significantly increase (OR=11.0[5.8-21.2]) the risk for
MMDE, which, however was also coupled to other relational
(impaired partner’s climax, patient’s hypoactive sexual desire,
HSD ; OR=2.7[1.4-5.0], 3.4[2.0-5.7] respectively) or intrapsychic (stress at work, OR=1.9[1.9-9.5]) factors. At multiple
regression analysis, some organic pathological conditions
(such as psychiatric disorders and hypogonadism) were also
associated to MMDE (OR=3.3[1.7-6.6] and 2.4[1.3-4.4]
respectively). In particular, hypogonadism (total testosterone
< 10.4 nmol/l) retained significance for DE even after
adjustment for HSD (Adj. OR= 2.08[1.11-3.89] ; p<0.05),
suggesting other effects of testosterone deficiency on the
ejaculatory reflex, besides reduced libido. Finally, patients
with MMDE showed higher SIEDY scale 2 and 3 scores when
compared with the rest of the sample (2.8±0.5 vs 1.9±0.1 for
scale 2 and, 5.9±0.3 vs 5.2±0.1 for scale 3, both p<0.05)
while no difference was observed in scale 1 score (4.2±0.4
vs 3.7±0.1 p=NS). On the other hand, patients with ASDE did
not show any significant difference in SIEDY scores in
comparison with the rest of the sample.
Conclusions : The present study demonstrates that multiple
psychobiological determinants are associated to DE, a still
obscure condition that substantially impairs psychosexual
equilibrium of the couple.
PO 105
PO 106
Sildenafil citrate and clomipramine versus
clomipramine alone in the treatment of premature
ejaculation
First report on ejaculation after brachytherapy for
localised proatate cancer
D. DELAVIERRE, B. DELAUNAY*, J. NOHRA*,
M. SOULIE*, J.M. BACHAUD‡, E. HUYGHE*
A. KHEIROLLAHI, M. BEIRANVANDI, M. BASHASHATI,
M.J. TARRAHI
Service d'Urologie et d'Andrologie, Centre Hospitalier
d’Orléans, France *Service d'Urologie et d'Andrologie,
Hôpital de Rangueil, Toulouse, France ‡ Institut Claudius
Regaud, Toulouse, France
Urology Department, Shohadaye-Ashayer Hospital,
Loreatan University of medical Sciences, Lorestan, Iran
Email : kheirollahi_ar@yahoo.com
Objective : Premature ejaculation (PE) is the most common
ejaculatory dysfunction. We compared the efficacy of sildenafil
citrate and clomipramine combination with clomipramine alone
to increase the time to ejaculation, and improvement of partner
satisfaction.
Objective : To evaluate ejaculation before and after (125)I
brachytherapy for localized prostate cancer using a selfadministered questionnaire.
Design : A randomized open-label clinical trial.
Materials and Methods : A total of 316 patients underwent
permanent implantation of (125)I seeds between July 2000
and May 2006 for localised prostate cancer. No patient
received supplemental external beam radiation therapy ; 29%
received adjuvant antiandrogen therapy for up to 4 months.
Of the 316 patients, 210 answered the questions about erection
and 198 (63%) completed the items concerning ejaculation.
Fourteen patients who were not sexually active were excluded.
Mean age was 66 years (43-80). Mean follow-up was 4 years
(2-6). The questionnaire focused on erectile function and
ejaculation.
Materials and Methods : A total of 80 patients with erectile
dysfunction were treated in a randomized, parallel-group
study conducted at outpatient clinic of Shohada-e-Ashayer
hospital in Khorram-Abad city of Lorestan province, the west
of Iran. Forty patients received clomipramine 25mg/po/day
[group A]. The latter group received 25 mg of oral clomipramine
per day, and 50 mg of oral sildenafile 60 minutes before each
intercourse [group B]. Each patient received drugs for 3
months. Intravaginal ejaculatory latency time (IVELT) and
rate of partners sexual satisfaction (scored: 0-10) were
recorded at the beginning and the end of the study.
Results : Mean IVELT at the beginning of the study was 2
±1.1 minutes and 2.6 ± 1.55 minutes in groups A and B
respectively (P=ns). After 3 months mean IVELT increased
to 3.42 ± 1.8 minutes, and 5.45± 1.4 in groups A and B
respectively. IVELT increasing was significantly higher for
those receiving sildenafile and clomipramine combination
(P=0.001). Partners of 8 patients in group A (20%), and 11
patients in group B (27.5%) satisfied of their intercourse at the
beginning (P=ns). At the end of the study, this rate changed
to 42.5% (17 partners) and 92.5 %( 37 partners) respectively
(P=0.001).
Conclusions : Sildenafil in combination with clomipramine
is more effective than clomipramine alone in patients with
premature ejaculation.
Design : Prospective non-randomised study with selfadministered questionnaire.
Results : Before treatment, 172 (87%) had no or mild erectile
dysfunction and 179 (90%) had ejaculation. Nine patients
described anomalies of ejaculation: pain in 1 case,
haemospermia in 2, light or fluid semen in 2, clotted semen
in 4 cases.
After treatment, 93 (55%) had no or mild erectile dysfunction
and 138 (70%) conserved ejaculation. However, 120 (87%)
reported a decrease in semen volume. Thirty-four patients
described anomalies of ejaculation : pain in 13 cases (9%),
haemospermia in 12 (9%), light or fluid semen in 5 (4%),
clotted semen in 4 cases (3%). Median time to appearance
of anomalies of ejaculation was 4 months (2-12).
Conclusions : This study is the first to focus on ejaculation
after prostate brachytherapy. It reveals that ejaculation is
conserved in more than two-thirds of patients, even if they
described changes of volume and consistency.
Support : None.
386
PO 107
Altered endocytotic pathways of epidermal
growth factor receptor in androgen receptor
positive prostate cancer cell lines
L. BONACCORSI, D. NOSI1, M. MURATORI, G. FORTI,
E. BALDI
route. These data highlight the role of AR in regulation of
EGFR endocytosis and signalling in PC cells. In view of the
role of EGFR signalling in invasion of PC cells, our data may
explain the lower invasive phenotype observed in AR-positive
cell lines.
PO 107 bis
Department of Clinical Physiopathology, Andrology Unit
and 1 Department of Human Anatomy, Histology and
Forensic Medicine*, University of Florence, Viale Pieraccini
6, 50139 Firenze, Italy. (l.bonaccorsi@dfc.unifi.it)
Although androgens and the androgen receptor (AR) are
involved in tumorigenesis of prostate cancer (PC) in initial
phases, less clear is their role in androgen-independent
disease. Recent reports indicate that re-expression of AR in
PC cell lines determines a less aggressive phenotype of the
cells. We have observed that re-expression of AR in the
androgen-independent PC cell line PC3 decreases invasion
of PC3-AR cells in vitro by interfering with signalling and
internalization of EGF receptor (EGFR).
Here we show that a reduced EGFR internalization (evaluated
both by immunofluorescence and flow cytometry techniques)
is also characteristic of AR positive PC cell lines LNCaP and
22Rv1. Our data demonstrate that the reduced EGFR
internalization in PC3-AR cells is due to an alteration of the
ability of the receptor to interact with 2 adaptor proteins that
play a key role in EGFR endocytotic signalling, namely Grb2
and c-Cbl. Indeed, by immunoprecipitation studies we found
that Grb2 and c-Cbl association with EGFR were highly
reduced in PC3-AR cells respect to the parental cell line. As
consequence of such reduced interaction, ubiquitination of the
receptor, which is mediated by the ubiquitin ligase c-Cbl, was
also found altered in PC3-AR cells.
In addition, we report evidence that expression of AR
determines a shift of internalization pathway from the clathrincoated pit one (which support full signalling and recycling of
the receptor) to the raft-mediated one (which is mainly involved
in lysosomal degradation of the receptor). Indeed, while
following internalization EGFR clearly co-localizes with early
endosome antigen-1, a marker of clathrin-coated pit pathway,
in PC3-Neo cells, such a co-localization was not observed in
PC3-AR or the other AR positive PC cell lines. Conversely,
when caveolin-1, a marker of raft-mediated endocytosis, was
studied, EGFR maintains co-localization with caveolin-1 after
EGF in PC3-AR cells but not in PC3-Neo.
We suggest that AR expression affects EGFR clathrinmediated endocytosis pathway, which plays a key role in the
signalling of the receptor, as many signalling pathways are
activated only when the receptor is internalized through this
387
Study of gene espression of recurrent fusion
between the androgen-responsive gene
TMPRSS2 and ets transcription factor erg in
prostate cancer
L. BONACCORSI1, F. NUTI1, C. KRAUSZ1, G. FORTI1,
S. SERNI2, EL. BALDI1
Department of Clinical Physiopathology, 1Andrology Unit
and, 2Department of Critical Care Medicine and Surgery,
University of Florence, Firenze, Italy
(l.bonaccorsi@dfc.unifi.it)
Prostate cancer (PC) is the second most commonly diagnosed
malignancy in American men and displays different clinical
behaviours from indolent to metastatic disease. It is not clear
which molecular events are responsible for the progression
of PC to a lethal form. Genes involved in carcinogenesis have
often been identified through analysis of recurrent
chromosomal rearrangements but they have not been identified
in carcinomas until recently, when Tomlins et al (2005) applied
bioinformatics techniques to identify oncogenic chromosomal
changes based on analysis of outlier gene expression.
The authors determined that two ETS transcription factors,
ERG and ETV1, were outliers in PC. They show recurrent
fusions of the 5’ untranslated region of the androgen-dependent
TMPRSS2 gene to ERG and ETV1 genes in the majority of
PC samples containing the outlier expression. We evaluated
the occurrence of the chromosomal rearrangement by profiling
gene expression of the above mentioned translocation on
chromosome 21 in 50 primary PC samples as well as in the
surrounding normal prostate tissue. To identify TMPRSS2:ERG
and TMPRSS2:ETV1 fusions we performed both standard
Reverse-transcription PCR (RT-PCR) followed by
electrophoresis of the products and quantitative PCR (QPCR)
by using SYBR Green dye on a Real Time PCR system.
Freshly frozen prostate surgical specimens were obtained
through radical prostatectomy from Urological Surgery in the
years 1998-2000 and carcinoma tissue was identified, excised
and immediately frozen in liquid nitrogen. Clinical data and
follow-up of patients are available for most of the subjects.
Our data confirm that this fusion gene product may play a key
role in the development, diagnosis, and treatment of PC.
0.001) than the control. Significant reduction (p <0.001) in
serum vitamin C levels were also observed in patients with
mPSA of 15.9 µg/L to 73.8 µg/L when compared to the control.
Precisely, serum vitamin C levels were decreased by 32% and
47% in patients with mPSA of 15.9 µg/L and 73.8 µg/L,
respectively. The extent of lipid peroxidation (LPO) in the
sera of patients was estimated by measuring the thiobarbituric
acid reacting substances (TBARs) formed. Serum LPO was
significantly elevated (p < 0.001) in patients with mPSA of 6.5
µg/L to 73.8 µg/L when compared to the control. Specifically,
LPO was elevated by 28%, 35% and 46% in patients with
mPSA of 6.5, 15.9 and 73.8 µg/L, respectively. Furthermore,
serum selenium levels were decreased by 35%, 34% and
38% in patients with mPSA of 6.5, 15.9 and 73.8 µg/L,
respectively.
PO 108
Conclusions : These results indicate an inverse relationship
between the non-enzymic antioxidant profile of prostate cancer
patients and their respective mPSA values. This relationship
should not be overlooked and will serve as basis to further
understand the biochemical aspect of the disease.
Our data confirm the high percentage of expression of the
fusion gene in PC. In particular, we found a percentage of
translocation expression of 70% in our cohort of patients.
The translocation was completely absent in the surrounding
normal tissue. In several samples we obtained a pattern of
expression characterized by bands with different molecular
weight. In some PC samples the expected band with
appropriate molecular weight was sequenced. Sequence
analysis confirmed the fusion of the complete exon 1 of
TMPRSS2 with the beginning of exon2 of ERG. No
translocations were found for the ETV gene in our cohort of
patients.
Support : Self-sponsored study.
Relationship between non-enzymic antioxidant
profile and mean prostate specific antigen
(mPSA) levels of known prosate cancer patients
PO 109
O.A. ADARAMOYE1*, O. AKINLOYE2, O.I. KAREEM3
An androgen-dependent PDE5 activity in bladder
might contribute to luts
1 Department of Biochemistry, College of Medicine,
University of Ibadan, Nigeria. 2 Department of Chemical
Pathology, Ladoke Akintola University of Technology,
Osogbo, Nigeria 3 Cancer Screening Unit (CSU),
University College Hospital (UCH), Ibadan, Nigeria.
*aoadaramoye@yahoo.com
S. FILIPPI1, A. MORELLI2, B. FIBBI1, P. SANDNER3,
L. VIGNOZZI1, M. MAGGI2
Objective : Oxidative stress has been implicated in the
etiology of several pathologies, prostate enlargement inclusive.
The present study was designed to relate the non-enzymic
antioxidant levels in prostate cancer patients with their mean
prostate-specific antigen (mPSA) values.
Patients and Methods : Participants were recruited (with
informed consent) from the Cancer Screening Unit (CSU),
University College Hospital (UCH), Ibadan, Nigeria. 120
prostate cancer patients were assigned into 3 groups on the
basis of mPSA values; group 1 with mPSA of 6.5 µg/L, group
2 with mPSA of 15.9 µg/L and group 3 with mPSA of 73.8 µg/L.
Patients had no recent hormone therapy and/or radiation
therapy. Likewise, 120 apparently normal subjects were
recruited as control and had mPSA value of 2.8 µg/L. The study
was approved by Ethical Committees of the UCH and Oyo
State Government of Nigeria.
Results : Patients with mPSA µ 6.5µg/L to 73.8 µg/L had
significantly lower serum uric acid and vitamin E levels (p <
388
1 Interdept.Lab of Functional and Cellular Pharmacology of
Reproduction, Depts. of Pharmacology and Clinical
Physiopathology ; 2 Dept. of Clinical Physiopathology,
Andrology Unit, University of Florence, Florence, Italy ; 3
Bayer HealthCare, Pharma Research EU, Wuppertal,
Germany (sandra.filippi@unifi.it)
Objective : BPH is the most common disease in male aging,
often associated to Erectile Dysfunction (ED). PDE5 inhibitors
(PDE5i) ameliorates LUTS in patient with ED and BPH. We
therefore studied their action in the rat and human bladder.
Design and method : We studied PDE5 expression and
activity in human bladder and effects of PDE5i using in vitro
(human and rat) and in vivo (rat bladder outlet obstruction,
BOO) models.
Results : In bladder tissues, we found high PDE5 mRNA
expression and an intense immunolocalization of PDE5 protein
in the vascular endothelium and smooth muscle cells. PDE5i
vardenafil, sildenafil and tadalafil, blocked 70% of the total
cGMP catabolizing activity in human bladder homogenates.
Vardenafil exhibited the highest IC50 (0.3 nM). In male rat,
the NO-donor sodium nitroprusside (SNP) only marginally
relaxed carbachol-precontracted bladder strips, while its
activity was strongly potentiated by vardenafil (100 nM) to
the level of the PDE-resistant cGMP analogue SP-8-Br-PETcGMPS. According to these results, in human bladder stromal
cells the anti-proliferative activity of SNP was strongly
potentiated by vardenafil up to the PDE-resistant cGMP
analogue. We also found that surgical castration in the rat
decreased, and T supplementation restored, PDE5 gene
expression (Real-time RT-PCR) and enzymatic activity.
Accordingly, bladder strips from castrated rats were more
sensitive to SNP induced-relaxation than strips from control
or T-replaced rats, while in presence of vardenafil, all the
groups showed the same SNP sensitivity. To elucidate whether
vardenafil affects in vivo bladder activity, the rat BOO model
was used. Chronic treatment with 10mg/kg/d vardenafil did
not influence bladder hypertrophy but significantly reduced
(47%, p<0.05 vs placebo) non-voiding contractions in the rat
to the level of tamsulosin (51%).
Conclusions : Overall, these results demonstrate that PDE5
in the bladder regulates smooth muscle tone, strongly limiting
the NO/cGMP signalling, and that vardenafil, by blocking
PDE5, might represent a possible therapeutic option for
bladder dysfunction.
PO 110
BXL-628, a vitamin D analog, decreases
RHO/ROK signalling in rat and human bladder
RhoA/ROK signalling activated in OAB. Design and Methods
: In vivo (2weeks; 30 mcg/Kg, Sprague-Dawley, SD, and
spontaneously hypertensive rats, SHR) and in vitro (human
bladder stromal cells, hBC) experiments were carried out to
investigate the BXL-628 effect.
Results : In SD bladder, BXL-628 did not affect maximal
responsiveness to carbachol but increased the lag time to
reach it, and reduced the maximal relaxant effect of the ROK
inhibitor Y-27632. In SHR bladders, that over-express
RhoA/ROK pathway and develop OAB, the Y-27632 effect was
higher than in normotensive control rats Wistar-Kyoto (WKY).
BXL-628 normalized SHR bladder sensitivity to Y-27632,
while did not significantly affect RhoA/ROK expression (qRTPCR) in rat bladder and in hBC. In immunokinase assay BXL628 significantly decreased ROK activity, by reducing Y-27632
effect, of hBC extracts immunoprecipitated with anti-ROK. In
SHR bladders ROK activity was higher than in WKY and
significantly reduced by BXL-628. We therefore investigated
whether BXL-628 impaired RhoA activation in hBC. Confocal
microscopy, using pancadherin (a membrane marker) and
RhoA immuno-labelling, revealed a reduction of RhoA
membrane expression, which indicates its activation state, in
BXL-628 treated hBC. Accordingly, BXL-628 significantly
reduced the activated, rhotekin-bound, RhoA fraction in hBC
(Western blot). Because RhoA is involved in cell motility, we
tested the effect of BXL-628 on hBC migration. BXL-628 even
at 0.01pM inhibited cell migration as other inhibitors of RhoA
(simvastatin, C3 exoenzyme). Calcitriol was effective at higher
concentrations (1nM).
Conclusions : in human and rat bladders, BXL-628 inhibits
RhoA/ROK signalling, suggesting a novel therapeutic
opportunity for OAB and LUTS treatment.
PO 111
The comparison of the three lobes of the rat
prostate in aging process and in
hyperprolactinemia induced by haloperidol
A. MORELLI1, S. FILIPPI2, L. VIGNOZZI1, G. FORTI1,
L. ADORINI3, M. MAGGI1
1 Andrology Unit, Department of Clinical Physiopathology ;
2 Interdepartmental Laboratory of Functional and Cellular
Pharmacology of Reproduction, Departments of
Pharmacology and Clinical Physiopathology, University of
Florence, Florence, Italy ; 3 Bioxell, Milan, Italy.
(a.morelli@dfc.unifi.it)
Objectives : BXL-628 is a non-hypercalcemic calcitriol analog
successfully tested in a phase IIa trial for benign prostate
hyperplasia therapy. Because part of low urinary tract
symptoms (LUTS) are generated by overactive bladder (OAB)
and bladder expresses the calcitriol receptor (VDR), we
investigated the BXL-628 effects on bladder contractility and
389
M. LASZCZYNSKA1, M. WYLOT2, M. PIASECKA2,
A. STARCZEWSKI3, A. BRODOWSKA3
1Laboratory of Embryology, 2Department of Histology and
Embryology, 3Department of Reproductive Medicine and
Gynecology, Pomeranian Medical University, Szczecin,
Poland e-mai l: Hyperlink "mailto :
laszcz@sci.pam.szczecin.pl" laszcz@sci.pam.szczecin.pl
Objective : The aim of the conducted studies was to establish
the influence of hyperprolactinemia on epithelium and stroma
in the three lobes of the rat prostate. We compared changes
in morphology and selected proteins expression in each lobe
of the rat prostate.
Design : Sexually mature 18-month-old male Wistar rats
were randomly divided into two groups: experimental and
control ones. Each of the groups consisted of eight animals.
The rats in the experimental group were intraperitoneally
administered haloperidol (HAL) in a dose of 2.0 mg/kg body
mass for 14 days.
Materials and methods : Immunoezyme method was used
for the measurements of hormones serum concentrations:
prolactin (PRL) (Spi-Bio, France), testosterone (T) (AxSYM
Testosterone test MEIA, USA) and estradiol (E) (DPC Immulite
2000, France). For light microscopic examinations, the slides
of each lobe of the rat prostate were routinely obtained,
prepared and stained with standard H-E and p.a.S. method.
Immunohistochemistry standard ABC techniques were used
to compare expression of prolactin receptor (PRLR) (Affinity
BioReagents, USA), androgen receptor (AR) (Novocastra
Lab, UK) and estrogen receptor β (ERβ) (Affinity BioReagents,
USA). To compare expression of proliferating cell nuclear
antigen (PCNA) and desmin and vimentin filaments, modified
immunohistochemical techniques were used with DAKO
EnVision System HRP (Dako/AS, Denmark).
Results : The rats in the experimental group (administered
HAL) showed about 2 times higher the mean PRL
concentration (50.23 ± 14.15ng/ml vs 24.53 ± 4.07ng/ml) and
increased E (30.38 ±11.30 pg/ml vs 21.02 ±2.28pg/ml),
whereas the mean concentration of T (0.46 ± 0.32 ng/ml vs
0.96 ± 0.31 ng/ml) was decreased in comparison to the mean
concentration in control group. Changes in morphology affected
each lobe of the prostate. Lateral and dorsal lobes revealed
hypertrophy of glandular ducts with dysplasia markers
especially in lateral lobe. Focal hyperplasia in epithelium
suggested digitate outgrowths and multistratified cells clusters,
and was confirmed with raised PCNA expression. In the
stroma of these two lobes, desmin concentrated surrounding
glandular ducts and forming thick sheath especially in dorsal
lobe. Ventral lobe, due to reduced T concentration, revealed
atrophic features – reduced proliferate activities and
smoothening of the glands surface. In the stroma there were
noticed inflammatory infiltrations with focal fibromatosis, and
strong expression of desmin and vimentin filaments especially
in dorsal and lateral lobes as well as changes in the
ultrastructure of the columnar epithelial cells.
Immunohistochemical study revealed expression of AR with
ERβ in the nuclei and expression of PRLR in cytoplasm of
columnar epithelial cells in each lobe of the rat prostate in both
experimental and control groups.
Conclusion : We postulate that prolactin in male rats,
especially in overnormal concentrations, affects the prostate
epithelium and stroma morphology as well as expression of
AR, ERβ and PRLR individually in each lobe of the prostate.
390
PO 112
A evidence based approach to the diagnosis of
late - onset hypogonadism (LOH) in men using
data from the european male ageing study
(EMAS)
A. TAJAR1, J.M. ARNOTT1, F.C.W. WU1, M. LUNT1,
J.D. FINN1, G. BARTFAI2, F. CASANUEVA3, G. FORTI4,
A. GIWERCMAN5, I. HUHTANEIMI6, K. KULA7,
M. PUNAB8, A.J.S. SILMAN1, D. VANDERSCHUEREN9,
AND THE EMAS GROUP
1 University of Manchester (UK) ; 2 Szeged University
(Hungary) ; 3 Universidade de Santiago de Compostela
(Spain) ; 4 University of Florence (Italy) ; 5 Lund University
(Sweden) ; 6 University of Turku (Finland) ; 7 Medical
University of Lodz (Poland) ; 8 Medical University of Tartu
(Estonia) ; 9 Katholieke Universiteit Leuven (Belgium)
Prevalence of ‘hypogonadism’ of ageing has been reported
at between 20-50% in 60-90 year old men using biochemical
thresholds alone. However, recent guidelines recommend
combining clinical features suggestive of androgen deficiency
together with low total testosterone (TT) to furnish a more
rational basis for diagnosis. However, there is no consensus
as to the nature, number or severity of symptoms to identify
these cases of ‘hypogonadsim’ in the ageing male population.
The present study aims to identify the most informative clinical
symptoms associated with testosterone deficiency. Our
analyses were based on data from The European Male Ageing
Study (EMAS), a population-based cohort study of men aged
40 to 79 years from 8 European cities (Manchester in the
United Kingdom, Malmo in Sweden, Tartu in Estonia, Szeged
in Hungary, Lodz in Poland, Leuven in Belgium, Florence in
Italy and Santiago in Spain). All 3369 men completed selfreported symptom questionnaires and had morning total T (TT)
and sex hormone binding globulin (SHBG) measured by
immunoassay, (Roche, Elecys E170) from which free T (FT)
was calculated. From a total of 31 possible symptoms
potentially related to T deficiency, only 5 (2 sexual and 3
physical symptoms) were independently associated with TT
and FT levels.
Using the relationships between these 5 symptoms with TT
and FT, we employed a statistical technique of piecewise
logistic regression and nonparametric regression LOWESS
(The LOcally WEighted Scatter plot Smoothing) to identify
testosterone thresholds below which a significant increase in
risk of developing a deficiency symptom was observed. This
revealed clear threshold levels of TT for the sexual symptoms
but thresholds were not as well-defined for physical symptoms.
FT levels showed less clearcut relationships with symptoms.
What is the Association between Testosterone
and Frailty in Ageing Men ?
respectively. Mean (95%CI) TT was lower in F and PF; 12.2
(11.1,13.4) and 13.9 (13.3,14.4) nmol/L respectively vs. 14.3
(13.7,14.9) nmol/L for NF (age adjusted p value, 0.037). Mean
fT was also lower in F and PF; 0.18 (0.17,0.2) and 0.22
(0.21,0.22) nmol/L respectively vs. 0.23 (0.22,0.24) for NF (age
adjusted p value, <0.001). F and PF men also had higher
levels of FSH and LH (age adjusted p values, 0.001 and
0.029 respectively) compared to NF. An inverse relationship
was observed between TT, fT and the number of frailty
components. In a multinomial logistic regression analysis at
a TT of <12 nmol/L, the age adjusted odds ratio (OR) (95%
CI) for being F was 1.77 (1.07, 2.95) (P = 0.02) when compared
to being NF. The presence of walk time criteria was significantly
associated with a TT <12 nmol/L with an OR (95% CI) of 2.9
(1.6,5.3) (P <0.001). Weight loss criteria was significantly
associated with a TT of <10 nmol/L with an OR (95% CI) of
1.7 (1.01, 2.9) (P=0.04).
U. SRINIVAS-SHANKAR1, J.A. OLDHAM2,
M.J. CONNOLLY1, F.C.W. WU1
Conclusions : Low T levels are associated with frailty and
some of its components (walk time and weight loss). However,
the role of testosterone needs to be assessed longitudinally
before a causal relationship can be attributed to testosterone
in the aetiology of frailty.
Our results highlight different dose-response relationships
between specific symptoms with TT and/or FT. Nevertheless,
we demonstrate that an evidence-based, clinically-pertinent
approach can be applied to establish objective criteria on
which a rational diagnosis of hypogonadism can be made in
symptomatic middle-aged and elderly men. These results
also suggest that the true prevalence of testosterone deficiency
may be considerably lower than previously estimated.
PO 113
1Department of Medicine and Endocrinology, Manchester
Royal Infirmary, United Kingdom 2Centre for Rehabilitation
Science, University of Manchester, United Kingdom.
Correspondence e-mail address:
usrinivas@manchester.ac.uk
Introduction : Frailty is a common cause of disability and
dependency in the elderly. The aetiology of frailty is
multifactorial. However, ageing-associated endocrine
dysregulation may play an important role. It is not known if
low testosterone (T) contributes to frailty, through its effects
on muscle (sarcopenia) and physical function (strength and
endurance).
Objective : To investigate the relationship between T and
frailty in elderly men by examining the associations between
T and various degrees and components of frailty.
Design : Cross-sectional observational study in communitydwelling men, who responded to invitation letters to participate
in a clinical trial of testosterone replacement therapy.
References : 1. Fried L.P., Tangen C.M., Walston J. et al. :
Frailty in older adults : evidence for a phenotype. J. Gerontol.
A Biol. Sci. Med. Sci., 2001, 56 : M146-156.
Support : Schering AG, Berlin.
PO 114
Testosterone Levels Among Octogenarians
U. SRINIVAS-SHANKAR1, J.A. OLDHAM2,
M.J. CONNOLLY1, F.C.W. WU1
1 Department of Medicine and Endocrinology, Manchester
Royal Infirmary, United Kingdom 2 Centre for Rehabilitation
Science, University of Manchester, United Kingdom.
Correspondance e-mail address :
usrinivas@manchester.ac.uk
Methods : One thousand and thirty one, men, median age
72 yr, (range 65-95) were screened for the presence of frailty
using Fried’s criteria1 (weight loss of 10 lbs or more in the past
year, self-reported exhaustion, decreased grip strength, slow
walking speed and reduced physical activity). Frail (F) men
were characterized by the presence of 3 criteria, prefrail (PF)
men by 1-2 criteria and non-frail (NF) men by the absence of
any criteria. Serum samples were obtained before 11.00 a.m
for the measurement of total testosterone (TT), sex hormone
binding globulin (SHBG), follicle stimulating hormone (FSH),
and luteinizing hormone (LH). Free testosterone (fT) was
calculated using the Verumulen formula.
Background : Circulating testosterone (T) levels decline
gradually with increasing age in men. However, the agerelated decline in several functional domains (e.g. frailty,
cognitive deficits, osteoporosis) accelerates in the eighth
decade of life. It is not known if the loss of function in the oldest
old is related to a more precipitous fall in T due to the relative
paucity of hormonal data in extreme old age.
Results : The prevalence of F and PF was 9% and 45 %
Objective : This study investigates the hypothesis that total
391
testosterone (TT) and free testosterone (FT) levels decline
more rapidly in men in the eighth compared to those in the
sixth and seventh decade. We also sought to determine, if
factors influencing T levels among octogenarians are different
from earlier decades.
PO 115
Post- vasectomy complications in Khorramabad
city, the west of Iran
Design : Cross-sectional observational study in communitydwelling men, who responded to invitation letters to participate
in a clinical trial of testosterone replacement therapy for frailty.
Methods : One thousand three hundred and twenty men,
mean age, 73 (range 65-95) years were included in this study.
Medical, drug and smoking histories were obtained by selfreport. Height and weights were measured and body mass
index (BMI) calculated. Serum samples were obtained before
11.00 a.m. for the measurement of total testosterone (TT), sex
hormone binding globulin (SHBG), follicle stimulating hormone
(FSH), and luteinizing hormone (LH) (Roche. Elecys E170).
Free testosterone (FT) was calculated using the Verumulen
formula.
Results : The entire cohort was divided into 4 age bands: B1
(65-69 years, n =510), B2 (70-74 years, n=357), B3 (75-79
years, n=258) and B4 (80-95 years, n=195). There was no
significant difference in TT in B4, compared to other age
bands (13.4 nmol/L vs.13.9, 13.7, 13.9 nmol/L for B1, B2 and
B3 respectively, p=0.63). Over 60% of men in B4 had a TT
>12 nmol/L, not significantly different from other age bands.
FT was significantly lower in B4, than in other age bands
(0.18 nmol/L vs. 0.23, 0.21, 0.20 nmol/L for B1, B2 and B3
respectively, p <0.001). The fall in FT with age was linear; there
was no rapid decline in FT in B4. B4 had significantly higher
SHBG, LH and FSH when compared to other age bands.
There was no significant difference in the prevalence of various
comorbidities among age bands except cerebrovascular
disease, which was higher in B4 (p=0.01). There was also no
difference in the number of comorbidities among various age
bands (mean [SD] 1.53 [1.4], 1.74 [1.40], 1.81[1.4], 1.66 [1.4],
[P=0.051] for B1, B2, B3 and B4 respectively). Men in B4
were more likely to be exsmokers (p<0.05) and less likely to
be current smokers p<0.01). Age, BMI, chronic obstructive
airways disease and cancer were independently associated
with TT (ß = - 0.06, -0.44, - 1.14 and -2.46 respectively) and
FT (ß = –0.003, -0.004 and -.018 respectively) in the entire
cohort. Increasing number of comorbidities were also
associated with falling TT levels (ß = -1.16, -1.17 and –1.80
for 2, 3 and >4 comorbidities respectively). A similar trend
was found for FT, but only in men with 3 or more comorbidities.
Analysis of the four individual age bands revealed that BMI
was associated with TT in all age groups. Chronic obstructive
airways disease was associated with falling TT only in B2
(ß= –1.72, p= 0.04). Cancer was associated with TT (ß =
–4.82 and –4.53) and FT (ß = –0.72 and –0.54) for B3 and
B4 respectively. In all age bands BMI was associated with FT,
except B4, where age, not BMI was significant (ß = –0.005).
Conclusions : TT does not fall at a faster rate in men in the
eighth decade and older. Factors associated with lower TT
and FT do not differ in octogenarians compared to younger
men.
Support : Schering AG, Berlin.
392
A. KHEIROLLAHI1, S. AHMADIPOOR1,
M. BASHASHATI1, M.J. TARRAHI1
Urology Department, Shohadaye-Ashayer Hospital,
Loreatan University of medical Sciences, Lorestan, Iran
Email : kheirollahi_ar@yahoo.com
Objective : Vasectomy as a permanent form of birth control
is the safest and easiest form of surgical sterilization, but
studies on complications which may arise from this procedure
are not enough reproduced. In this study we assessed the
frequency of some late complications of vasectomy in a
sample of Iranian patients.
Design : A cross sectional study.
Materials and Methods : At August 2004, invitation letter
were posted to 324 men whom had underwent vasectomy in
the health service center number III of Khorram-Abad city, in
western Iran between March 1998 and September 2002. We
asked them to come to urology clinic of the University for
Follow-up and checkup. Data including their libido, impotency,
ejaculation disorders, psycho-somatic and sleep disorders,
chronic orchalgia, granuloma formation, joint pain, procedure
failure, and surgical regretting were gathered.
Results : Within 6 months, 110 of invited men came to the
clinic. 40% of the patients had libido variations. 14.5% reported
impotency which occurred after the procedure. 10.9% had
premature ejaculation. Mood and sleep disorders were
detected in 10% and 4.5% of the patients respectively. 20%,
14.5%, and 9.8% of the patients showed chronic orchalgia,
granuloma formation, and joint pain respectively. 2.7% of the
patients regret vasectomy. There was no failure after clearance
in operated patients.
Conclusions : In comparison with studies from other regions,
chronic orchalgia and psychiatric problems were more common
in our patients. Whether it relates to socio-culture status or
vasectomy procedure needs to be assessed.
PO 116
Female Genital Mutilations and sexual troubles
S.M. GUEYE, L. NIANG, M. NDOYE, J.J. DIAW, I.
LABOU, M. JALLOH
Department of Urology and Andrology, Univerity Cheikh
Anta Diop and Grand Yoff General Hospital, PO Box 6039
Dakar-Etoile, DAKAR – SENEGAL smgueye@refer.sn
Objectives : The goal of this study was to describe female
genital mutilations and to assess their impact in sexual life
among a population of non selected women.
Patients and Methods : We underwent a prospective study
among a population of women randomly selected. All these
women filled a questionnaire. Therefore we realized a casecontrol study including 176 women victims of female genital
mutilations whom we compared to 352 women without a
history of genital mutilation.
We excluded of this study pregnant women, not healthy
women and women younger than 18 years.
The data studied were: sexual desire, sexual arousal, sexual
pleasure, dyspareunia and sexual satisfaction.
For the statistical analysis, we underwent simple and cross
tabulations. Chi square was used for data comparison. p was
significant when less than 0.01.
Results : In our study population, 71.8% of circumcised
women experienced sexual dysfunctions compared to 56.4%
of non circumcised women. Excision was found to increase
the risk of luck of sexual desire 2.9 fold while infibulation
increased the same risk 8.2 fold. Excision increases the risk
of feeling no pleasure 3.2 fold.
Conclusion : Female genital mutilations represent a significant
risk factor of developing sexual dysfunctions. More studies
should be carried out to better assess the negative impact of
that practice.
Keywords : Female genital mutilations, sexual dysfunctions,
cultural practice.
393
394
Abstracts of the Fourth European Congress of Andrology and
23rd Congress of the French Speaking Society of Andrology
Author Index
A
Aarabi, M. 345
Abdelmoula, A.. 339
Abdelmoula, N. 341, 368
Abel, M. 341
Abid, N. 320, 324, 325, 364
Abs, G. 362
Adamopoulos, D.A. 312, 319
Adaramoye, O.A. 388
Adorini, L. 376, 389
Affara, N. 341
Affara, N.A. 309
Agoulnik, A.I. 290
Ahmadipoor, S. 392
Ajina, M. 320, 344, 353, 364
Akbarzadeh, N.R. 343
Akhondi, M.A. 345
Akhondi, M.M. 343
Akinloye, O. 336, 388
Aknin-Seifer, I. 314, 340
Albert, M. 340, 366
Al-Hussaini, T.K. 364
Ali, K.A. 322
Ali, O. 323
Allam, J.P. 328
Allhoff, E. 332
Almont, T. 323
Aloisi, A.M. 379
Amaral, A. 316
Amirjannati, N. 345
Ammar-Keskes, L. 324, 325, 359
Amouri, A. 339, 341, 368
Angelopoulou, R. 350
Antonini, G. 378
Anzuini, A. 345
Arnott, J.M. 390
Arrus, J.A. 380
Athanasiou, E. 332
Attia A.M. 322
Attia, H. 359
Auger, J. 347
Ausmees, K. 325
Autio, R. 288
Axmon, A. 362
B
Bacer-Kermavner, L. 333, 373
Bachaud, J.M. 377, 377, 386
Bahloul, A. 324, 325
Bailly, M. 340
Baldi, E. 317, 351, 352, 352, 387
Balercia, G. 338, 354, 370, 381, 381, 382, 384, 385
Barbonetti, A. 365
Barros, A. 310, 336
Bartfai, G. 308, 390
Barthélémy, C. 373
Bashashati, M. 386, 392
Basma, H. 359
Baumann, T. 361
Bechoua, S. 315
Becker, S. 311, 344
Behre, H.M. 301
Beiranvandi, M. 386
Ben Abdallah, F. 359
Ben Jamaa, N. 320, 364
Benahmed, M. 289
Bengoudifa, B. 334, 335, 378
Bergère, M. 366
Bergmann, M. 291
Berthaut, I. 373
Besbes, S. 359
Beskorovainaya, T.S. 335
Bhushan, S. 327
Biarda, B. 365
Bizzaro, D. 307
Blasco, A. 380
Blumenaue, V. 363
Bocchio, M. 303, 349, 384
Bödeker, R.H. 319
Bonaccorsi, L. 387, 387
Bonde, J.P. 307
Bonefeld-Jørgensen, E.C. 307
Bongain, A. 304
Bonifacio, V. 302
Bontis, J. 312, 321, 332
Boscaro, M. 354
Botta, A. 347
Brehm, A. 336
Bresson, J.L. 373
395
Brodowska, A. 353, 389
Brucker-Davis, F. 304
Bujan, L. 323, 347, 370, 373
Buldreghini, E. 354
Bungum, L. 362
Bungum, M. 362
Burgoyne, P.S. 309
Degl’Innocenti, S. 315
Dejucq-Rainsford, N. 295, 329
Delannes, M. 377, 377
Delaunay, B. 377, 377, 386
Delavierre, D. 377, 386
Demir, R. 360
Denis, H. 329
Dennefeld, C. 291
Di Luigi, L. 368
Di Sante, S.... 375
Diaw, J.J. 393
Dinu, D. 321, 322, 334
Dobracheva, A.D. 358
Dolci, S. 375
Dondi, D. 288
Drobnic, S. 333, 373
Droupy, S. 295
Ducot, B. 304
Dumaine, A. 337
Dumitrache, C. 322
C
Calhaz, J. 313
Callies, C. 336
Calogero, A.E. 298
Cama, E. 345, 378
Carles, A. 288
Carlsson, L. 301
Carlucci, M. 379
Caron, C. 290
Carosa, E. 375
Carvajal, A. 380
Casanueva, F. 308, 390
Cauni, V. 321, 322, 334
Cavelot, P. 366
Cayli, S. 360
Chakraborty, T. 327
Chakroun, N. 324, 359
Chalet, M. 373
Chambon, P. 291
Chantot-Bastaraud, S. 337
Charlton, H. 341
Chausiaux, O. 341
Cherif, J. 368
Chernykh, V.B. 335
Chevreau, C. 371
Chouteau, J. 314, 340
Chuhrova, A.L. 335
Ciana, P. 288
Clavequin, M.C. 373
Clavier, B. 328
Clemenzi, A. 378
Comhaire, F. 331
Connolly, M.J. 391, 391
Cooper, T.G. 330
Cordeschi, G. 349
Corona, G. 381, 381, 382, 383, 384, 385
Cygankiewicz, I. 369
E
Ebbesen, T. 360
Eberhard, J. 306
Edgren, H. 288
El Ghezel, H. 344
El Shafey, E.N. 322
El-Garem, Y. 359
Elliesen, J. 301
Ellis, P.J.I. 309
Erenpreiss, J. 338, 360
Ernst, E. 360
Escalier, D. 350
Escoffier, E. 290
F
Farag, A.G.A. 322
Faure, A.K. 290, 314
Fechner, J. 349
Fénichel, P. 304
Ferguson, L. 309
Fernandes, A.T. 336
Fernandes, S. 310, 336
Fernandez, M. 380
Ferras, C. 310
Ferri, C. 303
Fibbi, B. 388
Fierro, V. 368
Fijak, M. 327
Filippi, S. 304, 348, 375, 376, 388, 389
Finn, J.D. 308, 390
Fisher, A.D. 381, 383, 385
D
Dacheux, J-L. 293
Dalantaeva, N.S. 358
Daudin, M. 306, 373
De Fleurian, G. 347
De Marco, E. 345, 378
396
Forti, G. 303, 304, 308, 315, 317, 338, 348, 351, 352, 370
376, 381, 382, 383, 384, 385, 387, 389, 390
Fraczek, M. 326
Francavilla, F. 365
Francavilla, S. 303, 349, 384
Frohnhoffs, F. 328
Furlong, R. 341
G
Gaczarzewicz, D. 353
Galan, J. 370
Garrone, F. 298
Geavlete, P. 321, 322, 334
Gerou, S. 312
Ghyselinck, N.B. 291
Giachini, C. 338
Gianfrilli, D. 302
Giannetta, E. 302
Gillling-Smith, C. 297
Giwercman, A. 306, 307, 308, 338, 360, 362, 372, 390
Giwercman, C. 307
Giwercman, Y. 306, 338
Giwercman, Y.L. 307
Glander, H.J. 361, 363
Godouet-Getti, B. 328
Golan, R. 311, 344
Gonçalves, J. 313
Gonçalves, R. 336
Goncharov, N.P. 358
Goulis, D.C. 321
Goulis, D.G. 312, 321, 332
Govin, J. 290
Granados, V. 340
Grillo, J.M. 347
Grimbizis, G. 312, 332
Grizard, G. 315, 357, 373
Gromoll, J. 336
Grunewald, S. 361, 363
Guarducci, E. 315, 338, 370
Guazzone, V.A. 327
Gudzenko, S.V. 335
Guerif, F. 362
Guerini, V. 288
Gueye, S.M. 323, 393
Guichaoua, M.R. 347
Guillou, F. 291
Gusmão, L. 313
Hanout, H.M. 322
Hassan, O.M. 364
Henkel, R. 349, 356, 359
Hennebicq, S. 290, 373
Hmeidan, F.A. 363
Hoppe, I. 356
Houlgatte, A. 371
Huhtaneimi, I. 308, 390
Humaidan, P. 362
Huszar, G. 360
Huyghe, E. 289, 306, 323, 334, 371, 377, 378, 386
I
Ihan, A. 363
Il’yna, E.V. 335
Iliadou, P.K. 321
Iosub, R. 327
Isidori, A.M. 302
J
Jaafoura, H. 341
Jalloh, M. 323, 393
Jannini, E.A. 375
Janny, L. 357
Jasatis, Y. 328, 331
Javadi, E. 343
Jeddi Tehrani, M. 343
Jedrzejczak, P. 326
Jégou, B. 329
Jones, R. 293
Jönsson, B.G.A. 307
Jouannet, P. 347
K
Kallioniemi, O. 288
Kamar, N. 334, 378
Kamieniczna, M. 326
Kammoun, S. 341
Kane, R. 323
Kantartzi, P.D. 321
Kapolla, N. 312
Kareem, O.I. 388
Kaufman, J.M. 331
Khantouche, L. 353
Kheirollahi, A. 386, 392
Khochbin, S. 290
Khosrowbeygi, A. 355, 355
Kilpinen, S. 288
Kleinstein, J. 332
Klug, J. 327
Kolbezen, M. 363
Koller, A. 305
H
Haidl, G. 328
Hamdy, F.C. 287
Hammoud, I. 340
397
Kopitar, A. 363
Koryakin, M.V. 358
Korrovits, P. 325
Koukkou, E. 312, 319
Koziol, K. 365
Kratzik, C. 305
Krausz, C. 315, 338, 370, 387
Kula, K. 308, 310, 369, 390
Kula, P. 369
Kurilo, L.F. 335
Kurpisz, M. 326
Mantas, D. 350
Mantero, F. 354
Maraee, A.H. 322
Marberger, M. 305
Marchiani, S. 351, 352, 352
Marchlewska, K. 310
Mark, M. 291
Marszal, H. 365
Mazerolles, C. 371
Mazzanti, L. 354
McElreavey, K. 314, 337
Meddeb, M. 339, 341
Meden-Vrtovec, H. 363, 373
Mehdi, M. 344, 353
Mehnert, C. 349
Meinhardt, A. 327, 327
Melun, M.C. 373
Menkveld, R. 356, 357, 359
Metzler-Guillemain, C. 373
Mhiri, N.M. 325
Michalakis, C. 319
Mieusset, R. 306, 323, 335
Mihalca, R. 349, 384
Mikos, T.H. 312, 332
Milazzo, J.P. 374
Mills, I. 288
Misrahi, M. 350
Mitios, G. 312
Mivsek, J. 333, 373
Modarressi, M.H. 345
Molina-Gomes, D. 340
Montañes, R. 380
Morelli, A. 303, 304, 315, 348, 375, 376, 388, 389
Morillon, M. 350
Motta, M. 288
Moubasher, A.A. 364
Mousset-Siméon, N. 328, 331, 373
Msaouel, P. 350
Muller, A. 347, 370
Muratori, M. 351, 352, 352, 387
Myachina, F.S. 335
L
La Vignera, S. 298
Labarthe, P. 371
Labou, I. 323, 393
Lackner, J.E. 305
Larsson, L. 301
Lass, H. 312
Laszczynska, M. 353, 389
Le Grand, R. 329
Le Tortorec, A. 329
Lenzi, A. 302, 345, 368, 375, 378
Lesovoy, V. 307
Lestrat, C. 209
Levy, R. 314, 340
Lewandowski, P. 365
Lewin, L. 311
Lewin, L.M.
Lindblom, A. 310
Lindenmeir, T. 332
Lombardi, A. 317
Lombardo, F. 294
Lotti, F. 383, 384
Lourenço, D. 337
Luconi, M. 303, 304, 317
Ludwicki, J.K. 307
Luetjens, C.M. 367
Lundberg Giwercman, Y 372
Lunt, M. 390
Lustig, L. 327
N
M
Navarro, P. 313
Ndoye, M. 323, 393
Neal, D. 288
Necozione, S. 303, 349, 384
Niang, L. 323, 393
Nickel, I. 332
Nicopoulou, S. 319
Nicopoulou, S.C. 312
Nieschlag, E. 283, 330, 336
Nohra, J. 334, 371, 377, 378, 386
Macé, B. 328, 331, 374
Maggi, A. 288
Maggi, M. 286, 303, 304, 348, 376, 381, 382, 383, 384,
385, 388, 389
Mahmoud, A. 331
Mancina, R. 303, 348
Mändar, R. 325
Mandelbaum, J. 337, 373
Manicardi, G.C. 307
398
Nosi, D. 387
Nuti, F. 315, 338, 370, 387
Rau, O. 332
Ravel, C. 337
Rebai, T. 324, 325, 339, 341, 368
Reiher, F. 332
Reinhardt, M. 361, 363
Rekik, R. 341
Relander, T. 372
Rieu, I. 315
Rignell-Hydbom, A. 306, 307
Rival, C. 327
Rives, N. 328, 331, 373, 374
Romanelli, F. 368
Romerius, P. 372
Ronquist, G. 301
Ronquist, K.G. 301
Rossi, A. 384
Rossi, C. 368
Rossi, S. 375
Rostaing, L. 334, 378
Rousseaux, S. 290
Royère, D. 362
Rylander, L. 307
O
O’Neill, T. 308
Oldham, J.A. 391, 391
Oltermann, I. 328
P
Paasch, U. 361, 363
Paolini, R. 379
Papadimas, J. 312, 321, 332
Papanicolaou, A. 312, 332
Pappa, A. 319
Pasquier, C. 296
Pasquier, G. 331
Passaquale, G. 303
Passavanti, G. 379
Pedersen, H.S. 307
Pelliccione, F. 303, 349, 384
Peltola, M. 288
Pereira, L. 313
Perrin, J. 347
Persu, C. 321, 322, 334
Petrone, L. 381, 381, 382, 383, 384, 385
Pflieger-Bruss, S. 319
Piasecka, M. 353, 389
Piccheri, C. 345
Piccolella, M. 288
Pierotti, S. 302
Pineau, C. 327
Pizzuti, V. 379
Plancha, C.E. 313
Plante, P. 306, 323, 371
Plastira, K. 350
Poch, O. 288
Poletti, A. 288
Polichronou, P. 312, 332
Polyakov, A.V. 335
Prakash, S. 346
Pratesi, G. 288
Primig, M. 292
Prisant, N. 350
Prithiviraj, E. 346
Publicover, S. 294
Pucci, C. 352
Punab, M. 308, 325, 390
S
Saad, A.Sadeghi, M.R. 320, 344, 353, 364
Sadeghi, M.R. 343
Saias, J. 373
Sakkas, D. 360
Sallemi Ben Hmida, A.
Sallemi, A. 339, 368
Salmonson, E.C. 306
Sandner, P. 388
Santucci, R.S. 365
Sari-Minodier, I. 347
Sassone-Corsi, P. 284
Sati, L. 360
Satie, A.P. 329
Sau, D. 288
Sauvalle, A. 373
Schalken, J. 288
Schatzl, G. 305
Schill, W.B. 319, 359
Schneider, E. 327
Schueller, S. 356
Schulman, C. 289
Schulmann, C. 381, 382
Schuppe, H.C. 319, 349
Sciarretta, F. 365
Sèle, B. 290
Sellami, A. 324
Sellami-Ben Hamida, A. 325, 341
Selva, J. 340, 366
Sermondade, N. 366
R
Radicioni, A.F. 345, 378
Rajmil, O. 380
Rajpert-De Meyts, E. 289
Ramalho-Santos, J. 316
399
Serni, S. 387
Sevastiadou, P. 332
Sgro, P. 368
Shochat, L. 311, 344
Sibert, L. 331
Siffroi, J.P. 337
Silman, A.J.S. 308, 390
Simoni, M. 330, 336
Sion, B. 315, 357
Slowikowska-Hilczer, J. 310, 369
Soffer, Y. 311, 344
Soltanghoraee, H. 345
Sorgentone, S. 365
Soufir, J.C. 350
Soulié, M. 377, 377, 386
Sousa, M. 310, 336
Spano, M. 307, 362, 372
Spira, A. 347
Srinivas-Shankar, U. 391, 391
Ståhl, O. 306, 372
Stalf, T. 349
Starczewski, A. 353, 389
Starker, W. 356
Stefanini, M. 285
Stepanova, A.A. 335
St-John, J. 316
Sullivan, R.
Suresh, S. 346
Sutcliffe, A.G. 284
Szermann, E. 373
Szumala-Kakol, A. 326
V
Vanderschueren, D. 308, 390
Vannelli, G.B. 375
Vanniez, J.P. 374
Varano, G. 317
Vaudreuil, L. 374
Velemir, L. 357
Venaki, E. 319
Vernet, N. 291
Vialard, F. 340, 366
Vicari, E. 298
Vicini, E. 285
Vignini, A. 354
Vignozzi, L. 303, 304, 348, 375, 376, 388, 389
Virant-Klun, I. 333, 363, 373
Von Wulffen, W. 327
W
Wagner-Mahler, K. 304
Walczak-Jedrzejowska, R. 310, 369
Waldinger, M. 294
Walschaerts, M. 306, 370, 371
Wasylyk, B. 288
Wember, F. 319
Werny, F.M. 330
Wesselmann, R. 367
Wolf, J.P. 340
T
Wolf, M. 288
Tachdjian, G. 350
Taha, E.A. 364
Tajar, A. 308, 390
Tamburrino, L. 352
Tarlatzis, B.C. 352, 332
Tarrahi, M.J. 386, 392
Tchatalbachev, S. 327
Thevenon, J. 290
Thomas, C. 373
Thonneau, P. 304, 306, 323, 347, 370, 371
Tiido, T. 307
Tinneberg, H.R. 349
Toft, G. 307
Tomazevic, T. 333, 373
Tortoreto, M. 288
Touraine, R.L. 314, 340
Trasler, J. 283
Trubacz, S. 365
Tsametis, C. 321
Tsarev, I. 338, 360
Tüttelmann, F. 330
Wolski, J.K. 365
Wranicz, J.K. 369
Wu, F.C.W. 308, 390, 391, 391
Wylot, M. 389
Y
Yogev, L. 311
Z
Zairi, A. 334, 378
Zanioti, K. 350
Zarghami, N. 355, 355
Zhang, X.H. 376
Zhou, X. 310
Zorn, B. 363
Zugaro, A. 365
Zvyezday, V. 307
400
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