Placental histomorphology in unexplained foetal loss with thrombophilia

Indian J Med Res 129, February 2009, pp 144-149
Placental histomorphology in unexplained foetal loss with
thrombophilia
S. Vora, S. Shetty, M. Khare* & K. Ghosh
National Institute of Immunohaematology (ICMR) & *Department of Histopathology, KEM Hospital
Mumbai, India
Received June 6, 2007
Background & objectives: Acquired and genetic thrombotic conditions, both organ and non organ
specific, are associated with increased foetal wastage. This study was carried out to examine the placenta
from women with abnormal pregnancies and a history of unexplained foetal loss, and to associate with
maternal thrombophilia status.
Methods: Placentas from eight women with history of unexplained foetal loss were analyzed for
histopathological characteristics. All the women were simultaneously screened for the common
acquired and genetic thrombophilia markers i.e., lupus anticoagulants ( LA), IgG / IgM antibodies
for anticardiolipin (ACA), β2 glycoprotein 1 (β2GPI) and annexin V, protein C (PC), protein S (PS),
antithrombin III (AT III), factor V Leiden ( FVL) mutation, prothrombin (PT) gene G20210A,
methylene tetrahydrofolate reductase (MTHFR) C 677T, endothelial protein C receptor (EPCR) 23 bp
insertion and plasminogen activator inhibitor ( PAI-1 4G/5G) polymorphisms.
Results: Six of eight women were positive for one or more thrombophilia markers. The placenta in all
the cases except one, showed the characteristic features of infarct fibrin deposition and calcification.
Among two women who were negative for thrombophilia, one showed clear evidence of thrombus in the
placental sections while the other did not show any characteristic infarcts in the placental sections.
Interpretation & conclusion: Our findings showed that the histopathological examination of the placentas
confirmed thrombophilia as the aetiological cause of thrombosis in 6 of the 8 women. The presence
of thrombus in a negative thrombophilia woman suggests yet unidentified thrombophilia markers or
probably non-haemostatic factors causing thrombosis.
Key words Antiphospholipid antibodies - inherited thrombophilia - recurrent spontaneous abortion
The placenta is a unique organ with dual
blood circulation, functioning throughout foetal
development. Placental trophoblasts express and
produce coagulation components, participating not
only in haemostasis but also in placental vascular
development and differentiation1.
For pregnancy to proceed normally the placenta
must be allowed to develop and grow appropriately so
that an adequate blood supply is available to support
and promote the growth of the developing foetus.
Failure of the normal uterine physiological changes to
occur and the development of intra-placental pathology
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VORA et al: THROMBOPHILIA, PLACENTAL PATHOLOGY & FOETAL LOSS
will ensure placental insufficiency and the features
that accompany are failing placenta, i.e., intra-uterine
growth retardation (IUGR), pre-term delivery, preeclampsia, and toxaemia of pregnancy2-4. Thrombotic
lesions of the placenta are a common feature in women
with recurrent foetal loss or with adverse pregnancy
outcomes. However, the aetiology of foetal loss or
other associated adverse conditions of the pregnancy
is unknown. It is believed that these are associated
with abnormal placental vasculature and haemostatic
disturbances leading to inadequate maternal foetal
circulation5-8.
The known thrombotic nature of the placental
lesions and the risk associated with both acquired
and genetic thrombophilias strongly suggest a causeeffect relationship between thrombophilias and severe
obstetric complications. The evidence of thrombosis
or infarcts in the placental sections suggests a
haemostatic abnormality with or without association
with thrombophilia. In the present study, the
histopathological analysis has been done in placenta
from eight women and subsequently compared to their
thrombophilia status to objectively confirm the effect
of thrombophilia on the placental vasculature.
Material & Methods
The study was carried out between July 2004 and
June 2007 at the Institute of Immunohaematology,
(IIH), Mumbai, in collaboration with the Department
of Histopathology, KEM Hospital, Mumbai. Placenta
from eight consecutive women having history of
unexplained foetal loss were collected at the time
of delivery for histopathological evaluation. These
women had either second or third trimester pregnancy
losses or both. The selection of these placenta from 8
women has been done based on the following criteria:
It was randomly selected and the number was restricted
by the large number of sections which were to be taken
and processed for each placenta so that important
pathological changes are not missed. The conventional
causes of pregnancy loss i.e., hormonal (T3, T4, TSH,
17 α-hydroxyprogesterone, estradiol, estriol, β hCG),
immunological (autoimmune markers – ANA, ds DNA,
rheumatoid factor) and karyotyping were excluded in
all women. The placental tissues were fixed with 10 per
cent formalin immediately after delivery and following
dehydration were embedded in paraffin immediately.
Microtome sections were subsequently prepared and
fixed on the slides using egg albumin. The slides
were then stained with hematoxylin and eosin and
145
mounted with DPX mountant for histopathological
analysis under an optical microscope (Olympus BX50
- SP-350). The study was approved by the Ethics
Committees of both the IIH and KEM Hospital.
Screening for thrombophilia: Ten ml of blood was
collected from each women by venipuncture into 3.18
per cent trisodium citrate (1:9 anticoagulant to blood)
and EDTA tubes. Plasma samples were stored in
-700 C until analysed. The cell pellet was preserved
at -200 C for DNA extraction. The samples were
collected 6 months after the miscarriage. As the natural
inhibitors of blood coagulation i.e., protein C, S and AT
do not get normalized immediately after the delivery
or a miscarriage. The general practice is to wait for a
minimum of 6 months before one collects for these
tests.
Screening coagulation tests i.e., prothrombin time
(PT), activated partial thromboplastin time (APTT)
and thrombin time (TT) were performed using
commercial reagents (Organon Teknika, Durham,
USA). Any sample showing >5 sec prolonged clotting
time (APTT) than the control sample, was analysed for
the presence of lupus anticoagulants (LAs). Fibrinogen
was measured by clotting assays using commercial kits
(Diagnostica Stago, Asniers, France).
Tests of antiphospholipid antibodies (APA): The plasma
was mixed with normal pool plasma (NPP). Kaolin
clotting time (KCT), dilute Russels Viper Venom
time (DRVVT) were performed as described earlier6,7
using commercial reagents (Dade Behring, Germany).
IgG/IgM antibodies for anticardiolipin (ACA), β-2
glycoprotein-1 (β2GP1), annexin V were measured
by ELISA using commercial kits (Varelisa, Freburg,
Germany). All the results were reconfirmed with a
fresh sample after a minimum duration of 4 months.
Tests for inherited thrombophilia: Protein C (PC)
and protein S (PS) were measured by ELISA using
commercial kits (Diagnostic Stago, Asniers, France).
Antithrombin III (ATIII) was measured by chromogenic
assays using commercial reagents (Diagnostica Stago,
Asniers, France). DNA was extracted from citrated cell
pellet using standard methods8.
Factor V Leiden (FVL) mutation was identified by
PCR amplification (Applied Biosystems, CA, USA) of
220 bp fragment using Mnl 1 enzyme for restriction
digestion9. The prothrombin (PT) gene G20210A
polymorphism was identified by Hind III cleavage of 322
bp PCR amplified product10. The C677 T polymorphism
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INDIAN J MED RES, FEBRUARY 2009
of methylene tetrahydrofolate reductase (MTHFR) was
detected using Hinf 1 cleavage of 175 bp PCR product11
while endothelial protein C receptor (EPCR) 23 bp
insertion was detected by PCR amplification without
any further restriction digestion12. Plasminogen activator
inhibitor-1 (PAI-1 4G/5G) polymorphism was detected
by allele specific PCR amplification using two sets of
primers as described13. In brief, the PCR protocols were
as follows : For FVL, 30 cycles of 940 C for 30 sec, 550C
for 40 sec and extension at 720C for 1 min; for MTHFR
and PT, 30 cycles of 940C for 1 min, annealing at 660C
for 1 min and extension of 1 min at 660C ; for EPCR, 30
cycles of 940 C at 1 min, annealing at 550C for 1 min and
an extension of 70 0C for 3 min; for PAI-1 4G/5G, 30
cycles of 940C for 1 min, annealing at 650C for 45 sec,
extension at 720C for 75 sec.
Results
The histological features in the placental
tissue sections and their association with maternal
thrombophilic status was shown in the Table.
Except two cases in all remaining cases more
then one thrombophilic risk factor was observed.
Three women were homozygous for PAI-1 4G/4G
polymorphism while one was homozygous for
MTHFR T677T polymorphism. While two were
positive for APA. Of the two women in whom
thrombophilia was not detected, one had low platelet
count, a history of portal venous occlusion, and clear
evidence of thrombosis in the histological analysis
of placental sections while in another women neither
thrombophilia nor thrombus in placental sections was
observed. A large area of subchorionic haemorrhage
was seen in the sections.
The primary pathology observed in all the
placentas was thrombosis. Placental infarction,
decidual vessel thrombosis, chronic villitis and
excessive perivillous fibrin deposition were seen in a
few sections. Multifocal uteroplacental thrombosis was
also a significant observation in some of the placental
sections (Figs 1-5).
Table. Placental histological and clinical features in association with the maternal thrombophilc status
Sr.
No
1
Patient ID
No. of
/Age
abortions/
IUFD*
(yr)
CY/ 29
2/2
Histological features
Villi with stromal fibrosis, a few foci of
calcification, chronic ischaemia, organized
thrombus stem vessels
Maternal clinical
features
Retro-placental
clot
Maternal
thrombophilia status
β2GP1 35MPL-U/ml
Anti-annexin V 25MPL-U/ml
Fibrinogen gene polymorphism
Arg/Lys
2
AD/ 27
0/1
3
PG /28
0/3
4
SJ / 23
0/3
5
MS / 29
0/4
6
CT /22
0/2
7
UN / 31
5/0
8
NP /22
0/1
Premature villi, focal haemorrhage, vessels
showing recent thrombosis.
Villi shows stromal fibrosis, chronic ischaemia,
infarct, placental insufficiency, thrombosis in
vessel.
Portal vein
thrombosis
PIH
Intervillous haemorrhage, infarct, foetal stems
arteries show organized thrombus.
PIH
Mild perivillous fibrin deposit, Mild neutrophilic
infiltrate, immature villi with retroplacental
haemorrhage, No thrombus.
Membrane shows normal amnion and chorion,
large area of subchorionic haemorrhage.
Areas of infarct, foci of extramedullary
haematopoiesis, polymorph infiltrate
suggestive of ischaemic changes with possible
chorioamnionitis.
Calcification, infarct
Excessive
bleeding
Not detected
Anti annexin V 22GPL-U/ml
PAI-1 4G/4G Homozygous
MTHFR C677T heterozygous,
PAI-1 4G/4G
Homozygous
MTHFR T677T homozygous
-
Not detected
Doppler
suggestive of mild
resistive flow in
left uterine artery
USG suggestive
of absence of
foetal movement
MTHFR T677T
IUFD, intrauterine foetal death; PIH, pregnancy induced hypertension; USG, ultrasonography
Homozygous
PAI-1 4G/4G
Homozygous
VORA et al: THROMBOPHILIA, PLACENTAL PATHOLOGY & FOETAL LOSS
Fig. 1. Placental tissue showing thrombus in foetal stem vessel.
(HE stain 100x). The arrow indicates areas of infarct in the
placental sections.
Fig. 3. Placental tissue showing many areas of calcification. (HE
stain 400x). The arrow indicates areas of infarct in the placental
sections.
147
Fig. 2. Placental tissue showing infarcted villi with coagulative
necrosis. (Avascular villi) (HE stain 400x). The arrow indicates
areas of infarct in the placental sections.
Fig. 4. Normal third trimester villi with syncitial knots and
dystrophic calcification. (HE stain 400x). The arrow indicates areas
of infarct in the placental sections.
villi, multiple vessel thrombosis, infarction, fibrosis
and decidual vasculopathy. Our findings are in line
with these findings. The studies show that 60-73 per
cent of the abnormal histopathological placentas were
positive for thrombophilia.
Fig. 5. Organized thrombus in foetal stem vessel. (HE stain 100x).
The arrow indicates areas of infarct in the placental sections.
Discussion
The reports of histomorphological changes in
placenta in women with recurrent pregnancy loss in
association with thrombophilia are relatively recent14-30
and all of them broadly show non vascularisation of
The primary pathology observed in the present
study was thrombosis. Some of the other pathological
features observed were placental infarction, decidual
vessel thrombosis, chronic villitis and excessive
perivillous fibrin deposition. Multifocal uteroplacental thrombosis was also a significant feature in
patients with thrombophilia.
Many physiological changes occur in the uterine
wall during pregnancy in order to allow the foetus to
grow. These include dilation and enormous growth
of the spiral arteries, destruction of the spiral artery
endothelium and decidual invasion by trophoblast31.
Only if an adequate blood supply is provided by the
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INDIAN J MED RES, FEBRUARY 2009
maternal blood vessels, normal placental growth and
development will continue. In case of thrombophilia
related foetal loss, where there is a compromised
blood supply, it will not be surprising that the placenta
exhibits significant pathological changes.
2.
Branch DW, Silver RM, Blackwell JC. Outcomes of treated
pregnancies in women with APS: An update of the Utah
experience. Obstet Gynaecol 1992; 80 : 614-20.
3.
Lubbe WF, Butler WS, Palmer SJ. Foetal survival after
prednisolone suppression of maternal lupus anticoagulant.
Lancet 1983; 1 : 1361-3.
The contribution of the foetal genotype in
determining pregnancy outcome demands further
investigation. The placenta receives two arterial
supplies (one maternal and one foetal). Placental
thrombosis occurs if any one of the vascular
supplies is compromised. It has been reported
that placental infarction was significantly more
when foetus carried the FVL allele compared with
normal factor V genotype32. Abruption, fibrosis
and hyper- vascularity of villi were the common
histological features observed in patients33,34. Of
the eight placentas analysed histologically in the
present study, in six cases maternal thrombophilia
could be attributed to foetal loss. Of these six, two
were positive for APA while four were positive for
inherited thrombophilia.
4.
Out H, Bruinse HW, Christiaens GCML. A prospective
controlled multicentre study on the obstetric risks of pregnant
women with APA. Am J Obstet Gynaecol 1992; 167 : 26-32.
5.
Branch DW, Porter TF, Rittenhouse L. Antiphospholipid
antibodies in women at risk for pre eclampsia. Am J Obstet
Gynaecol 2001; 184 : 823-34.
6.
Thiagarajan P, Pengo V, Shapiro SS. The use of the dilute
Russel Viper Venom time for the diagnosis of lupus
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7.
Exner T, Rickard KA, Kronenberg H. A sensitive test
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Sambrook J, Fritsch EF, Manniatis T. Molecular cloning: A
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One woman who had focal haemorrhage and
vessels showing recent thrombosis was negative for
all the thrombophilic markers studied and another who
showed subchorionic haemorrhage was negative for all
the thrombophilic markers studied. Thus, there could
be more thrombophilia markers, yet to be identified, or
as more markers are added to the testing panel, we may
get an answer for the cause of recurrent foetal loss.
Histology of placenta from miscarriages gives a
fair idea as to the cause of pregnancy failure, though
the precise pathogenesis of the thrombotic diathesis
associated with thrombophilia still remains unknown.
Analyzing previous placentas could have been helpful as
the same pathological process is repeated in subsequent
pregnancies, making it possible to decide a therapeutic
regime for individual patients. It is well known that the
thrombosis is due to a complex interaction between
the inherited, acquired and circumstantial risk factors.
The thrombosis was objectively confirmed in majority
of the placentas tested and who were also positive for
thrombophilia. Except APA we have not analysed other
acquired risk factors for thrombosis. A comprehensive
analysis of all these risk factors will probably provide
answers to all women with unexplained foetal loss.
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Reprint requests: Dr Kanjaksha Ghosh, Director, National Institute of Immunohaematology (ICMR), KEM Hospital
Parel, Mumbai 400 012, India
e-mail: [email protected]
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