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Institut de Recherches Interdisciplinaires en Biologie Humaine et Moléculaire (C.D., G.S., V.d.M., S.C., G.V.) and Laboratory of Research on Human Reproduction (Y.E., A.D.), Faculté de Médecine; and Fertility Clinic (Y.E., A.D.) and Medical Genetics Department (G.S., G.V.), Hôpital Erasme, Université Libre de Bruxelles, B-1070 Brussels, Belgium
Address all correspondence and requests for reprints to: Dr. Anne Delbaere, Clinique de Fertilité, Hôpital Erasme, 808 Route de Lennik, B-1070 Brussels, Belgium. E-mail: adelbaer{at}ulb.ac.be.
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Abstract |
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Introduction |
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Spontaneous forms of OHSS are very rare and are always reported during pregnancy. We and another group have recently described three different mutations in the FSH receptor (FSHr) of patients presenting with recurrent severe spontaneous OHSS (8, 9, 10). In vitro, the mutant receptors displayed abnormally high sensitivity to hCG, providing a satisfactory pathophysiological explanation for their implication in OHSS development in vivo. FSHr are usually not stimulated or are only very weakly stimulated during pregnancy, because pituitary gonadotropins fall to very low or undetectable levels in serum. In spontaneous OHSS patients, the mutated FSHr expressed in the developing follicles will be hyperstimulated by the pregnancy-derived hCG. Accordingly, the follicles will start growing, enlarge, and finally acquire LH/CG receptors on granulosa cells, which will also be stimulated by hCG, inducing follicular luteinization together with the secretion of vasoactive molecules responsible for the development of the syndrome (8, 11). Although these mutant FSH receptors are more sensitive to hCG than the wild-type FSHr, (8, 10), high concentrations of hCG are needed for their activation. It is likely that the effective threshold level of hCG will vary according to the type of mutation (11). hCG usually peaks between 8 and 10 wk gestational age (GA) and declines thereafter. Accordingly, the symptomatology of most spontaneous cases of OHSS develops after 8 wk of amenorrhea, culminating at the end of the first trimester of pregnancy.
In iatrogenic OHSS, the follicular recruitment phase is performed through ovarian stimulation with exogenous FSH. Thereafter, the numerous mature follicles undergo luteinization as a result of ovulation induction with exogenous hCG. These differences explain the early appearance of the symptoms in iatrogenic OHSS (between 3–8 wk GA) compared with spontaneous OHSS (between 8–13 wk GA) (8, 11).
However, spontaneous and iatrogenic OHSS share similar pathophysiological sequences: massive recruitment and growth of ovarian follicles, extensive luteinization, and oversecretion of vasogenic molecules (e.g. vascular endothelial growth factor and angiotensin) by luteinized corpora lutea, provoking a third space fluid shift (8, 11).
Both hCG and vasoactive mediators involved in the development of OHSS are essential for human pregnancy (12). An direct etiological treatment of OHSS is thus impossible, and conservative management of the syndrome is the general practice. Therefore, research should concentrate on the prevention of iatrogenic OHSS. Risk factors have been described for the development of the syndrome in IVF patients, such as young age, the presence of polycystic ovarian syndrome, an explosive response to gonadotropin stimulation [e.g. rapid increase of serum estradiol (E2) levels], and the development of multiple follicles (>20) before induction of ovulation (1, 2). However, there are no absolute predictive factors of the syndrome. Furthermore, prevention measures, including cancellation of the cycle or cryopreservation of the embryos, are usually decided at the end of the ovarian stimulation and are often accompanied by emotional distress. In consequence, we are facing the challenge of identifying patients likely to respond excessively to the ovarian stimulation before starting the treatments so as to adapt their treatment scheme.
Considering the pathophysiological sequence of OHSS, prevention of the syndrome could be addressed by identifying potential hyperresponders to FSH stimulation or potential hypersecretors of vasogenic molecules. Recent reports suggested that the FSHr genotype was associated with the ovarian response to FSH stimulation (13, 14, 15). These observations together with the identification of FSHr mutations in spontaneous OHSS led us to test whether coding polymorphisms of the FSHr (A307T and S680N) (16) could be associated with the development of iatrogenic OHSS.
No statistically significant differences were found in allelic and genotypic frequencies between the IVF control population and the OHSS patients. However, a significant enrichment in allele N680 was observed as the severity of OHSS increased (P = 0.034). These results suggest that the genotype in position 680 of the FSHr cannot predict which patient will develop OHSS, but could be a predictor of the severity of symptoms among OHSS patients.
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Subjects and Methods |
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Thirty-seven Caucasian patients, between 24 and 40 yr of age, presenting OHSS after multiple follicular growth for IVF at the Fertility Clinic of Erasme Hospital were included in the study. Depending on the severity of the symptoms, patients were classified by a single clinician into three groups according to the classification of Golan (3): 11 patients presented mild OHSS, 13 patients had moderate OHSS, and 13 patients had severe OHSS. Another group of 130 female Caucasian patients who attended our fertility clinic and were treated by IVF without developing OHSS served as the IVF control population. Ninety-nine women who did not attend the fertility clinic served as Caucasian controls. Patients originating from Africa, Asia, and Latin America were excluded from the study. A 10-ml EDTA blood sample was obtained from all subjects for DNA analysis. The study is part of a clinical research project approved by the ethics committee of Erasme Hospital.
Ovarian stimulation protocol
Ovarian stimulation for IVF was performed with GnRH agonists and gonadotropins using a long protocol as previously described (17). Briefly, pituitary desensitization was achieved by intranasal administration of buserelin acetate (Suprefact, Hoechst, Germany). Ovarian stimulation was performed with human menopausal gonadotropin (Humegon, Organon, The Netherlands; or Menopur, Ferring, Denmark). Follicular development was monitored by transvaginal sonography and serum E2 levels. Ovulation induction was achieved by the administration of 10,000 IU hCG (Pregnyl, Organon, The Netherlands) when at least two follicles reached 18–20 mm in diameter. Oocyte retrieval was performed through vaginal puncture under ultrasound guidance 36 h later. Luteal phase support consisted of daily im injections of 100 mg progesterone in oil (Sterop, Brussels, Belgium) or daily intravaginal administration of 3 x 200 mg micronized progesterone (Utrogestan, Besins International, Paris, France), starting the day after the oocyte retrieval.
Hormonal assays
E2 and progesterone (P) were measured by immunoassays (kit module E170, Basel, Switzerland). Intra- and interassay variations did not exceed 4% for E2 and 5% for P.
Genotyping
The genotypes were determined by DNA sequencing. DNA was extracted from a 10-ml EDTA blood sample using standard procedures. Regions encompassing A307T and S680N polymorphisms of exon 10 of the FSHr were amplified by PCR using Taq DNA polymerase (Invitrogen Life Technologies, Merelbeke, Belgium) and the following primers: FSHr exon10A forward, 5'-GCTATACTGGATCTGAGATG-3'; FSHr exon 10A reverse, 5'-ACCACTTCATTGCATAAGTC-3' (for nucleotide 919 genotyping; codon 307); and FSHr exon 10Dnew forward, 5'-ACATCGTGTCCTCCTCTAGTG-3'; FSHr exon 10Dnew reverse 5'-AATGTGTAGAAGCACTGTCAGC-3' (for nucleotide 2039 genotyping; codon 680). Purification of the amplification products was performed with the enzyme ExoSAP-IT as recommended by the manufacturer (U.S. Biochemical Corp., Cleveland, OH). Sequencing of the purified amplification products was realized using BigDye Terminator technology according to the manufacturer’s recommendations (Applied Biosystems, Foster, CA).
Statistics
Differences between groups were analyzed with the Kruskal-Wallis test in the case of continuous variables. The Pearson’s 
2 test was used for testing independence between unordered discrete variables; the P value was calculated by the asymptotic method or by the exact method when required. The Kruskal-Wallis test was used for detecting a difference in allelic or genotypic frequencies of codons 307 and 680 among the three ordered (mild, moderate, and severe) OHSS groups. Two-sided tests were used. Computations were performed with StatXact 5 (Cytel Software Corp., Cambridge, MA). P < 0.05 was considered statistically significant.
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Results |
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and 2.
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). The frequencies of genotypes at position 680 were 17% SS, 45% SN, and 38% NN for Caucasian controls; 25% SS, 51% SN, and 24% NN for IVF controls and 32% SS, 54% SN, and 16% NN for OHSS patients. Again the difference reached statistical significance between Caucasian and IVF controls (P = 0.046) and between Caucasian controls and OHSS patients (P = 0.035), but not between IVF controls and OHSS patients (P = 0.599; Table 3).
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). Allelic and genotypic frequencies for codon 307 of FSH according to the clinical severity of the OHSS are shown in Table 5. Allelic and genotypic frequencies for codon 680 of FSH according to the clinical severity of the OHSS are shown in Table 6. Allelic frequencies were 73% S and 27% N for the mild OHSS group, 58% S and 42% N for the moderate OHSS group, and 42% S and 58% N for the severe group (Table 6). These allelic frequencies were significantly different among the three groups (P = 0.035). In two by two comparisons, the difference in allelic frequencies (i.e. enrichment in N680 or impoverishment in S680) observed between mild and severe groups was statistically significant (P = 0.034), but it was not significant between mild and moderate or between moderate and severe groups (P = 0.278 and 0.267, respectively). The genotype frequencies were 46% SS, 55% SN, and 0% NN for the mild OHSS group; 31% SS, 54% SS, and 15% NN for the moderate group; and 15% SS, 54% SN, and 31% NN for the severe group. Comparison among the three groups did not reach statistical significance (P = 0.072) nor did the difference between mild and severe groups (P = 0.093). The difference between mild and moderate or between moderate and severe groups was not significant (P = 0.581 and 0.522, respectively).
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).
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Discussion |
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60% and 40%, respectively), and A307N680 and T307S680 are rare haplotypes (1% for each) (16). The presence of a serine in position 680 has been associated with higher levels of basal FSH on d 2–4 of the menstrual cycle and higher requirements of exogenous FSH for ovarian stimulation, suggesting that an FSHr with a serine in position 680 could be less efficient than an FSHr with an asparagine in that position (13, 14). A retrospective study in IVF patients has shown an association between the presence of Ser in position 680 and poor responses to gonadotropin stimulation, suggesting that the S680 allele was associated with a diminished sensitivity to FSH (15). However, in clomiphene citrate-resistant normogonadotropic anovulatory patients, altered ovarian sensitivity to exogenous FSH during ovulation induction could not be established (20). The potential association of the S680 allele with poor responders to ovarian stimulation for IVF led us to hypothesize that the N680 allele could be associated with hyperresponders, that is, patients at risk of iatrogenic OHSS.
The frequencies of the polymorphism at position 680 in our Caucasian control population (99 patients) were identical to previous reports (16). The control IVF population (130 patients) was enriched in the S680 allele compared with the Caucasian control population, with allelic frequencies at position 680 comparable to those observed in a population of ovulatory IVF patients (13). Our control IVF population was selected only on the basis that it did not develop OHSS after ovarian stimulation; therefore, it includes both ovulatory and anovulatory patients. The OHSS population (37 patients) tended to be even more enriched in the S680 allele than the control IVF population (although the difference from the IVF control population was not statistically significant), which was unexpected, because a higher prevalence of S680 allele was previously found in poor responders to ovarian stimulation (15). Confronted with this paradox, we studied the frequencies of the alleles according to the severity of OHSS. A significant difference in the allelic frequencies at position 680 (i.e. enrichment in N680 and impoverishment in S680) was observed among the three groups as the severity of OHSS increased (P = 0.035). This difference persisted when the analysis was performed among mild, moderate, and severe OHSS patients who were pregnant (P = 0.003).
The in vivo association of S680 with higher levels of basal FSH on d 2–4 of the menstrual cycle has not been explained in molecular terms (13, 14, 16). In vitro functional studies of both isoforms (AS or TN) could not show any difference in binding characteristics or in cAMP or inositol phosphate production upon FSH stimulation (14, 21). However, the S680N polymorphism is located in a portion of the C-terminal tail important for recycling of the human FSHr (22), and S680 is a potential site of phosphorylation. As previously suggested (13), a difference in receptor trafficking could explain the observed genetic association with poor responders to FSH stimulation, but this hypothesis has yet to be tested in vitro. Nevertheless, if such a difference exists, it is surprising that the frequency of the S680 allele was increased among OHSS patients, because they are characterized by a strong response to ovarian stimulation. The slight enrichment in rare haplotypes in the OHSS population cannot explain this increase in S680, because enrichments in both AN and TS chromosomes were observed. A possible explanation is that the S680 allele constitutes a risk factor for an abnormal response to a standard stimulation treatment, with the hypo or hyper character of the response depending on polymorphisms of other genes, i.e. the genetic background of each patient (23). A higher prevalence of the N680 allele was observed in severe forms of OHSS. It is generally considered that the severity of the syndrome depends on the number of granulosa cells and the capacity of luteinized granulosa cells to secrete vasogenic factors. Because the number of punctured follicles and serum E2 levels were comparable among the three groups, a difference in granulosa cell number is a priori very unlikely. This suggests that the S680N polymorphism could influence the secretion capacity of luteinized granulosa cells. It has been suggested that stimulation of the FSHr could increase the level of LH/CG receptor mRNA production via modulation of the protein kinase B pathway (24). The presence of an S or an N in position 680 of the FSHr could potentially modulate this pathway. It is also possible that the S680N polymorphism does not play any direct functional role in the development of OHSS, but is in linkage disequilibrium with other polymorphisms, which could account for our observations. Finally, one limitation of the present study is that because of the small sample size, there is a possibility of sampling biases.
From a clinical point of view, our OHSS population was typical, including young patients (mean age, 31 yr), a high frequency of polycystic ovarian syndrome (46%), a large number of follicles punctured (mean, 28), and high preovulatory E2 levels [mean, 3,897 pg/ml (14,300 pmol/liter)] (1, 2). All OHSS patients were recruited in the same fertility clinic, which limits variations in IVF stimulation protocols, OHSS diagnosis, and clinical evaluation. Interestingly, the unique difference we observed among mild, moderate and severe groups was the frequency of the alleles in position 680.
In conclusion, and bearing in mind the limitations due to the retrospective character of the study and to possible sampling biases, these results suggest that the genotype in position 680 of the FSHr cannot predict which patients will develop OHSS, but could be a predictor of severity in OHSS patients.
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Footnotes |
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1 A.D. and G.S. contributed equally to this work. 
Abbreviations: E2, Estradiol; FSHr, FSH receptor; GA, gestational age; hCG, human chorionic gonadotropin; IVF, in vitro fertilization; OHSS, ovarian hyperstimulation syndrome; P, progesterone.
Received June 2, 2004.
Accepted September 20, 2004.
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