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Complete Hydatidiform Mole Coexisting with Three Viable Fetuses in a

Quadruplet Pregnancy
Article in Journal of the College of Physicians and Surgeons--Pakistan: JCPSP · April 2016

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Nabia Tariq
Shifa College
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CASE REPORT
Complete Hydatidiform Mole Coexisting with
Three Viable Fetuses in a Quadruplet
Pregnancy
Nabia Tariq1, Usman Ghazali2, Zeeshan Uddin3, Kamran Rasheed4 and Hina Tariq3
ABSTRACT
We hereby report a case of quadruplet pregnancy with delivery of 3 viable infants and a complete mole. This was an
induced conception with clomiphene citrate. At 22 weeks, cystic structures were noticed in one of the placentae and a
suspicion of co-existant molar pregnancy was made. The case discussed with oncologist and pregnancy was continued
with close monitoring of β-hCG and Ultrasound. Her β-hCG at 23 weeks was 748 mIU/ml, which continued to rise until
the 29th week of gestation to a level of 305881.68 mIU/ml and declined gradually thereafter. Similarly, hydropic change in
placenta also continued to increase progressively. She was given steroid cover at 32 weeks and delivery was aimed at
34 weeks of gestation. The patient went into preterm labour at 33 weeks and 3 female infants delivered by lower
segment cesarean section (LSCS) followed by removal of 3 placentae along with copious molar tissue at the end. The
newborns were kept in the nursery, non-requiring assisted ventilation and discharged in satisfactory condition. The
histopathology and immunohistochemistry confirmed the diagnosis of a quadruplet pregnancy comprising of one
complete mole with 3 normal placentae.
Key Words: Complete hydatidiform mole. Quadruplet pregnancy. Multiple pregnancy.

a normal fetal karyotype, it is justifiable to await

INTRODUCTION
Cases of a hydatidiform mole (HM) coexisting with a
fetus have recently become more common as a result
of an increase in the incidence of multiple pregnancy
arising from ovulation induction therapy and in vitro
fertilization. HMs may be partial mole (PM) or complete
mole (CM) depending on their gross appearance,
immunohistochemistry and karyotype. PMs usually
have a triploid karyotype, derived from maternal and
paternal origins and they are positive for p57 antibody
on immune-histochemistry, whereas CMs are diploid
and have paternal origins only, therefore, they are
negative for p57 stain.
Coexistence of a viable fetus with a hydatidiform mole
is a rare condition with an estimated frequency of 1 in
22,000 to 100,000 pregnancies.1 Most cases suffer
severe complications such as pre-eclampsia, abortion
and preterm delivery or termination immediately after
diagnosis. Delivery of viable infants from this condition
is even rarer.2
Early diagnosis is very important because of the risk of
developing severe complications in pregnancy. In most
cases, termination of pregnancy is recommended when
the diagnosis is made in early pregnancy. 3 In the case
of Department of Obstetrics and Gynaecology1 / Medical Student2 /
Pathology3 / Oncology4, Shifa College of Medicine,
Shifa International Hospital, Islamabad.
Correspondence: Prof. Nabia Tariq, Consultant Obstetrician
Gynaecologist, Shifa College of Medicine, Shifa International
Hospital, Islamabad.
E-mail: nabiatariq_fcps@yahoo.com
Received: November 06, 2014; Accepted: September 21, 2015
developments in the absence of maternal complications.
CASE REPORT
A 27-year female in her second pregnancy, presented
for booking after having her pregnancy confirmed by
urine pregnancy test. Her present pregnancy was a
result of treatment with clomiphene citrate 50 mg daily
for 5 days, owing to failure to conceive for 4 years. At
13 weeks, ultrasound showed dichorionic-triamniotic
pregnancy (Figure 1).
The patient had severe nausea and vomiting throughout
her first trimester, for which she was given supportive
treatment from time to time. Ultrasound at the 22nd
week of gestation showed 3 alive fetuses and tiny cystic
spaces in the left half of anteriorly lying placenta.
Hydropic degeneration in one placenta was also noted
(Figure 2). Oncology consultant was sought for the
partial hydatidiform mole. Keeping in view normal
development and growth of all three fetuses and
absence of any maternal complications, pregnancy was
decided to be continued with close feto -maternal
surveillance and serial β-hCG monitoring. Her β-hCG at
23 weeks was 748 mIU/ml, which continued to rise until
the 29th week of gestation to a level of 305881.68
mIU/ml and declined progressively thereafter. Similarly,
hydropic change in placenta also continued to increase
progressively. The pregnancy continued well and it was
not complicated by any medical comorbid. At 32
weeks+6 days, she presented with complaint of severe
abdominal pain and vaginal leakage. Examination
confirmed triplet pregnancy with full dilatation, hence an
emergency cesarean section was planned.
Complete hydatidiform mole coexisting with three viable fetuses in a quadruplet pregnancy

and fetus can depend upon the clinical symptoms and

Figure 1: Early pregnancy scan
showing biamniotic trichorionic
pregnancy.
Figure 3: Gross appearance of the
placental discs with 3 umbilical
cords. The molar tissue is also
visible (arrow).
Three female babies with
Figure 2: 22 weeks scan showing
molar change in one placenta.
Figure 4: Microscopic picture
showing molar change involving all
of the chorionic villi, including villous
enlargement, cistern Formation
(small arrow) and trophoblastic
10X). Inset depicts p57 immuno-
signs, physical examination, sonographic findings, and
abnormal biochemical data. Clinically, the patient may
present with hyperemesis, hyperthyroidism, vaginal
spotting or even heavy bleeding, pregnancy-induced
hypertension and larger-than-gestational age uterus.
Our patient had no clinical symptom of molar pregnancy
and this was an accidental finding on ultrasound.
Although it is possible in most cases to diagnose CHM
from 11 - 12 weeks of gestation. Amano reported
diagnosis of CHM coexisting with multiple fetuses at 18
weeks of gestation. 4 Suspicion of molar change, in this
case, was made at 22 weeks, which is quite an usually
delayed diagnosis. Very frequent diagnosis is easily be
made during the first trimester scan. However,
diagnosing multiple pregnancy using ultrasound, the
focus is naturally on the fetuses, which may lead to
other possible findings being overlooked. Although the
present case resulted in a favourable outcome, a review
of the 14 reported cases suggests high fetal loss rate
(90%).5 In a large study by Vaisbuch et al., they
reported 130 cases of twins with CHMF (complete
hydatidiform mole and coexistent fetus) pregnancy of
which 41% were terminated because of the positive
probability of serious maternal complications.6
Once the suspicion of molar change was made on
ultrasound, serial β-hCG levels showed a rise from 748
mIU/ml at 23 weeks upto 305881 mIU/ml at 29 weeks

proliferation (large arrow) (H&E,

3 complete placentae histochemical
delivered, weighing 1.6 - molar tissue, showing Negative
nuclear staining in the cytotro-
1.8 kg each, none required phoblastic cells which is consistent
ventilator support. Copious complete hydatidiform (90X).
amount of trophoblastic
vesicular tissue was also removed at the end (Figure 3).
Dichorionic-triamniotic placenta with part of one
placental disc showing numerous vesicular structures
(Figure 3).
On microscopy, the normal appearing placenta showed
mature chorionic villi with normal fetal membranes and
umbilical cords. However, the vesicular structures
reveal diffusely dilated chorionic villi with cistern
formation and trophoblastic proliferation (Figure 4).
Nucleated RBCs were not identified. The trophoblastic
cells were negative for p57 immunostain (Figure 4
inset). Based on all these findings, a diagnosis of
complete hydatidiform mole complicating a quadruplet
pregnancy was made. The molar tissue was considered
to be derived from the fourth conceptus.
DISCUSSION
CHM (complete hydatidiform mole) occurring with
multiple living fetuses is very rare. There are very few
case reports of quadruplet pregnancy with complete
mole. However, all pregnancies ended up before 25
weeks, either due to obstetric or maternal
complications.4 Prenatal diagnosis of coexistent mole
staining done on and a gradual decline thereafter to non-pregnancy level
12 weeks postpartum. Duplicate immunohistochemical
staining by the polymer method was done for each of
the 14 equivocal cases, and the staining patterns were
compared with those of the control cases confirmed
genetically in our laboratory. Four cases (Cases 2, 5, 6,
and 9 indicated by asterisks in Table 1) showed a
clearly negative immunoreaction for p57kip2. T he others
stained positively.
Thus, we were able to differentiate these 4 cases
as complete moles among the 9 equivocal cases of
partial or
Women with hydatidiform mole are at risk of preterm
delivery (PTD). Neimann in 2007 revealed that the risk
of PTD after a diploid mole with a viable fetus is similar
to that after a singleton molar pregnancy. 7 Literature
review of previously reported cases involving
quadruplets or triplets with a complete hydatidiform
mole revealed all cases ended as premature non-viable
fetuses. However, our patient successfully completed
33 weeks of gestation and achieved viability with a
multidisciplinary approach. Hydatidiform moles can be
difficult due to a number of overlapping features.
Therefore, for a more definitive diagnosis,
immunohistochemical stain p57 was applied; p57
staining is helpful in separating a complete mole from a
partial mole. It is a paternally imprinted protein and
expressed predominantly.

Journal of the College of Physicians and Surgeons Pakistan 2016, Vol. 26 (4): 326-328 327
The histological distinction between partial and complete
In partial mole, maternal genes are present and
expressed. Therefore, p57 is immunoreactive in the
cytotrophoblastic cells lining the chorionic villi. 8 In this
case, p57 was negative confirming the morphological
diagnosis of complete hydatidiform mole.
The risk of persistent trophoblastic disease (PTD) is
the same as in the case of a singleton complete mole.
The patient was followed-up with serial β-hCG, which
became normal in 12 weeks postpartum. As a
preliminary study, we performed standard streptavidin-
biotin immunohistochemical staining of p57kip2 in our
DNA-established complete mole and hydropic abortion
cases to know how effective the reported method is for
differentiation of complete moles from hydropic
abortion [5, 6]. In several established moles, however,
we observed false positive stain-ing. In reading the
previous papers carefully, we learned that the
investigators also encountered a small percentage of
false positive staining.
With the standard streptavidin-biotin method,
endoge-nous biotin has a positive effect on the staining
pattern. So, we then used 3% hydrogen peroxidase
solution to quench the endogenous biotin activity. This
was 10 times the concentration reported by Jun et al.
[6], but we still
encountered false positive staining in several
established complete moles. Subsequently, we learned
that the poly-mer method of immunohistochemical
staining, in which a secondary antibody conjugated
with a polymer is used, is much more sensitive (10 to
100 times) than the standard streptavidin-biotin
method. Duplicated 4 m thick sections from the formalin-
fixed, paraffin-embedded blocks were obtained in each case.
Sections were deparaffinized in xylene and alcohol, washed,
and rehydrated in distilled water.
After endogenous peroxidase activity was
quenched with 3% hydrogen peroxidase solution,
antigen retrieval was performed. The sections were
immersed in 0.01 M citrate buffer (pH 7.0) with 0.1%
Tween-20, kept for 40 minutes at 98 ∘C. The sections
were allowed to cool for 20 minutes spontaneously.
Next, sections were immersed in 1 mM EDTA (pH 9.0),
for 40 minutes at 98∘C, and again allowed to cool. Next
the sections were immersed again in 1 mg/mL
protease XXIV (Sigma-Aldrich. St. Louis, MO, USA) in
PBS for 60 minutes at room temperature and then
washed in water and PBS. To block nonspecific
reactions, these sections were immersed with 5% gout
serum for 20 min-utes at room temperature. Mouse
monoclonal antibody for human p57kip2 protein, the
primary antibody, was applied to samples for overnight
incubation at 4∘C (Novocastra Liquid Mouse
Monoclonal Antibody for human p57 protein (Product
code: NCL-L-p57: Leica Biosystems Newcastle Ltd,
Newcastle, UK)).
Journal of the College of Physicians and Surgeons Pakistan 2016, Vol. 26 (4): 326-328
Peroxidase-labeled secondary antibody for anti-
mouse immunoglobulin conjugated with amino acid
polymer (Nichirei Co, Ltd., Tokyo, Japan) was applied
for 60 minutes at room temperature. Sections were
then washed three times for 5 minutes each with PBS.
The sections were incubated with diaminobenzidine
as a chromogen for 10 minutes, then washed in water,
and nuclear-counterstained with hematoxylin. Staining
patterns on the tissue sections were examined
microscopically and compared to those of control
sections. The control sections were prepared from the
DNA-established androgenetic complete moles, partial
moles (triploidy), and abortions of biparental origin and
prepared in the same manner as the cases’ sections.
The polymer method is easy and more sensitive,
and it is not affected by endogenous biotin.
The secondary antibody conjugated with a polymer
can be easily obtained commercially.
The polymer-based method is now described in
textbooks as an improved method.
We applied the polymer method to our DNA
analysis-established androgenetic complete moles and
confirmed that the polymer methods do not produce
false-positive or false-negative staining.
We found the method to be a reliable tool that can
be used to differentiate complete mole in equivocal
cases without the need for DNA analysis of each
specimen.
However, there is 1 report of a definitive
androgenetic complete mole that stained positively for
p57kip2 [3].
Of course, we must be vigilant, and we must realize
that immunoreaction is not always absolute.
DNA analysis should be done, whenever a case
remains questionable. However, there is no doubt that
the polymer method is sensitive and effective.
We believe this method to be a very useful tool for
differentiation of complete mole, when the results of
other tests are equivocal. We would like to recommend
that the polymer method of immunohistochemistry be
applied first as a routine examination in any equivocal
cases, especially for doctors who work in developing
countries, where DNA analysis is far too expensive or
even unfeasible. We may be able to avoid the cost of
DNA analysis.
Kihara et al. published the first report of perfect
concor-dance between negative p57kip2
immunoreactivity and molar tissue of androgenetic
origin [8]. They used a polymer system produced by
DakoCytomation (Glostrup, Denmark). Our study
independently supports their findings.
Conclusion
Polymer-based immunohistochemical staining of p57kip2
(paternally imprinted gene, expressed from maternal
allele) is a very effective method that can be used to
differentiate androgenetic complete mole from partial
mole and hydropic abortion. We might be able to avoid
the cost of DNA analysis.
328
Nabia Tariq, Usman Ghazali, Zeeshan Uddin, Kamran Rasheed and Hina Tariq
Ethical Approval
The authors obtained permission to conduct this study
from the ethics committee of Iino Hospital, and they
obtained informed consent from all 14 study patients.
Conflict of Interests
The authors declare that there is no conflict of interests
regarding the publication of this paper.
Acknowledgments
This paper was presented at the XVII World Congress
on Gestational Trophoblastic Diseases held in Chicago,
IL, USA, September 19–23, 2013. The authors express
their thanks
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Journal of the College of Physicians and Surgeons Pakistan 2016, Vol. 26 (4): 326-328