Hydatidiform Moles
Application of Flow Cytometry in Diagnosis
JANICE M. LAGE, M.D., SHIRLEY G. DRISCOLL, M.D., DEBRA L. YAVNER, M.D., AGATHA P. OLIVIER, B.S.,
STEVEN D. MARK, M.D., AND DAVID S. WEINBERG, M.D.
Hydropic chorionic villi are found in hydropic abortuses, partial hydatidiform moles (PM), and complete hydatidiform
moles. Partial and complete moles have the potential for persistent trophoblastic disease. The vast majority of partial
moles are triploid and generally follow a benign clinical course.
Complete moles are diploid and distant metastasis and choriocarcinoma may develop. The authors determined the nuclear
ploidy by flow cytometry of 31 placentas, 19 of which appeared
hydropic either on obstetric ultrasonography or gross examination. Of ten complete moles classified by histologic criteria,
ten were diploid, whereas five of seven histologically classified
PM were triploid. The remaining two cases classified as PM
were diploid; one most likely represented a regressing complete mole; the other a hydropic abortus. All 14 control placentas were diploid. In all cases in which karyotypic analysis
was performed, the flow cytometric determination of ploidy
was confirmed. It was concluded that DNA flow cytometric
analysis is a rapid, accurate, and cost-effective means for assaying nuclear ploidy in these tissues, and as such, offers an
informative supplement to the histological interpretation of
hydropic placentas. (Key words: Hydatidiform mole; Placenta;
Flow cytometry; Triploidy; Ploidy) Am J Clin Pathol
1988;89:596-600
GROSSLY EDEMATOUS chorionic villi are present in
all cases of partial hydatidiform mole (PM) and complete hydatidiform mole (CM) and, on occasion, in
some nonmolar spontaneous abortuses. Light microscopy can frequently distinguish these pathologic states
from one another. Classically, CMs are characterized by
diffusely edematous chorionic villi covered by hyperplastic trophoblast. PMs show focal villous swelling with
at least focal trophoblast hyperplasia. The hydropic
abortus (HA) also contains edematous villi; these, however, are covered by attenuated trophoblast. In some
cases nature is not so careful at preserving these simple
distinctions. At one end of the spectrum of difficult
cases is the placenta with prominent edematous villi and
florid trophoblast hyperplasia, but with some residual
Received June 15, 1987; received revised manuscript and accepted
for publication October 5, 1987.
Presented in part at the meeting of the United States and Canadian
Academy of Pathology, Chicago, March, 1987.
Address reprint requests to Dr. Lage: Department of Pathology,
Brigham and Women's Hospital, 75 Francis Street, Boston, Massachusetts 02115.
596
Department of Pathology and Division of Hematology,
Department of Medicine, Brigham and Women's Hospital;
Department of Pathology, Harvard Medical School, and
Division of Epidemiology, Harvard School of Public Health,
Boston, Massachusetts
population of normal villi. To decide whether such a
conceptus should be classified as a PM or a CM can be
vexing. An exactly symmetric dilemma occurs when the
villous and trophoblast changes are mild. Then the question becomes distinguishing a PM from an HA with
thickened polar trophoblast.
Given that no two of these three categories have identical prognostic and therapeutic implications, it is important that the pathologic classification be as accurate
as possible. Both the PM and CM need serologic followup of serum human chorionic gonadotropin (HCG) to
rule out persistent trophoblastic disease. Metastatic gestational trophoblastic disease and choriocarcinoma are
of concern following a CM, but neither has ever been
documented subsequent to a PM. Hormonal testing and
contraceptive counselling are of benefit to PM and CM
patients, but are unnecessary in patients with HA.
Recent cytogenetic evidence has shown that there are
major genetic differences between these pathologic entities. The PM has a triploid karyotype, 69,XXX or
69,XXY, 10 and as such, is genetically distinct from both
the CM which is diploid, 46,XX or 46,XY, and the HA
from a spontaneous abortion which commonly is also
approximately diploid.9 These findings have helped clarify the biologic distinctions that underlie the pathologic
categorization of hydropic gestations.'' However, the financial and temporal constraints of the karyotypic process limit its usefulness on a routine diagnostic basis. In
many tissues, DNA flow cytometry has been shown to
be a rapid and inexpensive technique for determining
nuclear ploidy.7 In view of the subjective nature of morphologic interpretations contrasted with the objective
nature of nuclear DNA content as analyzed by flow
cytometry, we undertook a study to evaluate whether
flow cytometry would be a reliable method of determining nuclear ploidy in placental tissues. In addition, we
Vol. 89 • No. 5
FLOW CYTOMETRY OF HYDATIDIFORM MOLES
wanted to assess whether these cytometric determinations would contribute to our histologic classification of
hydropic gestations.
Material and Methods
Placental tissues from 34 specimens were submitted
fresh for flow cytometric analysis of nuclear DNA content. Twenty cases consisted of all the tissues over a
one-year period that either had hydropic villi on gross
examination, or came with a clinical history of molar
gestation based on obstetric ultrasonography. Over the
same time interval, 14 controls were obtained from material destined to be discarded. Of these, ten were from
therapeutic abortions with no evidence of fetal or placental abnormality; three were from pregnancies terminated for reasons of fetal anomalies detected either on
ultrasound (one anencephalic) or by karyotype (two
Trisomy 21, one Trisomy 18); and one was from a spontaneous abortus in a patient with history of repeated
spontaneous abortions. Karyotype of this control was
46,XX. Karyotype analysis was performed on the hydropic specimens whenever possible. In all, 11 cases
with molar villi were submitted for karyotype analysis.
All cases were examined microscopically, and either the
entire specimen or at least ten blocks of tissue were submitted from each one with hydropic villi. Representative sections of the controls were examined. All histologic material was originally classified by senior staff
pathologists and subsequently reviewed independently
by one of us (J.M.L. or S.G.D.) without knowledge of
either the previous diagnosis rendered or the results
from either flow cytometry or karyotype analysis. One
of us (D.S.W.) analyzing theflowcytometric results was
kept unaware of the clinical history, gross findings and
microscopic diagnosis.
Flow Cytometry
Cell nuclear DNA content was determined by flow
cytometry using a modification of a method described
by Thornthwaite and associates.13 Fresh placental tissue
(approximately 0.1-0.5 g) was placed in a petri dish
containing 5-6 ml of nuclear isolation medium (NIM),
consisting of Hank's balanced salt solution (GIBCO
Laboratories, Grand Island, NY) with 0.2% bovine
serum albumin (Sigma Chemical Co., St. Louis, MO)
and 0.4% Nonidet (NP-40). Tissues were minced with
scalpels in NIM at room temperature for 1 to 2 minutes,
then filtered through 70 n nylon mesh (Nytex). Nuclei
were counted using a hemocytometer, and two aliquots
containing 1 X 106 and 0.5 X 106 nuclei each were
placed in plastic test tubes. To the second tube, 0.5 X 106
normal lymphocyte nuclei (prepared by suspending peripheral blood mononuclear cells in NIM) were added,
to a final volume of 0.5 ml in each tube. To each tube
597
were added 0.5 ml of propidium iodide (PI) solution (50
Mg/ml in NIM, Sigma) and 15 nl of RNase (final concentration 500 units/ml, ribonuclease A, bovine pancreas type III-A, Sigma). The cells were incubated at
room temperature for 20-30 minutes. If analysis was
delayed, the cells could be stored for 24 hours at 4 °C
without change in the DNA histogram. Immediately before analysis, the suspension was passed twice through a
26-g needle in order to disperse nuclear aggregates. Flow
cytometry was performed on a FACS Analyzer (Becton
Dickinson, Mountain View, CA) equipped with appropriate filters to excite fluorescence of PI at 488 nm and
to detect emission above 570 nm. Electronic volume
gates were left open in order to include all sizes of nuclei,
and cellular debris was excluded by using the PI fluorescence signal rather than the volume signal to trigger data
collection. Histograms were generated from 5,000 to
10,000 nuclei and displayed as linearfluorescence.DNA
histograms were generated for both placental nuclei
alone and placental nuclei with added control lymphocyte nuclei. For any DNA distribution, the euploid peak
was identified as that peak amplified by the addition of
normal control cells. The DNA index (DI) was calculated as the ratio of the placental Go/Gi peak channel to
that of normal lymphocytes, according to convention.4
A DI of 1.00 was assigned if a G0/Gi peak distinctly
different from normal lymphocytes was not detected.
Ideally, triploid DNA content would be detected as a
separate G0/Gi peak having DI = 1.50. The mean coefficient of variation (CV) of the G0/Gi diploid peak for the
31 specimens was 4.4%. The mean CV for the six triploid PM specimens was 3.9% for the G 0 /G| diploid
peak and 3.8% for the G0/Gi triploid peak.
Histology
The histologic diagnosis of HA was made on placental
tissues containing enlarged, swollen villi covered by a
thin and attenuated mantle of trophoblast. The histologic criteria for classification of PM have been well
described by Szulman and co-workers.10 Briefly, a PM is
a pathologic condition in which there are two populations of villi, some hydropic and some of normal size.
The villi are covered by focally hyperplastic trophoblast.
This hyperplasia involves principally the syncytium and
is the hallmark of a PM (Fig. 1). Additionally, evidence
of a fetus (fetal parts, nucleated erythrocytes in villous
vessels), scalloped villous outlines, and trophoblastic inclusions are often seen but are not pathognomonic of a
PM.10 CMs have diffusely hydropic villi which are
usually obvious grossly. Microscopic examination
shows enlarged, frequently cavitated villi covered by a
variable mixture of hyperplastic syncytiotrophoblast
and cytotrophoblast (Fig. 2). Although rarely seen, fetal
tissues are not usually present.
LAGE ET AL.
598
AJ.C.P. • May 1988
FIG. 1 (left). Cavitated villus of partial mole with trophoblast hyperplasia principally involving syncytium (arrow), Hematoxylin and eosin (X20).
FIG. 2 (right). Complete mole with cavitated villus evincing syncytial (S) and cytotrophoblast (C) hyperplasia, Hematoxylin and eosin (X20).
Results
Based on original microscopy of the 34 samples submitted for flow cytometry there were 10 CMs, 10 PMs,
and 14 normal placentas. Three of the PMs could not be
processed byflowcytometry: one was lost in transport to
the laboratory and in two specimens, the nuclei were too
autolyzed and fragmented for analysis of DNA content.
In all, 31 samples were analyzed by flow cytometry: 14
controls and 17 cases with grossly or ultrasonographically hydropic villi. Representative flow cytometric
DNA histograms are shown in Figure 3.
All placental specimens contained both maternal
(blood, endometrium) and fetal tissues, so that a component of normal diploid cells was present in all histograms. All 10 CMs as well as the 14 histologically normal placentas had a diploid DNA index of 1.0 (Table 1).
Of the seven cases with an original diagnosis of PM, five
had a triploid DNA index of 1.3-1.5 (Table 2). The
remaining two cases had a diploid DNA index of 1.0.
One had a confirmatory karyotype of 46,XX. Karyotype analysis had not been performed on the other case.
There was concordance between the flow cytometric
and karyotypic determinations of ploidy in all cases
where karyotypic results were obtained (two cases of
CM, five cases of PM, and three controls). Among our
cases of triploidy proven by karyotype analysis, the DI
ranged from 1.3-1.5 (Table 2). The range we observed
was partly due to instrument variability and deviation of
the sensing electronics from absolute linearity. Exami-
nation of nuclear suspensions which intentionally included aggregated control nuclei showed that the peak of
nuclear doublets was located at 1.8 to 1.9 times the
channel number of the singlet DNA value. The location
of the doublet peak varied depending on the channel
number of the antecedent diploid peak. A similar relationship was noted between the Gn/Gi peak and the
G2/M peak. No attempt was made to adjust mathematically for this nonlinearity since the abnormal peaks in
the triploid cases were near triploid in value and were so
easily distinguished from the diploid peak. If the purpose of this study had been to distinguish triploidy from
other types of aneuploidy, such a correction of the DI
would be warranted. No variation of the DI was observed during the generation of histograms and runs of
the same sample were always identical.
In four of the five PMs found byflowcytometry to be
triploid, chromosomal karyotypes confirmed that classification. Tissues from the fifth case of PM were contaminated in culture and failed to grow. That case had
fetal anomalies typical of triploidy including 3-4 syndactyly of the hands.3'14
Two of the seven specimens originally classified as
PM in this study showed a diploid DI of 1.0. One was
from a 25-year-old woman at 17 weeks by dates and 12
weeks by uterine size who was found on routine screening to have a low serum alpha fetoprotein. Subsequent
ultrasound examination showed a molar gestation. Preevacuation serum beta HCG was 263 mlU/ml. A curettage yielded approximately 12 ml of tissue including
FLOW CYTOMETRY OF HYDATIDIFORM MOLES
Vol. 89 • No. 5
rare, very small vesicular structures. Microscopy documented chorionic membrane, implantation site and
avascular, hydropic and sometimes necrotic villi with
focally thick, degenerating trophoblast mantles embedded in hyalinized tissue. No fetal tissues were seen. On
clinical follow-up beta HCG values fell spontaneously to
undetectable levels (less than 5 mlU/ml)1 within three
weeks postevacuation, remaining there for three subsequent biweekly determinations. This case most likely
represented a spontaneously regressing CM. The second
case histologically classified as PM with a subsequent
diploid DI of 1.0 was from a 23-year-old woman who
was 16 weeks by gestational age and whose uterus was
only 10 weeks by clinical examination. Obstetric ultrasonography showed a missed abortion with questionably hydropic villi raising the possibility of a PM. On
gross examination, an aggregate volume of nonvesicular
tissue measuring 5 X 4 X 4 cm was received. No fetal
599
Table 1. Comparison of Complete Hydatidiform
Moles, Partial Hydatidiform Moles and Controls
Histologic
Diagnosis
No. of
Cases
Complete
moles
Partial moles
Controls
Fetal
Tissue
DI
10
None
1.0
5
2
14
4 cases
None
All
1.3-1.5
1.0
1.0
Comment
46,XX (two cases)
Table 2
46,XX (one case)
Trisomy 21 (two cases)
Trisomy 18 (one case)
46,XX (one case)
tissues were found. Microscopy of the entire specimen
showed focally hydropic villi with inconspicuous trophoblast and a diagnosis of PM was made. Upon review
of this specimen the diagnosis was changed to HA based
on the absence of trophoblast hyperplasia.
Discussion
400'
-Go/Gl
Normal
Placenta
D. 1 = 1.00
t^A
S ,G2/M
200
4a
b.
Complete
Mole
D.I.= 1.00
3
1
o
40a
u
>\
1
T
p''-' 1 ' I
Partial
Mole
D.I. = 1.45
100
"^-n
Channel Number
FIG. 3. DNA histograms of (a) normal placenta, (b) complete mole,
and (c) partial mole. Ordinate indicates relative number of cells at each
channel of DNAfluorescence.Abscissa indicates fluorescence channel
number which stoichiometrically corresponds with DNA content.
Go/Gi peak contains noncycling diploid cells (G0) and cells resulting
from recent mitosis now in gap phase (Gi); S peak reflects cells synthesizing DNA for impending mitosis; and G2/M peak contains cells with
twice normal DNA composition (G2) and those undergoing mitosis
(M). Note: Triploid peak (arrow) on graph c.
Triploid conceptuses account for 1-2% of recognized
conceptions and about 20% of spontaneous abortions
with abnormal karyotypes.2 In one large series, 86% of
triploid placentas met histologic criteria for classification as PM.10
In a recent study designed to evaluate the intra- and
interpathologist variability in the routine diagnosis of
gestational trophoblast disease it was found that there
was only 75% agreement by referral pathologists with
the outside diagnosis of CM made by the submitting
pathologist.8 Furthermore, they found that the variability between the two study pathologists at the referral
center for the diagnosis of incomplete (partial) mole was
quite high, with one pathologist diagnosing 10 cases as
PM and the other diagnosing these same 10 cases as 3
CM's and 7 "not gestational trophoblast neoplasia."8 An
earlier study performed in England found similar results
on the interpathologist variability in the diagnosis of
CMs. In that study there was only 55% agreement between two pathologists in the diagnosis of CMs.6
In our series there was concordance between the flow
cytometric and karyotypic determinations of ploidy. All
our cases of CM had grossly distended villi and a diploid
DI of 1.0. Five of seven cases classified as PM had distinct triploid DNA peaks with the DI for those cases
ranging from 1.3-1.5. All five of these cases also had
grossly visible vesicles. All of our control placentas had a
diploid DI of 1.0.
The difficulty in diagnosis of PM was illustrated by
our remaining two cases which were originally diagnosed as PM by experienced obstetric pathologists. One
case had no vesicles grossly and the other only small
vesicles. Both had a diploid DI of 1.0, one of which was
confirmed by tissue karyotype. Upon review, one case
was reclassified as a HA and the other as a regressing
600
AJ.CP.-May 1988
LAGE ET AL.
Table 2. Partial Hydatidiform Moles by Histology Submitted for Flow Cytometry
Gross Exam
Histologic Diagnosis
Karyotype
Dl
Fetal parts
Fetal anomalies
No fetus
Fetal anomalies
Fetal anomalies
No fetus
No fetus
PM
PM
PM
PM
PM
PM
PM
69,XXX,-7,+t(7;9)/69,XXX
Contaminated
69,XXX
69,XXX
69,XXX
46,XX
Not performed
1.3
1.5
1.43
1.44
1.45
1.0
1.0
CM. Follow-up in both cases showed spontaneous resolution of HCG values to undetectable levels.
Our findings of two cases originally classified as PM
which subsequently showed a diploid DNA content
suggests that the diagnosis of PM even by experienced
obstetric pathologists may be more difficult than is generally suggested in the literature. There have been a few
rare case reports of diploid PMs12 and a single case of
PM with Trisomy 2.5 Upon review of our two cases of
PM with diploid DI, the diagnosis of PM was not substantiated histologically. Knowledge of the results of
nuclear ploidy analysis would have raised the possibility
of a possible misclassification, suggesting reconsideration of the diagnosis.
Subsequent to this series, we have seen a recent case in
which flow cytometry was useful in a confirmatory role.
On gross examination this specimen had hydropic villi
and no fetal parts. Microscopy showed diffuse and extensive trophoblast hyperplasia involving nearly all the
villi. There were, however, rare normal appearing villi.
On this basis, though with some hesitation, we made the
histologic diagnosis of PM. Flow cytometry demonstrated that this difficult case was indeed triploid.
We conclude that ploidy determinations are a useful
adjunct in diagnosing molar placentas and that flow cytometry is an accurate means of making those determinations. In this hospital the cost of flow cytometry is
one-eighth that of a cytogenetic study, and the results
are available within a few hours of obtaining the fresh
specimen rather than in the 2-4 weeks generally required for chromosomal analysis. However, we are not
suggesting that determination of ploidy is a substitute
for histologic diagnosis. At this stage it is best regarded as
a useful tool to quickly confirm the initial histologic
impression or, in cases of discordance between the histologic diagnosis and the results of flow cytometry, to suggest reexamination of the specimen and submission of
additional tissue if necessary.
Increasing the accuracy of classification of gestational
trophoblastic lesions has benefits beyond the immediate
therapeutic advantage that accrues to each individual
patient. Nondifferential misclassification decreases the
validity of statistics gathered on gestational trophoblastic disease and reduces the power of any study searching
Miscellany
Degenerating mole
No vesicles
Reclassified as hydropic abortus
for an association between antecedent factors and hydatidiform moles. Furthermore, it may be that not all
molar placentas are either diploid CMs or triploid PMs.
Perhaps there is more than one biologic aberration underlying what we presently designate as the PM. Careful
characterization of these tissues along all the relevant
dimensions reasonably obtainable will help us detect
any residual variability. Only then may one meaningfully correlate the pathological findings with some biologic marker of abnormal behavior and decide whether
there is yet another group of anomalous molar gestations hiding within the rubric of our present system of
classification.
References
1. Berkowitz RS, Goldstein DP, Bernstein MR: Natural history of
partial molar pregnancy. Obstet Gynecol 1983;66:677-681.
2. Boue J, Boue A, Lazar P: Retrospective and prospective epidemiologic studies of 1500 karyotyped spontaneous human abortions. Teratology 1975;12:11-26.
3. Doshi N, Surti U, Szulman AE: Morphologic anomalies in triploid
liveborn fetuses. Hum Pathol 1983;14:716-723.
4. Hiddemann W, Schumann J, Andreeff M, et al: Convention on
nomenclature for DNA cytometry. Cytometry 1984,5:445456.
5. Honore LH, Dill FJ, Poland BJ: The association of hydatidiform
mole and trisomy 2. Obstet Gynecol 1974;43:232-237.
6. Javey H, Behmard S, Langley FA: Discrepancies in the histological diagnosis of hydatidiform mole. Br J Obstet Gynaecol
1979;86:480-483.
7. Lovett EJ, Schnitzer B, Keren DF, Flint A, Hudson JL, McClatchey KD: Application of flow cytometry to diagnostic pathology. Lab Invest 1984,50:115-140.
8. Messerli ML, Parmley T, Woodruff JD, Lilienfeld AM, Bevilacqua L, Rosenshein NB: Inter- and intra-pathologist variability
in the diagnosis of gestational trophoblastic neoplasia. Obstet
Gynecol 1987;69:622-626.
9. Shepard TH, Fantel AG: Embryonic and early fetal loss. Clinics
Perinatal 1979;6:219-243.
10. Szulman AE, Philippe E, Boue JG, et al: Human triploidy: Association with partial hydatidiform moles and nonmolar conceptuses. Hum Pathol 1981;12:1016-1021.
11. Szulman AE, Surti U: The clinicopathologic profile of the partial
hydatidiform mole. Obstet Gynecol 1982;59:597-602.
12. Teng NNH, Ballon SC: Partial hydatidiform mole with diploid
karyotype: Report of 3 cases. Am J Obstet Gynecol
1984;150:961-964.
13. Thornthwaite JT, Sugarbaker EV, Temple WJ: Preparation of
tissues for DNA flow cytometric analysis. Cytometry
1980;1:229-237.
14. Wertelecki W, Graham JM, Sergovich FR: The clinical syndrome
of triploidy. Obstet Gynecol 1976;47:69-76.