FSH PLAYS A central role in oogenesis. It triggers the

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1 X/00/$03.00/0 Vol. 85, No. 9 The Journal of Clinical Endocrinology & Metabolism Printed in U.S.A. Copyright 2000 by The Endocrine Society Ovarian Response to Follicle-Stimulating Hormone (FSH) Stimulation Depends on the FSH Receptor Genotype* MARITZA PEREZ MAYORGA, JÖRG GROMOLL, HERMANN M. BEHRE, CLAUDIA GASSNER, EBERHARD NIESCHLAG, AND MANUELA SIMONI Institute of Reproductive Medicine (M.P.M., J.G., E.N., M.S.) and Department of Obstetrics and Gynecology (H.M.B., C.G.) of the University, D Münster, Germany ABSTRACT Because the ovarian response to FSH stimulation in assisted reproduction is variable, ranging from hyporesponse to hyperresponse, with the possible complication of ovarian hyperstimulation, it would be of great benefit to predict the response of the patients to FSH. To date, no clear-cut predictors of ovarian responsiveness to FSH have been identified. In this study, we investigated the role of two distinct FSH receptor (FSHR) variants, Thr307/Asn680 and Ala307/Ser680, in the response to FSH in women undergoing controlled ovarian stimulation. The FSHR polymorphism at position 680 was analyzed by restriction-fragment-length polymorphism in 161 ovulatory women below the age of 40 yr. With reference to the couple, infertility has been diagnosed as being attributable to male causes (76%), tubal factor (11%), or both (13%). The distribution was 29% for the Asn/Asn, 45% FSH PLAYS A central role in oogenesis. It triggers the maturation of follicles, e.g. the proliferation of granulosa cells, and induces synthesis of the androgen-converting enzyme aromatase (1). Furthermore, it plays a pivotal role in the recruitment of the dominant follicle. FSH action is mediated by the FSH receptor (FSHR), a member of the family of G-protein-coupled receptors expressed solely in granulosa cells, which mediates FSH signal transduction through the camp pathway (2). The cellular action of FSH is not well understood, but recent investigations showed that cell cycle factors, such as cyclin D2, are regulated by FSH (2). Given the crucial function of FSH action on gametogenesis, screening for mutations in the FSHR has been implemented in the search for causes of infertility (3 5). An inactivating mutation located in the extracellular domain of the FSHR could be identified, leading to a FSH resistance syndrome characterized by streak gonads and primary amenorrhea (3). However, primary follicles could be observed in ovarian biopsies of the affected patients, suggesting that FSH action is dispensable up to this stage of follicle maturation (6). It then became evident that partially inactivating mutations of the FSHR can cause arrest at the antral or later stages of follicular growth, indicating Received March 13, Revision received May 25, Accepted June 5, Address correspondence and requests for reprints to: Prof. Dr. E. Nieschlag, F.R.C.P., Institute of Reproductive Medicine of the University, Domagkstrasse 11, D Münster, Germany. nieschl64unimuenster.de. * Supported in part by the German Research Foundation (Ni-130/15-2). for the Asn/Ser, and 26% for the Ser/Ser FSHR variant. Peak estradiol levels, number of preovulatory follicles, and number of retrieved oocytes were similar in the 3 groups. However, basal FSH levels were significantly different among the 3 groups ( IU/L, IU/L, and IU/L for the Asn/Asn, Asn/Ser, and Ser/Ser groups, respectively, P 0.01). The number of FSH ampoules required for successful stimulation was significantly different among the 3 groups ( , , and for the Asn/Asn, Asn/Ser, and Ser/Ser groups, respectively, P 0.05). According to multiple linear regression analysis, the number of ampoules needed could be predicted from a linear combination of both the type of polymorphism and basal FSH levels (P 0.001). These clinical findings demonstrate that the ovarian response to FSH stimulation depends on the FSHR genotype. (J Clin Endocrinol Metab 85: , 2000) that different degrees of FSH-FSHR activity are required to promote maturation (6). In such cases, follicular growth seems to be arrested at various stages, depending on how severely FSH/FSHR interaction is impaired. Conversely, in preovulatory follicles, follicular development proceeds independently of the FSH concentration, once a given threshold concentration of FSH has been reached (6). During screening for mutations of the FSHR gene, two polymorphisms were identified: one located in the extracellular domain at position 307, occupied by either alanine (Ala) or threonine (Thr); and a second one, located in the intracellular domain at position 680, occupied either by asparagine (Asn) or serine (Ser). Both polymorphic sites are within exon 10 and give rise to two discrete allelic variants of the FSHR, i.e. Thr307/Asn680 and Ala307/Ser680 (7). No difference in the distribution of the two variants has been found when comparing infertile men or women to the normal population (7 9). In assisted reproduction programs, the response of ovulating women to exogenous FSH therapy is quite variable. Notwithstanding several years of clinical experience, the ovarian response to intense gonadotropin stimulation is difficult to predict. Patient characteristics, rather than the stimulation protocol, seem to determine the individual response. In young ovulating women undergoing in vitro fertilization (IVF) treatment, the standard stimulation protocol can result in either poor response (requiring adjustment of the FSH doses) or in ovarian hyperstimulation syndrome. This is a serious, potentially life-threatening complication of IVF char- 3365

2 3366 PEREZ MAYORGA ET AL. JCE&M 2000 Vol. 85 No. 9 acterized by enlarged ovaries and extravasation of fluid to the abdominal cavity, resulting in ascites, hypovolemia, and hemoconcentration (10, 11). Advance identification of patients who will elicit a poor response or hyperresponse to standard treatment would be of great clinical advantage. Several parameters have been postulated as predictors of the ovarian response (12 18), all of which strive to assess ovarian reserve. Of these, FSH seems to be the best predictive value, but a significant intraindividual variability from cycle to cycle has to be noted (19). Other factors proposed to affect ovarian response to FSH are the distribution of FSH isoforms (20) and the interference of circulating FSH binding inhibitors or FSH antibodies (21). Intraovarian interference, at the level of FSH binding to its cognate receptor, and the presence of FSHR isoforms with altered signal transduction have also been discussed (22, 23). However, none of these hypotheses have been proven up to now. In this paper, we demonstrate that the FSHR genotype is a major determinant of ovarian responsiveness to FSH in ovulation induction. Subjects Subjects and Methods One hundred sixty-one ovulatory women with infertility caused by male factor or tubal factor were included in this study. Ovulation was documented indirectly by midluteal progesterone levels. Eighty-five patients were recruited prospectively from the ongoing assisted-reproduction program. Another group of 115 women who had previously been treated in our IVF clinic, selected by the above mentioned inclusion criteria, was asked, by letter, to participate in the study. Seventy-six patients responded by returning an EDTA blood sample, together with a signed, written consent. A 3-ml EDTA blood sample was obtained from each woman and was stored at 20 C. Each patient received a full explanation of the purpose of the study and gave written informed consent. Treatment In all cases, controlled ovarian stimulation, according to standard protocols, was performed before genetic analysis. In general, follicular development was monitored by transvaginal sonography, after 6 days of stimulation and then every other day. In cases of insufficient follicular growth and low estradiol serum levels, FSH dosage was increased gradually. Sonography was performed daily when the leading follicle exceeded 14 mm in diameter. Ovulation was induced by 10,000 IU human CG (hcg), im, when at least one follicle was 20 mm in diameter. Oocytes were collected by transvaginal, ultrasoundguided aspiration. Conventional IVF was performed in 38 patients. To this end, up to 10 mature oocytes were inseminated, depending on sperm motility (24). A total of 32, ,000 sperm were incubated overnight with a single oocyte in a 100- L droplet of Medi-Cult universal IVF medium (Medi-Cult, Jyllinge, Denmark). Intracytoplasmic sperm injection was performed in 125 patients, according to the protocol of Van Steirteghem et al. (25). DNA isolation and polymorphism analysis All DNA analyses were performed after completion of ovarian stimulation. Genomic DNA was extracted from white blood cells with the Blood and Cell Culture DNA kit (QIAGEN, Düsseldorf, Germany), according to the manufacturer s instructions. A fragment of exon 10 of the FSHR gene, 10D to 10G (4), was amplified from genomic DNA by PCR and analyzed by electrophoresis in a 2% agarose gel. After analysis, the segment was subjected to phenolchloroform extraction. The purified fragment was digested by Bsr1 (Biolabs, Schwabach, Germany), and the fragments were run on a 2.5% agarose gel electrophoresis. The uncleaved fragment, indicating homozygosity for Asn, has a size of 755 bp; whereas the cleaved fragment, indicating homozygosity for serine, gives rise to 612- and 143-bp fragments (7). The presence of all three fragments indicated a heterozygous state. Singlestranded conformation polymorphism analysis was employed for the characterization of the polymorphism at position 307, in 20 samples, and was performed as previously described (7). Hormonal and clinical data Basal FSH levels (day 3 of the menstrual cycle) were obtained in one of the previous cycles before ovarian stimulation. The peak estradiol levels considered here were those of the day of hcg administration. Serum levels of FSH and estradiol were measured by standard specific immunoassays on the respective cycle days (Vitros EC, Ortho-Clinical Diagnostics, Schwabach, Germany). Statistical analysis Statistical analysis was performed by one-way ANOVA, applying the Sigmastat statistical software package, version 2.03 (Jandel, Erkrath, Germany). For data not normally distributed, the Kruskal-Wallis test was used. Multiple linear regression was used to evaluate the association of basal FSH levels, type of polymorphism, and number of FSH ampoules required. Data are presented as the mean sem and median and range, respectively. P values 0.05 were considered statistically significant. Results The frequencies of the polymorphism at position 680, grouped according to infertility diagnosis, are shown in Table 1. The overall frequency distribution was 29% for the Asn/Asn variant, 45% for the Asn/Ser variant, and 26% for the Ser/Ser variant. SSCP analysis of the polymorphic site at position 307 displayed a linkage of Thr307 to Asn680 and of Ala307 to Ser680 variants in all patients investigated (data not shown). Infertility was caused by impaired semen variables in 76%, by tubal pathology in 11%, and by both in 13% of couples. When the patients were grouped according to their FSHR genotype, the basal levels of FSH (day 3) were significantly different among the three groups (Fig. 1). The FSH concentrations were IU/L, IU/L, and IU/L for the Asn/Asn, Asn/Ser, and Ser/Ser groups, respectively (P 0.01). The age of the patients, the estradiol levels on the day of hcg administration, the number of preovulatory follicles, and the number of retrieved oocytes are shown in Table 2. No statistically significant differences among the three groups were observed, suggesting that the treatment was equally successful, independent of the FSHR isoform. Moreover, no TABLE 1. Distribution of FSHR isoforms in the patients subdivided according to the infertility diagnosis Asn/Asn Asn/Ser Ser/Ser All patients n % % 45% 26% Male infertility n % % 43% 29% Tubal pathology n % Male infertility and tubal pathology n % 13 38% 48% 14%

3 FSH RECEPTOR AND OVARIAN STIMULATION 3367 correlation between these variables and the FSHR genotype was found. However, the number of FSH ampoules required for achieving this effect was significantly different among the groups. As shown in Fig. 2, the number of ampoules was , , and for the Asn/Asn, Asn/Ser, and Ser/Ser groups, respectively (P 0.01) (Fig. 2). Multiple linear regression analysis revealed that the dependent variable (number of ampoules) could be predicted from a linear combination of the two variables, type of polymorphism and basal FSH level (P 0.001). Discussion This study demonstrates that the FSHR genotype plays a fundamental role in determining the physiological responsiveness of the target organ to FSH stimulation. This finding has important implications, both in terms of physiology of FSH action and for its possible clinical applications. The fact that a different ovarian response to FSH stimulation occurs, depending on the FSHR genotype, gives important insights into the hormonal regulation of reproduction. The patients investigated in this clinical setting were ovulating women with FSH levels within the normal range. Previous studies have shown that mutations in the FSHR are extremely rare, show a distinct phenotype, and occur only in severe cases of infertility. Therefore, the presence of such mutations in our patient population is highly improbable. Our results suggest that subtle genetic differences in the FSHR result in different hormone levels within the normal range. Obviously, the allelic variants must have slightly different activities in vivo, as reflected by the significantly different FSH levels observed when the patients were subdivided into the homozygous Ser, heterozygous Asn/Ser, and homozygous Asn groups (Fig. 1). This indicates a fine tuning in the feedback regulation of FSH, dependent on the FSHR genotype. Inhibin B might be the mediator of such precise control of FSH secretion. Unfortunately, because of the lack of serum, day 3 inhibin B could not be measured in this group of retrospectively selected patients. With regard to FSH, different isoforms have been described, characterized by variations in oligosaccharide structure, as well as in the number of terminal sialic acid residues. These isoforms can probably elicit different kinds of ovarian response (20). The FSHR isoforms might further contribute to such pleotropic answers by a molecular mechanism, which could reside in the potential for glycosylation/phosphorylation that each isoform possesses. It has been reported that the FSHR is capable of coupling with more than one G protein subtype, depending on the glycosylation pattern of the ligand and on the degree of receptor transducer activation (26), culminating in differential actions at the target site. The possible mechanism of different receptor activity, depending on the genotype, could be attributed to various aspects. For example, each isoform could show different FIG. 1. Basal FSH levels (day 3 of the menstrual cycle) in ovulatory women, grouped according to the FSHR genotype (AA, n 46; AS, n 72; SS, n 43). Both the individual and the mean SEM values are shown. Results for groups AS and SS were significantly different (P 0.05) vs. group AA, by Kruskall-Wallis test. FIG. 2. Number of FSH ampoules (75 IU/ampoule) required to achieve ovulation induction and oocyte retrieval in ovulatory women, grouped according to the FSHR genotype (AA, n 46; AS, n 72; SS, n 43). Both the individual and the mean SEM values are shown. Results for groups AS and SS were significantly different (P 0.05) vs. group AA, by Kruskall-Wallis test. TABLE 2. Age of the patients and parameters of ovulation induction subdivided according to FSHR isoform Parameter Asn/Asn n 46 Asn/Ser n 72 Ser/Ser n 43 Age b (yr) Peak estradiol c (pmol/l) ( ) ( ) ( ) Number of follicles c 11 (2 23) 11 (2 24) 11 (3 22) Number of oocytes c 11.5 (1 27) 11 (1 36) 12 (1 32) a Kruskall-Wallis test. Mean SEM. Values are median and range. P a

4 3368 PEREZ MAYORGA ET AL. JCE&M 2000 Vol. 85 No. 9 expression at the cell surface, determining the probability of interaction with the hormone. Moreover, each receptor isoform could show differences in the turnover or in the downregulation rate. Finally, each receptor isoform might have different affinity for more- or less-active FSH isoforms (23). The presence of an Asn residue at position 680 introduces a consensus sequence for glycosylation, which might be important for posttranslational receptor processing and expression at the cell surface; whereas a Ser residue contributes potential phosphorylation, which might be involved in the receptor turnover (27). All these hypotheses need to be tested in an appropriate in vitro system. In previous experiments, we were unable to find significant differences in binding characteristics and receptor activation between the two isoforms (7). Such experiments were performed in transiently transfected COS7 cells, i.e. in a cell system that does not necessarily reflect the physiological setting of granulosa cells. Because the expression reflects physiological diversity and is not pathological, it could be expected that FSHR isoforms will not possess drastically different binding and signal transduction characteristics. This challenges the experimental setting in which to study such isoforms. In fact, the use of cell lines, such as COS-7 cells, transiently transfected with different receptor constructs, completely neglects the presence of cell-specific elements such as the different G proteins or phosphodiesterases present in granulosa and Sertoli cells (2). In addition, camp production is the only parameter of receptor activation considered so far. There are indications that FSH action can be mediated by signal transduction pathways distinct from the camp/protein kinase A route, and it is tempting to speculate that a different combination of second-messenger systems is activated, depending on the nature of the FSH isoform-fshr isoform interaction. Investigations of subtle changes of receptor activity, using a cell system not fully representative of the cell that naturally hosts the receptor, might not lead to a proper evaluation. Therefore, we propose that, in the future, a granulosa cell-based system should be used for the functional characterization of FSHR isoforms. In addition, the different receptor activity observed in vivo might be mediated by cellular effects beyond, or other than, the camp production. The finding that allelic variants of the FSHR determine FSH sensitivity opens a new perspective in endocrinology and indicates that subtle genetic changes might fine-tune the hormonal regulation of reproduction. In an elegant study, Cargill et al. (28) recently showed the presence of singlenucleotide polymorphisms in nearly all the key players of the hypothalamo-pituitary-gonadal axis, i.e. the GnRH receptor, LH and its receptor, and FSH and the FSHR. This group also confirmed the presence of the polymorphisms of the FSHR described here. The presence of single-nucleotide polymorphisms in these genes points to individual genetic heterogeneity, resulting in individual hormone profiles. This concept of fine-tuning of FSH action in the ovary becomes more complex when considering that there are not only genetic variants of the hormone but also differently glycosylated isoforms that might have different bioactivity. Future studies have to take into account a more complex setting of FSH isoforms interacting with different FSHR isoforms. Our study also has important clinical implications. In agreement with previous studies, to improve ovarian response, we used a long stimulation protocol employing a GnRH agonist in the large majority of the patients (29). This protocol also aims at optimizing the response in patients expected to be poor responders, such as patients with high basal FSH levels (30). Indeed, the response to ovulation induction was similar among the three groups, although the group with higher basal FSH levels required more ampoules to reach the same result (Fig. 2). Basal day-3 FSH level has been used as a measure of ovarian reserve, with high levels predicting poor response (14, 31 33). However, high FSH levels can also be found in fertile patients, and the sensitivity of this parameter alone has been estimated to be only 8% (3). FSH levels in our patients showed a wide variation, as was also observed in young patients with normal ovarian function (34). It has been suggested that this feature could be related to intraovarian modification of FSH action owing to the presence of inhibitors and/or enhancers of the binding to the receptor (35), principally to growth factors (36) or to individual differences in the FSHR (22). Each receptor isoform might mediate a different level of intraovarian FSH action, explaining the parallelism between FSH levels and therapeutic requirements. Our data suggest that the different responsiveness of each FSHR genotype is reflected by the basal FSH levels, and only when a supraphysiological response is required, as in ovarian stimulation, does the different capacity of responsiveness becomes evident. Using standard stimulation protocols, a highly significant difference could be observed in the number of ampoules required to achieve ovulation. The marked difference, of more than 10 ampoules, between the homozygous Asn group and the homozygous Ser group is also reflected by the different FSH serum levels in these groups. However, because FSH values show wide interindividual variations, even within the same FSHR group, the subdivision of the patients solely according to their serum FSH concentrations is not clinically useful, whereas identification of the FSHR genotype permits better classification. This underlines the clinical significance of our findings, which goes far beyond the mere description of an indirect association of two markers, as shown in other polymorphism studies. Knowing the different sensitivity to FSH, depending on the FSHR genotype, could be important to prevent ovarian hyperstimulation syndrome, a complication with increasing incidence after the introduction of GnRH agonists, which involves the administration of higher doses of gonadotropins (10). It is difficult to prevent this complication because of the narrow therapeutic margin of the ovulation induction agents and because of the impossibility of predicting individual response. Knowledge of the FSHR genotype might provide a means of assessing this individual factor, assuming that pertinent modifications of the ovarian stimulation treatment can be made. The finding that the FSHR genotype determines ovarian response to FSH should have impact on the delineation of stimulation protocols. In the future, it should be possible to tailor FSH therapy to the patient s genetic background and to design an individualized ovarian stimulation protocol in

5 FSH RECEPTOR AND OVARIAN STIMULATION 3369 advance, adjusting not only doses administered but also the timing of stimulation. Among the possible benefits of adjusting stimulation protocols according to the expected response could be that the duration and the total amount of FSH needed decreases. Immediate implications are benefits not only from the economic point of view but also in terms of treatment acceptance. Acknowledgments We thank E. Pekel for excellent technical assistance and S. Nieschlag, M.A., for language editing of the manuscript. References 1. Simoni M, Nieschlag E FSH in therapy: physiological basis, new preparations and clinical use. Reprod Med Rev. 4: Simoni M, Gromoll J, Nieschlag E The follicle-stimulating hormone receptor: biochemistry, molecular biology, physiology, and pathophysiology. Endocr Rev. 18: Aittomaki K, Dieguez-Lucena JL, Pakarinen P, et al Mutation in the follicle-stimulating hormone receptor gene causes hereditary hypergonadotropic ovarian failure. 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