Genetics of ovarian hyperstimulation syndrome

RBMOnline - Vol 19. No 1. 2009 14-27 Reproductive BioMedicine Online; www.rbmonline.com/Article/4119 on web 20 May 2009

Symposium: Update on prediction and management of OHSS Genetics of ovarian hyperstimulation syndrome Botros Rizk MD MA FACOG FACS HCLD FRCOG FRCS Professor and Head of Reproductive Endocrinology and Infertility, Medical and Scientific Director of USA In Vitro Fertilization and Assisted Reproduction. He graduated from Cairo University Medical School. Dr Rizk completed his residency in Obstetrics and Gynaecology in London and fellowship in reproductive medicine at the Bourn Hallam Clinic in London and Cambridge under the leadership of Professor Robert Edwards and Professor Howard Jacobs. He is a leading authority on ovarian hyperstimulation syndrome, ovarian stimulation and endometriosis. He has published nine medical textbooks including ovarian hyperstimulation syndrome, endometriosis and a major textbook on infertility and assisted reproduction. Dr Botros Rizk Botros Rizk University of South Alabama College of Medicine, Department of Obstetrics and Gynecology, 251 Cox Street, Suite 100, Mobile, AL 36604, USA Correspondence: e-mail: [email protected]

Abstract Ovarian hyperstimulation syndrome (OHSS) is typically iatrogenic following the administration of gonadotrophins. Sporadic and familial cases of spontaneous OHSS have generated an interest in genetic mechanisms for OHSS independent of exogenous gonadotrophins. The genetic studies have addressed the genes and receptors for FSH and luteinizing/human chorionic gonadotrophin hormones. Mutations in the FSH receptor (FSHR) could be activating, leading to a predisposition to OHSS, or inactivating, resulting in sterility. Polymorphisms of FSHR have been investigated and, to date, 744 single nucleotide polymorphisms have been identified in the FSHR gene, of which only eight are located in the coding region, exons, with the rest being intronic. Ovarian response is dependent on FSHR genotype. Clinical studies on the p.N680S polymorphism of the FSHR gene have demonstrated the homozygous Ser/Ser variant to be less sensitive to endogenous or exogenous FSH in terms of oestradiol production. Polymorphism of the FSHR, Ser680Asn, in the FSHR gene is a predictor of the severity of symptoms in patients who develop OHSS. OHSS is characterized by leakage of intravascular fluids resulting in ascites and haemoconcentration. These pathological changes are mediated for the most part by vascular endothelial growth factor (VEGF). Targeting the VEGF system at different levels has been the focus of intense research for the prevention of OHSS. Keywords: FSH receptor, genetic mutations, HCG, OHSS, VEGF

Introduction

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Ovarian hyperstimulation syndrome (OHSS) is a complication that typically follows administration of gonadotrophins (Mozes et al., 1965; Golan et al., 1989; Rizk and Smitz, 1992). The administration of human chorionic gonadotrophin (HCG) results in the release of vasoactive substances such as vascular endothelial growth factor (VEGF) that causes vasodilation and leakage of fluids (McClure et al., 1994; Rizk et al., 1997; Pellicer et al., 1999; Rizk and Aboulghar, 2005; Busso et al., 2008). Genetic studies of OHSS have focused on the FSH receptor (FSHR) gene and its mutations as well as targeting VEGF receptors as a method to modulate OHSS. Primate studies have demonstrated interesting scientific studies regarding luteinizing hormone secretion

but have not yet impacted clinical management. Mutations in the FSHR can be inactivating, causing sterility, or activating, contributing to OHSS. During the last decade, several cases of spontaneous and familial OHSS have been reported, suggesting a possible genetic cause for OHSS. The discovery of FSHR mutations that made them sensitive to stimulation by HCG (Vasseur et al., 2003; Montanelli et al., 2004a) provided the first molecular explanation for OHSS (Kaiser, 2004). There is no rational scientific treatment for OHSS, the treatment remains empirical (Rizk, 1992, 1993; Rizk and Dickey, 2009). Targeting VEGF receptors may prevent OHSS and therefore has tremendous advantages to patients (Soares et al., 2008; Rizk et al., 2009).

© 2009 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK

Symposium - Genetics of ovarian hyperstimulation syndrome - B Rizk

Follicle-stimulating hormone Structure and function FSH is the central hormone of human reproduction necessary for gonadal development and gamete production (Simoni et al., 1997). FSH, LH and HCG consist of a common alpha subunit and a receptor-specific beta subunit (Figures 1 and 2). FSH acts by binding to specific receptors localized specifically in the gonads. Ovulation of a single healthy oocyte in a natural cycle is an elegantly orchestrated event modulated by secretion of several glycoproteins that act on the ovary to stimulate changes in functional morphology and steroidogenesis (Schuler and Scammell, 2008). Follicle-stimulating hormone is secreted from the anterior pituitary gland in response to the pulsatile

stimulus of gonadotrophin-releasing hormone from the hypothalamus (Ulloa-Aguirre et al., 2007). Circulating FSH stimulates follicular growth in the ovary via specific FSHR in the cell membranes of granulosa cells. FSH induces the proliferation of granulosa cells and the synthesis of the androgen-converting enzyme aromatase, and also plays a crucial role in secondary follicle recruitment and selection of the dominant follicle (Simoni et al., 2008) Initially, an antral follicle emerges and exhibits autocrine feedback to increase sensitivity to FSH, LH and HCG through an increase in receptors to these hormones. As the follicle grows and continues to secrete oestradiol, the remaining follicles in the ovary undergo atresia. This single follicle becomes a mature follicle, which will ovulate in response to a surge in LH, also secreted from the anterior pituitary gland. Prior to ovulation, it acts on specific LH receptors (LHR) in ovarian theca cells to stimulate oocyte maturation. After ovulation, it stimulates corpus luteum formation and supports corpus luteum function (Schuler and Scammell, 2008). Luteogenic hormone acts

Figure 1. Schematic representation of the primary structure of α- and β-subunits of the gonadotrophin family. HCG = human chorionic gonadotrophin; HFSH = human FSH; HLH = human LH. Reproduced with permission from Oxford University Press (Olijve et al., 1996).

Figure 2. The sequence of the common human α-subunit. Reprinted from Modern Assisted Conception, with permission from Reproductive Healthcare Ltd. (Edwards and Risquez, 2003). RBMOnline®

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through a VEGF-dependent mechanism to up-regulate alpha 5 beta 1 and alpha 5 beta 3 integrins, promoting the migration and survival of human luteinized granulosa cells (Rolaki et al., 2007).

FSH receptor The FSHR belongs to the family of G protein-coupled receptors (GPCR). The FSHR can be divided into three regions: the extracellular domain, a transmembrane region and the intracellular domain (Rizk, 2006a). It is leucine rich and characterized by seven hydrophobic helices inserted in the plasmalemma and intracellular and extracellular domains. Binding occurs through a ‘hand-clasp’ binding model (Schuler and Scammell, 2008). As the name suggests, a ‘hand-clasp’ binding model resembles the appearance of two hands clasped together: the FSH molecule fits into a notch in the curved receptor and the receptor itself wraps around the middle of the FSH molecule. FSH in complex with the FSHR has recently been crystallized (Schuler and Scammell, 2008). The intracellular portion of the FSH receptor is coupled to a Gs protein and, upon receptor activation by the hormonal interaction with the extracellular domain, initiates a cascade of events that finally leads to the specific biological effects of the gonadotrophin as described by Edwards and Risquez (2003) (Figure 3).

Signal transduction and post-receptor events Signal transduction of the FSHR is mainly mediated by the protein kinase A (PKA) pathway, activation of adenylate cyclase and elevation of intracellular cyclic AMP. The intramolecular mechanisms involved in the transduction of the activation signal from the binding step to the activation of the G protein have been the subject of intense investigations (Montanelli et al., 2004b) and, in most rhodopsins like GPCR, there is evidence for a direct interaction between agonists and serpentine domains. The

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models for the activation of gonadotrophin receptors suggest that binding of the hormones to the receptors would promote a conformational change in their ectodomains, transforming them into full agonists of the serpentine domain (VlaeminckGuillem et al., 2002; Montanelli et al., 2004b). Upon agonist binding, the activated FSHR stimulates a number of intracellular signalling pathways (Ulloa-Aguirre et al., 2007). In the classical, linear signalling cascade, occupancy of the FSHR causes activation of the heterotrimeric Gs protein, followed by dissociation into two molecules, the α-subunit and the β/γ-heterodimer. The α-subunit stimulates the effector adenylyl cyclase with the consequent increase in the synthesis of the second messenger cAMP, activation of PKA, phosphorylation of a number of transcriptional regulators, including the cAMP regulatory element-binding protein (CREB), and activation of transcription. The β/γ-heterodimer activates phospholipase C but this enzyme is also activated by alternate Gαs subunits (Simoni et al., 2008).

FSH receptor gene The FSHR as well as TSH and LH/HCG receptors have evolved from a common ancestral gene (Montanelli et al., 2004b). The chromosomal mapping of the FSHR gene has been performed by fluorescence in-situ hybridization using cDNA or genomic probes and by linkage analysis (Rousseau-Merck et al., 1993; Gromoll et al., 1994). The FSH receptor gene is located at chromosome 2p21–p16 in the human (Simoni et al., 1997, 2002; Themmen and Huhtaniemi, 2000; Lussiana et al., 2008). The LHR gene can be mapped to the same chromosomal location whereas the human TSHR is located on chromosome 14q31. The structure and organization of the FSHR gene has been investigated in humans and rats (Rizk, 2006a; Lussiana, et al., 2008). The FSHR gene is a single-copy gene and spans a region of 54 kbp in the human and 84 kbp in the rat, as determined by restriction analysis of genomic clones and size determination of PCR probes. It consists of 10 exons and nine introns. The

Figure 3. Attachment to FSH receptor and signal transduction mediated by the Gαs protein pathway in response to a gonadotrophic stimulus (ligand). C = catalytic subunits; R = regulatory subunits. Reproduced from Modern Assisted Conception, with permission from Reproductive Healthcare Ltd. (Edwards and Risquez, 2003). RBMOnline®

Symposium - Genetics of ovarian hyperstimulation syndrome - B Rizk

extracellular domain of the human receptor is encoded by nine exons ranging from 60–251 bp. The C-terminal part of the extracellular domain, the transmembrane and the intracellular domains are encoded by exon 10 with more than 1234 bp. The FSHR gene encodes 695 amino acids, including a signal peptide with 17 amino acids. The nine introns vary greatly in their corresponding sizes from 108 bp for intron 7 to 15 kbp for intron 1 (Simoni et al., 1997, 2002). The techniques used for research purposes are different according to the aim of the study, the gene sequence (FSHR gene), or the protein structure and function (FSHR protein). The genetic study (genotype analysis) is often followed or paralleled by a functional study of the protein (phenotype analysis). The FSHR gene has been investigated by detecting point mutations and their functional consequences at the protein level and by assessing the presence and frequency in a given population of allelic variants at known or suspected polymorphic sites (Lussiana et al., 2008; Simoni et al., 2008). The database of the National Centre for Biotechnology Information includes more than 700 single-nucleotide polymorphisms and mutations of the FSHR gene (Lussiana et al., 2008). As the study of these mutations is accomplished mainly on patients with altered reproductive function, mutations have been classified as activating, inactivating or neutral according to the FSHR activity level in each specific phenotype (Gromoll, et al., 1996; Themmen and Huhtaniemi, 2000; Lussiana, et al., 2008).

Ovarian response and FSH receptor FSH plays a central role in oogenesis. It triggers the maturation of follicles, proliferation of granulosa cells and the aromatase enzyme induction. Its role is pivotal in the recruitment of the dominant follicle. FSH action is mediated by the FSHR and therefore, screening for mutations in the FSHR have been pursued in the search for causes of infertility (Aittomaki et al., 1995; Whitney et al., 1995; Gromoll et al., 1996; Lussiana et al., 2008). The portion of chromosome 2, including the gene codifying the receptor of FSH, can display point mutations that cause variations in the amino acid sequence of the receptor protein. Some of these structural changes affect the receptor functional properties that may be enhanced or impaired (Aittomaki et al., 1995).

different polymorphisms in any given population is variable according to ethnic origin. In the Japanese population, the overall frequency of TN/TN, TN/AS was reported to be 41%, 46.9% and 12.1%, respectively, and no other allelic combination was found (Sudo et al., 2002; Lussiana et al., 2008). Among Korean patients undergoing IVF, the FSHR genotype distribution was 41.8% for variants homozygous for Asn680 (i.e. TN/TN), 12.5% for variants homozygous for Ser680 (i.e. AS/AS) and 45.6% for variants having heterozygous Asn/Ser at position 680 (i.e. TN/AS) (Jun et al., 2006). White women are homozygous for Ser at position 680 in 45.5% of cases, Asn/Ser heterozygous in 22.7% cases and Asn680 homozygous in 31.8% of cases (Loutradis et al., 2006). Interestingly in white women, the TN/AS variant was found to be significantly more frequent (66.7% of the subjects) among patients with polycystic ovaries (PCO) than in patients with normal ovaries (43.5%). Polymorphisms have been reported for some G proteincoupled receptors and functional consequences have often been observed (Lussiana et al., 2008). To date, the National Centre for Biotechnology Information(NCBI) single nucleotide polymorphism (SNP) database indicates 744 SNP in the FSHR gene, of which only eight are located in the coding region (exons), with the rest being intronic (Wunsch et al., 2007). One SNP is located in the 5ʹ untranslated region of the FSHR mRNA at position 29. Seven of the eight SNP within the coding region are found in exon 10 at codon positions 307, 329, 449, 524, 567, 665 and 680, and six of these are non-synonymous. The p.T307A and the p.N680S polymorphisms are those best characterized with respect to frequency (approaching 50%) and ethnic distribution and have been confirmed to be in linkage disequilibrium by the HapMap project (see http://www.hapmap. org) (Wunsch et al., 2007; Luissiana et al., 2008). FSHR gene polymorphisms at specific sites (e.g. codons 307 and 680) may influence FSHR protein responsiveness to exogenous FSH. The clinical interest in these polymorphisms is because they may affect the effectiveness of IVF treatment as well as the likelihood of developing severe OHSS (Jun et al., 2006; Wunsch et al., 2007; Binder et al., 2008; Lussiana et al., 2008; Simoni et al., 2008).

Activating mutations confer to FSHR a higher responsiveness to FSH, making it constitutively active even in the absence of the ligand, or render it able to non-specifically respond to other trophic hormones (e.g. TSH) (Lussiana et al., 2008). These mutations may predispose to OHSS (De Leener et al., 2006). Inactivating mutations reduce the receptor’s function up to a total block, altering either the formation of the receptor–ligand complex or FSH signal transduction. These mutations may cause amenorrhoea, infertility or premature ovarian failure.

Two common SNP within exon 10 of the human FSHR gene result in two almost equally common allelic variants exhibiting Thr or Ala at position 307 in the hinge region, respectively, Asn or Ser at codon 680 of the intracellular domain. Clinical studies have demonstrated that p.N680S polymorphism determines the ovarian response to FSH stimulation in patients undergoing IVF-embryo transfer treatment (Perez-Mayorga et al., 2000; de Castro et al., 2003). Patients with the Ser680 allele need more FSH during the stimulation phase to reach the serum oestradiol concentrations of Asn680 patients. A study investigating women with normal, mono-ovulatory menstrual cycles revealed that the Ser680/Ser680 genotype leads to higher FSH serum concentrations and a prolonged cycle.

Polymorphisms of the FSHR, which are changes at the nucleotide level of the FSHR gene, resulting in allelic variations and finally in an altered amino acid sequence on the receptor protein, do not occur in scattered individuals, but are frequent and distributed in a population with well defined proportions. They are distinct from point mutations (that are very rarely observed) and are called ‘polymorphisms’. The proportion of

Jun et al. (2006) investigated the association between FSHR gene polymorphism at position 680 and the outcomes of IVF– embryo transfer in Korean women. This study included 263 patients under 40 years of age who underwent IVF–embryo transfer procedures. The Korean investigators excluded patients with polycystic ovary syndrome, endometriosis, or a previous history of ovarian surgery. Following extraction of genomic

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Symposium - Genetics of ovarian hyperstimulation syndrome - B Rizk

DNA, the FSHR polymorphism at position 680 was determined by PCR analysis. The FSHR genotype distribution was 41.8% for Asn/Asn, 45.6% for Asn/Ser and 12.5% for Ser/Ser FSHR genotype groups. Although there was no difference among the three genotype groups in terms of the age and infertility diagnosis of study subjects, the basal concentrations of FSH (day 3) were significantly different (mean ± SEM: 5.7 ± 0.3 IU/l , 6.0 ± 0.3 IU/l and 8.2 ± 0.9 IU/l for Asn/Asn, Asn/Ser and Ser/Ser groups, respectively. The Ser/Ser group required a higher dose of gonadotrophins for ovarian stimulation and showed lower serum oestradiol concentrations at the time of HCG administration that the other two groups, although these differences did not reach statistical significance. The numbers of oocytes retrieved were different for the three groups (9.6 ± 0.6, 10.2 ± 0.6 and 7.9 ± 0.8 for Asn/Asn, Asn/Ser and Ser/ Ser groups, respectively). More interestingly, the clinical pregnancy rate was significantly higher in Asn/Asn, compared with the others (45.7 versus 30.5%, P = 0.013). In conclusion, the homozygous Ser/Ser genotype of FSHR polymorphism at position 680 may be associated with a reduced ovarian response to ovarian stimulation for IVF–embryo transfer, while Asn/Asn genotypes showed a higher pregnancy rate. Greb et al. (2005), in a study involving menstrual-cycle monitoring in women with normal, mono-ovulatory cycles, demonstrated that during the luteo-follicular transition, serum concentrations of oestradiol, progesterone and inhibin A were significantly lower and FSH started to rise earlier in women with the Ser/Ser genotype compared with women who carried the Asn/Asn genotype for the FSHR p.N680S polymorphism. FSH concentrations were steadily and significantly higher during the follicular phase in the Ser/Ser genotype group, whereas no differences were observed between groups with regard to oestradiol, inhibin B and growth velocity of the dominant follicle, showing that higher concentrations of endogenous FSH are necessary to achieve ovulation in carriers of the Ser/Ser genotype, with a difference of about 2 days between these women and those with the Asn/Asn genotype. The Ser/Ser genotype results in a higher ovarian threshold for FSH, decreased negative feedback to the pituitary and a longer menstrual cycle (Greb et al., 2005).

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Genetic analysis of animal models and previous molecular studies in women treated with FSH support the hypothesis that the FSH response is a complex trait that is dictated by both genetic and environmental factors (de Castro et al., 2003). Animal models have provided evidence of genetic control of FSH response in the hormone-induced ovulation rate. Six-fold differences in the ovulation rate while using hormones have been shown in different mouse strains (de Castro et al., 2003). A genome-wide search analysis of these mouse strains with different response patterns during ovarian stimulation has revealed at least five major genetic loci controlling folliculogenesis and ovulation rate. One of these loci, the CYP19 aromatase gene, was shown to be related to ovulation rate. Higher responses to gonadotrophins in mice have been associated with increased aromatase activity and oestrogen production. de Castro et al. (2003) analysed the clinical outcome of 102 ovarian stimulation cycles and the role of Ser680Asn in recombinant FSH. Although the results suggest Ser/Ser patients have a lower response to recombinant FSH during ovarian stimulation cycles and an increased risk of cycle cancellation, the presence of Asn/Asn homozygotes among poor responders and among patients whose cycles were cancelled

indicates that the Ser680 allele alone is not sufficient to cause a lower response to recombinant FSH. Furthermore, variables of ovulation induction were similar among genotypes suggesting that other factors such as age, ovarian reserve or other genes may contribute to the outcome (de Castro et al., 2003). Altmae et al. (2009) examined the genetic influences of CYP19A1 TCT trinucleotide insertion/deletion (TTTA)n and microsatellite intronic polymorphisms on ovarian stimulation outcome. Del/Del homozygous with shorter TTTA repeats exhibited decreased ovarian FSH sensitivity in ovarian stimulation. The authors postulated that this may reflect variations in aromatase gene expression during antral follicle development. Altmae et al. (2009) suggested a correlation between Del allele and shorter (TTTA)n repeat sizes with smaller ovaries and fewer antral follicles on days 3–5 of the menstrual cycle. Serum FSH concentrations are among the predictors of ovarian response (Sallam et al., 2008). Since the first report on the genotypic variance of the FSHR gene, the possibility has been considered as to whether a SNP of FSHR gene affects the ovarian response to exogenous gonadotrophins. That is to say, the ovarian responses to gonadotrophins for ovarian stimulation in IVF–embryo transfer cycles might differ according to FSHR gene genotypes (Aittomaki et al., 1995). Two non-synonymous polymorphisms have been described in exon 10 of the transmembrane region of the FSHR (Simoni et al., 2002). The first one is A919G (Thr307Ala) located just before the beginning of the first transmembrane helix and the second polymorphism is A2039G (Asn680Ser) located intracellularly at the end of the C permanent tail of the receptor. In Caucasian populations, four haplotypes have been described by Simoni et al. (2002). The common haplotypes are Thr307Asn680 and Ala307Ser680 (~60% and ~40%, respectively). The rare haplotypes are Ala307Asn680 or Thr307Ser680 (~1% each). Perez-Mayorga et al. (2000) evaluated the impact of FSHR genotypes at position 680 on the ovarian responses to FSH stimulation in 161 infertile women undergoing IVF–embryo transfer. Although no differences were observed in terms of the number of oocytes retrieved and serum oestradiol concentration on the day of HCG injection, the number of gonadotrophin ampoules and basal FSH concentrations were found to be higher in the Ser/Ser group. The presence of a serine in position 680 is associated with high basal concentrations of FSH on days 2–4 of the menstrual cycle and higher requirements of exogenous FSH for ovarian stimulation (Perez-Mayorga et al., 2000; Sudo et al., 2002). This means that a FSH receptor with a serine in position 680 is less efficient than a FSH receptor with an asparagine in position 680 (Perez-Mayorga et al., 2000; Sudo et al., 2002). de Castro et al. (2003) demonstrated an association between the presence of serine in position 680 to poor responses to gonadotrophin therapy in IVF patients and suggested that the Ser680 allele was associated with a diminished sensitivity to FSH. However, Laven et al. (2003) could not establish altered ovarian sensitivity to exogenous FSH during ovulation induction in clomiphene-resistant normogonadotrophic anovulatory patients. The in-vivo association of Ser680 with higher concentrations of basal FSH on days 2–4 of the menstrual cycle has not yet been explained in molecular terms (Perez-Mayorga. et al., 2000; Simoni et al., 2002; Sudo et al., 2002; Daelemans et al., 2004). Binder et al. (2008) reported that higher frequencies of wild-type RBMOnline®

Symposium - Genetics of ovarian hyperstimulation syndrome - B Rizk

FSHR Asn680Ser and Ile160Thr appear to improve fecundity but cannot predict the severity of OHSS. No polymorphisms could be demonstrated that may lead to hyperreactio luteinalis, a condition that is presumably closely linked to spontaneous OHSS. Typical manifestations are bilateral enlarged ovaries with multiple luteal cysts in early or late pregnancy but mostly without severe symptoms or ascites; hyperreactio luteinalis and OHSS could be entities in continuum (Haimov-Kochman et al., 2004).

Naturally occurring FSH receptor mutations and OHSS The association between naturally occurring FSHR mutations and spontaneous OHSS (D567N and T449I/A) (Figure 4) has opened new horizons in the understanding of the

pathophysiology of this syndrome (Kaiser, 2003). Activating mutations in the FSHR and OHSS have been reported in the last 5 years (Table 1) (Smits et al., 2003; Vasseur et al., 2003; Montanelli et al., 2004a: Delbaere et al., 2005; De Leener et al., 2006; Lussiana et al., 2008). Mutant FSH receptors displayed abnormally high sensitivity to HCG and, in addition, D567N and T449A displayed a concomitant increase in sensitivity to TSH and detectable constitutive activity (Smits et al., 2003; Vasseur et al., 2003; Montanelli et al., 2004a). These mutations broaden the specificity of the FSHR so that it responds to another ligand, HCG (Figure 4). Vasseur et al. (2003), identified a HCG-sensitive mutation in the FSHR as a cause of familial gestational spontaneous OHSS. In the interesting case, the patient developed OHSS during all of her four pregnancies that went beyond 6 weeks of gestation. The patient’s sisters who also had OHSS in their pregnancies

Figure 4. Pathogenesis of familial gestational spontaneous ovarian hyperstimulation syndrome. HCG = human chorionic gonadotrophin; FSH = follicle-stimulating hormone; LH = luteinzing hormone. Reproduced with permission from New England Journal of Medicine (Kaiser, 2003). RBMOnline®

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Table 1. Activating mutations of FSH receptor (FSHR) and ovarian hyperstimulation syndrome (OHSS). Reference

Mutation

Smits et al. (2003)

Asp567→Asn FSHR increased sensitivity to FSH or HCG Spontaneous or iatrogenic OHSS Thr449→Ile FSHR increased sensitivity to FSH, HCG or TSH Thr449→Ala Spontaneous or iatrogenic OHSS, due to hypothyroidism Ile545→Thr FSHR increased sensitivity to FSH, HCG, or TSH Spontaneous or iatrogenic OHSS, due to hypothyroidism

Vasseur et al. (2003) Montanelli et al. (2004a) De Leener et al. (2006)

Effect

FSHR = FSH receptor; HCG = human chorionic gonadotrophin; OHSS = ovarian hyperstimulation syndrome; TSH = thyroid-stimulating hormone.

had the same mutation but not another sister who did not develop OHSS. The mutation consisted of a substitution of thymidine for cytosine in exon 10 of the FSHR gene. This resulted in the replacement of threonine by an isoleucine at position 449 of the FSH protein (Figure 5). In-vitro characterization of the mutated receptor showed an increased sensitivity to HCG. Smits et al. (2003) identified another mutation in the FSHR gene in a patient with spontaneous OHSS during each of her four pregnancies. The mutation consisted of substitution of an adenine for a guanine at the first base of the codon 567 in exon 10 of the FSHR gene resulting in the replacement of an aspartic acid with an asparagine (Figure 6). The functional response of the mutant receptor when tested in vitro displayed an increased sensitivity to HCG. Montanelli et al. (2004a) described a familial case of recurrent spontaneous OHSS associated with a different mutation affecting the residue 449 of the FSHR. The affected women were heterozygous for a different mutation involving codon 449, where an alanine was substituted for threonine (Figure 7). Similar to D567N, the T449A FSHR mutant shows an increased sensitivity to both HCG and TSH, together with an increase in basal activity. De Leener et al. (2006) reported another novel FSHR gene variation, Ile545Thr, in a patient with spontaneous OHSS previously described by Chae et al. (2001).

How do FSH receptor mutations result in OHSS? HCG activity is normally limited to LH/HCG receptors expressed in the corpus luteum and assists in the maintenance of pregnancies (Rolaki et al., 2007). FSHR mutations result in the promiscuous stimulation by HCG of FSHR expressed on the granulosa cells of the ovarian follicles resulting in excessive follicular development. Kaiser (2003) postulated that excessive follicular recruitment in association with luteinization of the follicles mediated by LHR results in OHSS (Figure 4)

Unexpected location for the FSH receptor mutations

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The mutations in the FSHR led to reduction of ligand specificity permitting activation by HCG. It was very unexpected that the mutations were not in the hormone-binding ectodomain but rather in the serpentine domain that is responsible for the

activation of signalling (Kaiser, 2003; Montanelli et al., 2004a). The affinity for FSH was not affected and no direct binding of HCG could be detected. These findings argue against changes in ligand binding. Kaiser (2003) suggested that the mutations affect the specificity of ligand recognition by allowing the low affinity interaction of HCG with the ectodomain of the FSHR to be sufficient to ‘flip the switch’. This would result in inducing an active confirmation of the serpentine domain and downstream signalling (Figure 4). Furthermore, the mutation in the FSHR reported by Smits et al. (2003), was such that the ligand specificity was reduced to an even greater extent permitting downstream signalling induction by TSH in addition to HCG and FSH and also permitting constitutive activity in the absence of ligand (Kaiser, 2003).

Are there other clinical effects of the FSH receptor mutations? Kaiser (2003) discussed three interesting effects of the FSHR mutations that were discovered and associated with spontaneous OHSS. The first possibility is that such mutations may lead to increased susceptibility to iatrogenic OHSS in patients undergoing ovulation induction. The second predisposition is multiple pregnancy as a result of increased stimulation of ovarian follicular development. Finally, there is an unresolved concern that ovulation induction may increase the risk of cancer of the ovary, breast or uterus. Therefore, patients with activating mutations of the FSHR require careful and diligent follow-up, perhaps for a long time.

Spontaneous and iatrogenic OHSS The identification of mutations in the FSHR gene that display an increased sensitivity to HCG and are responsible for the development of spontaneous OHSS provides, for the first time, the molecular basis for the physiopathology of spontaneous OHSS (Delbaere et al., 2004). In these cases, the abnormal function of mutant FSHR in vitro provides a reasonable explanation for their implication in the development of OHSS in vivo. During pregnancy, there is a significant decrease in FSHR expression in the corpus luteum. However, the expression of FSHR in the granulosa cells of the developing follicles remains constant (Simoni et al., 1997). Since the pituitary gonadotrophins fall to very low concentrations in the serum, these receptors are not usually stimulated during pregnancy (Delbaere et al., 2004). Mutant FSHR expressed in developing

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Symposium - Genetics of ovarian hyperstimulation syndrome - B Rizk

Figure 5. FSHR mutation and spontaneous ovarian hyperstimulation syndrome. sequence of exon 10 of the FSHR in the proband. Arrow indicates the heterozygous position at 449. Reproduced with permission from New England Journal of Medicine (Vasseur et al., 2003).

Figure 6. FSHR mutation and spontaneous ovarian hyperstimulation syndrome. Nucleotide sequence traces of FSHR around codon 567 in a control subject and in a patient with recurrent spontaneous ovarian hyperstimulation syndrome. Reproduced with permission from New England Journal of Medicine (Smits et al., 2003).

Figure 7. FSHR mutation and spontaneous ovarian hyperstimulation syndrome. Detection of the T449A mutation. (A) Nucleotide sequence traces of exon 10 of the FSHR around codon 449 in a patient with spontaneous OHSS. (B) Family pedigree. Reproduced with permission from The Endocrine Society (Montanelli et al., 2004a).

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Symposium - Genetics of ovarian hyperstimulation syndrome - B Rizk

follicles could be stimulated by pregnancy-derived HCG and the follicles would start growing and enlarge. Finally, the granulosa cells acquire LHR which may also be stimulated by HCG (Delbaere et al., 2004). This would induce follicular luteinization and secretion of vasoactive substances that is implicated in the pathophysiology of the syndrome. The interaction between the FSHR and HCG is therefore a requirement for the development of spontaneous OHSS. Delbaere et al. (2004) highlighted the differences between spontaneous and iatrogenic OHSS and proposed a model to account for the different chronology between the two forms of the syndrome (Figure 8). In the iatrogenic form, follicular recruitment and enlargement occur during ovarian stimulation with exogenous FSH, while in the spontaneous form, the follicular recruitment occurs later through the stimulation of the FSHR by pregnancy-derived HCG. In both forms, massive luteinization of enlarged stimulated ovaries ensues, inducing the release of vasoactive mediators, leading to the development of the symptoms of OHSS (Rizk, 2006b). It is possible that the stimulation of the mutated FSH receptor occurs at the threshold HCG concentration. That threshold value could vary according to the type of mutation. In the first trimester of pregnancy, HCG peaks between 8 and 10 weeks and declines thereafter. It follows therefore, that the initiation of follicular growth by pregnancy-derived HCG could start between 6 and 10 weeks of amenorrhoea (Delbaere et al., 2004). If these follicles develop at the same rate as ovarian stimulation, the development of OHSS will occur at the time of massive follicular luteinization, between 8 and 12 weeks amenorrhoea (Figure 8).

Prediction of severity of symptoms in iatrogenic OHSS by FSH receptor polymorphism The association between FSHR gene polymorphism and ovarian responses to controlled ovarian hyperstimulation has been well investigated (Jun, et al., 2006; Wunsch, et al., 2007; Binder, et al., 2008; Simoni, et al., 2008). The proportion of different polymorphisms in any given population is variable according to ethnic origin, but in general it follows Mendelian laws as previously discussed (Lussiana et al., 2008). The potential association of the S680 allele with poor responders to ovarian stimulation for IVF (Perez-Mayorga et al., 2000; de Castro et al., 2003) led to the hypothesis that the Asn680 allele could be associated with hyper-responders, i.e. patients at risk of iatrogenic OHSS. In an elegant study published by Daelemans et al. (2004), no statistically significant differences were found in allelic frequencies between the IVF control population and the OHSS patients. These results have been confirmed by d’Alva et al. (2005) and Kerkela et al. (2007). However, Daelemans et al. (2004) observed a significant enrichment in allele 680 as the severity of OHSS increased (P = 0.034). The authors suggested that the genotype in the position 680 of the FSHR cannot predict which patient will develop OHSS but could be a predictor of severity of OHSS symptoms among OHSS patients.

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Luteinizing hormone receptor Lessons from New World monkeys Schuler and Scammell (2008) elegantly reviewed the discoveries regarding the LHR and pituitary hormones in New World monkeys. New World primates are thought to have separated from Old World anthropoids about 35 million years ago (Schrago, 2007). Living New World primates, which include primate species such as marmosets, squirrel monkeys and owl monkeys, likely appeared 15–20 million years ago and are distributed from Central to South America. The LHR from common marmoset testis has been cloned and sequenced (Zhang et al., 1997). Unlike human LHR mRNA that is transcribed from 11 exons, the LHR in marmoset testis lacks exon 10 at the mRNA level. More recent studies indicate that: (i) the absence of LHR exon 10 mRNA is also seen in testicular RNA from other New World primates including squirrel monkeys (Gromoll et al., 2003); (ii) exon 10 sequences are present in genomic DNA from New World primates (Gromoll et al., 2003); and (iii) the skipping of exon 10 results from alterations in nucleotides in the New World primate genes leading to failure of the splicing machinery (Gromoll et al., 2007). This form of the receptor has been termed the type-2 LHR. Expression of the type-2 LHR lacking exon 10 has also been observed in humans (Gromoll et al., 2000). The activity of the type-2 LHR was subsequently compared with that of wild-type LHR in vitro and found to be impaired in several aspects of receptor function. First, the type-2 receptor was transported less efficiently to the plasma membrane than wild-type LHR (Zhang et al., 1998). Once expressed at the cell surface, however, the type-2 LHR binds both natural ligands LH and chorionic gonadotrophin (CG) normally (Muller et al., 2003). The second defect in the type-2 receptor involves signal transduction. Whereas LH and CG are equipotent in generating the intracellular signal cyclic AMP in COS-7 cells transfected with wild-type receptor, activation of type-2 LHR by LH was significantly impaired compared with CG (Schuler and Scammell, 2008). These results suggest that type-2 LHR are refractory to stimulation by LH, but respond normally to CG. This is supported by a study with a patient with Leydig cell hypoplasia secondary to homozygous deletion of exon 10. This patient failed to respond to highly elevated serum LH concentrations, but treatment with CG achieved an increase in testosterone biosynthesis, an increase in testicular volume and complete spermatogenesis (Gromoll et al., 2000). How does LH complete its functions in New World primates if these primates naturally express an LHR type that is refractory to LH? The answer is simply that New World primates do not express LH, but rather CG is the relevant pituitary gonadotrophin. Attempts to amplify by reversetranscriptase polymerase chain reaction of the β-subunit of LH from marmoset pituitaries were unsuccessful. Rather, it was discovered that the β-subunit of CG is highly expressed in marmoset pituitaries (Gromoll et al., 2003; Muller et al., 2004). Primate research revealed that squirrel monkey and owl monkey pituitary glands similarly express the β-subunit of CG, but not LH (Scammell et al., 2008; Schuler and Scammell, 2008). Thus, CG is the only gonadotrophin with luteinizing function in the pituitaries of New World primates. Furthermore, New RBMOnline®

Symposium - Genetics of ovarian hyperstimulation syndrome - B Rizk

Figure 8. Chronological development of iatrogenic and spontaneous ovarian hyperstimulation syndrome. FSHR = FSH receptor; HCG = human chorionic gonadotrophin; LH/CGR = LH/chorionic gonadotrophin receptor. Reproduced with permission from Oxford University Press (Delbaere et al., 2004).

World primate CG possesses an amino acid sequence that may result in in-vivo behaviour different from HCG. As mentioned above, the carboxyl-terminal extension of the β-subunit of HCG possesses four O-linked glycosylation sites that are thought to confer a long circulating half-life, approximately 30 h. Using the NetOGlyc 3.1 Server on-line program (available at www. cbs.dtu.dk/services/NetOGlyc/), none of these four sites are predicted to be O-linked glycosylated in New World primate βCG, although the carbohydrate composition of marmoset βCG suggested the presence of one O-linked site (Amato et al., 1998). One of the O-linked sites in New World primate βCG is a recognition site for N-linked glycosylation (Simula et al., 1995; Maston and Ruvolo, 2002; Muller et al., 2004; Scammell et al., 2008). The different glycosylation pattern of New World primate βCG could result in a shorter half-life, which would be appropriate for a pituitary gonadotrophin released in a pulsatile manner. Further studies are required to determine the relative half-lives of human and New World primate CG and indeed to determine within New World primates whether patterns of glycosylation (and hence half-life) of placental CG are different from pituitary CG.

Luteinizing hormone receptor gene mutations Mutations of the LHR and FSHR genes provide natural models for studying the differential effects of LH and FSH on the gonads. Inactivating mutations of the LHR gene are

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recognized as a cause of amenorrhoea and infertility in genetic females and have recently been recognized as the cause of male pseudohermaphroditism resulting from Leydig cell hypoplasia in genetic males (Kremer et al., 1995; Laue et al., 1995; Latronico et al., 1996). Women who carry LHR gene activating mutations are apparently asymptomatic. In boys, heterozygous dominant activating mutations of the LHR gene result in premature Leydig cell activation that presents as gonadotrophin-independent precocious puberty. These findings are corroborated by the fact that HCG-producing tumours result in sexual precocity in males but not in females. Therefore, although the action of HCG and LH is enough to stimulate steroidogenesis in males, ovarian steroidogenesis apparently requires the action of FSH. In conclusion, activating LH mutations have not been clinically associated with syndromes in women undergoing infertility treatment or assisted reproduction. In males, the action of HCG and LH is necessary for the normal development of primary and secondary sexual characteristics and FSH might be necessary for normal spermatogenesis. In contrast, in females, the action of FSH is necessary for the development of mature ovaries and complete secondary sexual characteristics, and additional LH action is necessary for ovulation. These findings infer that the main gonadotrophin is LH in males and FSH in females and that both gonadotrophins are required for fertility in females and possibly also in males (Arnhold et al., 1999). LH receptor polymorphisms in the coding region have been studied by Kerkela et al. (2007) and found not to predispose patients to

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Symposium - Genetics of ovarian hyperstimulation syndrome - B Rizk

OHSS. It is possible that non-coding polymorphisms in the promoter region of these genes might still have functional significance in the pathogenesis of OHSS.

Vascular endothelial growth factor gene and isoforms and OHSS

Bone morphogenetic protein-15

Rizk et al. (1997) highlighted the importance of VEGF as one of the main angiogenic factors responsible for increased vascular permeability leading to extravasation of protein-rich fluid and, subsequently, the full appearance of OHSS. Both its vasoactive properties and its increased ovarian expression during the development of OHSS suggest that VEGF plays a major role in the development of this syndrome and it may be targeted to prevent OHSS (Pellicer et al., 1999; Rizk, 2001; Pau et al., 2006; Busso et al., 2008; Chen et al., 2008; Gutman et al., 2008; Soares et al., 2008; Rizk et al., 2009).

Bone morphogenetic protein-15 (BMP-15) is an important oocyte-derived growth factor which is essential for normal folliculogenesis and female fertility of mammals (Di Pasquale et al., 2004; Moore and Shimasaki, 2005). BMP15 is a member of the transforming growth factor β (TGFβ) superfamily. Within the ovary, BMP-15 mRNA is found exclusively in the oocyte (Juengel and McNatty, 2005). In the human, BMP-15 is detected in the oocytes of primordial follicles and progressively expressed by oocytes in growing follicles through folliculogenesis (Shimasaki et al., 2004). The in-vitro biological activities of BMP-15 demonstrate its role in promoting early follicle growth through the stimulation of granulosa cell mitosis while simultaneously restricting FSHinduced follicle development through the suppression of FSHR mRNA expression. The in-vivo relevance of the role of BMP-15 was established by the identification of naturally occurring BMP-15 mutations in sheep, which cause infertility in homozygous carrier ewes and, in striking contrast, increased fecundity in heterozygous carrier ewes due to an increase in ovulation quota (Moore and Shimasaki, 2005). Therefore, BMP15 appears to be associated with mechanisms of infertility and superfertility in a dosage-sensitive manner (Galloway, 2000). High BMP-15 in follicular fluid is also associated with high quality oocytes and subsequent embryonic development (Wu et al., 2007). Moron et al. (2006) performed a genetic association study of ovarian stimulation outcome in 307 unrelated women with normal ovarian function who underwent ovarian stimulation using recombinant FSH. Four single nucleotide polymorphisms located at the BMP-15 gene were analysed in order to investigate the role of this gene in relation to ovarian stimulation outcome. The results support the hypothesis that BMP-15 alleles predict over-response to recombinant FSH and ovarian hyperstimulation syndrome in humans. Further studies of BMP-15 may help to further confirm this hypothesis and be able to predict patients who are at risk for OHSS.

Other genetic markers that may predict poor response during ovarian stimulation

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As already discussed, the Ser680Asn polymorphism in the FSHR gene is associated with basal FSH concentration and elevated gonadotrophin requirements during ovarian stimulation. This has led other investigators to examine other genetic markers (Georgiou et al., 1997) studied PvuII and rare BstUI restriction fragment length polymorphisms within the ESR1 gene and suggested that ESR1 alleles might affect pregnancy rate and oocyte ratio during in-vitro fertilization. Sundarrajan et al. (2001) partially replicated this work and, moreover, they found that ESR1 PvuII PP carriers had lower follicle and oocyte numbers observed during ovarian stimulation. de Castro et al. (2004) also detected an oligogenic model, including specific FSHR, ESR1 and ESR2 genotype patterns, related to the poor response to FSH hormone during ovarian stimulation treatment.

VEGF gene expression The human VEGF gene (VEGFA, OMIM 192240) is located on chromosome 6p12. Several transcription factor-binding sites are found in the VEGF 5ʹ-untranslated region (5ʹ-UTR) and variation within the region increases the transcriptional activity (Zhao et al., 2008). The human VEGF gene is made up of eight exons. Exons 1–5 and 8 are always present in VEGF mRNA, while the expression of exons 6 and 7 is regulated by alternative splicing. This process allows the formation of various VEGF isoforms differing in length, all VEGF products having a common region. In humans, five different VEGF mRNA have been detected encoding the isoforms VEGF121, VEGF145, VEGF165, VEGF189 and VEGF206. Isoforms VEGF121 and VEGF165 appear to be mainly involved in the process of angiogenesis and are in fact the only ones that the ovary is able to secrete (Rizk, 2006a,b; Busso et al., 2008). The VEGF gene shows the same exonic structure in rodents and humans. Murine VEGF-expressed isoforms VEGF120, VEGF144, VEGF164, VEGF188 and VEGF205 have only one amino acid less length when compared with human VEGF isoforms and there is a 95% protein homology between these two. Similar to the human, hybridization studies in the rat ovary have demonstrated significant VEGF mRNA expression, seen mostly after the LH surge (Rizk et al., 1997; Rizk et al., 2009).

VEGF receptors VEGF receptors are present on the endothelial cell surface and belong to the tyrosine kinase receptor family. They are also present in the inner theca of human follicles. Two specific endothelial cell membrane receptors for VEGF have been identified, VEGFR-1 (Flt-1) and VEGFR-2 (Flk1/KDR). The receptor Flk1/KDR appears to be mainly involved in regulating vascular permeability, angiogenesis and vasculogenesis. VEGFR-1 is also produced as a soluble receptors (sVEGFR-1) by alternative splicing of the precursor mRNA, acting as a modulator of VEGF bioactivity. In fact, the soluble molecules compete with the full length VEGF-R1 for binding VEGF and inhibit vascular permeability. Targeting the Flk1/KDR receptor has been a goal for researchers working in gynaecological oncology. Different specific VEGFR-2 blockers have been used in animal models reducing tumour growth and ascites. The mechanism of ascites formation may be different in neoplasms and OHSS. Busso et al. (2008) presented their elegant research to reverse ascites formation in OHSS targeting RBMOnline®

Symposium - Genetics of ovarian hyperstimulation syndrome - B Rizk

the VEGF system. Future attempts may also make use of genetic expression of VEGF and its receptors to prevent OHSS or reverse its pathological manifestations (Soares et al., 2008; Rizk et al., 2009).

Conclusion A surge in scientific research into the genetic basis of OHSS has been observed in the last decade. The importance of the FSHR genotype for ovarian response in the physiological and therapeutic setting has clearly been demonstrated in vivo. Pharmacogenetics for ovarian stimulation will guide the clinicians. In the future, stimulation protocols that are carefully personalized to each woman’s individual needs according to the polymorphisms inherited might be a reality in ovarian stimulation. However, the FSHR genotype cannot predict the risk of iatrogenic OHSS. Interestingly, in patients who develop OHSS, the FSHR genotype may predict its severity. In primates, the discovery that the LHR that were present in the ovary are type 2 which are refractory to stimulation by LH but respond normally to CG has led to the discovery that New World primates do not express LH but rather CG. Therefore, further studies on gonadotrophin receptors may continue to yield new findings that finally will help us understand the different aspects of OHSS. Prevention of OHSS may be achieved by modulating the pathogenetic mechanisms involved leading to safer treatment options.

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Symposium - Genetics of ovarian hyperstimulation syndrome - B Rizk

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Declaration: The authors report no financial or commercial conflicts of interest. Received 18 December 2008; refereed 20 February 2009; resubmitted 20 April 2009; accepted 8 May 2009.

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