The FSHR gene is located on chromosome 2 p21-p16 and consists of 10 exons and 9 introns. The first 9 exons encode for the extracellular domain of the receptor, whereas exon 10 encodes for the C-terminal end of the extracellular domain, the entire transmembrane domain and the intracellular domain of the FSHR. Exon 10 is fundamental for signal transduction, but it is not necessary for ligand binding. Up till now around 1,800 SNPs of the FSHR gene have been reported in the National Centre for Biotechnology Information (NCBI) SNP database (http://www.ncbi.nlm.nih.gov). SNPs are located either in the coding regions (exons, 8 SNPs), or within intronic regions of exons. Only 1 SNP is located in the 5′ untranslated region of the FSHR mRNA position−29 (ss2189241). Of the eight SNPs within coding regions 7 are located in exon 10 at codon positions 307, 329, 449, 524, 567, 665, and 680. Six of the latter SNPs eventually result in amino acid substitution and are therefore non-synonymous. The two best characterized polymorphisms as far as their allele frequencies and ethnic distribution is concerned are the Ala307Thr (rs6165) and the Ser680Asn (rs6166). Both of these polymorphisms are linked to each other during recombination in a way that they always occur together (17).

Several studies aimed to correlate the FSHR polymorphism and ovarian function. The polymorphism at position 680 harboring Serine residues at both alleles is associated with higher endogenous FSH serum concentrations and a longer follicular phase length. This suggest that this FSHR variant is less sensitive to FSH. Indeed, women having the Ser/Ser variant needed more exogenous FSH during their ovarian stimulation phase during IVF treatment cycles. Moreover, Asn680Ser polymorphism was not associated with premature ovarian failure (POI). Finally, in women with PCOM the distribution of the two allelic variants greatly varied amongst different studies (17–20).

#### FSH RECEPTOR POLYMORPHISMS AND PCOS

#### FSHR Polymorphisms and PCOS Susceptibility

The first study ever which screened the entire coding region of FSHR gene for pathogenic mutations in 124 patients with

PCOS and found no mutations in these patients. The two well-known polymorphisms, Thr307Ala and Ser680Asn showed similar distributions of the allelic variations and protein isoforms in PCOS compared to 236 normal healthy control subjects. It appears from this study that mutations in the coding regions of FSHR gene are not a causative factor for PCOS in Chinese Singapore women (21). Similarly, Fu and co-workers recruited 384 unrelated PCOS patients and 768 healthy individuals from the Shaanxi province in the northern part of China. The Ala307Thr and Ser680Asn polymorphisms were studied together with clinical characteristics of the study subjects in a case-control setting. The frequency of FSHR Ala307Thr and Ser680Asn variants along with the haplotype were not significantly different between the PCOS patients and the controls although the Ser680 variants seemed to be associated with higher levels of endogenous FSH and low estradiol levels. This study suggests that the two variants of the FSHR gene are not a causative factor of PCOS in Northern Chinese Han women (22). In another study in Han Chinese women suffering either from PCOS (n = 215) or being healthy controls (n = 205) recruited from Shanxi Province in north China the Ala307Thr and Ser680Asn polymorphisms of FSHR were not associated with PCOS. However, the FSHR polymorphisms were related to the endogenous serum concentrations of FSH and Prolactin. No other PCOS-associated endocrine hormones as well as clinical pregnancy rates in PCOS patients were recorded in that study (23). Only one study in Chinese women revealed a significant between FSHR polymorphisms and PCOS. In a case-control sample using 60 PCOS patients and 92 healthy controls all being unrelated Han Chinese from Shanghai the haplotypes covering components Thr307Ala and Asn680Ser were studied. These authors observed a significant association of both polymorphisms and PCOS (24).

Ten case-control studies were included in the first metaanalysis addressing the relationship between the commonest FSHR polymorphisms and PCOS. This meta-analysis showed no consistent association between either the Thr307Ala polymorphism or the Asn680Ser polymorphism and susceptibility to PCOS. Stratified analysis of ethnicities also showed no association. The authors of this meta-analysis suggested that the FSHR polymorphisms were not associated with an increased risk of PCOS (25). In a second meta-analysis data from 11 studies were analyzed. The pooled odds ratio and 95% confidence interval were calculated using fixed- or random-effect model based on heterogeneity test in 5 genotype models analyses. This analysis showed that the Asn680Ser polymorphism was significantly associated with the reduced susceptibility to PCOS in a dominant model as well as in recessive, homozygote comparison, and allele contrast models. All Odds ratios were similar reducing the chance of having PCOS with about 20%. However, no significant associations were found between Thr307Ala and PCOS (26).

In a Korean study recruiting 235 PCOS patients and 128 control subjects, all within their reproductive age years, from Seoul. The FSHR polymorphisms Ser680Asn and Ala307Thr genotype frequencies were measured. They found that the Ser680Asn of FSHR was significantly more often associated with PCOS. In contrast the Ala307Thr variant was not at all associated with PCOS. However, their haplotype analysis revealed that both the Ser680Asn and Ala307Thr polymorphisms did not constitute a risk factor for PCOS (27). In another study genotyping was performed in 377 women with PCOS and 388 age-matched controls all from South Korean origin. Findings of this study suggest a significant association between FSHR gene polymorphisms Thr307Ala and the Asn680Ser allele frequencies and PCOS. The homozygote variant genotype results in significantly higher risk of PCOS (28). In a population of 522 Japanese women, the overall frequency of Asn/Asn, Asn/Ser, and Ser/Ser was 41.0, 46.9, and 12.1%, respectively. In polycystic ovary patients, the Asn/Ser population was significantly larger when compared with the spontaneously ovulating group (29). In a Pakistani study genomic results from 96 women with PCOS were compared with those from 96 healthy controls from the Punjab region in Pakistan. This study provides evidence of statistically significant associations between susceptibility to PCOS in Pakistani women and the gene polymorphisms in FSHR, the LHHCG-Receptor, LH-β chain and both ERα and ER-β receptor genes (30). In another study in 386 Thai women with chronic anovulation either without (121 women) or with PCOS (133 women) using 132 known ovulatory fertile women as controls no association between the FSHR gene polymorphism at codons 307 and 680 and PCOS was found (31).

The first published European study assessed the distribution of the two most common FSHR polymorphisms in 148 normogonadotropic anovulatory infertile women and in 30 normo-ovulatory controls. Normogonadotropic anovulatory infertile patients have a different FSH receptor genotype than do normo-ovulatory controls (32). In another study in Dutch women with PCOS FSHR variants were strongly associated with the severity of clinical features of PCOS. The findings in a discovery cohort of 240 women with PCOS were replicated in another independent sample of 185 women suffering from PCOS and showed that the Ser/Ser genotype was associated with higher endogenous levels of FSH, LH and testosterone (19, 20).

In a study in Turkish adolescent girls the possible association between SNPs of the FSHR was studied. Samples from 44 adolescent girls with PCOS and 50 healthy controls were compared. Polymorphic loci on the FSHR (A307T, N680S) genes did not reveal any significant differences between cases and controls. These data do not support an association between SNPs of the FSHR and the susceptibility to PCOS in Turkish adolescent girls (33). Another study in Caucasians included 294 premenopausal Caucasian patients with PCOS, and 78 women with regular menorrhea and without hirsutism. In this study in Polish women no differences in genotype and allele frequencies of the Ser680Asn and Ala307Thr polymorphisms between the case and the control groups. In addition, the two FSHR variants in exon 10 did not increase the risk for PCOS in Polish women (34).

To assess the cross-ethnic effect a meta-analysis of the Dutch data (703 Dutch PCOS patients and 2,164 Dutch controls) combined with results of previously published studies in PCOS patients from China (n = 2,254) and the United States (n = 2,618) has been performed. Overall, this study observed for 12 of 17 genetic variants mapping to the Chinese PCOS loci similar effect size and identical direction in PCOS patients from Northern European ancestry, indicating a common genetic risk profile for PCOS across populations. In this study two previously identified GWAS FSHR polymorphisms, i.e., rs2268361 and rs2349415 were significantly associated with PCOS (15). Another study trying to replicate the Chinese GWAS findings in Caucasians included 905 women with PCOS and 956 control women. The strongest evidence for association mapping to FSHR was observed with rs1922476. Furthermore, markers with the FSHR gene region were associated with FSH levels in women with PCOS. Fine mapping of the chromosome 2p16.3 Chinese PCOS susceptibility locus in a European ancestry cohort provides evidence for association with two independent loci and PCOS. The gene products of the FSHR gene are therefore likely to be important in the etiology of PCOS, regardless of ethnicity (35). An American study tried to replicate the FSHR variants from the Chinese GWA studies in case-control examination in a discovery cohort of 485 women with PCOS and in 407 controls from Boston. Replication was performed in women from 884 PCOS cases and 311 controls from Greece and in an additional cohort from Boston constituting 350 cases and 1,258 controls. One variant, rs2268361-T, in the intron of FSHR was associated with PCOS and lower FSH levels (36). In an effort to replicate the hits for the Chinese GWA studies 845 European subjects with PCOS and 845 controls were recruited into this study. Variants in DENND1A, THADA, the FSHR, and the INS-Receptor were associated with PCOS in Europeans. The genetic risk score, generated for each subject based on the total number of risk alleles, was associated with the diagnosis of PCOS and remained associated even after exclusion of the four variants individually associated with PCOS (37). Finally, in an Arab case-control study, involving 203 women with PCOS, and 211 age- and ethnically-matched control women. Significantly lower frequencies of a heterozygous FSHR variant (rs11692782) genotype carriers were observed between women with PCOS and controls (38). In summary most studies in Chinese and Thai women did not substantiate an increased susceptibility for PCOS based on FSHR polymorphisms. In contrast Studies in Korean and Pakistani women did find an increased susceptibility for PCOS based SNP's in the FSHR. Results from meta-analysis are contradictory whereas most studies in women from Caucasian descent revealed a clear-cut increased susceptibility for PCOS based on the FSHR genotype (**Table 1**).

### FSHR Polymorphisms and Clinical Features of PCOS

One study aimed to investigate whether the PCOS related SNPs in the FSHR gene are associated with PCOM. The 447 unrelated Han Chinese PCOS from south China were grouped into PCOM (n = 384) and non-PCOM (n = 63) women. Significant differences were found in the allele distributions of the GG genotype of rs2268361 between the PCOM and non-PCOM groups while no significant differences were observed in the allele distributions of the GG genotype of rs2349415. When rs2268361 was considered, there were statistically significant differences of serum FSH, estradiol, and sex hormone binding globulin between genotypes in the PCOM group. In case of the rs2349415 SNP, only serum sex hormone binding globulin was statistically different TABLE 1 | Showing the effect different single nucleotide polymorphisms have on either the prevalence of PCOS or on different phenotypical features of the syndrome.

(Continued)

TABLE 1 | Continued

between genotypes in the PCOM group (40). Similar findings were reported another study showing that the Ser/Ser genotype had significantly higher basal level of serum FSH was observed as compared with that in the Asn/Ser group (29). A study trying to replicate the GWAS findings of the first two Chinese studies in Caucasians found a similar relationship between FSHR polymorphisms and lower FSH levels (36). Several Dutch studies found similar relationships between the Ser/Ser FSHR variant and higher basal endogenous FSH serum concentrations (19, 20, 39). In one study it was also reported that the Ser/Ser variant was also associated with increased serum concentrations of LH and testosterone (39). There is only limited information available regarding the relationship between FSHR SNP's and the phenotype of PCOS. Some SNP's are associated with the PCOM whereas most data are showing a more strict relationship between higher basal FSH serum levels and the least sensitive FSHR (**Table 1**).

#### FSH RECEPTOR POLYMORPHISMS AND TREATMENT OUTCOME IN PCOS

In order to determine whether an allelic variant of the FSHR gene affects fertility parameters in women with PCOS a UK group of investigators studied 93 women with PCOS and compared those with 51 healthy controls. The allelic variant Thr307/Ser680 was found to be similarly prevalent in both study groups and had no phenotype in terms of fertility parameters in women with PCOS (41). In a study comparing 58 women with PCOS and 80 healthy ethnically matched female controls there was no evidence that the Asn680Ser FSHR genotypes were neither associated with PCOS nor with the response to clomiphene citrate (42).

In contrast in a population of 522 Japanese women the NS genotype was significantly more prominent in women with PCOS. In women with the SS genotype significantly higher basal serum levels of FSH were observed as compared with the NS group suggesting a lower sensitivity of this particular SNP. Indeed, higher doses of the exogenous FSH was required to achieve ovulation induction in the SS group. Moreover, after hCG administration, estradiol levels at the time of ovum pickup were significantly lower in the SS group as compared to the other allelic variants. In case the two receptor variants were over expressed in 293T cell line no differences could be found in either levels of FSH-stimulated cAMP production, PI turnover or ligand-binding affinity. These results suggest some FSHR variants might have clinical implications (29). Similar results have been reported in a large Dutch case control cohort of women with PCOS and healthy controls the Ser/Ser FSHR polymorphism was associated with some clinical features such as FSH and LH serum concentrations. Indeed, the Ser/Ser FSHR polymorphism seemed to be less sensitive to endogenous FSH thereby leading to higher serum concentrations of FSH. Surprisingly this variant was also associated with higher serum LH and testosterone levels (32, 39). In the same study the Dutch group revealed an association with clomiphene citrate resistance during ovulation induction treatment. A pooled analysis showed an 89% higher chance of being CRA in homozygous carriers of the Ser/Ser FSHR variant. Similarly, a lower chance of ongoing pregnancy [hazard ratio 0.51 (95% confidence interval 0.27–0.98)] was observed among these patients during clomiphene citrate treatment in two independent prospective cohorts (39). These data may be used to design a treatment algorithm that is more efficacious and better tailored to the individual patient (20).

Similar results were obtained from another Dutch group retrospectively studying a cohort of 193 patients all diagnosed with PCOS according to Rotterdam criteria and treated with ovulation induction. Significantly more patients with Ser/Serpolymorphism were resistant to CC compared with the Asn/Ser and Asn/Asn genotypes with an odds ratio for ovulation of 0.44. Patients with higher FSH levels, higher age and lower BMI were significantly more likely to ovulate in univariate analysis. In a multivariate logistic regression model, corrected for age, BMI, mean ovarian, volume, hyperandrogenism, and amenorrhea, only the FSHR genotype and basal FSH serum levels were predictive for ovulation (19). An Italian study compared 40 women with PCOS undergoing in-vitro fertilization (IVF) with 66 normo-ovulatory women. That study showed that the heterozygote FSH-R polymorphism Ala307Thr was significantly more frequent in women with PCOS compared to the normo-ovulatory subjects. Moreover, the Ala307Thr SNP was more frequently associated with a higher ovarian responsiveness to exogenous FSH (43). Although smaller studies in Caucasian women did not reveal a clearcut relationship between FSHR polymorphisms and treatment outcome the larger studies did reveal an increase in clomiphene citrate resistance and the least sensitive FSHR (Ser680Ser variant) (**Table 1**).

### OTHER GENETIC VARIANTS GONADOTROPHIC REGULATION THAT MIGHT BE ASSOCIATED WITH PCOS

#### FSH-β Gene Polymorphisms

In a cohort of 135 patients with PCOS and 105 normal control subjects no missense mutations were found in the functional units of the FSH-β gene in patients with PCOS. However, this study identified a thymine-cytosine substitution in exon 3 (codon 76, TAT to TAC) that led to creation of a so called AccI digestion site. The distribution pattern of this AccI polymorphism in the patients was significantly different from that in the control group since homozygous carriers were more often affected by PCOS. Within the PCOS patient group homozygosity for Accl was also associated with obesity. The latter finding correlated with significantly higher androgen levels in the obese patients. Hence the AccI polymorphism in FSHβ gene may be associated with PCOS in some women, especially those with obesity (44). A metaanalysis of Chinese GWAS data showed that the allele frequency difference of a SNP in the FSH-β gene (rs11031010) between PCOS and controls was genome-wide significant. PCOS women with AA and AC genotypes had a significantly higher LH serum levels compared to women carrying the CC genotype. Hence, variants in FSH-β gene are associated with PCOS and LH levels in Han Chinese women. The FSH-β gene is thus likely to play an important role in the etiology of PCOS, regardless of ethnicity (10) (**Table 1**).

#### LHCG-Receptor Polymorphisms

In a retrospective case-control study, involving 203 women with PCOS, and 211 age- and ethnically-matched control women LHCGR genotyping was done by allelic exclusion method. Significantly lower frequencies of heterozygous LHCGR rs7371084 genotype carriers were seen between women with PCOS vs. controls. Furthermore, an increased frequency of heterozygous homozygous LHCGR rs4953616 genotype carriers was detected between women with PCOS compared to control women. The authors of this study observed a significant increase of LHCG-Receptor variants (rs7371084, rs4953616) SNP's in women with PCOS (38). In a case control study in Hui ethnic women from China 51 patients with PCOS and 99 healthy women were involved. The frequencies of the genotype and allele frequency of rs13405728 in LHCG-Receptor gene were significantly different between the PCOS and the control women. Moreover, PCOS cases with TT genotype of the variant rs13405728 had higher serum level of total testosterone, triglycerides, and low-density lipoproteins (LDL) than those with the CC and CT genotypes. The authors concluded that the SNP rs13405728 in the LHCG-Receptor gene was associated with PCOS and some of its clinical features (45). An attempt to replicate the Chinese SNP's in an American cohort revealed that the LHCG-Receptor variant (rs13405728) was not informative in a white Americans from European descent. However, these authors identified and genotyped three markers (rs35960650, rs2956355, and rs7562879) within 5 kb of rs13405728. Of these, rs7562879 was nominally associated with PCOS. The gene products of the LHCRG-Receptor gene are therefore likely to be important in the etiology of PCOS, regardless of ethnicity (35).

In Indian study involving 204 women with PCOS and 204 healthy, sex-, and age-matched controls. This study demonstrated an association between LHCGR (rs2293275) polymorphism and PCOS. Moreover, a significant association of the GG allele with body-mass index, waist to hip ratio, insulin resistance, LH, and LH/FSH ratio was demonstrated in PCOS when compared with controls. Indeed this study also suggests that LHCG-Receptor polymorphism are associated with PCOS (46) (**Table 1**).

### SUMMARY

Data from Chinese studies regarding the relationship between FSHR polymorphisms and PCOS susceptibility are conflicting. Although the majority of data do not substantiate such a relationship (21–23) there is one report suggesting that FSHR might play a role in genetic susceptibility to PCOS (24). Two different meta-analysis revealed also contradictory results. The first one suggested that the FSHR polymorphisms were not associated with an increased risk of PCOS (25) whereas the last one did reveal a decreased susceptibility to PCOS for Thr307Ala variant carriers (26). Several Korean (27, 28) as well as Japanese (29) and Pakistani studies (30) do substantiate the FSHR variants as a susceptibility locus for PCOS. However, a study in Thai women did not reveal such a relationship (31). It seems that data from South-East Asia are different from those generated in Chinese populations. In European women data are similarly conflicting. Some Dutch studies revealed a straight on forward association between FSHR gene polymorphisms and PCOS (19, 20, 39) whereas others did not substantiate such susceptibility (17, 33, 34). Arab women carrying certain FSHR polymorphisms seem to be more often suffering from PCOS too (38). The most convincing evidence comes from larger replication studies and a cross ethnic meta-analysis that revealed a strong relationship between FSHR variants and PCOS susceptibility (15, 35–37).

In conclusion along with these more convincing data observations underpinning the association of FSHR polymorphisms with clinical features indicating differences in sensitivity of the different receptor genotypes. These receptor variants might generate several phenotypes with differences in basal serum FSH, LH, and testosterone concentrations. Some of the data are also pointing in a direction that at least some of these genetic variants have clinical implications in that they determine individual sensitivity for exogenous FSH (19, 29, 32, 39, 42, 43). The observation that treatment outcome is also partially determined by FSHR polymorphisms is another clue that genetic variants might play a role in the pathophysiology of PCOS (19, 20, 29, 39, 42, 43).

In conclusion SNP's in the FSHR gene causing genetic variants in the FSHR on the one hand do determine the susceptibility for PCOS and on the other had do also affect the sensitivity of the receptor for exogenous FSH during ovulation induction therapy. There is also limited evidence showing that SNP's in the LHCG-Receptor as well as those in FSH-β gene might also determine a women's susceptibility for PCOS.

#### AUTHOR CONTRIBUTIONS

JL delivered a substantial contribution to the conception of the work and the interpretation of data in the literature for the work.

#### REFERENCES

He drafted the work and agrees to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Laven. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# FSH Beyond Fertility

Daria Lizneva<sup>1</sup> , Alina Rahimova<sup>1</sup> , Se-Min Kim<sup>1</sup> , Ihor Atabiekov <sup>1</sup> , Seher Javaid<sup>1</sup> , Bateel Alamoush<sup>1</sup> , Charit Taneja<sup>1</sup> , Ayesha Khan<sup>1</sup> , Li Sun<sup>1</sup> , Ricardo Azziz <sup>2</sup> , Tony Yuen<sup>1</sup> and Mone Zaidi <sup>1</sup> \*

<sup>1</sup> The Mount Sinai Bone Program, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, United States, <sup>2</sup> Academic Health and Hospital Affairs, State University of New York, Albany, NY, United States

The traditional view of follicle-stimulating hormone (FSH) as a reproductive hormone is changing. It has been shown that FSH receptors (FSHRs) are expressed in various extra-gonadal tissues and mediate the biological effects of FSH at those sites. Molecular, animal, epidemiologic, and clinical data suggest that elevated serum FSH may play a significant role in the evolution of bone loss and obesity, as well as contributing to cardiovascular and cancer risk. This review summarizes recent data on FSH action beyond reproduction.

Keywords: FSH, FSHR, obesity, osteoporosis, BMI, cardiovascular risk

#### Edited by:

Livio Casarini, University of Modena and Reggio Emilia, Italy

#### Reviewed by:

Adolfo Rivero-Muller, Turku Centre for Biotechnology, Finland Giulia Brigante, Università degli Studi di Modena e Reggio Emilia, Italy

> \*Correspondence: Mone Zaidi<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Reproduction, a section of the journal Frontiers in Endocrinology

Received: 21 November 2018 Accepted: 13 February 2019 Published: 19 March 2019

#### Citation:

Lizneva D, Rahimova A, Kim S-M, Atabiekov I, Javaid S, Alamoush B, Taneja C, Khan A, Sun L, Azziz R, Yuen T and Zaidi M (2019) FSH Beyond Fertility. Front. Endocrinol. 10:136. doi: 10.3389/fendo.2019.00136 INTRODUCTION

Follicle-stimulating hormone (FSH) is long thought to exert its effects in gonadal tissues, mainly limited to Sertoli cells in testes and granulosa cells in ovaries (1).

Our group first demonstrated that by increasing bone resorption by osteoclasts, FSH regulates bone mass in mouse models (11). Moreover, we found that FSH exerts action on adipocytes.  These findings are consistent with large epidemiologic data. Indeed, the Study of Women's Health Across the Nation (SWAN) has shown significant reductions in bone mineral density (BMD) and high resorption rates ∼2–3 years prior to menopause, which was also associated with increased body weight and visceral adiposity (12, 13). It is important to note that these changes take place when serum FSH level is increasing and estrogen level remains normal (14). Emerging epidemiologic evidence also suggests a relationship between FSH and several cardiovascular risk factors such as coronary artery calcium deposition, carotid intima-media thickness, and the number of aortic plaques (15–17). In particular, FSH interacts with its receptor on monocytes, up-regulates RANK expression and promotes monocytic infiltration of atherosclerotic plaque (18).

With this new evidence, the view that FSH acts solely as a gonadal hormone has changed rapidly over the past decade. It also provides perspectives on new roles that FSH might have in the pathophysiology of certain diseases and how treatment approaches targeting FSH may open up new possibilities for prevention and treatment. For example, we showed that a FSH blocking antibody could prevent bone loss and visceral adiposity in various mouse models (10). These data provide the foundation for future human studies. Similarly, detection of vascular endothelial FSHR in various types of solid tumors and sarcomas has prompted a debate as to whether an anti-FSH antibody could serve as a treatment modality in future anti-cancer drugs (7, 19).

Here, we review the epidemiologic, molecular and animal data on FSH action in normal physiology and the pathophysiology of osteoporosis, obesity, cardiovascular disorders, and cancer.

#### The Role of FSH in Reproduction

FSH, luteinizing hormone (LH), thyroid-stimulating hormone (TSH) and human chorionic gonadotropin (hCG) are all glycoprotein hormones, which share the same alpha subunit and differ in their beta polypeptide units, specific for each of aforementioned molecules (20). Pulsatile release of gonadotropin-releasing hormone (GnRH) from the hypothalamus stimulates the release of FSH and LH. Inhibin B and estradiol are the primary inhibitors of FSH secretion (21– 23). Several other pituitary-regulatory proteins, such as activin and follistatin, have been implicated in FSH secretion and action (22). The activity of FSH is regulated in part by glycosylation.

FSH exerts its biological action via a G protein-coupled receptor, FSH receptor (FSHR). A stimulatory Gα<sup>s</sup> protein initiates signal transduction via the cAMP/protein kinase A (PKA) pathway (1, 24). This cascade of events leads to the activation of cAMP regulatory element-binding protein (CREB) (24). In addition to CREB, cAMP-activated PKA activates several other factors such as p38 MAP kinases, p70-S6 kinase and phosphoinositide-3 kinase (PI3K), PKB/Akt and FOXO1 and regulates gene expression in target tissues (25, 26). According to recent data, the effect of FSHR activation is not limited to the classical pathway, but also produces its action through Gα<sup>i</sup> (27), Gα<sup>q</sup> (28), and via other molecules, including β-arrestins (29) and an adapter protein having pleckstrin homology and phosphotyrosine binding domains together with a leucine zipper motif (30). In this case, the signal transduction is accomplished though inositol trisphosphate (IP3), Akt and ERK1/2.

FSH plays a pivotal role in the development and regulation of both the male and female reproductive systems by acting on the FSHR which is predominantly expressed in granulosa and Sertoli cells (24). In females, FSH induces follicular growth and maturation, and contributes to LH-triggered ovulation and luteinization (31–33). In males, FSH regulates the mitotic proliferation of Sertoli cells, supports their growth and maturation and prompts the release of androgen-binding protein, which regulates the overall process of spermatogenesis (34). Moreover, in testis, endothelial FSHR mediates FSH transport across gonadal endothelial barrier (35). Below, we will discuss the role of FSH on bone, fat, cardiovascular system and cancer cells.

#### Epidemiologic and Clinical Data Supporting FSH Action on Bone

Traditionally, bone loss in peri- and postmenopausal women has been attributed primarily to reduced estrogen production due to ovarian senescence. Estrogen replacement therapy has been considered a logical therapeutic choice in an attempt to slow postmenopausal bone loss and reduce fracture risk (36). However, FSH has been implicated in bone loss in reproductive and non-reproductive age women, as well as in women undergoing menopausal transition (37, 38).

While data from placebo-controlled randomized clinical trials is not available, the multi-center multi-ethnic cohort SWAN showed a compelling correlation between FSH action and bone loss during the menopausal transition. SWAN demonstrated that changes in bone turnover markers and bone mass density (BMD) in perimenopausal women undergoing menopausal transition were independent of serum estradiol, but were inversely related to changes in the FSH level. The levels of serum FSH over a 4-year time period predicted BMD reduction in these women (14, 39). Moreover, lower levels of bone loss in the lumbar spine during perimenopause were noted in women with higher estrogen-to-FSH ratio (40). All of these observations may suggest that bone loss during perimenopause is not solely dependent on estrogen, and may be due in part to FSH action on bone.

Epidemiological data from across the US, Europe, and China further substantiate findings from SWAN (41–46). The US NHANES III cohort study documented the relationship between serum FSH and femoral neck BMD among woman between the ages of 42 and 60 (41). Likewise, using univariate regression analyses, another US cross-sectional study confirmed the inverse relation of FSH to BMD in perimenopausal women (42). The Italian Bone Turnover Range of Normality (BONTURNO) study compared women undergoing menopausal transition, and showed significantly increased bone loss in the group with FSH>30 IU/L vs. age-matched controls, although both had regular menses (43). Yet another cross-sectional study conducted in Spain included 92 postmenopausal female participants and showed a positive correlation between serum FSH and Cterminal telopeptide of type I collagen (CTX) and serum osteocalcin, but no relation to estradiol. Several Chinese studies have reported a negative relationship between bone loss, bone turnover markers and serum FSH levels in perimenopausal women (45–47), with those in the highest quartile of serum FSH showing bone loss at a rate that was 1.3–2.3-fold higher than those in the lowest quartile (48).

The detrimental and deleterious effect of FSH on bone during a woman's reproductive years can be observed in instances of hypergonadotropic conditions. For example, lower lumbar spine bone density was reported in a hypergonadotropic amenorrheic group as compared to hypogonadotropic European patients under 40 years of age (49). Groups did not differ in estradiol or progesterone levels; however, in hypergonadotropic women, FSH levels had a negative relationship with lumbar spine BMD. Interestingly, females diagnosed with functional hypothalamic amenorrhea tend to develop less severe bone loss (50, 51).

Evidence from genetic studies further explores the function of FSHR in humans. In particular, women with an activating FSHRN680<sup>S</sup> polymorphism have an increased risk of developing postmenopausal osteoporosis, independent of circulating levels of FSH and estrogens (52). Likewise, in a multicenter study of postmenopausal Spanish women two-gene combinations of wild type IVS4 or 3′UTR markers of CYP19A1 with FSHR and BMP15 genes yielded skeletal protection (53). Therefore, epidemiologic data derived from several cross-sectional and cohort studies, together with genetic association studies, suggest a detrimental effect of FSH on bone.

In contrast, a couple of clinical studies in humans using GnRH agonists failed to demonstrate any effect of FSH suppression on bone. For instance, FSH suppression with leuprolide acetate in a group of postmenopausal women has not being associated with any significant changes in bone resorption markers (54). In another study, eugonadal men receiving goserelin acetate combined with daily topical testosterone gel did not demonstrate any changes in serum N-terminal telopeptide, C-terminal telopeptide, and osteocalcin compared to control (55). However, both studies were relatively small and the duration of the intervention was short (approximately 4 months).

#### Mechanistic Studies on FSH Action on Bone

In 2006, we were the first to observe the direct regulation of bone mass by FSH, which resulted mainly from osteoclastic bone resorption in rodents (11). Accumulating evidence now shows that FSH acts directly on bone via a specific shorter isoform of the FSHR (identified in humans), which then increases osteoclastogenesis and stimulates bone resorption (4, 11, 56–58). Studies failed to identify the expression of FSHRs on osteoclast lineage cells most likely used PCR primers designed to target the full-length gonadal FSHR (59, 60). FSH binding to the bone FSHR has subsequently been proven in vivo through the binding of fluorophore-tagged FSH to gonads and bone. A molar excess of unlabeled FSH displaced tagged FSH underscoring the specificity of FSH binding to bone (10, 61). The level of FSH glycosylation is important, as fully glycosylated (i.e., 24 kD) recombinant FSH isoform has a higher affinity to the bone FSHR, as compared to the partially glycosylated FSH molecule (i.e., 21 kD isoform), which is more active in gonads (62, 63).

FSH acts on FSHRs on osteoclasts, stimulating NFκB, MEK/Erk, and AKT pathways and, thus, promoting osteoclast formation, function and survival. The osteoclastic FSHR is coupled to Gαi2, so that its activation causes intracellular cAMP reductions, in contrast to the ovaries where the FSHR couples with a Gαs-protein and triggers an increase in cAMP. Blocking the aforementioned pathway or absence of Gαi<sup>2</sup> leads to bone unresponsiveness to FSH (11). Stimulation of osteoclasts by FSH also occurs via an indirect pathway—the upregulation of receptor activator NFκB (RANK) increases the synthesis of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα) proportionately to FSHR expression (64, 65). Moreover, FSH can interact with an immunoreceptor tyrosine-based activation motif (ITAM) adapter to enhance osteoclastogenesis (57).

In vivo FSH injection caused enhanced bone loss, whereas FSH inhibitor administration decreased bone resorption in ovariectomized rats (66, 67). Mice with an absent or deficient allele of FSHR or FSHβ had higher bone mass and diminished bone loss, which may be partially explained by high serum androgens (68). However, mice lacking aromatase, despite elevated androgen levels, still showed dramatic bone loss (69). Moreover, when FSH inhibitor was injected into male mice they also developed increased bone mass (9). To prevent confounding, generated by the opposite effects of FSH and estrogens on bone resorption, we developed a specific antibody to FSHβ (70, 71), which was shown to decrease osteoclastogenesis in vitro (10, 71), and decrease bone loss and stimulate bone formation in vivo (11, 70, 71). It is also known that FSH acts via the FSHR on mesenchymal stem cells to suppress their differentiation into osteoblasts (70).

### Epidemiologic and Clinical Data Supporting an Action of FSH on Body Composition

There is strong correlative evidence between high FSH and body fat in postmenopausal women. A Michigan sub-study of the SWAN, which included women undergoing menopausal transition, showed a positive relationship between fat mass and serum FSH. Participants with higher FSH had increased fat mass and waist circumference, even after adjusting for baseline measurements, and lower lean and skeletal muscle mass (72). In addition, the Oklahoma Postmenopausal Health Disparities Study, which included a large group of postmenopausal women, showed that the best predictors of waist-to-hip ratio were serum FSH, estradiol and body mass index (BMI) (73). A similar positive relation between FSH to central obesity in infertile females of reproductive age has also been reported (74).

 The Study of Women Entering and in Endocrine Transition (SWEET) found significantly higher lean mass in premenopausal Sub-Saharan African females, as compared to postmenopausal females, with a negative correlation between FSH and lean mass (76).

However, several groups reported an inverse relationship between FSH levels and BMI in women, particularly those in the reproductive age (77–82). This phenomenon can be explained by feedback FSH inhibition by estrogens arising from adipose tissue. For example, a study from France reported that nonobese reproductive-age females undergoing infertility workups had higher levels of gonadotropins and estradiol compared to obese women (78). Another study found an inverse relationship between FSH and BMI in reproductive age females over 326 IVF cycles (77). Overweight/obese fertile women from Italy had lower FSH, LH, estradiol and inhibin B in the early follicular phase (79). The same scenario was reported in postand perimenopausal females. For instance, Penn Ovarian Aging Study compared abdominal MR images and hormonal levels in women at different time points and demonstrated a positive relationship between estradiol and visceral fat, but a negative one was found between FSH and visceral fat (13). Furthermore, data from the 11-year follow-up SWAN study demonstrated that obesity is associated with low FSH trajectory in women of all ethnicities (80). According to The Pan-Asia Menopause (PAM) Study, gonadotropins and estradiol had a strong positive correlation with BMI. Interestingly, estrogen and LH levels were dependent on age, whereas FSH was not (81). Another study, conducted among 73 postmenopausal Serbian women, found higher FSH in normal weight individuals than in obese females (82).

These observations are consistent with those in girls, particularly among pubertal girls who underwent bioelectric impedance measures of body fat >29%. Sorensen and Juul demonstrated that girls within this cohort had significantly lower LH and FSH levels vs. normal weight comparators (83). Likewise, Bouvattier et al. observed a negative correlation between LH, FSH and GnRH responses regarding body mass index among perimenarchial and young adult girls (84).

No significant correlation between BMI and FSH was identified in observational studies of males regardless of age (85–88), except that one cross-sectional study reported that body mass index was negatively related to FSH, inhibin B, and testosterone levels in adult men (89). However, very recent data from a randomized clinical trial suggest that high serum FSH levels cause an increase in body fat in the absence of changes in other hormones. A two-arm open-label randomized clinical study included 58 men with prostate cancer, who were randomly assigned to orchiectomy or GnRH agonist treatment for 24 weeks (90). Notably, serum FSH levels increased after orchiectomy, while GnRH agonist injections inhibited FSH secretion (91). Men treated with orchiectomy experienced greater increases in total fat mass, subcutaneous adipose tissue mass, and weight at 48 weeks as compared to men treated with GnRH agonist (90). This is the first intervention study to demonstrate that FSH regulates body fat in human.

Limited data also suggests that serum FSH may be related to metabolic syndrome. For example, one cross-sectional study, of 320 Polish women reported FSH to be a better indicator of increased risk for metabolic syndrome than SHBG levels (92). Serum FSH also appeared to be more accurate in metabolic syndrome prediction compared with leptin or C-reactive peptide in menopausal females (93).

The role of FSH in non-alcoholic fatty liver disease (NAFLD) has not been well-established. However, a few studies have reported an association between serum FSH levels and fat deposition in the liver, detected by ultrasonography (94, 95). For example, the 2014 Survey on Prevalence in East China for Metabolic Diseases conducted among women over 55 years of age have revealed that serum FSH levels were negatively associated with NAFLD (94). In an adjusted model for waist circumference and HOMA-IR, FSH levels were not associated with mild hepatic steatosis, however the association of FSH with moderate-severe hepatic steatosis remained evident (P for trend <0.01) (94). Similarly, another cross-sectional study conducted among 71 elderly (i.e., 60 years of age or older) patients from China showed that the "normal" diurnal rhythm of FSH was independently associated with NAFLD (95).

#### FSH Action on Body Fat in Mice

There is compelling evidence for FSHR expression in chicken, murine and human adipocytes (9, 96, 97). FSH directly stimulates primary murine adipocytes and 3T3-L1 cells through Gαicoupled FSHR (**Figure 1**), resulting in the up-regulation of core fat genes, such as Fas, Lpl, and Pparg, and the induction of lipid biosynthesis (9). Moreover, FSHR activation leads to cAMP reduction and subsequently UCP1 inactivation in ThermoMouse-derived differentiated brown fat cells (9).

We have fine-mapped the receptor-binding epitope of FSHβ and developed a blocking antibody capable of binding to this

motif to prevent FSHβ/FSHR interaction (9, 10). Injection of this anti-FSHβ antibody in various murine models, including ovariectomized mice and mice either pair-fed on a high-fat diet or allowed ad libitum access to normal chow, caused a significant reduction in visceral, bone marrow and subcutaneous fat (9). The FSH antibody also significantly increased bone mass in ovariectomized mice (9, 70). These phenotypes were recapitulated in haploinsufficient Fshr+/<sup>−</sup> mice, indicating a dominant role of FSH in bone and body fat regulation. Interestingly, anti-FSHβ antibody failed to decrease adiposity in Fshr+/<sup>−</sup> male mice fed on a high-fat diet, proving FSH specificity (9). These observations were reproduced contemporaneously in other centers, using various laboratory methods (9, 100).

FSH blockade in mice also led to the up-regulation of brown adipocyte genes, such as Cox7, Cox8a, Ucp1, and Cidea, in visceral fat indicating "beiging," which, we found, was occurring independently of sympathetic tone (9). However, it is not clear as to whether the beige adipocytes were the product of transdifferentiation of white adipocytes or if they were formed from a specific precursor (101, 102). Using in vivo fluorescence imaging on the IVIS platform, FSH blockade in ThermoMice triggered UCP1 transcription in brown-fat-rich areas initially with increases in white fatpredominant regions. The production of mitochondria-rich, thermogenic "beige" adipose tissue with the anti-FSHβ antibody was further substantiated using PhAM mice, and by documenting elevated basal energy expenditure in metabolic cage studies (9). Interestingly, FSH blockade did not affect glucose or insulin metabolism (92, 93).

Other studies have found a direct correlation between abdominal fat mass and FSHR mRNA expression in female chickens (96). FSH was found to alter lipid metabolism by affecting the expression of Dci, Lpl, RarB, Rdh10, Dgat2, and Acsl3 genes, shifting fatty acid and retinol metabolism, and altering PPARγ signaling (96). Interestingly, FSH has also been shown to inhibit hepatic cholesterol metabolism. FSH was found to interact with FSHRs in HepG2 cells, reducing LDLR levels (103). Moreover, FSHR knockdown with specific siRNA in mice demonstrated lower LDLR (103), suggesting that FSH may be indirectly involved in the pathogenesis of NAFLD. Finally, in a Chinese cohort, rising FSH levels correlated positively with serum cholesterol and LDL levels in postmenopausal women (104).

#### FSH Action on Cardiovascular System

Males receiving androgen deprivation therapy (ADT) for prostate cancer have an increased risk of cardiovascular dysfunction, atherosclerosis and thrombosis (105–107). For example, it has been shown recently that FSH promotes the development of cardiovascular risk in ADT-treated males (18). Moreover, several studies in females have demonstrated effects of FSH on cardiovascular risk measures, such as coronary artery calcium deposition and carotid intima-media thickness. For example, the SWAN study showed that in 856 women who never reported a stroke or a heart attack, FSH trajectory was correlated positively with intima-media thickness(17). Furthermore, a substudy of the Prospective Army Coronary Calcium project, called the Assessment of the Transition of Hormonal Evaluation with Non-invasive Imaging of Atherosclerosis, showed that serum FSH levels were associated with the number of aortic plaques in 126 women undergoing menopausal transition using contrastenhanced CT angiography and carotid ultrasound (16). However, a 22-site population-based Survey on Prevalence in East China for Metabolic Diseases and Risk Factors, showed a negative association between FSH levels and cardiovascular risk (108). The study had a cross-sectional design and any causal relationship between FSH levels and cardiovascular risk factor factors could not be established.

Mice receiving ADT have been used to study the relation of serum FSH and cardiovascular disease (CVD) development. The interaction of FSH with FSHR on monocytes has been shown to up-regulate RANK expression and promote monocytic infiltration of atherosclerotic plaques (18). Th1 helper cells then release RANKL, which activates RANK on monocytes, leading to osteoclast formation. Osteoclasts resorb calcified areas and provoke atherosclerotic plaque instability, increasing the risk of rupture and thrombosis (18). In a second study, serum FSH levels were also found to be significantly lower in mice treated with GnRH antagonists, as compared to animals getting GnRH agonist or orchiectomized (109). The first group displayed less fat mass, at least a two-fold lower atherosclerotic plaque burden, high levels of high-density lipoproteins (HDL), and reduced serum low-density lipoproteins (LDL) compared to the latter two groups. Although all animals developed fatty changes in the aortic wall, the necrotic regions were dramatically smaller in the first group (109). This suggests that increased CVD risk in ADT cannot be explained solely by hypoandrogenemia, and may relate to changes in serum FSH. Furthermore, it has also been surmised that, as atherosclerotic plaque development is dependent on neovascularization (110), FSH may act by stimulating new vessel formation [as effectively as vascular endothelial growth factor (111, 112)] via FSHR present on vascular endothelial cells (7). The mechanism includes the stimulation of VCAM-1 synthesis by FSHR expressed on endothelial cells. VCAM-1 then recruits monocytes to affect their migration and differentiation into macrophages that accumulate lipid droplets and eventually become foam cells (17, 113). Finally, FSH may elevate production of cytokines, namely IL-6 and TNFα, from macrophages to cause low-grade inflammation, atherosclerosis development and insulin resistance (114). We have documented this direct action in osteoclasts (115).

#### FSH Action in Oncogenesis

FSH levels are elevated in ovarian cancer (116, 117). Furthermore, epithelial and endothelial FSHRs have been detected in various cancer types, including prostate (118, 119) and ovarian cancers (120–122), as well as in established cancer cell lines, namely prostate cancer cell lines DU145 (118) and PC-3 (118, 119), ovarian cancer cell lines including OVTOKO, CaOV-3, RNG1, OVCAR-3, and TOV-21G (121– 126). Endothelial FSHR was detected by immunohistochemical and immunoblotting analysis in samples obtained from >1,000 patients with breast, prostate, colon, pancreas, urinary bladder, kidney, lung, liver, stomach, testis, and ovarian cancer (7). Recent data indicates that these FSHRs are signaling-efficient. In particular, endothelial FSHR expression is associated with vascular remodeling and tumor angiogenesis (6, 7), whereas epithelial FSHR induces cell proliferation (118–120), migration, and cancer cell invasion (127).

Interestingly, murine T-cells directed against FSHR- positive ovarian cancer cells showed increased survival without causing toxicity (122). FSHR stimulation upregulated Oct4 expression via the Erk1/2 pathway in epithelial ovarian cancer (128). Epithelial-to-mesenchymal transition in ovarian cancer was also stimulated through PI3K/Akt-Snail signaling (129). It has been suggested that FSH stimulates ovarian cancer cell proliferation via FSHR isoform 3, which is not coupled with G-proteins and not associated with cAMP production, but activates the Erk pathway in a Ca2+-dependent manner (130, 131).

Cancer cells express abundant receptors to various growth factors, suggesting the potential possibility of restricting cancer growth through antibody-mediated blockade of these receptors (132, 133). Unfortunately, the delivery of antibodies through the endothelium is poor and high doses are prone to cause toxicity (134, 135). To avoid this problem, a different approach, notably targeting the tumor vasculature, has been proposed. However, two major groups of extensively studied agents targeting tumor vessels have proven limitations and lack efficacy. Antiangiogenic agents reduce the action of various growth factors inside the tumor, preventing new blood vessel formation (136–139). Their maximum effect is tumor shrinkage and these agents have failed to improve survival (140, 141). The second group of agents, namely vascular disrupting agents, affect mature vessels, rearranging the endothelial cytoskeleton and increasing vascular wall permeability (142), thus disrupting blood supply and leading to extensive central necrosis of a tumor (143); this nonetheless leaves viable peripheral neoplastic tissue that subsequently repopulates the necrotic area (144–146). A new promising direction for anticancer target therapy is to cause peritumoral infarction using truncated tissue factor (tTF) coupled to ligands that are highly specific for FSHR (147). Antihuman FSHR antibody, conjugated with tTF, binds the FSHR, which is abundant in peritumoral endothelium, initiating blood clotting with subsequent blood supply disruption and tumor necrosis (148). Interestingly, the vasculature of bone and fat has not been shown to express FSHRs: thus, such therapy will most likely cause no issues (8, 149). However, their presence in the female reproductive system may limit anti-FSHR-tTF treatment. This approach still needs extensive investigation in the future and

provokes extensive discussion on the development of a cancer therapies based on agents tethered to anti-FSHR antibodies (19).

#### CONCLUSION

In the transitional phase of a women's reproductive life to menopause, the risk for osteoporosis, obesity and CVDs increase concurrently. Along with declining estrogen levels, sharply rising FSH levels have now been implicated in the pathogenesis of these diseases. It is now well-known that bone loss begins even before estrogen levels are altered in the perimenopause (150).

Several key findings have emerged relating serum FSH to bone loss, obesity, and perhaps even cardiovascular risk and cancer. First, it is clear that FSH directly impacts bone cells—osteoclasts and osteoblast precursors. The underlying mechanisms include a direct action on osteoclasts through the enhancement of RANKL signaling, and indirect actions

#### REFERENCES

to increase the expression of RANK in osteoclasts (151) and stimulate the synthesis of pro-resorptive cytokines, including TNFα, IL1β, and IL-6. Studies also conclusively demonstrate the expression of functional FSHRs on adipose tissue (97), which, when blocked by an FSH antibody, result in a profound reduction of body fat and generation of thermogenic "beige" adipose tissue (152). Together, the studies form the framework for using a humanized FSH antibody for the simultaneous treatment of two public health hazards—obesity and osteoporosis—with a single agent. Admittedly speculative at this stage, an increased risk of cardiovascular event(s) among postmenopausal women may also be in part attributable to subclinical atherosclerosis promoted by sharply rising FSH levels (153). Finally, certain cancers, prominently ovarian tumors in which oncogenic signaling through the FSHR can be proven may be amenable to novel FSH-based therapeutic agents.

Thus, positive correlations between rising FSH levels and a plethora of illnesses like obesity, osteoporosis, cardiovascular pathology, and cancer changes our view of FSH from monogamously associated with fertility to a much broader view of the role of this "gonadotropin" in other medical conditions and in human physiology. It is therefore now conceivable that we question whether FSH is a true aging hormone. By developing new treatment approaches that target this gonadotropin, we may in the future be able to treat multiple age-related diseases perhaps even with a single drug.

#### AUTHOR CONTRIBUTIONS

DL wrote the manuscript with support from S-MK, AR, IA, SJ, BA, CT, AK, and LS. TY, MZ, and RA helped supervise the project. All authors provided critical feedback and helped shape the manuscript.

#### FUNDING

Work at Icahn School of Medicine at Mount Sinai was supported by the National Institutes of Health (NIH) by R01 Grants DK113627 to (MZ and LS), AG40132 (to MZ), AR65932 (to MZ), AR67066 (to MZ).

#### ACKNOWLEDGMENTS

The authors acknowledge Mount Sinai Innovation Partner for their collaboration on the actions of FSH on bone.

in endometriotic lesions. J Clin Endocrinol Metab. (2016) 101:2905–14. doi: 10.1210/jc.2016-1014

J Exp Clin Cancer Res. (2015) 34:12. doi: 10.1186/s13046-015- 0128-7

**Conflict of Interest Statement:** MZ is a named inventor on a patent related to FSH and bone, owned by Icahn School of Medicine at Mount Sinai. MZ will receive royalties and/or licensing fees per Mount Sinai policies, in the event the patent is commercialized. MZ also consults for Merck, Roche, and a number of financial consulting platforms.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Lizneva, Rahimova, Kim, Atabiekov, Javaid, Alamoush, Taneja, Khan, Sun, Azziz, Yuen and Zaidi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Extragonadal FSHR Expression and Function—Is It Real?

Marcin Chrusciel <sup>1</sup> , Donata Ponikwicka-Tyszko<sup>2</sup> , Slawomir Wolczynski 2,3 , Ilpo Huhtaniemi 1,4 and Nafis A. Rahman1,3 \*

1 Institute of Biomedicine, Research Centre for Integrative Physiology and Pharmacology, University of Turku, Turku, Finland, 2 Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn, Poland, <sup>3</sup> Department of Reproduction and Gynecological Endocrinology, Medical University of Bialystok, Bialystok, Poland, <sup>4</sup> Institute of Reproductive and Developmental Biology, Imperial College London, London, United Kingdom

Expression of the follicle-stimulating hormone receptor (FSHR), besides gonadal tissues, has recently been detected in several extragonadal normal and tumorous tissues, including different types of primary and metastatic cancer and tumor vessel endothelial cells (TVEC). The suggested FSH actions in extragonadal tissues include promotion of angiogenesis, myometrial contractility, skeletal integrity, and adipose tissue accumulation. Non-malignant cells within cancer tissue have been shown to be devoid of FSHR expression, which implies a potential role of FSHR as a diagnostic, prognostic, or even a therapeutic tool. There are shared issues between several of the published reports questioning the validity of some of the conclusion. Firstly, protein expression of FSHR was performed solely with immunohistochemistry (IHC) using either an unavailable "in house" FSHR323 monoclonal antibody or poorly validated polyclonal antibodies, usually without additional methodological quality control and confirmations. Secondly, there is discrepancy between the hardly traceable or absent FSHR gene amplification/transcript data and non-reciprocal strong FSHR protein immunoreactivity. Thirdly, the pharmacological high doses of recombinant FSH used in in vitro studies also jeopardizes the physiological or pathophysiological meaning of the findings. We performed in this review a critical analysis of the results presenting extragonadal expression of FSHR and FSH action, and provide a rationale for the validation of the reported results using additional more accurate and sensitive supplemental methods, including in vivo models and proper positive and negative controls.

Keywords: FSH, FSHR, extragonadal expression, cancer cells, tumor vessel cells

## INTRODUCTION

Follicle-stimulating hormone (FSH) is synthesized by the anterior pituitary gonadotroph cells, and it plays a critical role in controlling male and female gonadal function (1, 2). FSH acts through its specific receptor (FSHR), a member of the highly conserved family of class A G-protein-coupled receptors (GPCR) (3). In females, FSHR is expressed in granulosa cells and it regulates the maturation of Graafian follicles, granulosa cell proliferation and estrogen production (4). In males, FSHR is expressed in testicular Sertoli cells and it regulates their metabolic functions necessary for proper spermatogenesis and germ cell survival (5). FSHR activation may trigger a number of intracellularsignaling pathways that will be activated in parallel or sequentially (6, 7). The canonical

#### Edited by:

Ren-Shan Ge, Wenzhou Medical University, China

#### Reviewed by:

Alberto Ferlin, Università degli Studi di Brescia, Italy Terje Svingen, National Food Institute, Technical University of Denmark, Denmark

> \*Correspondence: Nafis A. Rahman<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Reproduction, a section of the journal Frontiers in Endocrinology

Received: 09 October 2018 Accepted: 16 January 2019 Published: 04 February 2019

#### Citation:

Chrusciel M, Ponikwicka-Tyszko D, Wolczynski S, Huhtaniemi I and Rahman NA (2019) Extragonadal FSHR Expression and Function—Is It Real? Front. Endocrinol. 10:32. doi: 10.3389/fendo.2019.00032 Gsα/cAMP/PKA signaling pathway, a key effector mechanism of FSH action, activates the cAMP response element-binding protein that modulates gene transcription (6, 7). However, in recent years, it has been shown that also Gαs-independent pathways, such as the PI3K/PIP3–AKT/mTOR pathway, βarrestin-dependent pathway or interaction of FSHR with PPL1, FoxO1a, and 14-3-3τ , are involved in FSH-dependent cellular responses [reviewed in (8)].

Recent studies have suggested the expression of FSHR in many normal extragonadal tissues, as well as in the tumors and tumor vessel endothelial cells (TVECs) (summarized in **Table 1B**). Considering these new findings on extragonadal FSHR expression, a potential role for FSHR as a diagnostic, prognostic and therapeutic tool has been suggested. However, when some of the extragonadal FSHR findings have been revisited no FSHR transcript (by in situ hybridization RNAscope study) or gene amplification (by TaqMan probes-based qPCR) has been found in the tissues presenting with FSHR protein immunoreactivity by IHC staining (9, 10, 16, 17).

This review will summarize and discuss the most important extragonadal FSHR expression findings and highlights some of the caveats involved in these data. We offer our remarks in constructive spirit, and hope they will be found useful in future research on the intriguing topic of extragonadal FSH/FSHR action.

#### DISCREPANCY BETWEEN FSHR mRNA AND PROTEIN EXPRESSION LEVELS

Surprisingly, in many studies, the high and clearly abundant FSHR expression at protein levels by immunohistochemistry or immunofluorescence was not associated with clear FSHR mRNA amplification (9, 11, 13). Nested polymerase chain reaction with gene-specific revers transcription, instead of traditional PCR, was needed to detect FSHR expression in umbilical cord, placenta, and uterus (9, 11, 13). This could indicate a short turnover time and/or rapid degradation of the FSHR transcripts or, not totally unlikely, nonspecific IHC staining results. Moreover, the lack of sequencing data of PCR products reduces the reliability of mRNA detection (9, 11, 13). In normal testis, there is ∼0.04 pg of FSHR transcripts per µg total RNA (43), whereas human ovary contains a small amount of endogenous FSHR protein (0.054 fmol/mg of total protein) (44), and thus its detection by Western Blot might be challenging (45). One explanation could be that FSHR mRNA turnover in extragonadal tissues is much faster than in gonads? Most likely not, considering that in mammals long half-life for RNAs is t(1/2) ≥ 4 h (e.g., housekeeping genes) and a short halflife for RNAs is t(1/2) < 4 h (regulatory genes); thus we should still be able to detect FSHR mRNA at a level relevant to protein levels (46).

### ANTI-FSHR ANTIBODIES

Antibodies are an essential tool for determining cellular protein localization and expression. However, the commercially available monoclonal and polyclonal anti-FSHR antibodies have been poorly or not at all validated. Basic information on selected popular FSHR antibodies is listed in **Table 1A**. The specificity of two frequently used antibodies, sc-7798 and sc-13935, from Santa Cruz Biotechnology, has been recently revisited (47). In a comprehensive study, the authors compared these antibodies with FSHR323 (44) and two potential therapeutic anti-hFSHRs Ylanthia <sup>R</sup> antibodies (Y010913, Y010916) for their suitability in IHC detection of FSHR expression in various tissues (47). Specificity of all antibodies was tested by their binding to native hFSHR from different sources and by IHC on paraffin-embedded Flp-In Chinese hamster ovary cells transfected with FSHR (47). Unfortunately, human ovary or testis tissues were not examined, which would have served as proper positive controls (47). The study showed that only the FSHR323 antibody was suitable for target validation of hFSHR in an IHC setting. Furthermore, the authors confirmed their earlier reports on specific overexpression of FSHR in peripheral tumor blood vessels but could not repeat the previously reported FSHR overexpression in ovarian and prostate cancer cells (24, 25, 33, 47).

From a rational point of study design, testing the specificity of FSHR antibodies using (transfected) cancer cell lines or extragonadal tissues cannot be considered sufficient. The most suitable tissues for the validation of specificity of reproducibility of an anti-FSHR antibody should be the human ovary and testis, the only tissues with undisputable FSHR expression. IHC for FSHR should also be tested on FSHR-negative control tissues. The FSHR323 antibody (44), suggested to be the most specific and reproducible, unfortunately has the major disadvantage of not being available either from the laboratory of its origin or commercially. The hybridoma cell line producing FSHR323 antibodies was removed from the American Type Culture Collection selection shortly after the milestone paper on FSHR expression in TVECs (26) was published in 2010, which created a major obstacle for independent validation of the recent studies on non-gonadal FSHR expression (26). If the authors/owners of this FSHR323 antibody are confident of its efficacy in detection of FSHR, they should make it available for other investigators, e.g., by re-depositing the hybridoma cell line to ATCC, so that independent verification of their data would become possible.

#### FSHR IN NORMAL EXTRAGONADAL TISSUES

Recent studies on FSHR localization have suggested its expression in many normal extragonadal tissues including umbilical vein, vessel smooth muscle cells (9), placenta and placental endothelial cells (12), fallopian tube (11), myometrium, endometrial stromal cells, endometrial glandular epithelium (16, 17), liver (48) bone osteoclasts (20, 49), and monocytes (50, 51) (**Table 1B**).

#### PLACENTA, UMBILICAL CORD VESSELS, AND HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS (HUVECs)

Human placenta and umbilical cord express FSHR, and these reports proposed that the expression is functional (details below) (9, 11). FSHR was found in the endothelial cells of the fetal TABLE 1A | Basic information on several commonly used FSHR antibodies for protein localization.

vasculature within the chorionic villi and villous stromal cells from 8 to 10 week of gestation until term, but not in trophoblast cells (11). In human umbilical cord, the location of FSHR was suggested in Wharton's jelly, and endothelial and smooth muscle cells of the vessels (9). Expression of a splice variant lacking exon 9 was detected in primary HUVECs (9).

Further functional studies showed that FSH-stimulated HUVECs responded with AKT activation, but not with cAMP production (9). HUVECs stimulated with a high pharmacological dose of rhFSH (600 µg/L, equals to 81.86 IU/L) induced significantly tube formation, wound healing, cell migration and proliferation, nitric oxide production and cell survival (9). This prompted the authors to propose that HUVECs express "functional" FSHR expression and that the FSH-FSHR activation promotes angiogenesis as effectively as the potent wellcharacterized angiogenic factor VEGF (9). However, opposite results were published by our group upon revisiting the same study concept and experiments (10). In this latter study, neither FSHR expression nor rhFSH stimulation of freshly isolated HUVECs could be shown (10). In our study, FSHR expression was analyzed with two kits for reverse transcription and qPCR systems, and both gave negative results for HUVECs and an immortalized HUVEC cell line (HUV-ST) (10). The PCR products from the positive control samples were sequenced in order to reconfirm their fidelity, which was not done in the earlier study (9). Numerous methodological uncertainties [presented in detail in (10)], made the results of the previous study hard to interpret and could explain the difference between these two studies (10). Consequently, our data (10) do not support the novel concept that FSH-FSHR activation is involved in the placental vasculature and its angiogenic process (11–13). Results showing that Fshr/FSHR is essential for normal angiogenesis of the fetal placental vasculature (12) are also contradictory because both males and females with inactivating FSHR mutations, thus obligatorily devoid of functional FSHR in their placenta, appear to develop normally in utero (52). Thus, caution is needed to interpret the existing data on the FSH-FSHR activation in placental vasculature and their proangiogenic process, and the functional expression of FSHR in HUVECs still remains a subject of uncertainty.

#### UTERUS (ENDOMETRIUM AND MYOMETRIUM)

Several studies suggest that FSHR is expressed in uterine tissues (11, 15–18, 53). One study appeared to detect positive FSHR signal in the endothelial cells of myometrium vessels and arterial smooth muscle of the normal non-pregnant myometrium, whereas signal in myometrial muscle fibers was weak or only traceable (11). Furthermore, FSHR staining was detected in the endothelial cells, arterial smooth muscle and muscle fibers of myometrium during pregnancy (11); however, the study group was small (n = 3). They also showed through PCR analysis that human myometrium from term pregnancy expresses fulllength FSHR mRNA, but the PCR product were not sequenced to reconfirm their fidelity (11). In another study, in term pregnancy non-laboring myometrium, FSH increased contractile activity, but the effects was observed only at supraphysiological doses of 100–1,000µg/L (136.43–1364.25 IU/L) (13). In contrast to findings of the former study (11), another recent study did not detect FSHR protein or FSHR transcripts in normal human myometrium (16).

Intriguingly, women with inactivating FSHR mutation, thus lacking both gonadal and extragondal FSHR, can maintain normal pregnancy until term following ovum donation, thus undermining the functional importance of extragonadal FSH action (54).

In human endometrial epithelial and stromal cells, cycledependent expression of FSHR was shown, and FSHR expression TABLE 1B | Summary of studies presenting FSHR expression and FSH action in extragonadal normal and malignant tissues.

(Continued)

IHC, immunohistochemistry; NA, not applicable; UC, umbilical cord; TVECs, tumor vessel endothelial cells.

increased during the endometrial secretory phase (15). Furthermore, it has been shown that FSH stimulation induced a decidual phenotype in stromal cells isolated from proliferative phase human endometrium (53). Microarray studies also proposed that FSHR mRNA expression was up-regulated in human endometrial stromal cells decidualized with progesterone and cAMP (55). Moreover, results of our study showed that FSH stimulation upregulated FSHR expression in human endometrial stromal cells undergoing decidualization in vitro (16). It has been shown that during pregnancy, FSHR is expressed in the decidual layer of the pregnant uterus in women (11). FSHR was localized in glandular epithelium and stromal cells of endometrial proliferative and secretive phases. A weak, mainly basolateral FSHR expression was localized in the microvascular endothelium of the normal proliferative endometrium (17). Our group confirmed FSHR expression at mRNA and protein levels in glandular epithelium and stromal cells of normal human secretive endometrium, but not in the proliferative endometrium (16). A very recent study also proposed the functionality of FSHR expression in human endometrium, as endometrial tissue produced cAMP upon FSHR stimulation (14). It seems that FSHR expression in human endometrium, mainly in secretive phase could be functional, but the concept that FSH/FSHR signaling may induce human endometrial stromal cell decidualization requires further evidence.

#### ADIPOSE TISSUE/OBESITY

The rise of FSH level in menopausal women is associated with increased visceral adiposity, and decreased bone density and energy expenditure (56). In human and mouse fat tissues and adipocytes, FSHR expression at mRNA and protein levels has been reported (23).  This antibody also reduced adiposity in ovariectomized mice. Immunostaining analysis showed the presence of FSHR in inguinal and visceral white adipose tissues and also in brown adipose tissues in mice. However, this study lacks the proper negative and positive (testis or ovary) tissue controls to confirm the specificity of the used FSHR antibody. To reconfirm the Fshr expression in adipocytes, the authors also sequenced full-length Fshr cDNA from primary mouse mesenchymal stem cells derived from ear lobes and mouse adipocyte-like 3T3.L1 cells (22). However, even though the expression profile of many genes was shown in white and brown adipose tissue in mice in this study, surprisingly no data on Fshr at mRNA levels was reported in those tissues. For proper FSHR expression assessment, the same tissues/cells should be used for both gene expression and protein analysis. Moreover, they showed by immunoprecipitation assay that their established FSH antibody binds to FSHβ, but the study lacks data on direct evidence for FSH binding to adipocyte FSHR (22). Another group of treatment with GnRH antagonists to block the gonadotropins (FSH) would have strengthened the findings. The most intriguing part of this FSH antibody-based data is that it contradicts with data from mice and humans using GnRH agonists/antagonists, where the endpoint is the same—blockage of gonadotropins. We did not observe any loss of body fat in transgenic mice treated by GnRH antagonist in conjunction with gonadal or adrenocortical tumor treatment (57, 58). FSH suppression concomitant with that of gonadal steroids did not decrease body fat in a clinical study on men (59). This group (22) has developed now an HF2 monoclonal antibody that targets human FSHβ, but no reports in humans have been shown yet (60).

### BONE MASS/OSTEOPOROSIS

It has been proposed that enhanced postmenopausal bone resorption is caused not by estrogen deficiency, but by increased FSH level (61). Direct effect of FSH-FHSR on bone loss in mice has recently been shown (20–22). FSHR was localized in mouse and human osteoclasts and mesenchymal stem cells (20, 62). The latest study reported that blockage of FSH action by a specific monoclonal antibody targeting FSHβ subunit increased bone mass and stimulated new bone formation by osteoblasts in mice (22). Moreover, osteoclastogenesis and expression of osteoclastspecific genes, and matrix metalloproteinase-9, were reduced after attenuation of FSH action (21). The authors showed the presence of FSHR in mouse bones only in vivo, through a specific binding of the fluorescently labeled FSH with near-infrared II fluorophore to FSHR (21). They did not perform any qPCR, immunohistochemistry or Western Blot analysis to demonstrate and/or directly localize FSHR expression in bone. Furthermore, as in the study of FSH action in adipocytes, there is no data showing direct FSH binding to FSHR in bone cells (21, 22).

Human studies are not able to replicate the data from the mouse models mentioned above. A direct interventional study showed that FSH does not regulate bone loss in postmenopausal women (63). Suppression of FSH secretion by GnRH agonist did not decrease the level of bone resorption markers, but rather tended to increase some of them (63). In addition, in normal adult men, FSH did not affect bone turnover and thus appears not to be an important regulator of skeletal metabolism (64). Another in vivo study in men showed that sex steroids modulate bone resorption independently of FSH action (65). Blockage of androgen action by GnRH agonist treatment in prostate cancer patients induced increased fracture risk and bone loss, with no positive effect of FSH suppression (66). Moreover, also some mouse studies showed opposite results (19, 67), in contrast to the earlier study (20). Furthermore, FSHR knockout mice have reduced bone mass (20, 67), and a dose-dependent FSH effects on increased bone mass through an ovary-dependent mechanism in female mice have been observed (19). In male mice FSH impact on bone loss has not been demonstrated (68). Taken together, further studies are needed to determine whether the FSH-FSHR system action in bone resorption is really functional, only speciesspecific, a probable indirect effect of inflammatory cytokines such as tumor necrosis factor α (TNF-α) (69) or an unidentified off-target effect of the FSH antibodies used.

### ENDOMETRIOSIS

Expression of FSHR has also been suggested in endometriosis (16–18). An immunohistochemical study described FSHR localization in the endometriotic glandular and stromal cells of the rectovaginal endometriotic nodules, ovarian endometriotic cysts, and peritoneal endometriotic lesions, as well as with robust vascular FSHR staining (17). Recently, our group showed functional FSHR expression in deep endometriotic lesions, where FSH through FSHR locally up-regulated aromatase expression and induced estrogen production, and FSHR localized at mRNA and protein levels in endometriotic glandular and stromal cells (16). Moreover, the ovarian pattern of differentiation with functional FSH-FSHR system, inducing production of the same steroidogenic cascade as in ovaries has been shown in endometriotic tissues (18) Based on these results, it appears that the expression of FSHR in endometriosis is functional (16, 18).

### FSHR IN CANCER AND TUMOR VESSEL ENDOTHELIAL CELLS (TVECs)

A number of studies (summarized in **Table 1B**) have shown the expression of FSHR in different types of tumor cells suggesting a potential role of FSH in tumorigenesis and suggesting FSHR as a potential target for cancer therapy. FSHR expression was suggested to be present in prostate, ovarian, thyroid, neuroendocrine pancreatic, and pituitary cancer cells and soft tissue sarcomas (**Table 1B**). A breakthrough in the study of extragonadal FSHR expression was, when the receptor expression was proposed in TVECs of different types of tumors (prostate, breast, colon, pancreas, urinary bladder, kidney, lung, liver, stomach, testis, and ovary), including their metastases (26, 27, 29). In the prostate cancer FSHR-positive arterioles, capillaries and venules (but not lymphatic vessels) were located at the periphery of the tumor core (26). Endothelial cells of the vessels present in normal-appearing tissues at a distance >10 mm outside the tumors or normal prostate were FSHR-negative (26). In subsequent studies, endothelial FSHR expression was shown in thyroid and neuroendocrine tumors (35, 37, 40). Several groups suggested that the FSHR observed in TVECs and cancer cells is functional (24–26, 70–72). FSH-stimulated proliferation, migration and invasion was observed in epithelial cancer cells (70). In TUVECs, FSHR mediated the FSH transport, tumor angiogenesis and vascular remodeling (26, 29). With regard to cancer cells and TVECs (primary and metastatic), it was suggested that FSHR might serve as a potential cellular marker of different tumors and provide a novel approach for targeted cancer therapy. If extragonadal FSHR expression and function could be proven by additional independent studies in the TVECs, as well as in the cancer cells, the finding would suggest for FSHR a great utility as a biomarker and/or medicinal target. This potentially important finding needs further verification by independent studies.

### PROPOSALS TO IMPROVE THE QUALITY OF FSHR LOCALIZATION—VALIDATION OF ANTI-FSHR ANTIBODIES

In light of the information reviewed above, it seems that the data on extragondal FSHR localization often relies on insufficiently verified antibodies, making interpretation of data difficult; more worryingly, to potentially erroneous conclusions. As an additional validation of FSHR IHC, we propose the use of RNAscope in situ hybridization (73) or other similar methods. This novel and sensitive method displays a single-transcript resolution and is now in common use. A set of validated, negative, and positive control probes (for genes with different expression levels), provided by manufacturer confirm the quality of the tissue used for hybridization. Using the FSHR323 antibody (donated to us by Dr. Ghinea), we were able to confirm FSHR expression in umbilical vein endothelial cells at protein level but not by in situ hybridization or qPCR on mRNA levels, leaving the question open why the expression at protein level is not supported by mRNA data (10). Recently it has been shown that endothelial cells and smooth muscle cells of umbilical cord vessels, despite clear FSHR immunoreactivity using FSHR323 antibody (9), did not express FSHR transcripts, even when confirmed by a gel electrophoresis of qPCR products (10). A similar discrepancy was observed in endometriosis samples where FSHR at protein level has been shown in endothelial cells of the endometroid tissue (17), but could not be reproduced by in situ hybridization at mRNA level (16).

#### IN VIVO AND IN VITRO MODELS TO STUDY EXTRAGONADAL FSH ACTION

To resolve the controversies of direct FSH action in extragonadal tissues proper in vivo mouse models would be very useful, as well as functional testing on the extragonadal receptors in vitro. Fshr null mice would be a good model for such studies; although these mice exhibit extragonadal defects, data from different scientific groups show contradictory results (20, 67). In vitro experiments on primary cell cultures from extragonadal tissues with purported FSHR expression have also yielded contradictory data (9, 10). Some in vitro studies could be performed on extragonadal tissue explants, allowing to examine mechanisms without disturbing tissue structure and tissue receptor status (16). However, to explain all inconsistencies in vivo and to prove that the extragonadal FSH-FSHR system is really functional in different extragonadal tissues, its actions should be evaluated through precise gene targeting, e.g., using the genome-editing CRISPR/Cas9–based method (74). Conditional knockout mouse models with tissuespecific deletion of FSHR via Cre-lox technology would allow us to directly analyze the loss of FSH-FSHR action in desired condition or extragonadal tissue type. A transgenic mouse model expressing human FSHR with a green fluorescent protein (GFP) or any other reporter marker expression under the FSHR promoter/enhancer could be also a very useful tool to track the FSHR expression. Thereafter, all data can be tested in vitro and in vivo with the same system to prove their functionality.

### GENDER DIFFERENCES PUTATIVELY AFFECTING THE EXTRAGONADAL FSHR EXPRESSION

Many of the studies on extragonadal FSHR function were carried out only on women (endometrium, endometriosis, HUVECs etc.) (9, 10, 12, 17, 18) or female mice (osteoporosis, bone metabolism, or the FSH action on the myometrium muscle contractions etc.) (11, 13, 21, 22, 63) or cells derived from females (for example HUVECs) (9, 10, 12).  Some studies do not state properly the sex of the individual or sample. One cannot rule out the option that some pathologies (for example osteoporosis or bone metabolism etc.) have strong sex differences and are influenced differently by sex steroids and in general the sexually dimorphic endocrine functions.

## CONCLUSIONS

FSHR expression in extragonadal tissues, despite numerous publications, remains controversial. We suggest a more critical analysis of such data, especially when the localization of FSHR in normal extragonadal and malignant tissues is based only on IHC data, without additional methodological confirmation. We suggest proper validation of antibody specificity and the reproducibility between lots. Unrestricted access to the currently existing non-commercial anti-FSHR antibodies would support the fairness of science. When planning experiments, more attention should be paid to inclusion of proper positive and negative controls. For functional studies, the doses of rhFSH should be standardized and restricted to physiologically meaningful concentrations. A proof of principle in human clinical trial with the monoclonal HF2 FSH blocking antibody with a comparison to a group of patients with GnRH antagonist is also needed. It is thus probably not yet time to re-write the textbooks with the functional implications of FSH/FSHR in extragonadal tissues or in cancer cells or TVECs. To this end, definitely many more studies are needed.

## AUTHOR CONTRIBUTIONS

MC, DP-T, SW, IH, and NR took part in designing, drafting, and finalizing of this invited review manuscript.

### FUNDING

This work was financially supported by Polish National Science Center grants 2012/05/B/NZ5/01867 (MC) and 2016/23/B/NZ5/01477 (NR).

#### Chrusciel et al. Follicle-Stimulating Hormone: Beyond Fertility

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Chrusciel, Ponikwicka-Tyszko, Wolczynski, Huhtaniemi and Rahman. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Frontiers in Endocrinology 5 November 2019 | Follicle-Stimulating Hormone: Fertility and Beyond