Abstract
Objective
To assess the pharmacogenetic potential of FSH for infertility treatment.
Design
Review of the literature and genomic databases.
Methods
Single-nucleotide polymorphism (SNP) assessed: rs6166 (c.2039A>G, p.N680S), rs6165 (c.919A>G, p.T307A), rs1394205 (c.−29G>A) in FSHR, and rs10835638 (c.−211G>T) in FSHB. Literature search via PubMed. Blast analysis of genomic information available in the NCBI nucleotide database. Comparison of allele frequency and haplotype distribution using the http://spsmart.cesga.estool.
Results
All these SNPs appear first in Homo, result in reduced FSH action, and are present with variable frequencies and combinations worldwide. Stringent clinical studies demonstrate that the FSHR genotype influences serum FSH levels and gonadal response in both sexes. Serum FSH levels depend on the −211G>T SNP, influencing transcriptional activity of the FSHB promoter. Genotypes reducing FSH action are overrepresented in infertile subjects.
Conclusions
Although the clinical relevance of the FSHR polymorphisms alone is limited, the combination of FSHR and FSHB genotypes has a much stronger impact than either one alone in both sexes. About 20% of people are carriers of the alleles associated with lower serum FSH levels/reduced FSHR expression or activity, possibly less favorable for reproduction. Prospective studies need to investigate whether stratification of infertile patients according to their FSHR–FSHB genotypes improves clinical efficacy of FSH treatment compared with the current, naïve approach. A relative enrichment of less favorable FSHR–FSHB genotypes may be related to changes in human reproductive strategies and be a marker of some health-related advantage at the cost of reduced fertility.
Invited Author's profile
Manuela Simoni is Full Professor of Endocrinology, Department of Biomedicine, Metabolism and Neurosciences, University of Modena & Reggio Emilia, Italy. Her research focuses on reproductive endocrinology and andrology (basic and clinic).
Introduction
Follicle-stimulating hormone (FSH) is fundamental for gamete maturation and is widely used in the treatment of hypogonadotropic hypogonadism and infertility in both sexes (1, 2). Both urinary FSH (in form of human menopausal gonadotropins (hMG) and highly purified FSH) and recombinant FSH (rFSH, and long acting rFSH) are commercially available and currently in use with very good results, especially in assisted reproduction. As unwanted infertility (defined as lack of conception after 1 year of unprotected intercourse) affects around 30% of couples, fertility treatment is very popular and the gonadotropin market florid, especially among gynecologists performing assisted reproduction technologies (ARTs) as well as endocrinologists and andrologists treating hypogonadism and male infertility.
With a steadily increasing demand for infertility treatment, therapies involving the use of FSH continue to evolve rapidly, mostly on empirical bases, so that ART protocols are often adjusted based on availability of new drug preparations despite the paucity of scientifically sound data. Systematic reviews (e.g. from the Cochrane collaboration) are regularly issued in order to compare different protocols but often fail to demonstrate the purported superiority of the new approaches (3, 4, 5, 6). On the other hand, new protocols of infertility treatment and ART are greatly needed, especially considering the constantly increasing age of women in couples undergoing ART (7), a factor which decreases the efficacy of the treatment by affecting both pregnancy and abortion rate. In such a scenario, pharmacogenetic approaches are appealing and have been proposed (8).
FSH works through binding to its specific receptor, the FSHR (FSHR, gene ID: 2492, location: 2p21–p16, OMIM: 136435) (9). In physiological conditions, serum FSH levels are under hypothalamic and gonadal control, depend on age and, in women, on the menstrual cyclicity. Research of the last two decades revealed the role of common genetic variants of FSHR and FSH beta subunit (FSHB, gene ID: 14308, location: 11p13, OMIM: 136530) in the determination of individual serum hormone levels and target organ response (10, 11). However, controversies exist concerning the impact of genetic polymorphisms of these genes on gonadotropin treatment and some contradictory findings have been published. Several good reviews appeared recently in the literature summarizing the current knowledge, covering various aspects of this topic (12, 13, 14, 15, 16, 17), and showing clearly that, currently, there is not enough evidence to provide practical clinical recommendations for the pharmacogenetic use of FSH.
In this article, we take a different approach and try to give a coherent interpretation to a rich literature containing partially contradictory and fragmented data: starting with the analysis of evolutionary aspects, considering the biological consequences of the genetic variants in vitro and in vivo under various pathophysiological conditions, and examining the possible explanations for the controversies in the literature, we propose that the true pharmacogenetic potential of FSH use in therapy becomes evident only when the genetic complexity of the FSH–FSHR system is considered in its whole. As the most studied genetic variants of FSHR and FSHB are associated with an overall lower FSH activity and appear first in the species Homo, we are facing an apparent evolutionary paradox, which suggests the opportunity of a wider view of the FSH role in lifetime reproductive success. We suggest that the genotypes of FSHR and FSHB both influence the final biological activity of FSH and should be considered together in future pharmacogenetic studies dealing with infertility.
Genetic variants of FSHR and FSHB: frequency, ethnic distribution, and evolution
The receptor for FSH belongs to the family of G protein-coupled receptors. Its three-dimensional structure was resolved recently, and the mode of FSH–FSHR interaction was illustrated (18). Figure 1 shows the genomic location of the FSHB and FSHR genes along with the position of the single-nucleotide polymorphisms (SNPs) considered in this article (Table 1), chosen because much information about their possible role in the biological response to FSH exist in the literature. As shown in Fig. 1, these SNPs are selected genetic markers among many others belonging to complex genomic regions: the HapMap Database (http://hapmap.ncbi.nlm.nih.gov) currently shows about 900 SNPs for FSHR and 24 for FSHB, respectively, organized in distinct linkage disequilibrium (LD) blocks, segments of the genome in which a given combination of alleles or genetic markers is inherited coordinately. The structure of FSHR and FSHB LD blocks in Caucasians is shown in Fig. 1. LD blocks differ in their structure between ethnic groups, resulting in various combinations of the different SNPs. This should be considered when assessing association studies obtained in different populations.
Intron–exon structure, SNP positions, and LD blocks in the human FSHR and FSHB genes. The LD blocks (dark triangles) were calculated by the Software Haploview (http://www.broadinstitute.org) in the selection of 121 samples obtained from Utah residents with Northern and Western European ancestry from the CEPH collection (CEU). The darkness of the squares indicates the strength of LD. The SNPs are indicated by black lines above the LD blocks (arrows). Gene name, chromosome number, and coordinates of the shown region, specified in the white boxes within each panel, were obtained from the HapMap Database (http://hapmap.ncbi.nlm.nih.gov). (A) Two hundred kilobase genomic region of chromosome 2 including the human FSHR gene. Exons are represented by boxes and the positions of SNPs rs6166 (c.2039A>G), rs6165 (c.919A>G), and rs1394205 (−29G>A) are indicated. (B) Two hundred kilobase genomic region of chromosome 11 including the human FSHB gene. The genomic region in the boxed area is enlarged in (C). (C) Five kilobase genomic region of chromosome 11 including the human FSHB gene and the position of SNP rs10835638 (−211G>T).
Citation: European Journal of Endocrinology 170, 3; 10.1530/EJE-13-0624
Intron–exon structure, SNP positions, and LD blocks in the human FSHR and FSHB genes. The LD blocks (dark triangles) were calculated by the Software Haploview (http://www.broadinstitute.org) in the selection of 121 samples obtained from Utah residents with Northern and Western European ancestry from the CEPH collection (CEU). The darkness of the squares indicates the strength of LD. The SNPs are indicated by black lines above the LD blocks (arrows). Gene name, chromosome number, and coordinates of the shown region, specified in the white boxes within each panel, were obtained from the HapMap Database (http://hapmap.ncbi.nlm.nih.gov). (A) Two hundred kilobase genomic region of chromosome 2 including the human FSHR gene. Exons are represented by boxes and the positions of SNPs rs6166 (c.2039A>G), rs6165 (c.919A>G), and rs1394205 (−29G>A) are indicated. (B) Two hundred kilobase genomic region of chromosome 11 including the human FSHB gene. The genomic region in the boxed area is enlarged in (C). (C) Five kilobase genomic region of chromosome 11 including the human FSHB gene and the position of SNP rs10835638 (−211G>T).
Citation: European Journal of Endocrinology 170, 3; 10.1530/EJE-13-0624
Intron–exon structure, SNP positions, and LD blocks in the human FSHR and FSHB genes. The LD blocks (dark triangles) were calculated by the Software Haploview (http://www.broadinstitute.org) in the selection of 121 samples obtained from Utah residents with Northern and Western European ancestry from the CEPH collection (CEU). The darkness of the squares indicates the strength of LD. The SNPs are indicated by black lines above the LD blocks (arrows). Gene name, chromosome number, and coordinates of the shown region, specified in the white boxes within each panel, were obtained from the HapMap Database (http://hapmap.ncbi.nlm.nih.gov). (A) Two hundred kilobase genomic region of chromosome 2 including the human FSHR gene. Exons are represented by boxes and the positions of SNPs rs6166 (c.2039A>G), rs6165 (c.919A>G), and rs1394205 (−29G>A) are indicated. (B) Two hundred kilobase genomic region of chromosome 11 including the human FSHB gene. The genomic region in the boxed area is enlarged in (C). (C) Five kilobase genomic region of chromosome 11 including the human FSHB gene and the position of SNP rs10835638 (−211G>T).
Citation: European Journal of Endocrinology 170, 3; 10.1530/EJE-13-0624
Nomenclature of genes and polymorphisms discussed in this article (http://www.ncbi.nlm.nih.gov/pubmed).
| Gene | refSNP | DNA nucleotide | Single-nucleotide variation | NCBI nucleotide reference sequence | Protein | NCBI protein reference sequence |
|---|---|---|---|---|---|---|
| FSHR | rs1394205 | −29G>A | g.49381585C>T | NT_022184.15 | ||
| FSHR | rs6165 | c.919G>A | g.49191041C>T | NT_022184.15 | p.A307T | P23945.3 |
| FSHR | rs6166 | c.2039A>G | g.49189921T>C | NT_022184.15 | p.N680S | P23945.3 |
| FSHB | rs10835638 | −211G>T | g.30252352G>T | NT_009237.18 |
Progress in genomic research and studies on human diversity produce public databases, which can be consulted freely online. We interrogated these databases (HapMap, Release #28; Perlegen, complete data set; CEPH, U. Stanford HGDP; CEPH, NIH-U. Michigan HGDP; 1000 Genomes, Phase I May 2011) to analyze, in different ethnic groups, frequency, distribution, and combinations of the SNPs considered in this article. The analysis was performed using the http://spsmart.cesga.estool, freely available online.
FSHR
Concerning the FSHR, the most studied, non-synonymous SNP is rs6166 (c.2039A>G, p.N680S), which shows a high degree of LD with rs6165 (c.919A>G, p.T307A) (10) in many ethnic groups (Table 2). Both SNPs are located in exon 10 and cause an amino acid exchange: rs6166 exchanges asparagine for serine in the intracellular domain of the receptor, introducing a potential phosphorylation site; rs6165 replaces threonine by alanine, i.e. it results in a change from a polar (T) to a nonpolar, hydrophobic (A) amino acid and removes a potential O-linked glycosylation site. Another common but less studied SNP (rs1394205) exchanges a nucleotide in the promoter region of the FSHR (−29G>A). The analysis of these SNPs using the http://spsmart.cesga.estool, extracting the genomic data present in the available databases (HapMap, Release #28; Perlegen, complete data set; CEPH, U. Stanford HGDP; CEPH, NIH-U. Michigan HGDP; 1000 Genomes, Phase I May 2011), reveals differences in their frequency between human populations.
Pairwise linkage disequilibrium (LD) parameters between the polymorphisms rs6165 and rs6166 in HapMap populations calculated by the Software Haploview (http://www.broadinstitute.org).
| HapMap populations | Sample size | rs6166 MAF | rs6165 MAF | D' | r2 |
|---|---|---|---|---|---|
| African ancestry in Southwest USA (ASW) | 53 | G 0.411 | A 0.325 | 1.000 | 0.362 |
| Luhya in Webuye, Kenya (LWK) | 110 | G 0.350 | A 0.209 | 1.000 | 0.143 |
| Maasai in Kinyawa, Kenya (MKK) | 156 | G 0.374 | A 0.436 | 0.979 | 0.445 |
| Yoruban in Ibadan, Nigeria (YRI) | 153 | G 0.493 | A 0.224 | 0.961 | 0.251 |
| Utah residents with European ancestry (CEU) | 121 | G 0.403 | G 0.403 | 1.000 | 1.000 |
| Tuscan in Italy (TSI) | 102 | G 0.475 | G 0.470 | 1.000 | 0.977 |
| Gujarati Indians in Houston, Texas (GIH) | 101 | A 0.490 | A 0.490 | 1.000 | 0.978 |
| Han Chinese in Beijing, China (CHB) | 139 | G 0.281 | G 0.312 | 1.000 | 0.865 |
| Chinese in Metropolitan Denver, Colorado (CHD) | 109 | G 0.303 | G 0.321 | 1.000 | 0.924 |
| Japanese in Tokyo, Japan (JPT) | 116 | G 0.348 | G 0.363 | 0.974 | 0.902 |
| Mexican ancestry in Los Angeles, California (MEX) | 58 | G 0.328 | G 0.316 | 0.956 | 0.876 |
Population names and their abbreviation (in brackets), sample size and minor allele frequencies (MAF) of rs6166 (FSHR c.2039A>G) and rs6165 (FSHR c.919A>G) were taken from the HapMap Database (http://hapmap.ncbi.nlm.nih.gov). The reference allele is placed before each MAF value. The two LD values D' and r2 are shown. D' is calculated by Haploview as D'=D/Dmax, where D is the deviation of the observed from the expected. r2 is the correlation coefficient between pairs of loci. The maximum values of D' and r2 are 1.000, which indicate complete LD or pairwise correlation between the loci respectively. D' and r2=0.000 indicate random coupling of the SNPs.
Looking at rs6166, its remarkable ethnic distribution is shown in Fig. 2, freely available at http://hgdp.uchicago.edu/cgi-bin/alfreqs.cgi?pos=49043425&chr=chr2&rs=rs6166&imp=false (19, 20) depicting the allelic frequency in 52 populations and demonstrating important interpopulation differences.
Geographic distribution of the rs6166 (FSHR c.2039A>G) allele frequencies. The world map with geographic coordinates shows the distribution of the alleles A and G in different countries by pie charts, calculated by the HGDP Selection Browser (http://hgdp.uchicago.edu). The panel on the right side shows the allele frequencies by bar charts for each population sample from different regions of the earth. The data on which the analysis is based are from the Stanford University SNPs selection from the Human Genome Diversity Project (http://www.hagsc.org/hgdp/files.html).
Citation: European Journal of Endocrinology 170, 3; 10.1530/EJE-13-0624
Geographic distribution of the rs6166 (FSHR c.2039A>G) allele frequencies. The world map with geographic coordinates shows the distribution of the alleles A and G in different countries by pie charts, calculated by the HGDP Selection Browser (http://hgdp.uchicago.edu). The panel on the right side shows the allele frequencies by bar charts for each population sample from different regions of the earth. The data on which the analysis is based are from the Stanford University SNPs selection from the Human Genome Diversity Project (http://www.hagsc.org/hgdp/files.html).
Citation: European Journal of Endocrinology 170, 3; 10.1530/EJE-13-0624
Geographic distribution of the rs6166 (FSHR c.2039A>G) allele frequencies. The world map with geographic coordinates shows the distribution of the alleles A and G in different countries by pie charts, calculated by the HGDP Selection Browser (http://hgdp.uchicago.edu). The panel on the right side shows the allele frequencies by bar charts for each population sample from different regions of the earth. The data on which the analysis is based are from the Stanford University SNPs selection from the Human Genome Diversity Project (http://www.hagsc.org/hgdp/files.html).
Citation: European Journal of Endocrinology 170, 3; 10.1530/EJE-13-0624
The ancestral A allele is most predominant in Southeastern Asia, while the derived G allele is highly prevalent in other populations and reaches its higher frequencies in Kalash (North-Western Pakistan), Yakuts (Siberia), Suruì Paiter (Mato Grosso, Brazil), and Melanesians (Oceania). It is interesting to notice that these ethnicities are geographically isolated and genetically well distinct, and posses a number of other genetic peculiarities. For instance, Melanesians display genomic signs of direct introgression of Neanderthal genome in modern humans after migration from sub-Saharan Africa as a consequence of strong positive selection (21); Yakuts are characterized by very low genetic diversity (22); Kalash and Suruì Paiter are very small populations (only a few hundred individuals left) at extinction risk. In these ethnic groups the enrichment of the rs6166 G allele of the FSHR may be a consequence of insulation or the result of genetic drift.
Considering all genomic data available in the public databases together (Fig. 3A, summarizing genomic data of 3228 individuals), the ancestral A allele has a minor allele frequency (MAF) of 0.6 in sub-Saharan Africans, which may be considered closer to the ancient human population. The G allele is enriched in Europeans, Middle East, Central-South Asia, and Oceania, while it shows the lowest frequency in Far East Asia and North America. The fixation index (FST) value, a measure of the population differentiation due to genetic structure, calculated for rs6166, is high in East Asians (0.0525) compared with Europeans (0.0195) and Middle East (0.007) populations, a phenomenon seen with genetic variants in several other genes (the so-called East Asian sweep pattern) of uncertain meaning (23). In any case, these ethnic differences should be considered when we turn to genetic association, and clinical studies based on this SNP, as they might explain some heterogeneity in the results.
Allele and genotype frequencies of the FSHR exon 10 SNPs rs6166 (c.2039A>G (A)) and rs6165 (c.919A>G (B)) in different geographic regions. The sample size is indicated above each bar; data were analyzed by SPSmart (http://spsmart.cesga.es) using the databases HapMap, 1000 Genomes, HGDP CEPH Stanford selection, and Perlegen as data sources. NA, data not available.
Citation: European Journal of Endocrinology 170, 3; 10.1530/EJE-13-0624
Allele and genotype frequencies of the FSHR exon 10 SNPs rs6166 (c.2039A>G (A)) and rs6165 (c.919A>G (B)) in different geographic regions. The sample size is indicated above each bar; data were analyzed by SPSmart (http://spsmart.cesga.es) using the databases HapMap, 1000 Genomes, HGDP CEPH Stanford selection, and Perlegen as data sources. NA, data not available.
Citation: European Journal of Endocrinology 170, 3; 10.1530/EJE-13-0624
Allele and genotype frequencies of the FSHR exon 10 SNPs rs6166 (c.2039A>G (A)) and rs6165 (c.919A>G (B)) in different geographic regions. The sample size is indicated above each bar; data were analyzed by SPSmart (http://spsmart.cesga.es) using the databases HapMap, 1000 Genomes, HGDP CEPH Stanford selection, and Perlegen as data sources. NA, data not available.
Citation: European Journal of Endocrinology 170, 3; 10.1530/EJE-13-0624
rs6166 is evolutionarily recent. As shown in Supplementary Fig. 1, see section on supplementary data given at the end of this article, FSHR c.2039A>G (p.N680S) is not present in nonhuman primates and, in most animal genomes analyzed so far, the Fshr gene carries an Asn at the amino acid position corresponding to position 680 of the human FSHR. The analysis of the Neanderthal genome (http://neandertal.ensemblgenomes.org) shows the presence of the G allele (Ser) in the three samples analyzed so far. This suggests that the new allele was already present in an extinct hominid branch very close to the modern human. Since, as it will be discussed below, the FSHR allele carrying a Ser at amino acid position 680 is functionally ‘resistant’ to FSH both in women and in men, the evolutionary advantage of this allele is still unclear.
Population data about rs6165 are less abundant and show some difference compared with rs6166 (Fig. 3B, summarizing genomic data of 2287 individuals). In particular, the c.919A>G ancestral G allele is predominant in sub-Saharan African populations, with a MAF of 0.274, while the MAF is grossly similar to that of rs6166 in the other ethnic groups. This suggests that the two SNPs are not in LD in Africans and, to a minor extent, in other ethnicities as well (Table 2). Practically, in all other species sequenced so far, the G allele is the rule and Ala is the amino acid occupying the position corresponding to 307 of the human FSHR (Supplementary Fig. 2, see section on supplementary data given at the end of this article). Therefore, both amino acids 307 and 680 are highly conserved across species but, interestingly, the nonhuman FSHR haplotype is Ala307–Asn680, which is rarely found in modern humans, with the notable exception of Africans. We consider this as the ancestral haplotype and assume that it changed through two independent mutational events, one introducing rs6166 (c.2039A>G; p.N680S), with the ancient allele encoding Asn still predominating in most populations (Fig. 3); the other event, rs6165 (c.919A>G; p.T307A), now results in a predominance of the derived allele (encoding Thr) in all ethnic groups except Africans. As a result, these changes are now in LD in most people and form the two major exon 10 haplotypes, Thr307–Asn680 and Ala307–Ser680.
The third SNP in the FSHR considered in this article is rs1394205 (c.−29G>A), located in a separated LD block (Fig. 1) and found with different frequencies and independently of the exon 10 haplotype (24). Ethnic differences in the distribution of this SNP have been known already for some time (25) and are confirmed by the current database collections (Fig. 4A, summarizing genomic data of 2288 individuals). Comparative alignment analysis suggests that the dominant G allele is the ancestral allele (Supplementary Fig. 3, see section on supplementary data given at the end of this article).
Allele and genotype frequencies of the FSHR and FSHB promoter SNPs rs1394205 (−29G>A (A)) and rs10835638 (−211G>T (B)) in different geographic regions. The sample size is indicated above each bar; data were analyzed by SPSmart (http://spsmart.cesga.es) using the databases HapMap, 1000 Genomes, HGDP CEPH Stanford selection, and Perlegen as data sources. NA, data not available.
Citation: European Journal of Endocrinology 170, 3; 10.1530/EJE-13-0624
Allele and genotype frequencies of the FSHR and FSHB promoter SNPs rs1394205 (−29G>A (A)) and rs10835638 (−211G>T (B)) in different geographic regions. The sample size is indicated above each bar; data were analyzed by SPSmart (http://spsmart.cesga.es) using the databases HapMap, 1000 Genomes, HGDP CEPH Stanford selection, and Perlegen as data sources. NA, data not available.
Citation: European Journal of Endocrinology 170, 3; 10.1530/EJE-13-0624
Allele and genotype frequencies of the FSHR and FSHB promoter SNPs rs1394205 (−29G>A (A)) and rs10835638 (−211G>T (B)) in different geographic regions. The sample size is indicated above each bar; data were analyzed by SPSmart (http://spsmart.cesga.es) using the databases HapMap, 1000 Genomes, HGDP CEPH Stanford selection, and Perlegen as data sources. NA, data not available.
Citation: European Journal of Endocrinology 170, 3; 10.1530/EJE-13-0624
FSHB
As far as FSHB is concerned, a detailed populations genetics study identified two major haplotypes possibly influencing conception (26). Most clinical studies performed so far consider only the SNP rs10835638 (c.−211G>T) and genomic databases contain information about 1093 individuals (Fig. 4B). The MAF of the −211G>T varies between 0.028 (in Africans) and 0.145 (in Europeans). It appears that this SNP increased in frequency after migration out of Africa and is epidemiologically relevant only in Europe and USA, with 20–25% of people carrying at least one T allele. Despite being rare, this SNP has a significant functional importance (11), and it is located in an element of the FSHB promoter, which binds the LHX3 homeodomain transcription factor influencing gene transcription (27). Alignment of the corresponding genomic region in several species demonstrates a large predominance of G (Supplementary Fig. 4, see section on supplementary data given at the end of this article), which therefore represents the ancestral allele.
Genetic variants of FSHR and FSHB: in vitro effects
In clinical studies, the variants of FSHR described above have been associated with changes in the sensitivity to FSH and the −211G>T SNP of the FSHB gene was associated to reduced FSH serum levels, as will be considered in the next section. Some in vitro studies were devoted to ascertain the molecular mechanism causing such changes in the levels/activity of FSH.
FSHR
As a number of clinical studies suggest a different sensitivity to FSH of the two FSHR exon 10 haplotypes in vivo (see next chapter), some experiments were dedicated to clarify the possible mechanism at the molecular level. The molecular consequences of the combination of p.T307A and p.N680S (Ala307–Ser680) have been studied in transiently transfected cell lines and in human granulosa–lutein cells (hGLC) naturally expressing one of the two receptor variants (28, 29, 30, 31). Intriguingly, no difference between the receptor activation depending on the FSHR haplotype could be demonstrated when very early events of cellular response, such as cAMP and IP3 production, were studied in various transiently transfected cell lines (28, 29, 30, 31). hGLC homozygous for each FSHR haplotype responded identically in terms of cAMP, progesterone and estradiol (E2) production, measured at saturation (31). Another experiment suggested that hGLC homozygous for Asn at amino acid position 680 only ‘tended’ toward higher FSH-induced FSHR expression, whereas the expression of other well-known FSHR-dependent genes, such as LHCGR and CYP19A1, was not affected (32).
We recently have analyzed the effects of FSHR exon 10 SNPs in vitro more in depth, looking at the kinetics of response and at different signal transduction pathways. Our data show that the two FSHR variants, studied in hGLC, respond by activating the different signal transduction pathways with different kinetics, suggesting that the final biologic response involves different mechanisms. As an example of these novel experiments, we show in Fig. 5 that ERK1/2 activation is blunted in hGLC naturally expressing the combination of p.T307A A and p.N680S S allele (Ala307–Ser680). These data, repeatedly confirmed in our laboratory (L Casarini , V Moriondo, Marino, Adversi, Capodanno, Grisolia, A La Marca, La Sala, M Simoni, unpublished observations), suggest for the first time that the Ala307–Ser680 FSHR is indeed less ‘active’ in vitro, providing a molecular explanation for the clinical data.
Kinetics of phospho-ERK1/2 activation in the absence (left panels) and in the presence (right panels) of 50 nM r-FSH (Gonal-F, Merck–Serono S.p.A.) in p.N680S homozygous -N or -S human primary lutein granulosa cells (hGLC), detected by western blotting. Cell preparation was performed as previously described (31) and specified in Supplementary Methods, see section on supplementary data given at the end of this article. One representative of three independent experiments is shown. hGLC carrying the p.N680S N homozygous genotype shown potent and rapid ERK1/2 FSH-dependent phosphorylation. hGLC homozygous for the p.N680S S genotype response to FSH was much lower and delayed in terms of ERK1/2 activation. PMA indicates the ERK1/2 activator phorbol myristate acetate (positive control); U0126 is a MEK (MAPKK) inhibitor (negative control).
Citation: European Journal of Endocrinology 170, 3; 10.1530/EJE-13-0624
Kinetics of phospho-ERK1/2 activation in the absence (left panels) and in the presence (right panels) of 50 nM r-FSH (Gonal-F, Merck–Serono S.p.A.) in p.N680S homozygous -N or -S human primary lutein granulosa cells (hGLC), detected by western blotting. Cell preparation was performed as previously described (31) and specified in Supplementary Methods, see section on supplementary data given at the end of this article. One representative of three independent experiments is shown. hGLC carrying the p.N680S N homozygous genotype shown potent and rapid ERK1/2 FSH-dependent phosphorylation. hGLC homozygous for the p.N680S S genotype response to FSH was much lower and delayed in terms of ERK1/2 activation. PMA indicates the ERK1/2 activator phorbol myristate acetate (positive control); U0126 is a MEK (MAPKK) inhibitor (negative control).
Citation: European Journal of Endocrinology 170, 3; 10.1530/EJE-13-0624
Kinetics of phospho-ERK1/2 activation in the absence (left panels) and in the presence (right panels) of 50 nM r-FSH (Gonal-F, Merck–Serono S.p.A.) in p.N680S homozygous -N or -S human primary lutein granulosa cells (hGLC), detected by western blotting. Cell preparation was performed as previously described (31) and specified in Supplementary Methods, see section on supplementary data given at the end of this article. One representative of three independent experiments is shown. hGLC carrying the p.N680S N homozygous genotype shown potent and rapid ERK1/2 FSH-dependent phosphorylation. hGLC homozygous for the p.N680S S genotype response to FSH was much lower and delayed in terms of ERK1/2 activation. PMA indicates the ERK1/2 activator phorbol myristate acetate (positive control); U0126 is a MEK (MAPKK) inhibitor (negative control).
Citation: European Journal of Endocrinology 170, 3; 10.1530/EJE-13-0624
Concerning the FSHR −29G>A, this SNP is located in the promoter region, in a consensus sequence for the cellular homolog to the viral E26 transformation-specific sequence (cETS-1). Our early experiments did not show statistically significant difference in the activity of the promoter in the two different cell lines COS7 and SK11 (25). However, others could demonstrate, using CHO cells, that this single base exchange resulted in a significant 56% decrease in the transcriptional promoter activity of the A allele (33). It has been shown that promoter activity by reporter assay can vary consistently depending on the cell line used (34), possibly explaining this discrepancy. The reduced promoter activity in vitro, as shown by Nakayama et al. (33), fits well with the clinical findings.
FSHB
The SNP rs10835638 (−211G>T) in the FSHB promoter falls in a binding element for the LHX3 homeodomain transcription factor, capable of influencing gene transcription. The −211G>T T allele decreased transcriptional promoter activity in vitro by about 50% in the LβT2 gonadotrope cell line (27). This confirmed earlier studies in vitro showing a reduction in promoter activity varying from 46% (in JEG3 cells) to 58% (in TE671 cells), and 86% (in HEK293T cells) (34). The FSHB promoter region containing SNP rs10835638 is located in a putative hormone-responsive element, but recent experiments have revealed that, unlike the murine that of Fshb, progestins and androgens are unable to induce FSHB transcription (27). Therefore, in the human, circulating progesterone levels are not expected to modulate serum FSH directly at the pituitary level via this mechanism.
In summary, the evolutionarily more recent SNPs, both in the FSHR and in the FSHB genes, are associated in vitro with changes either in signal transduction (FSHR exon 10) or in transcriptional activity (FSHR and FSHB promoter) resulting in an overall reduced FSH action.
FSHR and FSHB polymorphisms influence serum FSH levels and reproductive parameters: studies in women
FSHR
The most popular model to study whether FSHR polymorphisms have any effect on FSH levels/action is represented by women with seemingly normal ovarian function undergoing ART for infertility in couples due to a male or tubal factor. These women are treated with FSH to induce multiple follicle development. Classically, they receive between 2000 and 5000 IU of FSH over 7–15 days of stimulation with a remarkable interindividual variability in ovarian response. In these women, the measurement of basal serum FSH levels, the amount of exogenous FSH needed to reach multi-follicular development, and the levels of serum E2 at the time of human chorionic gonadotropin (hCG) administration for final follicular maturation can be taken as parameters of FSHR sensitivity.
The first observation that the FSHR haplotype consisting of the two SNP in exon 10 could be a determinant of serum FSH levels and ovarian response to FSH dates back to 2000 (10). In normoovulatory women undergoing ART, we showed that the FSHR exon 10 haplotype p.T307A A and p.N680S S allele (Ala307–Ser680) was less sensitive to FSH, as these women had significantly higher basal serum FSH levels and required significantly more FSH to achieve multiple follicular maturation (10). This observation immediately opened the questions whether FSHR genotype might be relevant for pharmacogenetic FSH therapy and for menstrual cycle physiology. A study involving the very stringent monitoring of the menstrual cycle in young (mean age: 25 years), normally cycling, ovulatory women of European origin, indeed demonstrated the FSHR genotype to be instrumental in the determination of serum levels of FSH, menstrual cycle length, and dynamics of reproductive hormones (35).
Following our original observation, a large number of studies have been published, mostly in women undergoing ART and reporting partially conflicting results. The reasons for these discrepancies will be analyzed in this study. In Supplementary Table 1, see section on supplementary data given at the end of this article, we summarize the main data of the studies published so far concerning FSHR genotype frequency distribution (rs6166), basal FSH levels, and FSH dosage subdivided by genotype. In addition, from each study, we extracted the information regarding the age of the study subjects and whether a cutoff of basal serum FSH levels was considered an inclusion criterion, two parameters crucial to interpret the results. In fact, age per se affects serum FSH levels in women and results obtained in young ovulatory women may well be different for (usually) older, infertile patients undergoing ART.
The data reported in Supplementary Table 1 show that some studies confirmed our original results (29, 36, 37, 38, 39, 40) but others did not, especially those conducted in women of advanced age (41, 42, 43, 44). Serum FSH levels were not significantly different between FSHR genotypes in older women (41, 42, 43, 44) and in conditions of very high FSH concentrations, such as in postmenopausal women (45) and in women with premature ovarian failure (46). Patient inclusion criteria are important as well, because studies excluding (young) women with basal FSH levels >10 IU/l, potentially excluding p.N680S S carriers, did not reveal any genotype-related difference (37, 47, 48). Some studies included women with ovarian and/or unexplained infertility (e.g. (49)). Finally, most studies analyzed only rs6166 (p.N680S), assuming perfect LD with rs6165 (p.T307A), but as shown in Fig. 3 this may not always be the case and represents another reason for inconsistency.
Many studies in women undergoing ART tried to assess whether the FSHR genotype is useful to predict response and/or decide the FSH starting dose. This would result in personalized ovarian stimulation with lower incidence of side effects (e.g. ovarian hyperstimulation syndrome) and saving of resources. These studies, reviewed recently in detail (12, 13, 14, 15, 16, 17, 50), are extremely heterogeneous (Supplementary Table 1) and provided partly conflicting results. For instance, several studies were unable to confirm that women carriers of the FSHR c.2039A>G G allele (Ser680) have higher basal serum FSH levels compared with Asn carriers (42, 43, 44). Nevertheless, a recent meta-analysis of seven studies including 1421 patients undergoing ART confirmed a significant difference in basal FSH levels depending on the FSHR genotype: carriers of one or two c.2039A>G A alleles (Asn 680) showed significantly lower FSH levels, with a weighted mean difference of – 1.57 IU/l (C.L. −2.51/−0.64 IU/l) (51), therefore quantitatively small. Again, in individual studies involving women undergoing ART, failure to detect increased basal FSH levels in c.2039A>G G allele carriers is likely to depend both on age (practically all ART patients are over 25 and several over 40, with a mean age varying in the individual studies between 30 and 38 years) and on heterogeneity of the patients included (Supplementary Table 1).
The importance of the woman's age in assessing the impact of the FSHR c.2039A>G on serum FSH levels has been recently proven by a very elegant study involving only fertile, young women (mean age of about 25 years) undergoing controlled ovarian hyperstimulation (COH) within an oocyte donation program (52). In this study, including 355 CHO cycles in 145 well-characterized, healthy, and homogenous oocyte donors, basal FSH levels, total FSH dose, antral follicle count, and number of eggs retrieved were significantly different between the genotypes, with the homozygous c.2039A>G G allele confirmed to be less sensitive to FSH stimulation. This study demonstrated once more that, in young, ovulatory women, the FSHR polymorphism c.2039A>G is indeed a major determinant of ovarian sensitivity to FSH.
A conservative but reasonable conclusion from the studies performed so far is that the FSHR genotype is a physiological determinant of basal FSH levels evident in young, normo-ovulatory women. This is not necessarily the case in older women of infertile couples especially in the presence of nonoptimal ovarian function and/or reduced reserve, which per se result in increased serum FSH level. Finally, lack of consideration of the FSHR −29G>A and/or FSHB −211G>T effect might also be one reason why not all studies have been able to demonstrate FSHR-dependent differences in FSH levels in women.
Concerning the FSHR −29G>A, associated with reduced transcriptional activity in vitro(33), this SNP received less attention so far. This SNP was not associated with serum FSH levels in women (25, 41), but Indian carriers of the FSHR −29G>A A allele needed significantly more FSH for multiple follicle maturation in ART compared with carriers of the FSHR −29G>A G allele (41). This might be due to reduced FSHR expression: the less active −29G>A A allele was reported to result in significantly lower levels of FSHR mRNA and protein in granulosa cells obtained from women undergoing ART (53). The first attempt to assess the combined effect of FSHR c.2039A>G and FSHR −29G>A genotypes revealed that the amount of exogenous FSH required for ovarian stimulation and the frequency of poor responders were the highest in double homozygous carriers of the FSHR −29G>A A and FSHR c.2039A>G G (Asn) combination (54). This is intriguing because, while the FSHR −29G>A A allele is transcriptionally less active, the FSHR c.2039A>G G (Asn) is expected to be more sensitive to FSH. Most probably, a much higher number of subjects are needed to reach conclusive evidence when combined genotypes are considered. Therefore, these observations require independent confirmation in different ethnic groups, but the concept of genotype-dependent FSHR expression levels is interesting and increases the complexity of the FSHR genotype influence on FSH action.
FSHB
After the demonstration of a strong FSHB −211G>T effect on serum FSH levels in men ((11), see below), the role of this polymorphism was studied in women. An intriguing retrospective study involving 365 normally cycling women undergoing ART suggested that the presence of T allele, i.e. that showing lower promoter activity in vitro and associated with the lowest FSH levels in men, was unexpectedly associated with significantly higher serum FSH concentrations in the follicular phase and lower progesterone levels in the luteal phase (55). Unable to explain this finding, the authors concluded for a gender-specific difference in the control of gonadotropin secretion. Granted that gonadotropin regulation is different in the two sexes, some factors could have affected the results, such as luteal insufficiency in 20.5% of the patients, inclusion criteria (FSH levels comprised between 3 and 15 IU/l; no age limit), and, perhaps more importantly, the lack of stratification by the FSHR genotype. In fact, considering both FSHR and FSHB polymorphisms together, we observed in women the opposite results. In a prospective study involving a homogeneous group of 193 women with regular cycles, age <40 years and normal antral follicle count and serum AMH levels, day 3 FSH concentrations were significantly lower in carriers of T allele, when stratified by the FSHR genotype. Interestingly, women carriers of the T allele did not show the age-related increase in serum FSH levels observed in GG homozygotes. This shows the importance of FSHB genotype in women, something deserving attention especially when FSH levels are evaluated as a marker of ovarian reserve (56). This study suggested that, in women, not considering the FSHR genotype might mask the FSHB polymorphism effect (56). In support of this concept, a very recent study in a longitudinal cohort of peripubertal girls has confirmed that the combined effect of FSHB −211G>T G allele and FSHR c.2039A>G A allele was associated with a more effective FSH action, with a tendency to anticipate puberty entry and a significant reduction in serum AMH (57). More studies, however, are necessary in order to confirm the interaction between the two genotypes in women. So far, no data are available about the possible role of the FSHB −211G>T polymorphism, alone or in combination with FSHR, on ART outcome.
Pharmacogenetic studies
Are these polymorphisms in FSHR/FSHB relevant for ART? Can we improve ART outcome using a pharmacogenetic approach based on their genotype? To date only one prospective, randomized, controlled study was conducted, in which women homozygous for the c.2039A>G of the FSHR were treated with fixed doses of FSH to assess ovarian response, measured as serum E2 levels on the day of hCG triggering (47). This study demonstrated that homozygous FSHR p.N680S S women produced less E2 compared with homozygous p.N680S N women treated with the same dose of FSH and this difference could be overcome by increasing the FSH dose. While demonstrating, in a clean way, that the FSHR genotype effectively impacts ovarian response in an interventional, prospective, controlled design, the study did not answer the question whether this effect is clinically relevant for the success of an ART program. In fact, the study was not powered to assess possible effects on pregnancy rate and live births, i.e. the real, clinically relevant end points of each ART intervention. In addition, it completely disregarded heterozygous women, i.e. the majority of patients, and did not consider the other SNPs in the promoter of FSHB and of FSHR, the relevance of which was never studied in prospective, interventional studies. Answering the question whether a pharmacogenetic approach based on the FSHR and/or FSHB genotype may improve ART outcome in terms of live births and/or reduced side effects would require a very large, multicenter effort, involving thousands of well-selected women. Given the socio-economical aspects currently involved (pressure of the infertile couple to achieve a pregnancy, financial aspects) and pathophysiological variables (woman's age, male factor, ovarian reserve, and unexplained infertility), it is unlikely that such a study will ever be performed and the question is whether it would be worth the trouble. There is, however, a plethora of studies analyzing retrospectively the relevance of FSHR and other gene polymorphisms for ovarian response (variably defined) in ART programs based on different types of patients. To mention only the most recent ones, some studies confirm the impact of the FSHR c.2039A>G SNP (40), some do not (49) and this reflects faithfully the results of over a decade of literature summarized in recent reviews (12, 13, 14, 15, 16, 17, 50). The heterogeneity of the study designs, patient characteristics, and primary end points, often in the absence of power analysis, together with relatively advanced age of women in ART programs are the possible reasons for this inconsistency (Supplementary Table 1). In addition, other genes (e.g. the ESR2, AMH, AMHR2, and MTHFR) have been variably shown to be associated with ovarian response, to which they could contribute (15, 17).
FSHR and FSHB polymorphisms influence serum FSH levels and reproductive parameters: studies in men
FSHR
Several studies assessed the association of FSHR exon 10 polymorphisms and reproductive parameters in men. However, for a long time, no effect of FSHR genotype on serum FSH levels could be demonstrated (reviewed in (58)). This was surprising because, meanwhile, many papers were published supporting the role of FSHR genotype in the determination of serum FSH levels in women (10, 12, 13, 14, 35, 38, 40, 59) (Supplementary Table 1). As a possible explanation, gender-specific differences in the feed-back regulation of FSH secretion were assumed.
The significant role of the FSHR p.N680S polymorphism in the male was very recently demonstrated by a study involving a very large number of Baltic men (60). Thanks to the large dimension of the study, performed on 1790 men, and to the meta-analytical approach it was possible to demonstrate for the first time the effects of the FSHR pN680S polymorphism alone on testis volume, serum FSH, inhibin B, and testosterone levels. The effect was of small entity (effect of the Ser allele on testis volume: −1.40 ml) but significant. One reason why it was not demonstrated earlier could be that, apart from the sample size effect, it might have been confounded by the FSHB −211G>T SNP effect in the previous studies based on much smaller subject numbers (61). No data concerning the FSHR −29G>A SNP in normal males are available so far.
FSHB
The −211G>T SNP in the FSHB promoter was demonstrated to have a strong effect in vitro on transcription by luciferase reporter assay, with the T allele showing a relative activity which was only half of that of the G allele (34). Together with the evidence that the haplotype structure of the FSHB gene might be subjected to balancing selection (26) and, thereby, influence reproductive parameters, and the high evolutionary conservation across species of the promoter region including rs10835638, this induced the analysis of whether FSHB −211G>T polymorphisms could be associated with serum FSH levels. A study conducted to determine the semen parameters in young male volunteers of Baltic origin demonstrated that this SNP indeed influenced serum FSH levels and other reproductive parameters. In particular, FSH levels in homozygous FSHB −211G>T T carriers were significantly reduced compared with both heterozygous and homozygous G carriers (11). This was then amply confirmed in German and Italian men (62, 63). In accordance with the epidemiological and in vitro data reported above, homozygous carriers of the minor allele represent only 1.5% of men with normal semen parameters but have serum FSH levels which are about 25% lower than homozygous, major allele carriers (11, 62, 64). In addition, this polymorphism is associated with lower testicular volume, lower sperm count, lower testosterone, and higher LH serum levels (11, 62, 63, 64, 65).
The association of FSHB −211G>T polymorphism with serum FSH and other reproductive parameters in men prompted to a reassessment of the (mild) effects of exon 10 FSHR polymorphisms in males. In an elegant study, Tüttelmann et al. (61) showed that when men are stratified by the FSHB genotype, significant differences in serum FSH levels and testicular volume between carriers of the different FSHR genotype become evident. This study demonstrated for the first time that the FSHR polymorphism effect is indeed present also in the male, although it can be masked by the FSHB polymorphism, suggesting, in addition, that the interplay between polymorphisms in hormone and receptor is of relevance under physiological conditions. Taking into account both FSHR and FSHB and considering their allelic frequencies, the authors suggested that carriers of the potentially ‘unfavorable’ allele combination (i.e. those associated with lower testis volume and serum FSH levels), represent about 45% of all males, have smaller testes and could be at risk for reduced reproductive fitness (61).
In summary, the current evidence shows that both FSHR and FSHB genotypes are physiologically very relevant and interact with each other to determine gonadotropin levels in both sexes. So far only few studies assessed the effects of the combination of rs6166 in FSHR and rs10835638 in FSHB (56, 57, 61). This combination results in nine different genotype combinations, which have been demonstrated to be associated with significantly different serum FSH levels (56, 61). The matter is further complicated by the possible effects of the FSHR, −29G>A SNP, influencing levels of FSHR expression: only one study considered the combination of rs6166 and rs1394205 in women (54). The combination of these three SNPs would result in 27 combinations (Table 3). Among them, those predicted to be associated with the ‘less favorable’ FSHB/FSHR genotype combinations (in terms of serum FSH levels and FSHR expression/sensitivity) are expected in about 20% of Caucasians. A very large number of subjects will be necessary to address systematically the pathophysiological relevance of these allele combinations and to attempt a meaningful pharmacogenetic approach.
Expected genotype frequencies in Caucasians of the 27 allele combinations resulting from rs6166 (FSHR p.N680S), rs1394205 (FSHR −29G>A), and rs1083563 (FSHB −211G>T) frequencies collected from the HapMap Database (http://hapmap.ncbi.nlm.nih.gov) based on 121 CEU subjects.
| rs6166 | AA | GA | GG | ||||||
|---|---|---|---|---|---|---|---|---|---|
| rs1394205 | GG | GA | AA | GG | GA | AA | GG | GA | AA |
| rs10835638 | |||||||||
| GG | 13.82 | 10.26 | 2.85 | 19.01 | 14.12 | 3.93 | 6.21 | 4.61 | 1.28 |
| GT | 4.15 | 3.08 | 0.86 | 5.71 | 4.24 | 1.18 | 1.86 | 1.38 | 0.38 |
| TT | 0.20 | 0.15 | 0.04 | 0.27 | 0.20 | 0.06 | 0.09 | 0.07 | 0.02 |
Bold indicates the FSHB/FSHR genotype combinations associated with lower serum FSH levels and lower FSHR expression and activity.
The FSHR and FSHB genotypes associated with lower FSH levels and/or less sensitive FSHR are enriched in human infertility
Ovulation disorders
Polycystic ovary syndrome (PCOS) represents an intriguing and frequent form of infertility and anovulation, and is an object of intensive research. Given the role of gonadotropins in ovarian physiology, the association with the FSHR gene SNP c.2039A>G has been repeatedly investigated in several populations. Overall, there seems to be a selective enrichment of the c.2039A>G G allele in PCOS and a meta-analysis including 1028 PCOS and 3587 controls demonstrated a mild protective effect of the homozygous c.2039A>G A allele (66). There is evidence that the c.2039A>G G genotype is more frequent in ovulation disorders, infertility, and poor or exaggerated FSH response in ART (36, 38, 42, 58, 59, 67, 68, 69, 70, 71, 72) (Supplementary Table 1). Interestingly, two genome-wide association studies in Chinese women identified, among other interesting candidates, the FSHR and the LHCGR as risk loci for PCOS (73, 74). Another, very carefully conducted, genetic association study based on selected, haplotype-tagging SNPs confirmed these findings in women of European origin (75). In the case of the FSHR, both a SNP 5.3 kb upstream of the gene and rs6165 (p.T307A) were nominally strongly associated with PCOS. Therefore, evidence is accumulating that the genomic region of chromosome 2 encompassing FSHR, LHCGR, and GTF2A1L (general transcription factor IIA, 1-like involved in gametogenesis) confers susceptibility to PCOS and deserves further intensive, functional, and genetic research. Experimentally, none of the existing mouse models recapitulate the whole spectrum of the human PCOS but, remarkably, overexpression of LH/hCG activity, androgen excess, and reduced aromatization/estrogen action reproduces the ovarian cystic phenotype in mice (76). Together, these evidences identify (maybe not surprisingly) altered gonadotropin action as the most plausible determinant of the ovarian phenotype of PCOS. Women with PCOS may be treated with clomiphene or gonadotropins for ovulation induction. However, no prospective, pharmacogenetic trial on ovulation induction in PCOS based on the FSHR genotype has been performed so far.
Concerning the FSHR c.−29G>A, an association was reported in Indian patients with poor ovarian response (41) as well as with primary and secondary amenorrhea (77). No data are available about the possible role of the FSHB c.−211G>T in female infertility. Understanding the molecular events related to the genetic FSHR (and LHCGR) variants will be essential for a pharmacogenetic approach to PCOS treatment.
Male infertility
Most studies failed to find any correlation between FSHR genotype and male infertility phenotype (reviewed in (78) and (79)). Conversely, the rare, FSHB −211G>T homozygous T genotype was reported to be significantly overrepresented in infertile men (65). Whether the FSHR −29G>A SNP is of any relevance in the male remains controversial. A small effect of FSHR −29G>A A allele on testis volume was reported in a small group of infertile men (80) but not replicated so far. The original description of an association of FSHR haplotypes including −29G>A, 919A>G, and 2039A>G with male infertility (81) has not been confirmed by the subsequent literature (79) and, for the time being, there is no evidence that the study of the −29G>A SNP adds any useful information to the FSHB −211G>T and FSHR c.2019A>G combination in the male.
Pharmacogenetic studies in men
Male idiopathic infertility remains a pathophysiological dilemma and a therapeutic challenge. In the ART era, most forms of male infertility are empirically resolved by ICSI but, given the burden (not only economical) of the procedure, the question is whether sperm quality can be improved by a less invasive, medical intervention. Owing to the role of FSH in spermatogenesis, FSH treatment has always been tempting and is actively prescribed by many doctors in spite of the lack of evidence of its superiority compared with placebo. Several studies suggest that FSH treatment might be useful in a subgroup of normogonadotropic infertile men (82), purportedly those without spermatogenic blockade, but these men are impossible to identify beforehand. Could knowledge of the FSHB and/or FSHR genotype be helpful here?
So far only two studies addressed the question whether a pharmacogenetic approach would be helpful in identifying responders to FSH treatment. One study showed that patients with at least one FSHR c.2039A>G G allele had a significant increase in total sperm count after 3 months of treatment with rFSH (150 IU/thrice per week) (83). Another study considered the FSHB −211G>T genotype and showed that TT homozygotes, representing 25% of men with oligozoospermia and low FSH levels, could significantly benefit from FSH treatment (64). No study has addressed the combination of the two genotypes so far. As both FSHB and FSHR genotypes affect testis volume and serum FSH levels (61), it would be interesting to investigate whether normogonadotropic, oligozoospermic men, carriers of the less favorable SNP combinations, could improve fertility upon FSH treatment. However, sperm parameters are naturally quite variable within the same individual and the problem of the correct (and robust) primary end point in such a study remains unresolved. Studies are ongoing assessing whether sperm DNA fragmentation, believed to be ameliorated by FSH, changes upon FSH treatment in dependence of the genotype (EudraCT 2010-020240-35).
Conclusions and outlook
In the light of the actual knowledge, is there any potential for the pharmacogenetic use of FSH in infertility treatment (male and female)? Overall, the available in vitro an in vivo data support the physiological relevance of the considered SNPs for FSH action. The genetic complexity thereof, illustrated by the four SNPs considered in this study, is probably much higher than supposed so far and only studies considering the FSHB/FSHR genotype combinations with numbers of subjects large enough in dependence of the frequency of each genotype in a given ethnic group will be able to assess this issue. Efforts should be dedicated to identify subjects with the ‘less favorable’ variant combination, expected to be overrepresented among infertile patients, and assess whether they would be candidates for FSH treatment and/or usage of higher FSH doses in case of ART or ovulation induction. This, however, needs to be proven in large, prospective studies, objectively difficult to conduct in the ICSI era. Nevertheless, FSH therapy might become an interesting medical option, especially for treatment of selected cases of male infertility, in which the reduction of sperm parameters is combined with a ‘sloppy’ FSHB/FSHR genotype combination and the female partner has normal ovarian function and a good reserve. Interventional trials should be started with such couples, because successful medical treatment of the man would relieve the woman of carrying the burden of male infertility.
There is another interesting and intriguing aspect to consider. When epidemiological, functional, and clinical data are considered together with the evolutionary aspects illustrated above, it appears that an overall trend toward a less efficient FSH–FSHR system emerges in the human species. This poses an interesting evolutionary biology question: which environmental conditions select the evolutionarily more recent FSHB and FSHR genotypes and are there unrecognized advantages for lifetime reproductive success for such genotypes which, counterintuitively, reduce fertility/fecundity? Another example of evolutionary paradox is PCOS, a common polygenic condition linked to both infertility and metabolic disturbances, which is steadily increasing in epidemiological relevance in spite of reduced fertility (84). As evolution maximizes reproduction other aspects may come into play.
In a recent evolutionary study, the FSHR gene was identified as a determinant of human birth timing (85), suggesting its association in as yet unclear processes involved in shortening gestation time and accelerating parturition in the human species. Although a driving evolutionary role of the less ‘sensitive’ FSHR c.2039A>G G allele was postulated (35), there is no evidence that specific polymorphisms of the FSHR gene influence duration of fertile life. The possible role in pregnancy duration remains intriguing. Other studies described the association of the FSHR and its variants with an increasing number of noncanonical and nonobviously FSH-related pathophysiological events, such as osteoporosis (45), vasculogenesis (86), hypertension (33), ovarian cancer (87), and testis cancer (88). All these suggestions would question the dogma of the unique action of FSH at the gonadal level. The expression of the FSHR in extragonadal tissues is currently a matter of vivid debate and the possible role of FSH in noncanonical target organ remains enigmatic. Should perhaps the FSHR gene be viewed as a marker of health at large and these apparently weird associations reconsidered with a wider, evolutionary vision?
FSHR exon 10 homozygous Ser680 genotype was significantly associated with a lower risk of developing sporadic Alzheimer disease (89) and with surviving over the age of 90 years in women but not in men (90), independently of previous fertility, age of menopause, or other known risk factors, suggesting a role of the FSHR genotype as a determinant of longevity. In our study in normal menstrual cycle physiology (35), we observed differences in menstrual cycle length between carriers of the two FSHR haplotypes, resulting in about one menstrual cycle less per year in homozygous p.N680S S compared with homozygous p.N680S N carriers. As no difference in the age of menopause was demonstrated so far, women with the homozygous p.N680S S genotype would experience 30–40 cycles less than women with p.N680S N genotype during their reproductive life and would be exposed to a lower incidence of pregnancies and related risks. In the absence of medical assistance, maternity-related lifetime risk, still relevant in underdeveloped countries, must have been very high in the earlier times of human evolution, possibly making pregnancy-related death an important factor in the determination of an evolutionary selection of this genotype. We speculated that fewer menstrual cycles during the reproductive life span might represent an evolutionary advantage, provided that the fertility of the species is maintained (35). Medical progress, better nutrition, contraception, and the recent dramatic changes in women's role in modern society are modifying the current human reproductive strategies, so that the less fertile genotype becomes epidemiologically relevant, especially in couples attempting to conceive in their late thirties.
In conclusion, the current evidence suggests that the combination of FSHR and FSHB genotypes is predicted to have a much stronger impact than either one alone, both on male and female gonadal function, because about 20% of people are carriers of the allele combinations associated with lower serum FSH levels and lower FSHR expression/activity (Table 3). The advent of powerful, sensitive, and inexpensive techniques assessing several SNPs simultaneously will be very helpful in the identification of the patients at infertility risk. Interventional, prospective studies are needed to investigate whether stratification of patients according to their FSHR–FSHB genotype combination is of any advantage in the treatment of male and female infertility compared with the current, naïve approach. In the absence of this, every pharmacogenetic approach to the clinical use of FSH is empirical and there is no hard evidence that it would be superior in terms of better outcome, reduced side-effects, and/or pharmaco-economic impact.
Supplementary data
This is linked to the online version of the paper at http://dx.doi.org/10.1530/EJE-13-0624.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the review reported.
Funding
This work was supported by a grant of Italian Ministry of University and Research (PRIN grant number: 2010C8ERKX) and of the Fondazione CRM, Modena (grant number: 2006/0320).
Author contribution statement
M Simoni and L Casarini selected and reviewed the literature and prepared the manuscript. L Casarini performed the in silico analyses and the in vitro experiments.
Acknowledgements
M Simoni dedicates this paper to her mentors: Prof. E Nieschlag, for his strenuous support, vision, and lifetime dedication to reproductive endocrinology and Prof. C Carani for his example of fortitude, determination, and bright commitment to the future of endocrinology. This paper would not have been written without years of collaboration and lively and fruitful discussions at the University of Münster with the colleagues and friends Prof. J Gromoll, Dr F Tüttelmann, and Dr J Wistuba, who are thanked for the critical reading of this manuscript.
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