Assessment of cells in the ascitic fluid of women with ovarian hyperstimulation syndrome: the clinical implications for subsequent ovarian malignancy
Reproductive Biology and Endocrinology
Assessment of cells in the ascitic fluid of women with ovarian hyperstimulation syndrome: the clinical implications for subsequent ovarian malignancy
Ioannis Hatzipetros 0
Peter M Gocze 0
Katalin Cziraky 1
Kalman Kovacs 0
Endre Kalman 1
Balint Farkas 0
0 Department of Obstetrics and Gynaecology, University of Pecs, Clinical Centre , Edesanyak Str. 17, 7624 Pecs , Hungary
1 Department of Pathology, University of Pecs , Pecs , Hungary
Background: Although some studies have reported a potential connection between ovulation induction therapy (OIT) and malignant ovarian diseases, the results have been inconclusive. In the present study, we sought to determine whether women undergoing OIT at our in vitro fertilization (IVF) clinic, especially those with severe ovarian hyperstimulation syndrome (OHSS) and suspicious cytologic findings, were at risk for developing malignant ovarian tumours after treatment. Methods: Patients who underwent OIT at our IVF clinic were enrolled in this study and assessed for any evidence of malignant ovarian tumours. Patients who developed severe OHSS as a result of OIT were treated with a culdocentesis. Cells from the ascitic fluid were cytologically scored for abnormality and malignancy. Peripheral blood samples were obtained from patients with severe OHSS to determine serum levels of the tumour markers (CA-125 and HE4) that were used to calculate the Risk for Ovarian Malignancy Algorithm (ROMA) index. Results: Follow-up data were available for 1,353 of the 1,587 patients (85%) who underwent OIT at our IVF clinic between January 2006 and December 2012. Twenty-three patients (1.4%) were hospitalized with OHSS. Culdocentesis was performed 16 times in nine patients with severe OHSS (age range, 23-34 years; mean, 27.1 years). Although cytological examination of the ascitic cells of these patients suggested malignant ovarian neoplasia, over the course of the observation period, the ovarian volume gradually decreased and became normal. Subsequent cytological and histological examinations failed to find evidence of any malignant tumours in these nine patients. None of the 1,353 participants who underwent OIT developed any malignant ovarian tumours during the study period. Moreover, none of the 462 patients who were in our ovarian tumour registry were also participants in the IVF program. Conclusions: The presence of atypical cells in the ascitic fluid of women with severe OHSS does not likely indicate malignancy; therefore, radical surgical intervention is not justified. The risk of malignancy is minimal shortly after OIT. At our centre, OIT has not been associated with any cases of ovarian tumour.
Ovarian hyperstimulation syndrome (OHSS) is an
iatrogenic complication of ovarian induction therapy (OIT)
that may be observed after stimulation with human
chorionic gonadotropin or after the spontaneous
luteinizing hormone peak. In OHSS, increased vascular
permeability leads to subsequent fluid accumulation,
especially in the abdominal cavity [
], with symptoms
appearing 5 to 10 days after gonadotropin
administration. The risk factors for OHSS include young age,
polycystic ovarian syndrome, and a medical history of
hyper-response to gonadotropins [
]. Early prediction of
OHSS is crucial for prompt treatment. Diagnostic
measures for predicting OHSS include an antral follicle
count of ≥14 on a transvaginal ultrasound (82%
sensitivity and 89% specificity) [
] and a basal anti-Müllerian
hormone serum level of ≥ 3.5 ng/mL (90.5% sensitivity,
81.3% specificity) [
There are three forms of OHSS that can be
distinguished by clinical signs and laboratory findings. Mild
OHSS is a relatively common side effect of controlled
ovarian stimulation that affects up to one-third of
patients undergoing in vitro fertilization (IVF). Moderate
and severe forms of OHSS have a combined incidence
ranging from 3% to 8% [
]. The clinical consequences of
mild and moderate OHSS are very minor. However,
severe OHSS is a potentially life-threatening condition
with symptoms that include ovarian enlargement,
hydrothorax, hemoconcentration, salt and water dysregulation,
oliguria, thromboembolic disease, and coagulation
abnormalities. Approximately 1.4% of OHSS cases are
severe, and severe OHSS is associated with a mortality risk
of 1 in 450,000 to 500,000 [
Several studies have suggested a possible connection
between OIT and ovarian tumours. For example,
researchers have observed cases of struma ovarii [
], serous papillary carcinoma [
], serous papillary cystadenoma [
epithelial ovarian carcinoma [
], and cystadenocarcinoma
] during and/or after OIT. In a case–control study
performed in Israel between 1990 and 1993, Shushan
et al. [
] concluded that OIT with human menopausal
gonadotropin might increase the risk of epithelial ovarian
malignancies, specifically borderline ovarian tumours.
However, in some studies, hyperstimulation-induced
reversible histological changes may have been grouped with
malignant disease [
In light of the potential confounders present in
previous data, further research is needed to clarify the
relationship between OIT and ovarian malignancy. The aim
of this study was to determine whether women
undergoing OIT at our IVF clinic, especially those with severe
OHSS and suspicious cytologic findings, were at risk for
developing malignant ovarian tumours after treatment.
Cells from the ascitic fluid recovered from patients with
severe OHSS were characterized to determine if they
were cytologically abnormal and whether the cytology
indicated the presence of an ovarian malignancy.
Patients and study design
This prospective study was approved by the University
of Pecs Institutional Ethical Review Board. Patients were
included in this study if they were treated with OIT at
the Clinical Centre of the University of Pecs Department
of Obstetrics and Gynaecology/Reproductive Centre
between January 2006 and December 2012 and provided
their written informed consent to participate. Patients
were questioned in person or surveyed by a mailed
questionnaire about any current or past treatments for
malignant ovarian tumours.
Evaluation of the abdominal fluid
From January 2006 to December 2012, nine IVF clinic
patients developed severe OHSS. Ovarian hyperstimulation
was classified into three grades according to the severity
of the symptoms, signs, and laboratory findings (Rizk and
Aboulghar, 1999). These patients were treated with
standard drug therapies, including a macrolide, intravenous
fluids, clexane, and aspirin, as well as
ultrasoundcontrolled culdocentesis [
]. During the culdocentesis,
ascitic fluid was obtained from these patients for further
The ascitic fluid was placed in a centrifuge tube on ice
and centrifuged at 400 × g for 10 minutes. Most of the
fluid was decanted, and the resulting pellet was
suspended with the remaining small amount of ascitic fluid
using a shaker. A smear was made according to the
usual procedures. The smear was fixed for 30 to 60
minutes in a 1/1 (v/v) mixture of ether and ethyl alcohol.
After dehydration, the smears were stained with
GIEMSA, evaluated by the Papanicolaou method [
and analysed in the Clinical Cytological Laboratory. The
samples were then analysed with visual light microscopy.
The presence of abnormal cells was based on the
assessment of the cellular shape (flat, sheet-like appearance
and well-defined borders) and nuclear and nucleolar size
differences in comparison to normal cells. The
histological examination of the ovaries was carried out at the
Department of Pathology of the Clinical Centre of the
University of Pecs. The following cytologic grading
system was used: P I, no abnormal or atypical cells; P II,
atypical cells present, but with benign cytological
appearance; P III, atypical cells suspicious for malignancy;
P IV, cells diagnostic for malignancy; and P V, a large
numbers of malignant cells.
Measurement of serum levels of tumour markers
Peripheral blood samples were obtained from patients
after the diagnosis of severe OHSS but before any
interventions were performed. The serum concentrations of
CA-125 (Fujirebio Diagnostics, Malvern, PA, USA;
Catalogue # 400–10, Lot # 29192) and HE4 (Fujirebio
Diagnostics; Catalogue # 404–10, Lot # 28374) were determined
by a quantitative sandwich enzyme-linked immunosorbent
assay (ELISA) according to the manufacturer’s protocol.
Serum concentrations were calculated with the Optima
2.10 R2 built-in data calculator software.
Risk for Ovarian Malignancy Algorithm (ROMA) index
The ROMA index is based on the serum levels of HE4
and CA-125 as measured by ELISA or calculated with
an Excel spreadsheet using pre-set formulas to generate
the predictive index (PI) for epithelioid ovarian cancer
according to the following equation for premenopausal
women: PI = −12 + 2.38 × ln [HE4] + 0.0626 × ln
[CA125]. The ROMA value is calculated as follows:
ROMA value (%) = exp (PI) / [1 + exp (PI)] × 100.
According to the manufacturer’s manual (Fujirebio
Diagnostic Inc., Malvern, PA, USA), a ROMA index equal to
or greater than 13.1% is associated with a high risk of
epithelioid ovarian cancer in premenopausal women [
Between January 2006 and December 2012, a total of
1,587 patients underwent OIT in 4,892 cycles at our IVF
clinic. Of these patients, 23 (1.4%) were hospitalized with
severe OHSS. We obtained follow-up data from 1,353
(85%) patients who underwent OIT and all 23 who
developed OHSS. Of the 1,353 patients who underwent
OIT at our clinic and were followed-up in this study,
none developed a malignant ovarian tumour during the
study period. A review of the local institutional registry
revealed that none of the 462 registered patients with
malignant ovarian tumours had participated in our IVF
Nine of the 23 patients who developed OHSS
underwent culdocentesis for severe OHSS. These patients
ranged in age from 23 to 34 years old (mean, 27.1 years).
Ascitic fluid was obtained from these patients for further
analysis. The cytological findings for these patients
suggested the presence of ovarian malignancy (Table 1) with
cytologic grades of P III and P IV in four cases each and
intermediate findings (P III - P IV) in one case (Figure 1).
None of the nine patients had ultrasonographic evidence
of a possible ovarian tumour before starting treatment;
therefore, we did not perform immediate surgical
intervention or histological sampling and elected to follow
the patients with supportive therapy.
Figure 1 shows a photograph from a representative
aspiration from case no. 2, a 23-year-old woman who
underwent OIT in preparation for homologous insemination.
Cells sampled from the ascitic fluid were graded as P IV.
In all cases, the volume of the ovarian ascitic fluid
gradually decreased. To ensure that no malignancy existed, a
laparoscopic examination was performed at 8 to 12 weeks.
After close inspection of the abdominal cavity, eluents
from the Douglas pouch were sampled and histological
samples were obtained from the ovaries. All cytological
and histological tests of these follow-up examinations
were benign (Table 1). Follow-up laparoscopies were not
performed for these three patients, who were instead
followed clinically. During the follow-up period, none of
the nine patients displayed signs of an ovarian malignancy.
Peripheral blood serum levels of CA-125 and HE4
tumour markers were also evaluated for the nine
patients with severe OHSS. The mean (± SD) value of
CA125 was increased (105.81 ± 161.55 U/mL) compared to
the reference range of 0 to 39 U/mL. However, the mean
serum level of HE4 (42.89 ± 4.88 pM) was within the
normal range of 0 to 150 pM. The ROMA predictive
index was determined based on the concentrations of
these two tumour markers and the subject’s
premenopausal status. The ROMA index was very low (5.63% ±
1.24%), which indicated that the patients were not at a
high risk for developing ovarian malignancies (Table 1).
Our data over a 6-year period indicate that there is no
relationship between OIT and subsequent malignant
ovarian tumour development among patients at our
centre. Although the cytologic results of the ascitic cells
The dates of the Douglas puncture and control histology tests are shown.
OIT Ovarian induction therapy, CC Clomiphene citrate, hMG Human menopausal gonadotropin, hCG Human chorionic gonadotropin, GnRH-a
Gonadotropinreleasing hormone analogue, FSH Follicle-stimulating hormone.
*See Methods for a description of the cytologic stages P I-P V.
#Negative histology, normal ovarian tissue.
The reference value for CA-125 is 0–39 U/ml and for HE4 is 0–150 pM.
from patients with severe OHSS were initially suggestive
of malignancy, these patients did not develop any
evidence of malignant ovarian tumours.
Because the risk of malignant ovarian tumours in is
higher in nulliparous women, greater vigilance is
necessary when treating this group. Bimanual examination
and vaginal ultrasonography are essential. The best
method to examine for enlarged ovaries is
colourDoppler ultrasound. Rarely, laparoscopy or laparotomy
may be necessary. Therapy should only begin after
malignant ovarian tumours have been ruled out and/or
benign tumours have been removed.
Tumour markers, although useful, cannot differentiate
ovarian enlargement caused by overstimulation from
enlargement due to a malignancy. For example, the serum
CA-125 level closely correlates with the volume of the
ovary and is not indicative of the underlying pathology
]. Previous studies found no statistically significant
differences when comparing serum CA-125 levels
between spontaneous and stimulated cycles or between
pregnant and non-pregnant patients [
follow-up of patients with sequential determinations of
their tumour markers may be helpful for an accurate
assessment. During the follow-up of patients with
hyperstimulated ovaries, the serum concentration of the
CA125 tumour marker declines and eventually normalizes. In
patients with malignant disease, the serum levels remain
elevated or gradually increase.
To overcome the relatively low specificity and
sensitivity of risk assessment by a single tumour marker, Moore
et al. introduced the ROMA index as an accurate
predictive index for ovarian cancer (76.4% sensitivity and
96% specificity) [
]. Our results confirmed that the
single biomarker determination of CA-125 was not
sufficient to reliable evaluate ovarian malignancy in OHSS.
When the combination of CA-125 and HE4 was used,
despite the high levels of CA-125, HE4 remained under
the reference value and indicated no obvious signs of
malignancy. This observation was demonstrated by the
low ROMA scores (Table 1).
Epidemiologic follow-up data of infertile patients
demonstrates an increased life-long risk for high-grade
or borderline malignant ovarian malignancies. However,
the exact reason for this increased risk is unclear.
Whereas some authors believe it is a result of the
infertility itself [
], others suggest that ovulation induction
is associated with cancerogenesis [
patients undergoing OIT may be at an increased risk of
developing ovarian tumours, studies have shown
reassuring results in terms of hormone treatment and
the incidence of invasive epithelial ovarian cancer .
However, exogenous hormone treatment is associated
with an increased risk of borderline ovarian malignancy
]. OIT has not been shown to increase the risk of
breast, uterine, or invasive ovarian cancers, although the
risk of borderline ovarian tumours might increase [
Moreover, the risk of cancer has been shown to be
similar in children conceived by artificial reproductive
therapies and those conceived naturally [
]. It should also
be noted that, due to close medical surveillance,
malignancies are overdiagnosed in the female population; this
may also augment the early detection of cancers [
Our data suggest that even when the cytological
evaluation of ascitic cells obtained in patients diagnosed with
OHSS indicates abnormality and possible malignancy,
radical surgical intervention is not clinically indicated.
Instead, these patients should be closely followed and
monitored. If the ovarian size remains abnormal, then
the aetiology of the enlargement should be determined
by histological sampling via laparoscopy, and the
histologist should be informed of the previous OIT. Surgery
may still be required for abdominal bleeding, ovarian
torsion or rupture, or extra-uterine pregnancy.
We observed a minimal risk of ovarian malignancy shortly
after OIT at our IVF clinic. Large population-based studies
will be required to determine if ovarian induction is
associated with tumourigenesis over the long-term.
The authors declare that they have no competing interests.
IH has made substantial contributions to the conception and the design of
this study. EK carried out the histopathologic analysis of the ascitic samples
along with KC, who also participated in the acquisition of data and the
pathologic assessments. KK provided the clinical data of the patients who
participated in the assisted reproduction program and helped in the
statistical analysis. BF participated in the design of the study and carried out
the immunoassays; in addition, he has also been involved in drafting the
manuscript. PMG conceived the study, participated in its design and
coordination, and helped to draft the manuscript. All authors read and
approved the final manuscript and have given final approval of the version
to be published.
We wish to thank the staff of the Reproduction Centre of the University of
Pecs, the Department of Obstetrics and Gynaecology, and the In Vitro
Fertilization Laboratory, especially Dr. A Varnagy, for allowing us to obtain
data and documentation. We thank Dr. B Barna for the histological
photographs. This work was supported by a Hungarian National Research
Grant (OTKA T 023656) and private funding (to IH). We also wish to thank
the editors of Elsevier English Language Editing for their professional
reediting of this manuscript.
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