Location
Type
Amino acid involved
Effect
Reference
Exon 3
Missense
Gln54 to Arg
Absence of spontaneous puberty in male
Weiss et al. (1992) [17]
Exon 2
Missense
Trp8 Arg
Ile15 Thr
Delayed pubertal progression in male and infertility in female
Petterson et al. (1991) [18]
Furui et al. (1994) [19]
Haavisto et al. (1995) [20]
Exon 3
Missense
Ala −3 Thr
Normal bioactivity
Jiang et al. (2002) [21]
Exon 3
Missense
Gly102 Ser
Infertility in male, menstrual disorders in female
Liao et al. (1998) [22]
Ramanujam et al. (1999) [23]
Pettersson and Söderholm [18] were the first to describe this common variant of LH (v-LH) as an immunologically anomalous form of LH. The occurrence of the v-LH varies according to geographic areas (Fig. 14.1) [24]. v-LH is due to two point mutations in the β subunit gene, both altering the amino acid sequence (Trp8Arg and Ile15Thr). v-LH has elevated bioactivity in vitro but significantly shorter (5–9 min) half-life in circulation when compared with the wild type LH (wt-LH) (12–22 min). As the pulse frequency of the v-LH is normal, this results in an overall LH action that is more potent at the receptor site but shorter in duration in vivo.
The v-LH is common worldwide, with carrier frequency varying from 0 to 52 % in various ethnic groups. Its incidence in Italy ranges between 12 and 13 %. The v-LH differs functionally from wt-LH, and it seems to predispose its carrier to mild aberrations of reproductive function menstrual irregularities causing infertility [19] and recurrent pregnancy loss [26].
In our observational trial, 60 normogonadotrophic patients undergoing a GnRH-a long downregulation plus r-hFSH for IVF/ICSI, and in whom at least five oocytes were retrieved, were divided into three groups: 22 women requiring a cumulative dose of r-hFSH >3,500 IU constituted group A, 15 patients requiring 2,000–3,500 IU were included in group B, and 23 women requiring <2,000 IU served as control group (group C). The presence of the v-LH was evaluated using immunoassays able to detect both wt-LH and polymorphism. Group A showed a significantly lower (p < 0.05) number of oocytes retrieved when compared with group B and C (7.3 ± 1.5, 11.7 ± 2.4, and 14.7 ± 4.1 in the three groups, respectively). Seven carriers (32 %) of v-LH were found in group A, whereas only one variant (7 %) was observed in group B; no variant was detected in group C. This study suggested, for the first time, an association between a less bioactive LH and a higher FSH requirement. In addition, it supports the idea that hyporesponders represent a specific subgroup of patients. In fact, all women requiring >3,500 IU of FSH had at least five oocytes retrieved and showed peak estradiol >500 pg/ml, which in turn would have lead physicians to classify them as normal responders. Nevertheless, they had a statistically significant reduction of the number of oocytes retrieved and estradiol levels when compared with woman requiring lower FSH doses.
On the basis of these finding we further investigated the relationship between v-LH and ovarian response to FSH [27, 28] in a Danish population. v-LH was present in 11 % of patients, whereas the allelic frequency was 12 %. Patients were divided into two groups according to their LH genotype. Group A included 196 wt/wt women, and group B was constituted by 24 individuals with v-LH (21 heterozygous and 3 homozygous). The mean number of oocytes retrieved, fertilization rate, and pregnancy rate per cycle were similar in the two groups. Group B received a significantly higher cumulative dose of r-hFSH than group A (2,435.86 ± 932.8 IU versus 1,959.8 ± 736.45; P = 0.048). LH genotype had a statistically significant effect (P < 0.01) on the cumulative dose of r-hFSH, showing a progressive increase from wt/wt (1,959.8 ± 736.45 IU) to v-LH heterozygotic (2,267.5 ± 824.3) and homozygotic women (3,558.3 ± 970.9). These results confirmed that carriers of v-LH have hyposensitivity to exogenous FSH during COS.
14.3.2 LH Receptors (Mutations and Polymorphisms)
The luteinizing hormone/choriogonadotrophin receptor (LHCGR) is a member of the superfamily of guanine nucleotide-binding protein-coupled receptors (GPCRs) and belongs to the glycoprotein hormone receptors [29]. LHCGR is expressed in Leydig cells and in ovarian theca, granulosa, as well as luteal cells. These receptors exert a fundamental role in reproductive process since puberty [30]. Several mutations have been identified in LHCGRs, and some of them have been related to reproductive disorders such as male-limited gonadotrophin-dependent precocious puberty, Leydig cell hypoplasia, and anovulation/amenorrhea [30]. In addition, some authors have observed an increased risk for endometrioid adenocarcinoma when rs13405728 mutation in gene LHCGR is associated with SNPs rs2479106 in gene DENND1A [31].
LHCGR mutation can be didactically divided into two categories:
1.
Activating mutations (such as missense Leu368Pro, missense Asp578His), which were associated with precocious puberty and Leydig cell neoplasia
2.
Inactivating mutations, characterized by pseudohermaphroditism and in some cases (such as deletion of exon 10) by normal sexual development with no sign of puberty [32]
In addition to the LHCGR mutations, more than 200 single nucleotide polymorphisms have been discovered. One of the most widespread polymorphisms is due to the presence of a two-amino acid insertion at position 18 in exon 1 (insLQ) and has been detected in breast cancer patient with lower survival rate [33]. Subsequently, another group have analyzed the same polymorphism in PCOS patients, but found no significant association [34].
A detailed phenotype of novel homozygous inactivating nonsense and missense mutations of the LH-receptor gene (Arg 554 stop codon 554 [TGA] and Ser 616 → Tyr 616, respectively) has been described in a woman with compromised ovulation and luteinization processes but apparent normal pubertal feminization [35]. This aforementioned patient presented with high LH and FSH levels and normal estradiol end progesterone values [36].
Evidence about the relationship between LHCGR and reproductive outcome during COS is scarce. In addition to the previously mentioned Kerkala et al.’s observations [34], some authors have recently observed that a higher expression of LH receptors by human cumulus granulosa cells is associated with lower fertilization rate [37].
14.4 The FSH System: From Physiology to COS
14.4.1 FSH Receptor (Mutations and Polymorphisms)
The FSH receptor (FSH-R), likewise its homologue LH, is a glycoprotein hormone receptor that belongs to subfamily of G protein-coupled receptors (GPCRs). FSH mutations have been extensively studied with more than 1,000 polymorphic variants identified to date [38]. Like LHCGR, FSH-R mutations are categorized in “activating” or “inactivating” mutations.
The first “activating” FSH-R mutation was discovered in a hypophysectomized man who surprisingly showed normal spermatogenesis despite undetectable FSH levels [39].
Other two peculiar cases of constitutively activated FSH-R were characterized by heterozygous Thr449Ile and Asp567Asn mutations. Both affected women had a history of spontaneous OHSS syndrome during pregnancy. The probable explanation for this phenotype is linked to the altered ligand site, which becomes activated in the presence of high hCG levels as normally seen during pregnancy [40, 41].
Carriers of “inactivating” mutations are usually affected by hypergonadotrophic hypogonadism, primary or early-onset secondary amenorrhea, variable sexual development, arrest of follicular maturation between primordial and preantral stage, and poor semen quality. While severe phenotypes have been described in carriers of Ala189Val and Pro348Arg mutations, mild forms have been detected in patients with a compound heterozygous mutation of Ala189Val and Ala419Thr [42–44].
The most investigated variant of the FSH-R consists in the replacement at position 680 of the amino acid asparagine by serine (Fig. 14.2) [32]. This polymorphism has been associated with higher basal FSH levels and an increased number of antral follicles during the early follicular phase [45]. In an observational trial, Perez Mayorga et al. [46] evaluated the relationship between the presence of the Ser/680 FSH-R variant and ovarian response to COS in 161 normo-ovulatory women undergoing IVF. All women were below 40 years. The distribution of genotypes in the study population was 29 % for the Asn/Asn, 45 % for the Asn/Ser, and 26 % for the Ser/Ser FSH-R variant. Both estradiol levels at the day of human chorionic gonadotrophin (hCG) and number of retrieved oocytes were similar in the three groups. Conversely, basal FSH levels were significantly different among the three groups (6.4 ± 0.4 IU/l, 7.9 ± 0.3 IU/l, and 8.3 ± 0.6 IU/l for the Asn/Asn, Asn/Ser, and Ser/Ser groups, respectively, P < 0.05). In addition, the mean number of FSH ampoules required for successful stimulation was significantly different among groups (31.8 ± 2.4, 40.7 ± 2.3, and 46.8 ± 5.0 for the Asn/Asn, Asn/Ser, and Ser/Ser groups, respectively, P < 0.05). These clinical findings demonstrated that ovarian response to FSH stimulation depends on the FSH-R genotype. Following these observations, Behre et al. [47] tested whether the same daily dose of FSH resulted in lower levels of estradiol in women homozygous for the Ser/Ser and whether the difference could be overcome by higher FSH doses. Fifty-nine women undergoing COS for IVF or ICSI and homozygous for the FSH-R polymorphism Ser/680 were randomly allocated in three groups. Group I (Ser/Ser, n = 24) received a daily FSH dose of 150 IU/day, and group II (Ser/Ser, n = 25) received a FSH dose of 225 IU/day. In group III (Asn/Asn, n = 44), FSH dose was 150 IU/day. Age and basal FSH levels were not different between groups. Total FSH doses were comparable in group I (1,631 ± 96 IU) and group III (1,640 ± 57 IU) but significantly higher in group II (2,421 ± 112 IU) (P < 0.001). Peak estradiol levels were significantly lower in group I (5,680 ± 675 pmol/l) compared to group III (8,679 ± 804 pmol/l) (P < 0.05). Increasing the FSH dose from 150 to 225 IU/day overcame the lower estradiol response in women with Ser/Ser (group II, 7,804 ± 983 pmol/l). The authors concluded that patients with the Ser/Ser FSH-R variant have lower FSH receptor sensitivity, which can be overcome by higher FSH doses. This study represented the first case of a pharmacogenomic approach to COS.
Fig. 14.2
Human FSH receptor mutations. The Ser680Asn is in linkage disequilibrium with Thr307Ala (green) (From Huhtaniemi and Themmen [32])
Recently, we have evaluated the occurrence of the Ser/680 FSH-R variant among women classified as “hyporesponders” (Alviggi et al. 2013). Forty-two normogonadotrophic patients in whom at least five oocytes were retrieved after GnRH-a long downregulation protocol followed by stimulation with r-hFSH for IVF/ICSI were retrospectively studied. On the basis of the total r-hFSH consumption, patients were divided into two groups: 17 women requiring a cumulative dose of r-hFSH >2,500 IU constituted group A, whereas 25 patients requiring <2,500 IU served as controls (group B). DNA was analyzed to determine the FSH receptor genotype. Estradiol peak levels were significantly lower in group A (997 ± 385 pg/ml) when compared with group B (1,749 ± 644; P < 0.001). The number of oocytes retrieved was also significantly lower in group A compared with group B (7.1 ± 1.5 versus 9.6 ± 2.4; P < 0.001). Homozygous Ser/Ser receptor variant at codon 680 was observed in 47.0 % of women of group A and in 28.0 % of women of the control group. The homozygous Asn/Asn receptor variant was found in 23.6 and 20.0 % of patients in the two groups, respectively. Heterozygosis Ser/Asn was detected in 29.4 % of patients of group A and in 52.0 % of patients of group B. These results indicated that FSH-R Ser 680/variant is more frequent in women with hyporesponse to r-hFSH.
Although some investigators found a positive association between pregnancy rate and presence of Ser680 genotype [48, 49], a recent meta-analysis confirmed that Ser/Ser genotype carriers have significantly higher basal FSH levels and require higher exogenous FSH doses for COS [50].
Nakayama et al. in 2006 identified another polymorphic variant of FSH-R with possible implication in COS [51]. It consisted of a polymorphism in the 5′-UTR of the FSH-R gene (position 29 A/G; rs1394205), which seems to be associated with a lower luciferase activity compared with G/G 29 allele. Subsequently, Desai and colleagues observed a reduced FSH-R expression in granulosa cells of AA genotype carriers [52].
In women undergoing assisted reproduction, variants A/A have been associated with poor ovarian response with respect to number of oocyte retrieved and doses of exogenous FSH for COS [53].
Lastly, the impact of a new FSH-R polymorphism has been investigated in a female Indian population. Specifically, 50 patients undergoing ART and 100 fertile patients have been recruited. The authors observed that Ala307Ala carriers required lower amount of exogenous FSH for ovulation induction in comparison with Thr307Thr and Thr307Ala subjects. Estradiol levels and incidence of OHSS were higher in the former [54].
14.4.2 FSH Mutations and Polymorphisms
Several β subunit mutations of FSH have been identified in the literature. Most of them inactivate the FSH effects. In females, primary amenorrhea, impaired fertility, and compromised pubertal development are the most frequent clinical manifestations. In contrast, FSH mutations in males do not affect sexual maturation although they result in azoospermia. Most of FSH mutations interfere with a specific cysteine knot region that is crucial for dimerization with α subunit and biological activity [32].
Unlike LH β, most of FSH polymorphic variants have been found in noncoding regions confirming that FSH β is strongly conserved in the human species [32, 59].
To date, only a single nucleotide polymorphism located into FSH β promoter-211G/T seemed to influence the FSH concentration in males [60, 61]. In addition, it seems that Ser 680 Asn polymorphism may influence serum FSH levels in the male population [62]. The same effects were also reported in the female population. Higher FSH serum levels were observed in women with the FSHB-211 GT + TT/associated with FSHR2039 AA genotype [59]. The impact of FSH polymorphisms and their combination with different FSH-R genotypes is yet to be evaluated.
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