There are several clinical conditions under which FSH expression or signaling via its receptor is increased, resulting in higher circulating levels and ultimately creating a FSH "gain of function" effect. These include ovarian hyperstimulation syndrome (23–26), certain ovarian cancers (27–29), and the recent discovery of non-gonadal FSH actions on bone in transgenic mouse models with elevated human FSH levels (30). GOF effects of FSH in ovaries were also occasionally noted in patients with FSH hypersecreting pituitary adenomas (31). The existence of such conditions highlights the importance of generating animal models that closely mimic human disease phenotypes, allowing us to expand the medical knowledge of these conditions and ultimately providing opportunities to learn how to treat them. While mouse models do not always accurately mimic human pathology, they provide a quick genetic test to address the function of human proteins in a physiological context that cannot be reliably achieved using in vitro experimental approaches. In this review, we highlight and describe previously generated FSH gain of function animal models and how they can potentially be used to develop new approaches for treating clinical conditions involving FSH.

#### GOF Mouse Models for FSH

Several GOF genetic models have been generated and used to study the physiological consequences of FSH. These models are described below in detail and summarized in **Table 1**.

#### Expression of Human FSHB in vivo

A transgenic mouse model harboring a 10 kb HFSHB transgene was the first mouse model generated to test cell-specific expression of FSH in gonadotrope cells and to identify that species-specific differences exist in FSH regulation (32). The HFSHB transgene was cloned into an EcoR1-Sph1 genomic fragment and microinjected into fertilized one-cell embryos. The resulting transgenic mice exhibit only pituitary-specific HFSHB transgene expression, with no ectopic expression in non-pituitary tissues (32). Expression of hFSHβ was found to be localized to only gonadotrope cells in the anterior pituitary gland. The FSH heterodimer presumably incorporated the mouse-α subunit, creating an interspecies hybrid heterodimer with hFSHβ, because no free hFSHβ was detected in serum (32). FSH dimer secretion and pituitary HFSHB mRNA expression were higher in both transgenic and normal males than in their female counterparts. The retention of normal gonadotrope-specific expression of FSH and its function in mice expressing hFSHβ demonstrates conservation of regulatory elements and transcription factors for this subunit gene in both mice and humans (32). This mouse model provided a novel approach for studying molecular mechanisms and regulatory elements that are involved in control of the human FSHβ-encoding gene and its expression.

### Gonadal Steroid Regulation of HFSHB

The same transgenic mouse model described above was also used for experiments designed to analyze steroid regulation of HFSHB in vivo (32, 33). This study included several experimental groups including castrated male and ovariectomized female mice. Castration resulted in elevated serum FSH levels in both normal controls and transgenic males. Similarly, increased serum FSH levels were observed in ovariectomized normal and transgenic females (33). Testosterone replacement after castration in male transgenic mice resulted in suppressed serum FSH levels. Estradiol (E2) replacement in ovariectomized females similarly resulted in suppressed serum and tissue FSH content. The sexually dimorphic pattern previously observed, in which both normal and transgenic males exhibiting greater tissue and serum FSH levels than the corresponding females, was also observed in these studies (33). These studies highlight the species-specific differences and suggest that the elements responsible for continued synthesis and secretion of hFSHβ in response to androgens are not present in the mouse pituitary environment (32, 33).

#### GnRH-Independent Androgen Inhibition of HFSHB Transgene

To further elucidate the direct roles that steroid hormones play in regulation of hFSHβ at the pituitary level, HFSHB transgenic mice were used to observe the effect of androgen in the presence or absence of gonadotropin-releasing hormone (GnRH) using both in vitro and in vivo approaches (34). Since there was an apparent species-specific difference in FSH secretion and molecular mechanisms in response to androgens, GnRH was identified as a possible key regulatory site in the androgen response of human FSHβ. For in vitro studies, primary pituitary cultures were obtained from GnRH-deficient hypogonadal (hpg)

**Abbreviations:** BAC, Bacterial Artificial Chromosome; cAMP, 3′ ,5′ -cyclic adenosine monophosphate; DCG, Dense Core Granules; E2, Estradiol; FSH, Follicle-stimulating hormone; FSHR, FSH-receptor; FoxL2, Forkhead box L2; FR-I, Fshb type - I genetic rescue; FR-II, Fshb type - II genetic rescue; GnRH, Gonadotropin releasing hormone; GOF, Gain-of-function; GPCR, G-protein Coupled Receptor; hCG, Human chorionic gonadotropin; HPG, Hypothalamus-Pituitary-Gonadal; hpg, Hypogonadal; LH, Luteinizing hormone; LHR, Luteinizing hormone receptor; mMT-1, mouse metallothionein- 1; RIP, Rat insulin II promoter; RT-PCR, Reverse transcription- polymerase chain reaction; T, Testosterone; Tg, Transgenic; TSH, Thyroid-stimulating hormone; m, mouse; h, human; p, porcine; o, ovine; WT, Wild-type.

TABLE 1 | Major phenotypes of FSH gain-of-function genetic models.

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mice (9, 52) carrying the HFSHB transgene (34). Testosterone treatment in the absence of GnRH resulted in suppression of HFSHB mRNA and confirmed the inhibitory action of androgens directly at the pituitary level independent of GnRH (34). In vivo experiments in the hpg HFSHB mice included daily GnRH injections, which induced HFSHB expression in both males and females. Simultaneous administration of testosterone propionate in males completely blocked the stimulatory effect of GnRH, whereas simultaneous E2 administration in females only partially inhibited GnRH effects (34). These results demonstrated direct effects of testosterone and E2 on hFSHβ subunit expression at the pituitary level as well as an indirect suppression of GnRH as an additional regulatory mechanism (34). Additional hypothalamic site of E2 action cannot also be ruled out based on the above data.

Having established that the 10 kb HFSHB transgene is appropriately targeted to and hormonally regulated in mouse gonadotropes, a series of deletions were made on the 10 kb HFSHB transgene (35). Several independent transgenic lines expressing 5′ and 3′ truncated versions of HFSHB transgene were produced and systematically analyzed. These in vivo models helped to identify that truncation of sequences upstream of 5 ′ promoter region retained expression of hFSHβ in mouse gonadotropes, truncation of poly-A sequences downstream of 3′ stop codon in exon 3 resulted in complete loss of expression. Replacement of 3′ poly-A sequences with heterologous sequences (for example, lacZ reporter sequences) similarly failed to confer expression (35).

Since FSH is normally released from the pituitary in response to GnRH, it is of great interest to observe the physiological response to targeted expression of FSH in non-pituitary tissues. Accordingly, mouse models have been generated that drive expression from either specific or multiple ectopic tissues.

#### Use of HFSHB Transgenes to Achieve Genetic Rescue of Fshb Null Mice

An FSH-deficient mouse model was created in 1997 through targeted mutation (Fshbm1 ) in exon 3 of the FSHβ-encoding gene (53). Mice that were homozygous, i.e., Fshb null (Fshbm1/ Fshbm1 ), and therefore FSH-deficient, were generated by intercrossing heterozygous mice. Fshb null males displayed decreased testis size, yet were fertile (53). Sperm number was decreased by 75%, however, viability remained unchanged. In contrast, Fshb null females with the Fshbm1/ Fshbm1 genotype were infertile, with small ovaries and thin uteri. Ovaries had arrested follicular development at the secondary stage, and lacked any corpora lutea (53).

Genetic rescue of FSH-deficient mice was achieved using two independent methods (36). The type 1 genetic rescue (FR-I) consisted of targeting the previously described 10-kb HFSHB transgene specifically to pituitary gonadotrope cells. This genetic strategy resulted in complete rescue of both males and females lacking endogenous Fshb (36). Testis size and sperm counts in FR-I males were restored to those observed in wild-type values. Similar results were obtained in FR-1 females, as uterine and ovarian sizes also returned to wild-type values. Normal follicular development, restored estrous cycles, and production of normal litter sizes were observed in FR-I females.

Low- level ectopic expression of HFSHB was achieved in multiple tissues using a mouse metallothionein (mMT-1) gene promoter with the goal of genetically restoring reproductive phenotypes in FSH-deficient mice, designated as type 2 rescue (FR-II) (36). The mMT-1 promoter was used to drive ectopic expression of both a hCGα-encoding minigene and a hFSHβencoding gene, thereby resulting in expression of hFSH dimer in multiple tissues (36). Male mice expressing ectopic hFSH (FR-II) showed complete restoration of testis size and sperm counts. However, restoration of normal reproductive phenotypes was incomplete in FR-II females. Only 3 out of 10 FR-II females were able to conceive, and litter sizes were small. Arrested folliculogenesis was frequently observed in FR-II females. The small number of FR-II females that were able to become pregnant produced one litter, had obvious corpora lutea, yet small antral follicles (36). The results with the type 2 genetic rescue suggest that ectopic expression of human FSH can completely rescue Fshb null male mice, yet only partially rescue Fshb null females (36).

#### Ectopic Overexpression of HFSH Dimer in Transgenic Mice

Overexpression of FSH may lead to high serum levels and clinical conditions that negatively affect fertility. A transgenic mouse model ectopically expressing human FSHβ using an mMT-1 promoter resulted in male and female mice overexpressing hFSH in several tissues (24). Mice expressing either only transgenic MT-α subunit or only MT-FSHβ were crossed to obtain mice expressing hFSH ectopically (24). Founders expressing hFSH dimer at very high levels were chosen for further analysis. Both males and females were infertile; males showed enlarged seminal vesicles and elevated testosterone levels yet normal testis size and spermatogenesis (24). These high level FSH expressing males were infertile, presumably due to male sexual behavioral deficits secondary to excess testosterone. However, this behavioral phenotype was not tested in these studies. Females displayed arrested folliculogenesis, along with increased serum estrogen, progesterone, and testosterone concentrations. Females also developed urinary tract obstruction and hemorrhagic and cystic ovaries, yet exhibited no signs of tumors (24). Their symptoms were comparable, but not identical to human conditions such as polycystic ovarian and ovarian hyperstimulation syndromes. Most females died between 6 and 13 weeks of age. (24). The overexpression of hFSH in multiple tissues gave insight into these clinical conditions and provided a model that may be used in the future for developing treatments.

#### Ectopic Expression of FSH in hpg Mice

The role of FSH in gonadal physiology was investigated using a mouse model similar to the models described above that carry a HFSHB transgene (39). However, this model expressed transgenic human-FSH (tg-FSH) on a gonadotropindeficient hypogonadal (hpg) background to observe FSH effects independent of LH. The HFSHB transgene was cloned into a vector containing the rat insulin II promoter (RIP) and injected

into mouse oocytes (39). RIP directed ectopic expression of tg-FSH to the pancreas. Hypogonadism was accomplished by breeding tg-FSH mice to an hpg strain containing a truncating mutation that caused GnRH depletion, thereby creating tg-FSH+ hpg mice (39, 52). Varying serum tg-FSH levels were found in different strains of mice, allowing for analysis of a range of circulating FSH concentrations. Tg-FSH seemed to have no effect on androgen levels in the tg-FSH+ hpg mice, which appeared to be due to underdeveloped epididymis and seminal vesicles. In male tg-FSH+ hpg mice, testis size increased as compared to non-tg-FSH hpg controls, however, this was only observed in males exhibiting high serum FSH levels (>1 IU/liter). Tg-FSH+ hpg female mice secreting high levels of FSH exhibited dosedependent, elevated inhibin B secretion. Ovaries of tg-FSH+ hpg females were also enlarged and exhibited increased follicular development to the antral stage (39).

Additional studies were performed using female tg-FSH+ hpg mice to determine the effect of FSH alone on primordial follicle reserve and the role of FSH in early follicular development (40). Partial disruption of follicular development was observed in non-tg hpg ovaries. Although development past the primary follicle stage occurred, there were small numbers of early antral follicles. In contrast, tg-FSH+ hpg females showed advanced follicular development up to the antral stage, although no corpora lutea were observed in any tg-FSH+ or non-tg FSH hpg ovaries due to the absence of LH (40). Significant increases in total primordial and secondary follicle numbers were seen in tg-FSH+ hpg females as compared with both non-tg hpg and wild-type mice. The total antral follicle count was 15-fold higher in tg-FSH+ hpg ovaries than non-tg FSH hpg levels, which restored values to wild-type levels (40). The findings from this study indicate an important role of FSH in early follicular development, showing an increase in primordial follicle reserve and stimulation of follicle growth.

Interestingly, when tg-FSH+ female mice alone with progressively rising hFSH levels (2.5–10 IU/ml) were monitored across the life span, age-specific phenotypes were observed. Whereas, tg-FSH+ female mice < 22 week of age delivered increased litter sizes, those that were older (>23 week of age) produced decreased litter sizes despite increased ovulations and demonstrated premature infertility due to embryo resorptions and parturition failure. Thus, this model provided a novel in vivo scenario in which age-related rise in FSH contributes to female reproductive aging and infertility by a post-implantation defect (embryo-fetal resorption) without directly affecting the ovarian reserve (41).

Contrary to the proposed deleterious and direct effects of FSH on bone osteoclasts in mice (54), ectopic human FSH expression in the above described genetic model caused an increase in bone mass in female mice (30). Similar phenotypes were also observed when the HFSHB transgene was expressed on the hpg genetic background with a total suppression of endogenous gonadotropins and E2. Expression analysis indicated osteoclasts did not express Fshr mRNA and the bone phenotypes manifest only when ovaries were intact. Further studies indicated that bone volume in these transgenic mice positively correlated with ovary-derived inhibin A and androgens. Thus, ectopic human FSH expression in this model suggests FSH acts indirectly to enhance bone function in an ovary-dependent and LHindependent manner (30). The controversy with regard to nongonadal actions of FSH is ongoing and has been recently described in detail (55).

### Rerouting of FSH Into the LH Secretion Pathway

Transcriptional responses of FSHβ and LHβ encoding genes are different and dependent on GnRH pulse frequencies (10, 11, 56). In immortalized gonadotrope cells, Fshb gene transcription is favored by slow GnRH pulses whereas Lhb gene transcription is dependent on fast GnRH pulses (10, 11, 56). Once the heterodimers are assembled, FSH is largely released constitutively, while LH is released as pulses via a regulated secretory pathway (10, 11). LH contains a carboxy terminal (C′ ) heptapeptide that directs its secretion via this regulated pathway. A novel mouse model took advantage of this heptapeptide to observe the physiological response to a mutant FSH that contained this peptide (37). Human transgenes encoding either a wild type (HFSHBWT) or mutant (HFSHBMut) FSHβ were introduced onto an Fshb-null genetic background.

The presence of interspecies heterodimers of mouse-α-and WT or mutant FSHβ subunits in different mouse lines was confirmed by Western blot analysis (37). LH is stored in densecore granules (DCG) prior to release (57–59). To determine whether mutant FSH was secreted via the same pathway as LH, co-localization of mutant FSHβ subunit and DCGspecific Rab27 was evaluated in gonadotrope cells. Interestingly, the number of mutant FSHβ subunit and Rab27 co-localized gonadotropes was 6 to 8- fold higher than seen in gonadotropes of control mice, where FSH is secreted via the constitutive pathway (37). Co-localization of mutant FSHβ and a chaperone protein chromogranin-A (Chr-A), which is found in the Golgi network in gonadotropes and important for regulated release of LH, was also examined. Co-localization of mutant FSHβ and Chr-A was higher when compared to both control and Fshb null mice expressing a HFSHBWT transgene and was similar to levels seen with co-localization of LHβ (37). These results suggest that the engineered mutant FSHβ (containing the C′ heptapeptide normally only found in LH)- containing FSH heterodimer successfully entered the regulated secretory pathway.

As described above, mice deficient in FSH are infertile, have small ovaries and thin uteri, along with disrupted folliculogenesis (53). HFSHBMut transgene was able to genetically rescue Fshbnull mice, restoring ovarian and uterine size as well as estrus cycles leading to the presence of antral follicles and corpora lutea in ovaries. Progesterone levels were higher in HFSHBMut– expressing mice than in control and wild-type mice (37). Interestingly, the number of ovulations was increased from 9 to 10 per cycle in control mice to 55 per cycle in mutant FSH–expressing Fshb null mice (37). This was accompanied by the presence of more pre-antral follicles and reduced follicular atresia in ovaries. Mice expressing HFSHBMut also demonstrated increased follicle size and enhanced granulosa cell proliferation, leading to a longer reproductive lifespan and follicle survival (37). This mouse model provided a novel approach to studying differential secretory pathways of FSH and LH, and demonstrated a potential role of rerouted FSH in treating age-associated reproductive conditions (37).

### Expression of a Transgene Encoding the Non-glycosylated Human FSHβ Subunit

One of the characteristic features of glycoprotein hormones, including FSH is the presence of N-linked sugar chains on both the α- and β-subunits. FSH possesses four (2 on each subunit) potential N-glycan attachment sites (1–4, 60, 61). The presence or absence of both N-glycans on FSHβ subunit contributes to macroheterogeneity, significantly affects serum half–life and may alter bioactivity (60, 61). To genetically determine the role of N-glycans on the human FSHβ subunit, the nucleotides corresponding to two Thr residues following Asn residues on which N-glycans are added, Asn7 and Asn24, were mutated to Ala, thereby abolishing N-glycosylation events at these two sites (38, 60).

The mutant cDNA transgene encoding the N-glycosylation double mutant FSHβ subunit was targeted to gonadotropes in transgenic mice first and the transgene was subsequently introduced onto Fshb null genetic background using the wellestablished genetic rescue scheme. An HFSHB WT transgene similarly was introduced onto Fshb null genetic background and the resultant Fshb null mice carrying the HFSHB WT transgene were used as positive controls (38, 60). Biochemical studies confirmed that the mutant FSHβ subunit inefficiently assembled with the endogenous mouse α-subunit and very little FSH heterodimer was present in pituitary extracts (38). Moreover, media collected from short term pituitary organ culture experiments and serum from mutant FSHβ-expressing Fshb null mice showed very low or undetectable levels of FSH by radioimmunoassays. These data indicated that the double Nglycosylation mutant FSHβ subunit was secretion incompetent (38). Moreover, the mutant transgene, unlike the HFSHB WT transgene, did not rescue Fshb null mice, confirming that even if secreted in low levels, the mutant FSHβ-subunit containing FSH dimer was biologically inactive (38). Thus, these studies provided in vivo genetic evidence that N-glycosylation on FSHβ subunit is critical for FSH heterodimer assembly, secretion and action (38).

### Expression of FSH in the Mammary Gland

Ectopic expression of bovine FSH has been achieved in a model targeting its expression in milk secreted from mouse mammary glands (42). This mouse model was created using a rat βcasein gene promoter driving expression of bovine α and FSHβ subunits. The β-casein promoter drives targeted expression in only the mammary gland, making it possible to observe the effects of ectopic bovine FSH expression in a single tissue (42). Transgenic (Tg) mice expressing the transgene were created either by microinjection of both subunits, or by breeding of mice that expressed each one of them separately (42). The presence of tg-FSH was confirmed by northern blot and radioimmunoassay. Bioactivity of tg-FSH was also confirmed by measuring granulosa cell counts and the ability of the cells to produce estrogen (42). The amount of tg-FSH positively correlated with granulosa cell number and estrogen production, and therefore suggesting successful bioactivity of the transgene-derived FSH (42). No other overt phenotypes were observed in transgenic mice. This mouse model provided a novel approach for expressing FSH in mammary glands and releasing it into milk.

A similar model was later generated that also ectopically expressed FSH in mammary glands, but in this case, the transgene encoded human FSH. In these studies, the investigators used the bovine β-casein gene promoter to specifically express human FSH in mammary glands of transgenic mice (43). Milk was collected from tg-hFSH mice to determine FSH concentration using an enzyme-linked immunosorbent assay (ELISA). Two mouse lines showed nearly undetectable levels of hFSH, as compared to one line that had high levels (300 mIU/mL). This variation in hFSH concentration in different lines was most likely due to differing transgene copy numbers (43). In vitro biological activity of hFSH was determined by measuring cyclic-AMP (cAMP) production after exposing hFSH receptor-transfected cells to sterilized milk from each cell line. Milk containing hFSH increased cAMP production in the assay, indicating receptor binding and intracellular signaling, and therefore, biological activity of hFSH (43).

Blood cell counts were performed to analyze the effects of any hFSH leakage into the circulation. Several of the transgenic lines showed increased white blood cell and platelet levels as compared to normal mice, red blood cells in the affected animals were also smaller in size (43). Ovarian and breast tumors were observed in one transgenic line, along with collapsed alveoli within the lactating glands. Human FSH also seemed to have a stimulatory effect on endogenous mouse FSH, as mFSH levels were higher at all estrus cycle phases in transgenic mice (43). This unique mouse model displayed distinct physiological responses to ectopic expression of hFSH in mammary glands and secretion into milk. Many of them negatively affected blood circulation and reproductive health. Ectopic hFSH leaking into the bloodstream appeared to lead to overproduction of endogenous mouse FSH, possibly contributing to the observed ovarian and breast tumors by increased estrogen production as a secondary consequence (43).

### Expression of Porcine FSH in Mice

Transgenic expression of porcine FSH using bacterial artificial chromosome (BAC) methods has been achieved using a gainof-function mouse model (45).  BAC clones containing pFSH α and β were then digested and microinjected into fertilized mouse zygotes (45). Transgenic (Tg) mice expressing pFSH were identified by PCR and Southern blot. After further breeding of Tg mice to wild-type mice, both pFSHβ and pFSHα BACs were transmitted in identical Mendelian ratios to offspring, indicating proper hybridization of the Tg subunits. Expression of Tg pFSH mRNA was confirmed by RT-PCR and northern blot, with expression localized specifically to the pituitary gland. Circulating pFSH was confirmed by evaluation of serum samples by ELISA, showing levels ranging from 6.36 to 19.83 IU/L, which were within the physiological range (45).

Female fecundity was analyzed in both Tg and WT mice. Interestingly, Tg females had litter sizes that were significantly larger than WT females as well as the total number of pups than WT littermates. This increase in fecundity in Tg females seemed to be due to enhanced ovulation, as histological examination revealed a significant increase in the number of corpora lutea at 14 and 28 weeks of age in Tg mice compared to WT. Serum hormone levels were analyzed to determine if increased ovulation was due to differential hormonal regulation (45).

Endogenous mouse FSH levels were elevated in Tg mice, as was estradiol. Serum levels of LH and testosterone were significantly lower in Tg mice compared to WT. Pituitary expression of LHβ mRNA was also lower in Tg mice than in WT mice (45). The increased estradiol levels could be due to greater aromatization of androgens to estrogens due to elevated levels of endogenous mFSH in addition to transgene-derived pFSH (45). This enhanced conversion of testosterone to estrogen as a result of increased aromatase activity could explain the lower serum levels of testosterone. However, the mechanism for reduced LHβ mRNA and serum LH despite low levels of serum testosterone is unclear. The results from this study provided confirmation of successful expression of porcine FSH in a transgenic mouse model with no reproductive effects in males but enhanced ovulation in female Tg mice (45).

## Expression of Transgenic Porcine FSH in Large White Boars

Porcine FSH was further analyzed by the same group that produced BAC pFSH subunits from Chinese Erhualian pigs, and introduced them into Large White Boars (44). As the Large White Boar previously showed poor reproductive performance, the investigators sought to determine if overexpression of pFSH from a highly prolific pig breed could improve fertility. Successful integration of pFSH into transgenic (Tg) boars was confirmed using genomic PCR as well as RT-qPCR analysis to determine mRNA expression of porcine FSHα and FSHβ. Expression of FSHα was observed in multiple tissues including heart, liver, spleen, kidney, brain, testis, and epididymis of both Tg and wild type (WT) boars, with higher expression in Tg than WT.

Expression of FSHβ was localized specifically to the pituitary and was significantly higher in Tg than WT boars (44). Higher serum levels of both FSHα and FSHβ were observed in Tg boars suggesting overexpression of pFSH. Male reproductive performance was measured by evaluating semen volume, sperm motility, sperm concentration, and total sperm number per ejaculation (44). There was no significant difference between semen quality parameters of Tg and WT boars. However, the number of germ cells per seminiferous tubule was significantly higher in Tg boars than WT (44). The elevated germ cell counts in seminiferous tubules suggested increased spermatogenesis capacity, but the lack of significant results from semen evaluation leaves room for further analysis to confirm this possibility (44).

In a third study done using the same BAC system containing pFSH α and β, analysis of reproductive phenotypes in female transgenic (Tg) Large White Boars were analyzed. Methods for pFSH BAC transfer were the same as previously described for this model (44). The specific integration site of pFSH into Large White Boars was determined using whole-genome sequencing, identifying exogenous FSHα/β genes at 140, 646, 456 bp on chromosome 9 (46). The transgene integration site was mapped to perhaps rule out that the integration itself did not result in modification of any endogenous loci that regulate fertility.

Analysis of Tg female boars revealed elevated levels of serum FSH and FSHβ protein in the pituitary, but Tg females produced reduced numbers of total newborn piglets as compared to WT. Reduced expression of Fshr, Lhr, Esr1, and Esr2 was also observed using RT-qPCR in Tg boars as compared to WT at 300 days of age. Reproductive organ weights, blood cell counts, and histological analysis revealed no differences between Tg and WT boars in overall reproductive health (46). The reduced expression of mRNAs encoding receptors for FSH, LH, and estrogen suggest a possible negative effect of pFSH overexpression in female pigs as well as the observation of reduced total newborn piglets (46). Further studies using this model are needed to confirm the effects. However, these studies provided novel information on the physiological role of porcine FSH in vivo in the homologous species.

### Inhibin Knock Out Mouse Model

Important regulators of FSH production and secretion are gonadderived dimeric growth factors, activin and inhibin. Inhibin is a heterodimer, consisting of an α and β subunit, whereas activin is a homodimer, consisting of various combinations of the two homologous β subunits (βA and βB) (21). The inhibin and activin subunits are expressed in multiple tissues throughout the body. Inhibins suppress and whereas activins promote FSH synthesis and secretion. To investigate the role of inhibin in reproduction and general physiology, a knockout mouse model with a targeted deletion of the α-inhibin–encoding gene was achieved using homologous recombination technology in mouse embryonic stem cells (47). Targeted deletion of only the α-subunit of inhibin ensured successful inhibin deficiency without unwanted deletion of the activins.

Male and female heterozygous mice produced normal litter sizes and were fertile. However, homozygous males and females proved to be infertile when crossed with wild-type mice, despite developing normal external genitalia (47). In addition to infertility, all homozygous mice tested showed gonadal tumors when examined histologically, which were evident as early as 4 weeks of age. Testicular enlargement was visible in males, along with gradual regression of spermatogenesis and a decrease in Leydig cell count starting at 5–7 weeks of age (47). In females, ovarian tumors disrupted follicular development and morphology from 7 to 16 weeks of age. FSH levels in both males and female homozygous, inhibin-deficient mice were 2 to 3-fold higher when compared to both heterozygous and WT control mice (47). The results of this study suggested a novel secreted tumor suppressor role for inhibin that was highly specific to the gonads. Development of normal external sexual organs and gametes followed by regression and disruption at a later age indicated normal embryogenesis and early sexual development, therefore suggesting gonadal tumors as the cause for infertility (47).

To further investigate the role of inhibin and FSH in gonadal tumorigenesis, a double-mutant knockout approach was taken. Since the inhibin-deficient mice developed aggressive gonadal tumors accompanied by elevated FSH levels, the contribution of FSH to tumorigenesis was directly assessed using a genetic strategy. To achieve this, double-homozygous mutant mice were created by intercrossing heterozygotes for each knockout mutation (Inham1 and Fshbm1 ) to generate mice deficient in both inhibin and FSH (Inham1 /Inham1 ; Fshbm1 /Fshbm1 ) (24). The first parameter examined in the double-knockout mice was weight, as the previous study showed that mice deficient in inhibin only, exhibit a severe cachexia-like syndrome and die by 12 weeks of age (24). Most of double-knockout mutant males survived for 1 year and showed no dramatic weight loss or testis phenotypes. Approximately, 95% of the inhibin-deficient female mice died by 17 weeks. In contrast, double null mutant females, about 70% survived past 17 weeks, but these all eventually died by 39 weeks and all of them exhibited severe weight loss (24).

Gonadal tumor progression was also altered in the doublemutant mice, as development of tumors was slower and less aggressive than in mice deficient in inhibin alone (24). In 12 week old double mutant males, the tumors were small, there were no signs of hemorrhage, and tubule morphology was also unaltered by tumor growth, despite proliferation of tumor cells (24). Beyond 1 year of age, some males showed no tumor development, as compared to inhibin-deficient males which all had tumors as early as 4 weeks (24).

In female double-mutants, ovaries appeared morphologically normal at 12 weeks of age. Histological analysis revealed hemorrhage, cysts, as well as granulosa cell tumors. However, these tumors in the double-knockout females appeared less invasive and developed more slowly than in inhibindeficient mice. Both male and female double-mutant mice showed reduced serum levels of activin A and estradiol as compared to mice lacking only inhibin (24). In addition to this, aromatase mRNA expression levels were reduced in double-mutant mice compared to those in inhibin-deficient mice. These results confirm the role of inhibin in gonadal tumorigenesis and identify FSH as an important modifier in the progression and aggressive growth of inhibin-deficient gonadal tumors (24).

#### FSH Receptor Gain of Function

The FSH receptor (FSHR) is a transmembrane, G-protein coupled receptor expressed on testicular Sertoli cells and ovarian granulosa cells in males and females, respectively (2, 4). Signaling via FSHRs results in steroidogenesis (production of

estrogen) and is essential for gonadal development in both sexes. A novel approach to studying the gain-of-function effects of FSH receptor was undertaken by generating a mouse model exhibiting constitutively active FSH receptor action on an hpg genetic background (48). The use of the hpg genetic background allowed observation of the effects of active FSHR completely independent of endogenous gonadotropins, FSH and LH.

The gain of function receptor mutation (FSHR+) was a single amino acid substitution (Asp567Gly) that was specifically expressed in Sertoli cells by using the rat androgen binding protein (rABP) promoter (48). Bioactivity of the ligandindependent FSH receptor was confirmed by measuring cAMP production, which was significantly higher in Tg-Sertoli cells than in non-Tg-hpg Sertoli cells in vitro (48). The in vivo effects of the FSHR+ mutation were first examined by measuring testicular weight. Tg-FSHR+ hpg testis weights were increased up to 5-fold, with an average of a 2-fold increase as compared to non-Tg hpg controls. Histological examination of FSHR+ testes showed round and elongated spermatids and signs of Sertoli cell maturation, as compared with non-FSHR+ hpg controls that lacked mature Sertoli cells and exhibited

blocked spermatogenesis. Tg-FSHR+ mice also had elevated testosterone levels, and undetectable levels of LH and FSH, suggesting that the observed physiological responses to the constitutively active FSH receptor was indeed gonadotropinindependent (48).

To determine whether the results from the FSHR+ mutation were solely due to the amino-acid substitution or if they were in response to overexpression of mutant FSH receptor, a comparative study was performed using the same mouse model in parallel with a transgenically overexpressed wild-type human FSH receptor (TgFSHRwt) (49). Transgenic male and female mice overexpressing TgFSHRwt were created by microinjection of a human FSHR DNA construct into mouse oocytes. Overexpression of TgFSHRwt was confirmed by measuring radioactive 125I-FSH binding to testicular membranes. Significantly elevated 125I-FSH binding was observed in TgFSHRwt testes as compared to non-Tg controls, similar to levels in FSHR+ mice, thereby confirming overexpression.

TgFSHRwt mice displayed no difference in testis weights or serum testosterone (T) levels, as compared to FSHR+ mice, which had larger average testis weights and elevated T concentrations when compared to non-Tg controls. TgFSHRwt Sertoli cells showed higher cAMP activity in vitro, however basal levels did not exhibit the same increased activity as FSHR+ cells (49). Receptor ligand-specificity was decreased in FSHR+ mice, as exposure to human chorionic gonadotropin and TSH resulted in intracellular signaling. However, the same response was not observed in TgFSHRwt mice. Steroidogenic enzyme-encoding mRNAs such as Cyp11a1 and Star, were elevated in FSHR+ mice, suggesting increased steroidogenic potential (49). However, this increase was also not observed in TgFSHRwt mice. Sertoli and germ cell maturation that was observed in FSHR+ mice was also absent in TgFSHRwt mice, as they exhibited immature development similar to non-Tg hpg mice. Together, all of these results suggest that the physiological responses to constitutively active mutant FSH receptors in FSHR+ mice were due to the mutation itself and not the result of receptor overexpression (49).

Activating mutations in human FSHR are very rare. To identify the phenotypic consequences of such mutations in humans, mouse models harboring mutant versions of Fshr were first developed (26, 50). The rationale was that phenotypic analysis of these mice would provide information to look for similar phenotypes in patients carrying analogous mutations. Two independent Fshr point mutants D580H and D580Y were created and expressed using human AMH promoter to achieve ovary-specific expression in transgenic mice. Additionally, an Fshr BAC clone was engineered to carry the D580Y mutation and knocked-in to the endogenous Fshr locus in ES cells first and subsequently, the knock-in mutant mice were generated (26, 50).

Both D580H and D580Y mutants displayed increased basal receptor activity and they both demonstrated FSH binding. D580H mutant FSHR was neither ligand-dependent nor promiscuous toward LH/CG stimulation (26, 50). Granulosa cell-specific expression of mFshr D580H resulted in multiple ovarian abnormalities in transgenic female mice. Ovaries in the majority of transgenic females displayed hemorrhagic cysts, accelerated loss of immature follicles, increased granulosa cell proliferation and E2 biosynthesis, and luteinized but unruptured follicles, and teratomas (26, 50). A subset of the most severely affected transgenic females were infertile due to disrupted estrus cycles, and decreased gonadotropin, and increased prolactin levels. The increase in E2 and PRL levels led to secondary abnormalities including mammary gland enlargement and hyperplasia, pituitary adenoma formation, and defects in adrenal glands (26, 50). In contrast to phenotypic consequences of mFshr D580H expression, either transgenic or knock-in expression of mFshr D580Y mutant resulted in milder phenotypes, mostly hemorrhagic cysts in ovaries (26, 50). Thus, these GOF Fshr mutant mice resulted in distinct changes in ovarian function and proved valuable in the search of similar mutations in humans.

The most recent FSH receptor gain of function model challenged the traditional dogma that testosterone is essential for spermatogenesis (51). A mouse model was created that possessed a constitutively active mutant FSH receptor on an LH receptor null background (Fshr-CAM/Lhr−/−) (51). As LH regulates testosterone production via binding of LH receptors (LHRs) in Leydig cells, it was hypothesized that an Lhr knockout approach would eliminate testosterone action. However, testosterone production persisted in the initial Fshr-CAM/Lhr−/− male mice, as serum levels recovered to about 40% of wild-type concentrations. Therefore, the rescue of testicular size and spermatogenesis was probably due to normal testosterone actions (51). To eliminate any basal T activity, a treatment using the antiandrogen, flutamide, was employed. In WT control mice that had no Fshr-CAM, reduction in the size of testes and seminal vesicles was observed as well as arrested spermatogenesis. Interestingly, the Fshr-CAM/Lhr−/− males had only reduced seminal vesicle size after anti-androgen treatment, with no change in testicular size, and normal spermatogenesis (51). In addition to this, expression of androgen-dependent genes (Drd4, Rhox 5, Aqp8, and Eppin) were tested in the Fshr-CAM/Lhr−/− males. Anti-androgen treated WT males showed reduced expression profiles of these genes, whereas mutant Fshr-CAM/Lhr−/− showed no reduction (51). These results suggested that even in the absence of testosterone, constitutively active FSH receptor alone is able to maintain androgen-dependent gene expression as well as normal spermatogenesis and testicular development.

### CONCLUSIONS AND FUTURE DIRECTIONS

The first GOF mouse model for FSH was generated by our group more than 25 years ago (32). Since then investigators have used different promoters to achieve gonadotrope-specific as well as ectopic expression of the FSH ligand. Most commonly, human FSHB gene or individual subunit-encoding cDNAs (CGA or FSHB) were used in these experiments. More recently, BACS, or genes encoding porcine FSHβ subunit were also used to create transgenic pigs. GOF models for FSH action were generated using constitutively active FSH receptor–expressing mice and the reproductive consequences were studied either in these models directly or on an Lhr null genetic background (**Figure 1**). Combination of Fshb null mice and FSH GOF models resulted in genetic rescue that was used as an efficient in vivo functional assay for testing bioactivities of FSH and FSH analogs (**Figure 2**). A summary of the major phenotypes observed in each model is listed in **Table 1**.

Promoters used to generate FSH GOF models have also proved useful to express different reporters specifically in the gonadotrope cell lineage (62, 63). Recent advances in temporally regulated gene expression (64–66) will allow us in the future to tightly control FSH expression in desired tissues at desirable times across the life span of a mouse. Such refined genetic models will be useful to identify age-dependent gene/protein networks in FSH target tissues. These genetic models to conditionally "turning on" FSH in desired cells will also allow us to test if FSH receptor expression in non-gonadal cells has any physiological significance (55).

#### REFERENCES

#### AUTHOR CONTRIBUTIONS

RM wrote the first draft of the manuscript and created the Figures. CS generated the entire Table. TK edited the manuscript, Table, and Figures. He also wrote the abstract, about Glycosylation, and Conclusions, and Future Directions. He created and formatted the entire Bibliography.

#### FUNDING

Work reported in this Chapter was supported in part by NIH grants CA166557, AG029531, AG056046, HD081162, and The Makowski Endowment (to TK). RM is a Makowski Summer Scholar in the Kumar lab and is a graduate student supported by the Integrated Physiology program at the University of Colorado Anschutz Medical Campus, Aurora, CO, USA.

#### ACKNOWLEDGMENTS

We thank Dr. George Bousfield for critically reading the manuscript and many helpful suggestions.

spontaneous ovarian hyperstimulation syndrome. J Clini Endocrinol Metabol. (2004) 89:1255–8. doi: 10.1210/jc.2003-031910

transgenic mice. Transgenic Res. (2007) 16:65–75. doi: 10.1007/s11248-006- 9031-5

**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 McDonald, Sadler and Kumar. 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.

# Modulation of Gonadotropins Activity by Antibodies

Elodie Kara1 \*, Laurence Dupuy 1 , Céline Bouillon1,2,3,4,5,6, Sophie Casteret 1 and Marie-Christine Maurel 1

1 Igyxos SA, Nouzilly, France, 2 Service de Médecine et Biologie de la Reproduction, CHRU de Tours, Tours, France, 3 Biologie Intégrative de l'Ovaire, INRA, UMR85, Physiologie de la Reproduction et des Comportements, Nouzilly, France, 4 CNRS, UMR7247, Nouzilly, France, 5 Université François Rabelais, Tours, France, 6 IFCE, Nouzilly, France

Gonadotropins are essential for reproduction control in humans as well as in animals. They are widely used all over the world for ovarian stimulation in women, spermatogenesis stimulation in men, and ovulation induction and superovulation in animals. Despite the availability of many different preparations, all are made of the native hormones. Having different ligands with a wide activity range for a given receptor helps better understand its molecular and cellular signaling mechanisms as well as its physiological functions, and thus helps the development of more specific and adapted medicines. One way to control the gonadotropins' activity could be the use of modulating antibodies. Antibodies are powerful tools that were largely used to decipher gonadotropins' actions and they have shown their utility as therapeutics in several other indications such as cancer. In this review, we summarize the inhibitory and potentiating antibodies to gonadotropins, and their potential therapeutic applications.

Keywords: potentiating antibodies, inhibitory antibodies, follicle-stimulating hormone, luteinizing hormone,

## INTRODUCTION

chorionic gonadotropin, signaling

Gonadotropins, namely follicle-stimulating hormone (FSH), luteinizing hormone (LH), and chorionic gonadotropin (CG) are heterodimeric glycoproteins, constituted by an alpha- and a beta- subunit. The alpha subunit is common to all glycoprotein hormones, including thyroid stimulating hormone (TSH) (1). FSH and LH/CG receptors are G-protein coupled receptors (GPCR), mainly expressed in granulosa cells in female ovaries and in Sertoli cells in male testis for FSH receptor (FSH-R) (2), and in granulosa and theca cells in female and Leydig cells in male for LH receptor (LH-R) (3).

Because of their role in reproduction, gonadotropins are routinely used in fertility treatments in men and women for assisted reproductive technologies (ART) (4, 5). In women, it consists on daily injections of FSH or a mixture of FSH/LH, for 8–12 days, to grow and mature follicles. The final maturation is then completed with an injection of human CG (hCG) 36 h after the last injection of FSH. In men, FSH and hCG injections 2–3 times a week for several months are used to treat hypogonadotropic hypogonadism and induce spermatogenesis (6–9). Currently, the preparations used are either endogenous FSH extracted from post-menopausal women urine (human menopausal gonadotropins, hMG), highly purified urinary FSH, or recombinant FSH. The first recombinant versions of FSH (follitropin alpha and beta, corifollitropin) and all their

#### Edited by:

Livio Casarini, Università Degli Studi di Modena e Reggio Emilia, Italy

#### Reviewed by:

Marco Bonomi, Istituto Auxologico Italiano (IRCCS), Italy Luca De Toni, University of Padova, Italy

> \*Correspondence: Elodie Kara<EMAIL_ADDRESS>
#### Specialty section:

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

Received: 30 November 2018 Accepted: 10 January 2019 Published: 18 February 2019

#### Citation:

Kara E, Dupuy L, Bouillon C, Casteret S and Maurel M-C (2019) Modulation of Gonadotropins Activity by Antibodies. Front. Endocrinol. 10:15. doi: 10.3389/fendo.2019.00015

**90**

biosimilars were produced in Chinese hamster ovary (CHO) cells. The follitropin delta approved in Europe in 2015 (RekovelleTM ) and follitropin epsilon still under development are produced in human cell lines: PER.C6 for follitropin delta (10) and GlycoExpress for follitropin epsilon (11). For LH and hCG, recombinant versions produced in CHO cells are also available. In animals, equine CG (eCG), formerly named pregnant-mare serum gonadotropin (PMSG), is widely used to induce ovulation in small ruminants (12). Porcine pituitary extracts are used for current superovulation treatments in cattle (13), whereas ovulation in swine herds is induced with a mixture of hCG and eCG (14).

Despite all the preparations that are on the market, the only ligands available for gonadotropin receptors as therapeutics are native hormones. New biased ligands or ligands with different potencies and efficacies on gonadotropin receptors can help better understand receptor signaling, decipher the implication of the different signaling pathways in physiological and pathophysiological mechanisms, and finally bring to the market new molecules to improve ART treatments. In 2015 in USA, 182,111 ART procedures were performed leading to 59,334 live-birth deliveries (15). Among other strategies like small molecule ligands, one way to modulate the gonadotropins' activity could be the use of antibodies, targeting either directly the receptor itself, or its ligand to modulate hormone's activity.

Antibodies are useful tools that help to better understand gonadotropins' structure by epitope mapping (16–18) and their function by neutralizing the effect of endogenous gonadotropins (19–22). They also allow their quantification by the development of radioimmunoassays (RIA) and enzymelinked immunosorbent assays (ELISA) (23–27). Finally, antibodies permitted the development of specific purification methods for gonadotropins, making them safer to use as therapeutic agents (28–30).

The effect of antibodies, if any, is expected to be inhibitory on antigen activity by impairing its interaction with its receptor. Surprisingly, some of them were described as being able to increase the activity of their antigenic protein. Potentiating polyclonal antibodies directed against epidermal growth factor (EGF) and insulin were first described by Shechter et al. in 1979 (31, 32). A few years later, potentiating monoclonal antibodies (mAbs) were described for ovine (o) and human (h) growth hormones (GH) (33–35). When hypopituitary Snell dwarf mice were treated with hGH in complex with a mAb, the actions of hGH on growth and body composition were enhanced compared to animals treated with GH alone (34, 35). The same kind of antibodies were described for TSH: mAbs directed against TSH were able to enhance its biological activity in vivo in Snell dwarf mice (36), suggesting that glycoprotein hormones' activity may be modulated by mAbs. Anti-receptor antibodies with stimulating activities were also described for TSH receptor (37, 38).

In this review, we give a brief overview of antibodies modulating gonadotropins' activity, either positively or negatively.

### STRUCTURE AND FUNCTION OF GONADOTROPINS

Alpha- and beta-subunits of gonadotropins are non-covalently linked. The alpha-subunit is common to all glycoprotein hormones in a given species, and presents two major sites of Nglycosylation (1). The specificity of each hormone is conferred by the beta-subunit, that contains 2 N-glycosylation sites. hCG, eLH and eCG present a longer beta-subunit with an additional carboxy-terminal peptide (CTP) that is ∼30 amino-acids long and contains multiple O-glycosylation sites.

eCG originates from uterine endometrial cups and is extracted from pregnant mare serum (39, 40). eLH and eCG beta-subunits are encoded by a single gene (41) but they differ in glycosylation. They both exhibit N-glycans on alpha- and beta-subunits, and O-glycans on the carboxy-terminal peptide (CTP) constituted of the last 29 amino-acids of the beta-subunit (beta 121–149) (42). With a carbohydrate content higher than 40% (43) and N-glycan chains terminated by sialic acids, eCG is the most heavily glycosylated glycoprotein hormone and has a longer in vivo half-life than other gonadotropins (∼60 h) (44, 45). eCG binds to LH receptors in equine, but exerts FSH and LH actions in non-equine species by stimulating FSH and LH receptors respectively (46–51). Its dual FSH/LH activity and its longer half-life were the reasons why eCG was widely used since decades to induce ovulation in breeding animals, especially in goats and ewes for out-of-season breeding to allow artificial insemination.

hCG is mainly produced during pregnancy, by syncytiotrophoblast cells and a hyperglycosylated isoform is produced by cytotrophoblast cells. These two isoforms are implicated in implantation and early embryo development. Regular hCG for example promotes progesterone secretion by corpus lutea, angiogenesis of uterine vasculature, or growth, and differentiation of fetal organs, whereas hyperglycosylated hCG stimulates implantation by invasion of cytotrophoblast cells or stimulates growth of placenta (52). However, hCG can also be produced in non-pregnant women: it is produced at low levels by gonadotrope cells of the anterior pituitary, and seems to have an LH-like activity during menstrual cycle. Free beta-subunit of hCG is produced by multiple non-trophoblastic cancers. It is elevated in most cancers such as bladder, renal, prostate, gastrointestinal, lung, breast, neuroendocrine, gynecological, head and neck, and hematological cancers (53) and promotes their growth and malignancy by blocking apoptosis in cancer cells (52). hCG beta-subunit is thus used as a tumor biomarker usually associated with poor prognosis (53).

hCG, unlike eCG, has an LH like activity only and does not bind FSH-R (1, 54). CG and LH exert their effects via the same receptor, LH/CG receptor (LHCGR), that is coupled to Gs and Gq in granulosa cells and theca cells (55). However, the receptor is able to differentiate the binding and activity of these two hormones (56), and hLH and hCG differentially regulate signaling pathways (57, 58).

FSH plays an important role in reproduction. In females, it is implicated in follicular growth. FSH-R is expressed in granulosa cells and it is mainly coupled to Gs, which activates adenylyl cyclase and induces the secretion of cyclic adenosine monophosphate (cAMP), but it is also known to be coupled to Gq. In males, FSH regulates spermatogenesis. FSH-R is expressed in Sertoli cells and signals via Gs and Gi (59). In HEK 293 cells expressing FSH-R, FSH stimulates Gs/cAMP/PKA signaling pathway as in granulosa or Sertoli cells, but also signals via a beta-arrestin-dependent pathway, leading in both case to extracellular signal-regulated kinases (ERK) 1/2 phosphorylation but with different kinetics (60).

FSH-R is also expressed in other tissues than reproductive organs, such as osteoclasts (61) or adipose tissue (62), suggesting that FSH may play other physiological roles. In adipose tissue, FSH-R is coupled to Gi. An increase in Ca2+ influx induces the phosphorylation of cAMP-response-element-binding protein, which in turn activates an array of genes involved in fatty acids and triglycerides biosynthesis (63). FSH is thus implicated in lipid biosynthesis and its storage in adipocytes, which may contribute to age-related obesity and diseases due to high FSH levels in aging populations (62, 63). The first paper mentioning the role of FSH in bone mass regulation was published in 2006 (61). The authors proposed a mechanism where FSH was able to increase osteoclasts formation and function via a Gi2acoupled FSH-R expressed in these cells and their precursors (64), suggesting that high circulating FSH levels were responsible for post-menopausal osteoporosis.

### ANTIBODIES MODULATING THE ACTIVITY OF CHORIONIC GONADOTROPINS

#### Antibodies Modulating the Activity of eCG

Most of the antibodies against eCG were developed for structural analysis purposes. Maurel et al. identified an antibody able to inhibit eCG binding to LH and FSH receptors (65). Chopineau et al. analyzed the affinity and the specificity of 14 mAbs directed against eCG (66). The aim of this study was to analyze the epitopic sites of eCG and permitted to draw a topographic map of antigenic and functional sites of this hormone. The affinity of antibodies for eCG was ranging between 10−7 and 10−11 M. Ten of them were alpha-subunit specific because they recognized both native eCG and free alpha-subunit, but not free beta-subunit. One antibody exhibited a higher affinity for alpha-subunit than for the native eCG, and 13 mAbs exhibited a better affinity for the dimer than for the free subunits. The effect of these mAbs was then tested on FSH and LH bioactivities of eCG in in vitro bioassays. One beta-subunit specific, one alpha-subunit specific and one native alpha/beta dimer specific antibodies did not show any effect on FSH and LH bioactivities. Nine alpha-subunit specific antibodies either weakly or strongly inhibited both bioactivities. Finally, two mAbs were potentiating FSH bioactivity of eCG: one was beta-subunit specific and the other was native dimeric eCG specific. They did not inhibit eCG binding to LH-R. The degree of inhibition of inhibitory antibodies was correlated with their affinity for eCG, but it wasn't for the two antibodies potentiating FSH bioactivity of eCG. These data suggest that the inhibitory or potentiating effects of mAbs on eCG bioactivities neither depend on their specificity nor their affinity. Moreover, the two antibodies potentiating the FSH bioactivity of eCG were either not affecting or inhibiting weakly the LH bioactivity of eCG, demonstrating that the effect on both bioactivities can be opposite (inhibitory or potentiating), with different degrees of activity (none or weak) on hormone bioactivities, highlighting the potential multiple mode of action of antibodies.

The high carbohydrate content makes also eCG highly immunogenic. Repeated injections of eCG for ovulation induction decrease the fertility of goats from 60 to 40% (12, 67). Roy et al. detected an immune response in animals treated with eCG for ovulation induction (68, 69), and demonstrated that the secreted antibodies from a previous treatment were inhibiting the action of eCG injected for the following treatment. The goats with high antibody levels at the time of eCG administration did have a much lower kidding rate (41%) than the other females (66%). This immune response was thus altering the fertility of these animals by delaying both the onset of estrus and the preovulatory LH surge. However, a deeper analysis revealed that some of the goats secreting high levels of anti-eCG antibodies did have a fertility beyond expected, ovulating and getting pregnant after each treatment, even after four treatments. The antibodies from the plasma of hypo-fertile or hyper-fertile goats secreting high levels of antibodies were purified and the IgG fractions were analyzed for their effect on FSH bioactivity in Y1 cell line derived from a mouse adrenal cortex tumor stably expressing human FSH-R, and for their effect on LH bioactivity in rat Leydig cells. The plasmas and the corresponding IgG fractions from different eCG treated goats exhibited either inhibitory, enhancing or no effects on FSH activity of eCG by modulating progesterone secretion by Y1 cells, and on LH activity of eCG by modulating testosterone secretion in Leydig cells (70). As expected, antibodies were mainly recognizing carbohydrate chains of eCG. Twenty-one either inhibitory, potentiating or neutral antibodies for LH and/or FSH bioactivities of eCG were analyzed. None of the antibodies cross-reacted with totally deglycosylated eCG or alpha-subunit of eCG. Interestingly, the inhibitory or stimulatory effects of these antibodies were not correlated with the affinity of the tested antibody for eCG (70). All together, these data demonstrated that gonadotropins' in vivo bioactivity and animals' fertility can be modulated with antibodies, especially since these antibodies are naturally occurring.

Later, Wehbi et al. (71) analyzed the effect of eCG in complex with three of these antibodies on FSH receptor signaling pathways. The eCG/antibody complexes effect was tested on HEK 293 cells expressing mouse FSH receptor and on granulosa cells punctured from slaughterhouse cows for their effect on cAMP production. The tested antibodies were differently modulating cAMP production: two of them were potentiating and one was slightly inhibiting eCG effect. In contrast, all three antibodies were enhancing ERK1/2 phosphorylation in HEK 293 cells expressing mouse FSH-R. Deeper analysis revealed that the antibodies were potentiating eCG signaling preferentially via beta-arrestin pathway, via cAMP/PKA pathway or via both. An antibody complexed to eCG was thus able to change the full agonist effect of eCG into a biased agonist effect, modulating differentially the balance between the signaling pathways induced by this hormone. This paper was also the demonstration that these antibodies were achieving the same in vivo effect in goat (i.e., high prolificity) via different signaling pathways. That was the first report of biased agonism at FSH-R and the authors suggested that such antibodies could help optimize glycoprotein hormones' bioactivities and thus the development of new therapies.

At the very beginning of infertility treatments, eCG was also used to treat women. The first successful treatment with eCG was described in 1945. Although its use lasted more than 30 years, scientists realized very early that women treated with eCG extracts, like animals, did produce "antigonadotrophic substances" which neutralize hormone's effect over time and after repeated injections. The immune response induced by eCG and the arrival of less immunogenic pituitary gonadotropin extracts led to the market withdrawal of eCG [reviewed in (4)].

### Antibodies Modulating the Activity of Human Chorionic Gonadotropin (hCG)

As for other gonadotropins, anti-hCG antibodies were essentially developed for epitope mapping and variant specific mAbs permitted the development of immunoassays, leading in fine to pregnancy tests (72–74).

Naturally occurring endogenous antibodies were also reported: patients treated with exogenous gonadotropins can develop anti-hCG antibodies that impair fertility. They were detected in young men with hypogonadotropic hypogonadism treated with hCG (75, 76). These antibodies, detected in a 15 year-old patient following a secondary resistance during a third treatment to hCG, were low affinity but high binding capacity antibodies (76). A few years later, seven additional young men with hypogonadotropic hypogonadism, aged between 11 and 18 years, and resistant to classical hCG regimen were tested for the presence of anti-hCG antibodies. Four of them showed antibodies, but the neutralizing effect of hCG could be counter passed by increasing the doses of hCG used (75). The same kind of antibodies were described in women (77, 78). Immune response against hCG impairs fertility of women and induces pregnancy loss within the first trimester of pregnancy. To thwart this negative effect, Muller and collaborators described a treatment that was successful in three women positive for hCG antibodies. This treatment combined membrane plasmapheresis, prednisolone, and intravenous immunoglobulin therapy (78).

Anti-hCG auto-antibodies were also detected in sera of men and women that never received any injection of exogenous hormone (79). These antibodies were low affinity and did not interfere with hormone activity. However, few years later, antibodies with high affinity and the capacity to neutralize hCG and LH activities were detected in a patient with a history of spontaneous abortion, that was never exposed to exogenous hormone therapy (80).

### ANTIBODIES MODULATING THE ACTIVITY OF FSH OR LH

#### FSH and/or LH Neutralization With Antibodies

Several inhibiting antibodies were described for FSH and most of them were used to better understand its physiological functions in vivo. Antibodies permit to block reversibly the action of one or several hormones, at a precise time of the lifespan of the studied model, rather than suppressing a whole organ like in hypophysectomy or a gene like in transgenic animals. For example, for female studies, in 1969, Goldman and Mahesh used an anti-sera obtained by rabbit immunization with ovine LH, that neutralized FSH as well as LH, to study the role of these hormones in ovulation (81). In 1970, the same group published data on the effect of the same anti-sera on neonatal rats (82). In 1971, Eshkol and Lunenfeld used the strategy of neutralization with antibodies to demonstrate the crucial role of FSH and LH in ovarian development during the first 2 weeks of life in rodents. FSH was found to be responsible for the stimulation of granulosa cell proliferation, organization and structure. FSH plus LH initiated secretory activity of granulosa cells, increased intrafollicular spaces, antrum formation, enrichment and maintenance of the theca layer, and development of the vascular system (19). At the same time, it was shown that LH anti-sera could block ovulation in rat, but not FSH anti-sera (83–86). Schwartz et al. suggested that FSH neutralization during estrus cycle could have a deleterious effect on follicles destined to grow and ovulate in following cycles (86). Several other studies have confirmed the role of LH as the indispensable trigger of ovulation, whereas FSH was required for the recruitment of antral follicles at the start of a new cycle in rat and hamster (87–91). The neutralization of FSH or LH with antibodies also permitted to study the role of these hormones in the synthesis of estrogen (92), on ornithine decarboxylase activity (93) and on gonadotropin surge-inhibiting factor/attenuating factor bioactivity (20) in rat and/or hamster. In monkey, the antibody neutralization of FSH highlighted the importance of FSH during follicular growth and showed that the mature follicle becomes less sensitive to FSH about 48 h before ovulation (94).

For male studies, Wickings and Nieschlag actively immunized Macaca mulatta against ovine FSH and observed a spermatogenesis suppression over a period of 2 years, confirming the importance of FSH for spermatogenesis (21). The importance of FSH in spermatogenesis was further confirmed by active immunization of Macaca radiata (95). In rat, even if the first studies obtained with immunoneutralization of FSH were controversial on the role of FSH in spermatogenesis (96, 97), later works with either passive (98) or active (22) immunization against FSH confirmed its crucial role in the maintenance of spermatogenesis. For active immunization, peptides from region 19–36 of rat FSH beta-subunit were used (22). Altogether, these results suggest that immunoneutralization of FSH could be used as a contraception in men by suppressing spermatogenesis. However, Nieschlag recommended to abandon the approach of immunization as a contraception because a complete suppression of spermatogenesis was not achieved even after 4.5 years of immunization (99). Nevertheless, Moudgal and collaborators carried out a pilot study in 1997 where five male volunteers were immunized with ovine FSH isolated from sheep pituitaries (100). The subjects only responded to the first two immunizations (day 1 and 20), and did not respond to the boosters given at day 40 and 70. Ovine FSH vaccination generated antibodies against human FSH, but only 25–45% of the antibodies generated against ovine FSH were able to bind human FSH and the sperm count reduction was around 30–64%, which is not enough to consider this method as a contraception.

Anti-FSH antibodies were also detected in women. Two types of antibodies have been identified: naturally occurring anti-FSH antibodies (101, 102) and anti-FSH antibodies resulting from exogenous gonadotropins (103–106). First, Haller et al. found naturally occurring anti-FSH antibodies in patients with endometriosis or polycystic ovary syndrome (PCOS) and none of these patients had undergone ovarian stimulation for IVF. They also detected anti-FSH antibodies in healthy non-pregnant women but at lower rates than for patients with endometriosis or PCOS (101). Likewise, Shatavi et al. found spontaneous antigonadotropin antibodies in 27% of patients with unexplained infertility and never treated with gonadotropins, but only in 8% of women in the general population (102). To explain the presence of such spontaneous antibodies, it was supposed that an alteration of the immune system might be necessary and that the antigen responsible for their production could be either the circulating FSH from the female organism or the FSH in seminal fluid that may upregulate the anti-FSH immune response in females (101). In in vitro fertilization (IVF) patients, Haller et al. demonstrated that anti-FSH antibodies increase in infertile women with common autoantibodies (against nuclear antigens, smooth muscle, gastric parietal cells, b2-glycoprotein I, cardiolipin, and thyroid peroxidase) and with a history of IVF stimulation (103, 104). Shatavi et al. also found that anti-FSH antibodies were more recurrent in infertile patients with history of gonadotropin treatment than in infertile patients never treated with exogenous FSH or in women in the general population (102). Anti-FSH antibodies could also be associated with anti-ovarian antibodies (AOA) in patients with history of gonadotropin treatment (102, 107). The association of anti-FSH antibodies and AOA was also detected in infertile patients never treated by FSH and in women in the general population but at a lower frequency (102). Other studies have investigated the consequences of the presence of anti-FSH antibodies on the results of controlled ovarian stimulation (COS). Some studies found that anti-FSH antibodies were associated with poor ovarian response to IVF stimulation (104, 105). Thus, anti-FSH antibodies might have an inhibitory effect on FSH by preventing the binding of the hormone to its receptor or by trapping FSH in immune complexes (101, 107). On the contrary, Reznik et al. identified a higher proportion of anti-FSH antibodies in patients with a good response to COS compared to patients with a poor response (106). Antibodies produced in patients with a good response might have either no effect or a potentiating effect on the action of FSH. However, in humans, no in vitro study of the inhibitory or potentiating action of anti-FSH antibodies on FSH receptor signaling has been published yet. In human FSH, one of the major epitopes seems to be the 78–93 amino acid sequence of the β-chain (101, 107). This region contains a loop called cysteine noose which plays a role in the specificity of FSH receptor binding (101). Therefore, it was supposed that the binding of the hormone to its receptor could be modulated by antibodies directed against this region (101). To explain why some infertile patients develop antigonadotropin antibodies, some studies focused on the Major Histocompatibility Complex (MHC) Class II (103). The role of the MHC Class II is to present exogenous proteins to immune cells, which leads to a humoral immune response. In IVF patients, only anti-FSH IgA were associated with HLA-DQB1∗ 03 (103). However, the development of anti-FSH antibody response to exogenous FSH treatment remains controversial. Indeed, a recent study conducted in healthy oocyte donors and infertile women has concluded that repeated gonadotropin treatments for IVF do not induce an immune response to FSH (108).

#### Antibodies Potentiating the Activity of FSH

The first anti-FSH mAbs described were directed against human FSH (16). Their binding specificities were well-characterized, but their effect on FSH activity was not investigated. The second anti-FSH mAb described in the literature was directed against bovine FSH (29) and was beta-subunit specific. It did not cross react with ovine or porcine FSH, indicating that it recognizes an area of bovine FSH not homologous to ovine or porcine hormones (29). Glencross et al. tested this antibody later on for its effect on bovine FSH bioactivity in hypopituitary Snell dwarf mice (109). The mAb injected in complex with FSH increased uterine weight whereas FSH alone or the mAb alone at the same concentrations did not have any effect, showing for the first time a potentiating effect with an anti-FSH mAb.

Holder's group described anti-sera directed against peptides derived from the beta-subunit of bovine FSH (110). When injected to hypopituitary Snell dwarf mice concomitantly with ovine FSH, these anti-sera produced by sheep immunized with peptides corresponding to 31–45 and 38–49 amino-acid regions of bovine FSH beta-subunit were able to enhance FSH activity, as measured by an increase in uterine and ovarian weight, and an increase in the percentage of keratinized cells in vaginal smears. The authors hypothesized that the administration of anti-peptide anti-sera precomplexed with FSH or active immunization of breeding animals with these peptides should result in a superovulatory response, and that the potentiating anti-sera strategy, in the case of FSH, could be used for several clinical situations, such as treatment of ovarian disorders related to low FSH secretion, induction of estrus and treatment leading to increase spermatogenesis.

### ANTI-GONADOTROPIN ANTIBODIES AS THERAPEUTIC AGENTS

### Therapeutic Antibodies in General

MAbs, initially developed for scientific purposes, now take part of the human therapeutic arsenal. Over the last three decades, they have grown to become more than 55% of the overall biotherapeutic market of the drug industry sales (111).

The regulatory story of therapeutic mAbs started in 1986 with the first FDA-approved therapeutic mAb, the murine mAb Orthoclone OKT3 R (Muromonab CD3) indicated for the prevention of kidney transplant rejection. Unfortunately, the development of murine mAbs has been hindered because of the risk of immunogenicity in humans due to their murine elements. Replacing the constant region of murine mAb by human sequences resulted in the generation of the chimeric antibodies (∼30% murine content). The first-approved one, Rituxan R (Rituximab) in 1997, was used for the treatment of low grade B cell lymphoma. To overcome immunogenicity risk even further, new technologies for the generation of predominately or entirely human origin mAbs were developed. The humanized mAbs (5–10% murine content) are tailored by replacing all sequences by human except antigen binding complementary determining regions, which were derived from the mouse. The humanization technology developed by Sir Winter lead to the first humanized therapeutic antibody CAMPATH-1H R (Alemtuzumab, approved in 2001). Sir Winter was awarded the 2018 Nobel Prize in Chemistry along with George Smith for this technology and the fully humanization using phage display. Fully human mAbs (last generation) were developed by replacing the whole of the rodent sequences by human sequences. Humira R (Adalimumab) is the first fully human antibody approved in 2004, for the treatment of rheumatoid arthritis. Thanks to these new technologies, the rate of approval and mAbs available on the market for the treatment of various diseases has increased dramatically. In 2017, the FDA and the European Medicines Agency broke the record and approved 10 new therapeutic antibodies (112). Currently, more than 70 mAb products are available on the market, most of them being humanized (32%) or fully human (54%) (113).

To date, approved mAbs are from different isotypes, but the preferred mAbs in clinical use are of the IgG1 isotype (80%) (114). Additionally, there are five monovalent antibodyfragments on the market, four antigen-binding fragments (Fab) and 1 single chain variable fragments (scFv) (114). More sophisticated forms have been engineered, such as Fc-modification, IgG2/IgG4 hybrid Fc, glyco-engineered mAbs, bispecifics, or antibody-drug conjugates. These types of sophisticated mAbs reach more and more clinical trial studies (114, 115). According to Zhou and Mark (116), common mechanism of action proposed for mAb drugs include: (i) disruption of ligand–receptor interaction; (ii) target cell elimination via antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP); (iii) engagement of cytotoxic T cell by bispecific Abs; (iv) receptor downregulation by enhanced internalization and degradation; and (v) targeted drug delivery.

The indication of treatment for ∼80% of the therapeutic mAb drugs could be classified into oncology and immune diseases. The last ∼20% are used for the treatment of infection and cardiovascular diseases, orthopedic, eye and rare diseases (113). Despite the high treatment cost, the success of therapeutic mAb has recently reached the veterinary health with the launch in the European Union, in 2017, of Cytopoint R (Lokivetmab), a treatment for atopic dermatitis in dogs. Notwithstanding all the therapeutic mAbs developed to date, so far, none of them have succeeded in the field of fertility, and none of them are potentiating antibodies.

### Therapeutics Involving Anti-gonadotropin Antibodies

#### Neutra-PMSG®, an Antibody Against eCG/PMSG

The unique antibody commercialized until now in the field of animal fertility is Neutra-PMSG R . This anti-eCG mAb is alpha-subunit specific and inhibits both LH bioactivity on small bovine luteal cells and FSH bioactivity on granulosa cells of bovine follicles (66). It was developed and marketed for cattle to limit the adverse effects of PMSG to improve the embryo production after superovulation treatment (117, 118). Due to a long half-life, PMSG had the disadvantage to cause a prolonged stimulation of follicular growth after preovulatory LH peak, inducing a poor response to superovulation treatment for cattle (117, 119, 120), sheep (121), and goat (122). Neutra-PMSG R injected 1–2 days after PMSG in superovulation treatment neutralized the adverse effects of PMSG by reducing its half-life in systemic circulation, improving embryo production. This mAb did not recognize endogenous gonadotropins in treated animals. Therefore, Neutra-PMSG R was highly specific for PMSG (117). Currently, most cows are superovulated using pituitary extracts containing FSH and LH (13), mainly because the pharmaceutical company (Intervet, The Netherlands) that developed the Neutra-PMSG R mAb stopped production in the 1990s.

#### Active Immunization Against hCG

A vaccination against hCG was also considered in the 1970s as a contraception method to avoid pregnancy in women (123, 124). The aim is to induce the secretion of antibodies that will bind hCG and block its activity, thus impeding pregnancy. Because an immunization with the whole dimeric hCG (alpha+betasubunits) was raising antibodies not only against hCG but also against human LH (125), a special preparation of betahCG was made by processing it against heterologous anti-LH immunosorbents (124) and conjugating it to purified tetanus toxoid as an immunogenic carrier (123). This processed molecule was able to induce an immune response with antibodies specific to hCG. The antibodies produced were able to abrogate the binding of hCG to its receptor and its biological effects in in vivo bioassays. Moreover, the antibody titer declines over time, indicating that the vaccination is reversible (126). This vaccination system went through phase 1 clinical trials in several countries (India, Finland, Sweden, Chile and Brazil) under the International Committee on Contraception Research of Population Council. A slightly different preparation consisting of a dimer of hCG beta-subunit non-covalently associated with ovine LH alpha-subunit conjugated to tetanus and diphtheria toxoids (127) underwent phase 1 and phase 2 clinical trials in several centers in India. Eighty percent of treated patients generated sufficient antibody titer (>50 ng/ml) to be protected against pregnancy, and only one pregnancy was recorded over 1,124 cycles in fertile and sexually active women with an antibody titer higher than 50 ng/ml. After 12 years of inactivity on this project, Talwar and his collaborators are now working on a genetically engineered recombinant vaccine that is expected to go through clinical development in the next few years (128).

A similar approach was also tested for colorectal and pancreatic cancer treatments. In 2000, AVI BioPharma collaborated with SuperGen for the clinical development and marketing of Avicine, a synthetic vaccine constituted of the C-terminal peptide of hCG (CTP-37) conjugated to diphtheria toxoid. The vaccine went through several clinical trials until phase 3 pivotal study. In phase 2 efficacy study for colorectal cancer, 73% of treated patients developed an immune response against hCG and this response was associated with an improved median survival time (129, 130). As far as we know, the product has not reached the market yet. Recently, another group proposed another vaccine, where one residue in the amino-acid sequence of hCG beta-subunit is substituted (hCGβR68E) to eliminate cross reactivity with LH and conjugated to heat shock protein (Hsp70) as carrier to increase its immunogenicity (131). This vaccine has not been tested in humans yet.

#### Other Potential Therapeutic Antibodies Antibodies Targeting FSH

To consolidate the assumption that high circulating FSH levels were responsible for post-menopausal osteoporosis, Zhu et al. showed that blocking FSH action attenuates bone loss in ovariectomized mice via two mechanisms: by inhibiting bone resorption and by stimulating bone formation (132). To do so, they used a mouse polyclonal antibody targeting a 13-amino acid sequence (LVYKDPARPNTQK) of mouse FSH beta-subunit that is a receptor-binding domain.

Liu et al. hypothesized that a pharmacological blockade of FSH action could also reduce body fat mass. In fact, they have shown that the same polyclonal antibody targeting the receptorbinding domain of FSH beta-subunit, injected daily for 8 weeks in mice, prevented the gain of body fat induced by the diet in male and female mice (133).  For the purpose of potential therapeutic application in human, this team has developed a mAb targeting the same epitope in human FSH beta-subunit (LVYKDPARPKIQK) that had the same effects on body fat and thermogenesis on the mouse as the mouse polyclonal antibody directed against a sequence that is 2 amino-acids different (LVYKDPARPNTQK) (135, 136). The modulation of FSH activity by anti-FSH antibodies may be considered as therapeutic means to reduce the risk of obesity in elderly people with high levels of FSH (133). It was thus proposed that the same antibody could both inhibit bone loss and body fat gain (133, 136). However, while some of the other studies published on the subject supported the role of FSH in bone mass regulation (137–141), some others were contradictory (142, 143). Moreover, clinical studies on human subjects reported that FSH suppression with GnRH agonist had no effect on bone resorption in women (144). In men, this suppression either increased bone loss (145), or had no effect when a testosterone supplementation was given (146). Furthermore, body fat mass is also increased in men treated with GnRH analog (147). Altogether, these data suggest that further investigations are needed to better understand the mechanisms underlying the role of FSH in bone mass and body fat regulation before a therapeutic approach can be envisaged (148). A therapeutic antibody to prevent osteoporosis is on the market since 2010. Receptor activator of nuclear factor-kB (RANK) ligand (RANKL) is a cytokine necessary for the development and the activity of osteoclasts. A fully human antibody, denosumab (Prolia R , Amgen) prevents RANKL binding to its receptor RANK. Denosumab, when given subcutaneously twice yearly for 36 months, reduces the risk of vertebral, non-vertebral, and hip fractures in women with osteoporosis (149), demonstrating that the strategy of therapeutic antibody can be used in this indication.

#### Antibodies Targeting hCG

Antibodies able to inhibit hCG activity were first described in 1980 (150). They were specific of the beta-subunit of hCG and did not cross react at all with other gonadotropins. Recently, one of these antibodies (mAb PIPP) was expressed recombinantly in tobacco leaves in different formats (scFv, diabody and entire antibody) and tested, after their extraction and purification, for their efficacy to neutralize hCG. The three formats of the same antibody were able to inhibit in vitro testosterone production induced by hCG in Leydig cells. In vivo, the entire mAb was able to block uterine weight gain in mouse model (151). These antibodies were envisaged as a contraception method by passive immunization in women, and were considered as a better method than active immunization where the response may be variable between patients, and a sufficient titer determined as the protective level of antibody was observed in 80% of the patients only. These anti-hCG antibodies have not entered a clinical development so far.

The same antibody was used for the development of an immunotoxin targeting hCG-expressing cancer cells (152). The VH and VL domains of the full antibody were linked together to form the scFv fragment (scFv PIPP). This scFv's gene was then fused with a gene expressing Pseudomonas exotoxin (PE38) and expressed in Escherichia coli as a recombinant protein (scPiPP-PE38). Once purified and tested on cancer cells, the immunotoxin showed 90% killing of hCG beta expressing histiocytic lymphoma, T-lymphoblastic leukemia, and lung carcinoma cells in vitro. However, further studies are needed to evaluate the potential of scPiPP-PE38 as a therapeutic agent for management of cancer cells expressing hCG or its subunits.

### CONCLUSION

Many different antibodies against gonadotropins were developed and have proven to be very useful tools for many applications. They can also be naturally secreted due to a humoral immune response to endogenous or exogenous gonadotropins. With the same structure, immunoglobulins can have inhibitory or potentiating effects depending on their paratope defined by CDRs and their epitope (binding site) on the antigen. In the case of eCG, its naturally occurring potentiating antibodies have demonstrated that a differential activation of signaling pathways of FSH-R could lead to the same in vivo effect, i.e., high prolificity in goats (71). The development of antibodies with a range of modulating effects on the potency and the efficacy of FSH on its signaling pathways could help deciphering the importance of each pathway for FSH roles in reproduction, bone mass and body fat regulation. Moreover, these antibodies can represent potential therapeutics, targeting one pathophysiological or physiological condition in particular. Several applications for anti-gonadotropin antibodies have already been proposed and are under exploration, like osteoporosis, obesity, contraception, or cancer. All of these indications require inhibition of gonadotropins' action. On the other hand, in small ruminants, the naturally occurring

#### REFERENCES

anti-eCG potentiating antibodies induced a better fertility and prolificity demonstrating that it is possible to improve fertility by potentiating gonadotropins' activity during several estrus cycles, without any side effects. All these studies demonstrated that it is possible to target each gonadotropin very specifically despite their similarities.

To conclude, the development of antibodies modulating gonadotropins' activity could not only provide new tools to better understand their roles in different physiological processes, but could also bring to the market innovative drugs. Taking into account the state of the art and the clinical development time, there is a long way to go until a therapeutic antibody targeting a gonadotropin can reach the market.

#### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

hormone, FSH, and chorionic gonadotropin) in male and female rats. Endocrinology (1984) 115:1821–7. doi: 10.1210/endo-115-5-1821

on alpha- and beta-subunits. Biochim Biophys Acta (1992) 1159:74–80. doi: 10.1016/0167-4838(92)90077-Q

women with osteoporosis. N Engl J Med. (2009) 361:756–65. doi: 10.1056/NEJMoa0809493

**Conflict of Interest Statement:** EK, LD, SC, and M-CM were employed by Igyxos SA.

The remaining 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 Kara, Dupuy, Bouillon, Casteret and Maurel. This is an openaccess 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.

# Glycosylation Pattern and in vitro Bioactivity of Reference Follitropin alfa and Biosimilars

Laura Riccetti 1 , Samantha Sperduti 1 , Clara Lazzaretti 1,2, Danièle Klett 3 , Francesco De Pascali 3 , Elia Paradiso1,2, Silvia Limoncella1 , Francesco Potì 4 , Simonetta Tagliavini 5 , Tommaso Trenti 5 , Eugenio Galano6 , Angelo Palmese6 , Abhijeet Satwekar 6 , Jessica Daolio7 , Alessia Nicoli 7 , Maria Teresa Villani 7 , Lorenzo Aguzzoli 7 , Eric Reiter 3 , Manuela Simoni 1,3,8,9 and Livio Casarini 1,8 \*

1 Unit of Endocrinology, Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy, 2 International PhD School in Clinical and Experimental Medicine, University of Modena and Reggio Emilia, Modena, Italy, 3 PRC, INRA, CNRS, IFCE, Université de Tours, Nouzilly, France, 4 Unit of Neurosciences, Department of Medicine and Surgery, University of Parma, Parma, Italy, 5 Department of Laboratory Medicine and Pathological Anatomy, Azienda USL, NOCSAE, Modena, Italy, 6 Analytical Development Biotech Products, Merck Serono S.p.A. (an affiliate of Merck KGaA, Darmstadt, Germany), Rome, Italy, 7 Azienda Unità Sanitaria Locale—IRCCS di Reggio Emilia, Department of Obstetrics and Gynaecology, Fertility Center, ASMN, Reggio Emilia, Italy, 8 Center for Genomic Research, University of Modena and Reggio Emilia, Modena, Italy, 9 Unit of Endocrinology, Department of Medical Specialties, Azienda Ospedaliero-Universitaria, Modena, Italy

#### Edited by:

David Gregory Mottershead, Keele University, United Kingdom

#### Reviewed by:

George Russell Bousfield, Wichita State University, United States Nebojsa Andric, University of Novi Sad, Serbia

> \*Correspondence: Livio Casarini<EMAIL_ADDRESS>
#### Specialty section:

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

> Received: 07 May 2019 Accepted: 11 July 2019 Published: 24 July 2019

#### Citation:

Riccetti L, Sperduti S, Lazzaretti C, Klett D, De Pascali F, Paradiso E, Limoncella S, Potì F, Tagliavini S, Trenti T, Galano E, Palmese A, Satwekar A, Daolio J, Nicoli A, Villani MT, Aguzzoli L, Reiter E, Simoni M and Casarini L (2019) Glycosylation Pattern and in vitro Bioactivity of Reference Follitropin alfa and Biosimilars. Front. Endocrinol. 10:503. doi: 10.3389/fendo.2019.00503 Recombinant follicle-stimulating hormone (FSH) (follitropin alfa) and biosimilar preparations are available for clinical use. They have specific FSH activity and a unique glycosylation profile dependent on source cells. The aim of the study is to compare the originator (reference) follitropin alfa (Gonal-f®)- with biosimilar preparations (Bemfola® and Ovaleap®)-induced cellular responses in vitro. Gonadotropin N-glycosylation profiles were analyzed by ELISA lectin assay, revealing preparation specific-patterns of glycan species (Kruskal-Wallis test; p < 0.05, n = 6) and by glycotope mapping. Increasing concentrations of Gonal-f® or biosimilar (1 × 10−3 -1 × 103 ng/ml) were used for treating human primary granulosa lutein cells (hGLC) and FSH receptor (FSHR)-transfected HEK293 cells in vitro. Intracellular cAMP production, Ca2+ increase and β-arrestin 2 recruitment were evaluated by BRET, CREB, and ERK1/2 phosphorylation by Western blotting. 12-h gene expression, and 8- and 24-h progesterone and estradiol synthesis were measured by real-time PCR and immunoassay, respectively. We found preparation-specific glycosylation patterns by lectin assay (Kruskal-Wallis test; p < 0.001; n = 6), and similar cAMP production and β-arrestin 2 recruitment in FSHR-transfected HEK293 cells (cAMP EC50 range = 12 ± 0.9–24 ± 1.7 ng/ml; β-arrestin 2 EC50 range = 140 ± 14.1–313 ± 18.7 ng/ml; Kruskal-Wallis test; p ≥ 0.05; n = 4). Kinetics analysis revealed that intracellular Ca2+ increased upon cell treatment by 4µg/ml Gonal-f®, while equal concentrations of biosimilars failed to induced a response (Kruskal-Wallis test; p < 0.05; n = 3). All preparations induced both 8 and 24 h-progesterone and estradiol synthesis in hGLC, while no different EC50s were demonstrated (Kruskal-Wallis test; p > 0.05; n = 5). Apart from preparation-specific intracellular Ca2+ increases achieved at supra-physiological hormone doses, all compounds induced similar intracellular responses and steroidogenesis, reflecting similar bioactivity, and overall structural homogeneity.

Keywords: FSH, biosimilar, gonal-F, bemfola, ovaleap, glycosylation, assisted reproduction (ART)

#### INTRODUCTION

Follicle-stimulating hormone (FSH) is a heterodimeric glycoprotein hormone produced by the pituitary and acting on the gonads (1). In fertile women, FSH controls reproduction supporting ovarian granulosa cell proliferation and follicular growth by binding to its G protein-coupled receptor (FSHR) (2).

FSH shares a 92-amino acid residue α subunit with other glycoprotein hormones and has a 111-amino acid residue, hormone-specific β subunit (3). Two N-linked heterogeneous oligosaccharide populations are bound to each protein backbone subunit and are involved in hormone folding and half-life, receptor binding, and activation (4, 5). After gonadotropin binding, FSHR conformation rearrangements occur, triggering intracellular signal transduction. Gαs protein signaling leads to adenylyl cyclase stimulation and cyclic-AMP (cAMP)/protein kinase A (PKA)-pathway activation (6, 7), resulting in cAMP-response element binding protein (CREB) (8, 9) and extracellular-regulated kinase 1/2 (ERK1/2) (10) phosphorylation. These phospho-proteins are key players modulating steroidogenesis, proliferation and survival/apoptosis (8, 11), all molecular events underlying reproductive functions (12). Upon ligand binding, FSHR recruits other heterotrimeric Gα proteins, including Gαq and Gαi (13–16), as well as other interactors (17), linking FSH action to multiple intracellular signaling pathways, such as the rapidly-activated, phospholipase C-dependent (18), cytosolic calcium cation (Ca2+) release (19). FSHR internalization and recycling is mediated by βarrestin 1 and 2, which triggers G protein-independent ERK1/2 signaling (20, 21).

FSH exists in a number of isoforms differing in content and composition of oligosaccharides attached to the protein backbone (22). FSH glycoforms were proposed as biased receptor ligands (5, 23, 24) due to isoform-specific contact with FSHR (25) and intracellular signaling (26). Glycosylation is a post-translational process influencing the isoelectric point (pI) and half-life of the gonadotropin (27). In women, more glycosylated and acidic FSH isoforms, mainly due to sialylation, exhibit a prolonged in vivo half-life due to reduced kidney clearance and are secreted mostly during the early and midfollicular phase, compared to FSH basic glycoforms, which are predominant before ovulation (28, 29). Highly acidic FSH isoforms are produced more after the menopause than during the fertile lifespan (30), suggesting that glycoform composition of circulating hormones is dynamic and might have a physiological role.

Several formulations of exogenous FSH may be used in assisted reproductive technologies (ART) to induce multiple follicle development. Both urinary and recombinant FSH and other gonadotropin preparations are commercially available, as well as follitropin alfa biosimilar drugs, which are recombinant compounds similar to the originator (31–33). Previous studies attempted to address effects of these preparations on ART outcomes, given their different glycosylation states featured as post-translational modifications by the cellular source and/or purification processes (31, 34, 35). In fact, previous analyses by mass spectrometry found preparation-specific pattern of glycans bound to the FSH β-subunits (36, 37).

In this study, the biochemical composition and hormoneinduced cell response of the originator follitropin alfa and two biosimilar preparations were analyzed in vitro. Glycosylation pattern was assessed in regard to cAMP production, Ca2+ release, β-arrestin 2 recruitment, CREB, and ERK1/2 phosphorylation and steroid (i.e., progesterone and estradiol) synthesis, which were analyzed in human primary granulosa-lutein cells (hGLC) and HEK293 cells transiently transfected with the human FSHR cDNA.

#### MATERIALS AND METHODS

#### Follitropin Alfa Reference Preparation (Gonal-f®) and Biosimilars

The reference follitropin alfa and two biosimilar preparations were analyzed: Gonal-f R provided by Merck KGaA (Darmstadt, Germany), Ovaleap R purchased from Teva Pharmaceutical Industries (Tel Aviv, Israel) and Bemfola R from Finox Biotech (Kirchberg, Switzerland). Different batches of each preparation were tested by performing both biochemical and functional evaluations, as follows: two batches of Gonal-f R (AU016646, BA045956), two batches of Ovaleap R (S27266, R38915), and three batches of Bemfola R (PPS30400, PNS30388, PNS30230). Additional Gonal-f R (199F005, 199F049, 199F051) and Ovaleap R (S06622) batches were used for glycopeptide mapping. Comparison of hormone induced-signaling in vitro were performed by stimulating cells with gonadotropins concentrations expressed by mass rather than International Units (IU), since the latter depends of the in vivo activity in rats (38). Gonal-f R and biosimilar dosages were determined starting by the batch concentration declared by providers, consisting of 44µg/ml for Gonal-f R , Ovaleap R and Bemfola R . Recombinant human choriogonadotropin (hCG; Ovitrelle R , Merck KGaA) was used as a negative control where indicated.

#### Silver Staining and Western Blotting Analysis

According to gonadotropin quantification provided by the producers, 300 ng of each compound were subjected to 12% SDS-PAGE. Gel electrophoresis was performed under denaturing-reducing or non-denaturing-non reducing conditions, followed by silver staining and Western blotting. Denaturing conditions consisted of boiling samples 5 min at 100◦C, while reducing conditions were obtained by adding 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA), disrupting disulfide bonds (39). Silver staining was performed after acrylamide gel electrophoresis, as previously described (40, 41). Briefly, fixation was performed by incubating gels 1 h in 50% ethanol buffer, in the presence of 12% acetic acid and 5 × 10–4% formalin (all from Sigma-Aldrich). After washes, gels were stained with 0.2% AgNO3 buffer 30 min-treatment and signals were developed by 3% Na2CO3 buffer, 0.0005% formalin and 4 × 10–4% Na2S2O3 before to be stopped. Originator follitropin alpha and biosimilars were evaluated by Western blotting using a rabbit anti-human polyclonal primary antibody against FSHβ/FSH (SAB1304978; Sigma-Aldrich), while the secondary antibody was anti-rabbit human horseradish peroxidase (HRP)-conjugated (#NA9340V; GE HealthCare, Little Chalfont, UK). Recombinant hCG (Ovitrelle; Merck KGaA) was used as a negative control. Signals were developed with ECL (GE HealthCare) and acquired using the VersaDoc Imaging System (Bio-Rad Laboratories Inc., Hercules, CA, USA).

### Lectin ELISA Assay and Glycopeptide Mapping

The technique was described previously (41, 42) and adapted to preparations used in this study. A 96-well-microtiter plate was coated overnight at 4◦C with the anti-human gonadotropin α subunit monoclonal antibody HT13.3 (43), which recognizes all human glycoprotein hormone α subunits, in 0.1 M sodium carbonate/hydrogen carbonate buffer (pH = 9.6). Plates were washed with a saline buffer (TBS-T; 25 mM Tris, 140 mM NaCl, 3 mM KCl, 0.05%, Tween 20; pH = 7.4) and non-specific sites were saturated by 1 h-treatment at room temperature (RT) using TBS-T containing 2% polyvinylpyrrolidone K30 (Fluka, Sigma-Aldrich). Duplicate 5 ng samples of each hormone preparation were then incubated over-night, in 100 µl/well of the saturation buffer. After washing, biotinylated lectins (Vector laboratories Ltd, AbCys Biologie, Paris, France) were placed into wells and incubated for 2 h at RT. Lectins used were: Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), Artocarpus Polyphemus lectin (jacalin), Ricinus communis agglutinin (RCA-1, ricin), Datura stramonium agglutinin (DSA), wheat germ agglutinin (WGA), Phaseolus vulgaris agglutinin (PHA-E) (**Supplemental Table 1**). They were diluted in saturation buffer containing 1 mM CaCl2, 1 mM MgCl2, and 1 mM MnCl2. Plates were washed and peroxidase labeled NeutrAvidinTM (Pierce, Interchim, Montluçon, France) was added in each well (100 µl in TBS-T), for 1 h at RT. After incubation with TMB ELISA peroxidase substrate standard solution (UP664781; Interchim, Montluçon, France) 20 min at RT, reactions were stopped by adding 50 µl/well of 2 N H2SO4, and absorbance measured at 450 nm wavelength using a spectrophotometer. Blank values, consisting of samples maintained in the absence of hormones, were subtracted to obtain ELISA data.

Additional information about reagents, glycopeptide mapping, hydrophilic interaction chromatography, and mass spectrometry analysis is provided in the supplemental section (**Supplemental Material and Methods**).

#### Cell Culture and Transfection

HEK293 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS, 4.5 g/l glucose, 100 IU/ml penicillin, 0.1 mg/ml streptomycin, and 1 mM glutamine (all from Sigma-Aldrich). Transient transfections were performed in 96-well plates using Metafectene PRO (Biontex Laboratories GmbH, München, Germany), in order to obtain exogenous FSHR and cAMP CAMYEL-, β-arrestin 2 or aequorin Ca2+-BRET biosensor protein expression (15), as previously described (41). For cAMP evaluation, 50 ng/well of FSHR-expressing plasmid were mixed together with 0.5 µl/well of Metafectene PRO in serum-free medium and incubated 20 min. A 50 µl aliquot of cAMP CAMYEL biosensorexpressing plasmid-Metafectene PRO mix was added to each well-containing 1 × 105 cells, in a total volume of 200 µl/well, and incubated 2-days before stimulation with gonadotropins. One hundred ng/well of FSHR-Rluc8- and 100 ng/well of βarrestin 2 biosensor-expressing plasmids were used for evaluating β-arrestin 2 recruitment. One hundred ng/well of FSHR- and 100 ng/well of aequorin biosensor-expressing plasmids were used to prepare cells for measure changes in intracellular Ca2+. All samples were prepared in duplicate and BRET measurements were performed using 2-day transfected cells, in 40 µl/well PBS and 1 mM Hepes.

Human primary granulosa lutein cells (hGLC) were isolated from ovarian follicles of about twenty donor women undergoing oocyte retrieval for ART, following written consent and with local Ethics Committee permission (Nr. 796 19th June 2014, Reggio Emilia, Italy). Patients had to match these criteria: absence of endocrine abnormalities and viral/bacterial infections, age between 25 and 45 years. Cells were recovered from the follicular washing fluid using a 50% Percoll density gradient (GE Healthcare, Little Chalfont, UK), following a protocol previously described (7, 44, 45). In order to restore expression of gonadotropin receptors (46), hGLC were cultured 6 days, then serum-starved over-night before use in experiments. Cells were cultured at 37◦C and 5% CO2 in McCoy's 5A medium, supplemented with 10% FBS, 2 mM L-glutamine, 100 IU/ml penicillin, 100µg/ml streptomycin and 250 ng/ml Fungizone (Sigma-Aldrich).

#### BRET Measurement of cAMP Production, and β-arrestin Recruitment and Intracellular Ca2+ Increase

Intracellular cAMP and Ca2+ increase, and β-arrestin 2 recruitment were evaluated following a previously described procedure (15, 41, 47). Cyclic-AMP production and Ca2+ increase were evaluated in transiently transfected HEK293 cells using the FSHR-expressing plasmid, together with the BRETbased cAMP biosensor CAMYEL (48), or the aequorin Ca2+ biosensor expression vector (49), respectively, while BRET experiments cannot be performed in hGLC due to sub-optimal transfection efficiency and the high mortality rate in this cell model. Recruitment of β-arrestin 2 was assessed after transient transfection of HEK293 cells with the C-terminal, Rluc-tagged FSHR cDNA plasmid (provided by Dr. Aylin C. Hanyaloglu, Imperial College, London, UK) and N-terminal, yPET-tagged β-arrestin 2 (provided by Dr. Mark G. Scott, Cochin Institute, Paris, France). Cells were incubated 30 min in 40 µl/well PBS and 1 mM Hepes, in the presence or in the absence of increasing concentrations of Gonal-f R or biosimilars (1 × 10−3 - 1 × 103 ng/ml range), and intracellular cAMP increase and β-arrestin 2 recruitment were measured upon addition of 10 µl/well of 5µM Coelenterzine h (Interchim). A 4 × 103 ng/ml hormone concentration-induced intracellular Ca2+ increase was evaluated over 100 s in transfected cells. Recombinant follitropin alfa or biosimilar addition occurred at the 25 s time-point. Light emissions were detected at 475 ± 30 and 530 ± 30 nm wavelengths using the CLARIOstar plate reader equipped with a monocromator (BMG Labtech, Ortenberg, Germany).

#### Evaluation of ERK1/2 and CREB Phosphorylation

Hormone-induced ERK1/2 and CREB phosphorylation was analyzed by Western blotting following a protocol previously described (50). Human GLCs were seeded in 24-well plates (1 × 105 cells/well) and treated for 15 min with increasing concentrations of gonadotropin (1 × 101 -1 × 103 ng/ml range). Cells were immediately lysed for protein extraction in icecold RIPA buffer along with PhosStop phosphatase inhibitor and a protease inhibitor cocktail (Roche, Basel, Switzerland). Cell lysates were subjected to 12% SDS-PAGE and Western blotting, while pERK1/2 and pCREB activation were evaluated using specific rabbit antibodies (#9101 and #9198, respectively; Cell Signaling Technology Inc., Danvers, MA, USA). Sample loads were normalized to total ERK1/2 (#4695; Cell Signaling Technology Inc.). Membranes were treated with secondary antirabbit HRP-conjugated antibody (#NA9340V; GE HealthCare) and signals developed with ECL (GE HealthCare). Signal detection employed the VersaDoc system using the QuantityOne analysis software (Bio-Rad Laboratories Inc.). Protein density volumes were semi-quantitatively evaluated by the ImageJ software (U. S. National Institutes of Health, Bethesda, MD, USA) (51).

#### Gene Expression Analysis

Hormone 50% effective concentrations (EC50s) were calculated from the cAMP dose-response curves and used for hGLC treatments before FSH-target gene expression analysis. Cells were seeded at 5 × 104 cells/well in 24-well plates and exposed to gonadotropins for 8 h, and RNA was then extracted using the automated workstation EZ1 Advanced XL (Qiagen, Hilden, Germany). Equal amounts of total RNA were retrotranscribed by iScript reverse transcriptase (Bio-Rad Laboratories Inc.), according to a previously validated protocol (52). The expression of STARD1 and CYP19A1 genes encoding steroid-acute regulatory protein (StAR) and aromatase enzymes, respectively, was evaluated by real time PCR (7, 44) using specific primer sequences and protocols previously validated (7). Target gene expression was normalized to ribosomal protein subunit 7 (RPS7) gene expression using the 2−11Ct method (53). Experiments were recorded as the mean value of duplicates.

### Steroid Hormone Stimulation Protocol and Measurement

Human GLCs were seeded in 24-well plates (4 × 104 cells/well) and treated 8 or 24 h with increasing hormone concentrations (1 × 10−3 -1 × 103 ng/ml). Where appropriate, 1µM 4-androstene-3,17-dione (androstenedione; #A9630; Sigma-Aldrich) was added, as a substrate to be converted to estrogen by the aromatase enzyme. Stimulations were terminated by freezing samples and total progesterone or estradiol was measured in the cell media by an immunoassay analyzer (ARCHITECT second Generation system; Abbot Diagnostics, Chicago, IL, USA).

#### Statistical Analysis

Data were graphically represented using box and whiskers plots, histograms, X-Y graphs and tables, and indicated as means ± standard error of means (SEM). Western blotting results were normalized to total ERK signals. Intracellular Ca2+ increase was represented as kinetics of acceptor emissions measured at 525 ± 30 nm, and area under the curve (AUC) values were extrapolated for comparisons between preparations. Dose-response curves for cAMP and β-arrestin 2 were obtained by data interpolation using non-linear regression. BRET data were represented as induced BRET changes by subtracting the ratio of donor/acceptor biosensor emissions of the untreated cells from the values of the stimulated cells. Data distributions were analyzed by D'Agostino and Pearson normality test, while differences were evaluated by Kruskal-Wallis or Friedman test with Dunn's multiple comparison post-test and considered significant when p < 0.05. Statistics were performed using the GraphPad Prism 6.01 software (GraphPad Software Inc., San Diego, CA, USA).

### RESULTS

### Western Blotting and Silver Staining Analysis

Samples comprising 300 ng/well of non-denatured and denatured Gonal-f R and biosimilar preparations were loaded onto a 12% acrylamide gel and separated by SDS gel electrophoresis under denaturing-reducing and non-denaturing-non reducing conditions. Denaturing conditions refer to 100◦C-boiled samples, while reducing conditions were obtained by adding 2-mercaptoethanol. While no signals were detected under non denaturing-non reducing conditions by Western blotting (data not shown), two bands corresponding to the reference follitropin alfa and biosimilar preparations were revealed under denaturing-reducing conditions (**Figure 1A**). Ovaleap R and Gonal-f R preparations featured an ∼20 KDa band, while a band corresponding to about 23 KDa molecular weight characterized

Bemfola R . All preparations displayed a 15 KDa band of varying intensity. Recombinant hCG served as a negative control, providing no signal using the anti-FSH antibody.

Analysis by silver staining under non denaturing-non reducing conditions revealed that all preparations shared an overall similar protein pattern characterized by a single band at about 37 KDa molecular weight (**Figure 1B**). hCG resulted in a 40 KDa band. All samples displayed signals at about 20 KDa molecular weight (**Figure 1C**). Interestingly, no 15-KDa signals were detected, oppositely to that demonstrated by Western blotting, likely to be attributed to the low amount of FSHβ bound by the antibody and undetectable using silver staining due to suboptimal sensitivity of this method (54). Three 35–20 KDa bands corresponding to recombinant hCG Ovitrelle R were detected, as previously described (41).

### Reference Follitropin Alfa and Biosimilar Reactivity to Lectins

The carbohydrate structure of follitropin alfa and biosimilars was investigated by ELISA, using a panel consisting of seven lectins characterized by specific recognition of different glycan features (**Supplemental Table 1**). Batches of each hormone were considered as experimental replicates and absorbance values measured at 450 nm were compared (**Table 1**).

Bemfola R displayed structural peculiarities and variability, emerging by lectin analysis (**Supplemental Table 2**), due to significantly higher reactivity against ricin than other preparations (Kruskal-Wallis test; p < 0.05; n = 16; **Table 1**). Moreover, lectin assay revealed higher affinity of Bemfola R to DSA than Gonal-f R (Kruskal Wallis test; p < 0.05; n = 6). Ricin recognizes Galβ(1,4)GlcNAc monomers with higher affinity in the absence of sialylation in the terminal galactose, while DSA lectin binds Galβ(1,4) linked N-acetylglucosamine oligomers and a branched pentasaccharide sequence, including two N-acetyl lactosamine repeats linked to a mannose (55). No signal was detected with SNA lectin regardless of the hormone tested, indicating that sialic acid of the α(2,6) type is absent (56), likely due to the absence of galactoside α(2,6) sialyltransferase enzyme expression by CHO-K1 cells (57). Sialic acid of α(2,3) type is detected by MAA lectin in all samples (58), without any significant preparation-specific pattern. Jacalin failed to produce any signal, demonstrating the absence of O-glycans of the Galβ1-3GalNac or GalNac type (59). PHA-E lectin recognizes bi-antennary complex-type N-glycan with outer Gal and bisecting GlcNAc sequences (60), while WGA lectin reacts with GlcNAc sequences and sialic acid (61). Antennarity (**Table 2**), sialylation (**Table 3**), and sialic acid (**Table 4**) distribution were analyzed by glycopeptide mapping of Gonal-f R and Ovaleap R batches.

These features were similarly represented among preparations (Kruskal-Wallis test; p ≥ 0.05; n = 6), as well as among batches (**Supplemental Results**), at least in Gonal-f R and Bemfola R (Chi-square test; p ≥ 0.05), which appeared to be homogeneous, overall (**Supplemental Tables 3**–**5**).

aKruskal Wallis test and Dunn's post-test.

aKolmogorov-Smirnov test.

#### Evaluation of Intracellular cAMP Increase and β-Arrestin 2 Recruitment

Transfected, FSHR-expressing HEK293 cells were used to compare intracellular cAMP increases and β-arrestin 2 recruitment induced by 30-min treatment with increasing doses of reference follitropin alfa and biosimilars. Different batches of each preparation were tested and dose-response curves obtained by plotting cAMP and β-arrestin 2 levels in a semi-log X-Y graph (**Supplemental Figure 1**), in order to calculate and compare EC50 values obtained from the individual dose-response-curves (**Figure 2**).

Although preparation-specific carbohydrate structures were detected (**Table 1**), no significant differences were found TABLE 3 | Sialylation distribution in reference and biosimilar follitropin alfa preparations.

aMann-Whitney's U-test.

TABLE 4 | Sialic acid distribution in reference and biosimilar follitropin alfa preparations.

aKolmogorov-Smirnov test.

between Gonal-f R and biosimilars' EC50 required for activating cAMP (**Table 5**; 12.9 ± 2.5–24.2 ± 6.0 ng/ml range; Kruskal-Wallis test, p ≥ 0.05; n = 4; **Figure 2A**) and β-arrestin 2

hormones. EC50 values were extrapolated by non-linear regression. Data are represented as box and whiskers graphs (Kruskal Wallis test, p ≥ 0.05; n = 4).

(**Table 5**; 140.7 ± 42.6–278.6 ± 56.9 ng/ml range; Kruskal-Wallis test, p ≥ 0.05; n = 4; **Figure 2B**), consistent between batches (cAMP: 10 ± 0.0–28 ± 0.0 ng/ml range; β-arrestin 2: 64 ± 0.0–610 ± 0.2 ng/ml range; Kruskal-Wallis test, p ≥ 0.05; n = 4; **Supplemental Figure 1**) and confirming similar potencies in vitro.

#### Analysis of pERK1/2 and pCREB Activation

The phosphorylation of ERK1/2 and CREB was evaluated in hGLC, which naturally express endogenous FSHR. Cells were treated for 15 min with increasing hormone concentrations, and phospho-protein activation was evaluated by Western blotting and semi-quantitatively measured (**Figure 3**). Total ERK served as a normalizer.

Similar ERK1/2 and CREB phosphorylation patterns were observed after stimulating cells with increasing doses of different batches of each preparation (**Supplemental Figure 2**). Mean results from batches of Gonal-f R , Ovaleap R , and Bemfola R were calculated and average hormone-specific pERK1/2 and pCREB activation results were reported (**Figure 3**). All preparations induced protein phosphorylation within the 1.5–15 ng/ml range (Kruskal-Wallis test, p < 0.05; n = 4), consistently between different batches of each preparation (Friedman test, p ≥ 0.05; n = 4). While no statistically significant differences between TABLE 5 | Efficiency (EC50) of 30 min-cAMP and β-arrestin 2 production induced by reference and biosimilar follitropin alfa preparations in transfected, FSHR-expressing HEK293 cells.

aKruskal-Wallis test.

Gonal-f R and biosimilars' patterns of ERK1/2 phosphorylation were detected, pCREB activation occurred upon cell treatment by 0.5 ng/ml Gonal-f, differently to that obtained using both biosimilars (Friedman test, p < 0.05; n = 4). Interestingly, cell treatment by Gonal-f R and Bemfola R maximal concentrations (15 ng/ml) resulted in slightly decreased levels of CREB phosphorylation, not differing, however, significantly from the plateau levels of pCREB activation.

### STARD1 and CYP19A1 Gene Expression Analysis

Expression of FSH target genes was analyzed by real time PCR in hGLC. For this purpose, cells were stimulated 12 h by Gonalf R , Ovaleap R or Bemfola R . Hormones were administered at the EC50 calculated from cAMP data (12 ng/ml Gonal-f R and Ovaleap R , 24 ng/ml Bemfola R ). Total RNA was reversetranscribed to cDNA and used for STARD1 and CYP19A1 gene expression analysis by real-time PCR. Data were normalized over the RPS7 gene expression and represented as fold-increase over unstimulated cells in a bar-graph as means ± SEM (**Figure 4**).

Gonal-f R , Ovaleap R , and Bemfola R resulted in about 15 fold STARD1 and 3-fold CYP19A1 increase compared to the basal level (Kruskal-Wallis test, p < 0.05; n = 4). In particular, Ovaleap R -induced CYP19A1 expression level lower than what was obtained by Bemfola R treatment (Kruskal-Wallis test, p < 0.05; n = 4). Treatment using different batches did not to affect STARD1 and CYP19A1 expression levels, since no significant differences between lots of any preparation occurred (Kruskal-Wallis test, p ≥ 0.05; n = 4; data not shown).

#### Steroid Synthesis Analysis

Progesterone production and androgen-to-estrogen conversion were evaluated in hGLC treated for 8 or 24 h with hormones. For this purpose, cells were maintained under continuous stimulation by increasing gonadotropin concentrations (1 × 10−3 -1 × 103 ng/ml range) until reactions were stopped by freezing cell plates. To evaluate estradiol synthesis, androstenedione was added into wells as a substrate for the aromatase enzyme. Eight- and Twenty-four hours progesterone and estradiol dose-response curves were obtained and evaluated by non-linear regression, EC50 values calculated, and compared (**Table 6**).

Reflecting cAMP accumulation, cell stimulation with Gonalf R , Ovaleap R , and Bemfola R resulted in similar 8- and 24-h

progesterone and estradiol production curves (Kruskal-Wallis test; p ≥ 0.05; n = 5), confirmed using different batches (Kruskal-Wallis test; p ≥ 0.05; n = 5; data not shown), as well as in similar progesterone and estradiol plateau levels (Kruskal-Wallis test; p ≥ 0.05; n = 5; **Supplemental Table 6**).

## Intracellular Ca2+ Increase

Kinetics of intracellular Ca2+ increase was evaluated in a transiently transfected HEK293 cell line that co-expressed both FSHR- and Ca2+-biosensors, by BRET. Cells were monitored for over 100 s and 4 × 103 ng/ml hormone addition occurred at the 25 s time-point (**Figure 5**). A 10–20-fold supra-physiological FSH concentration was used, compared to FSH serum levels described in cycling women (62), due to the lack of an intracellular Ca2+ signal at lower hormone concentrations (data not shown). Thapsigargin and vehicle treatment were used as positive and negative controls, respectively. Data were represented as means ± SEM. AUC values were calculated to compare preparation-specific intracellular Ca2+ increase.

Addition of vehicle failed to induce any intracellular Ca2+ increase, confirming the lack of activity exerted by the solvent used for hormone dilution on calcium response. After confirming the absence of batch-specific results (Kruskal-Wallis test; p ≥ 0.05; n = 3; data not shown), cell treatment by Gonal-f R induced rapid intracellular Ca2+ increase, which was about 230 fold higher than vehicle (Kruskal-Wallis test; p < 0.05; n = 3) and occurred within 1–2 s after hormone addition. Bemfola R and Ovaleap R induced only a minimal, not significant intracellular

FIGURE 4 | STARD1 and CYPA19A1 gene expression analysis. The expression of STARD1 (A) and CYPA19A1 (B) gene was evaluated in hGLC stimulated for 12 h with the EC50 of Gonal-f® or biosimilars (12 ng/ml Gonal-f® and Ovaleap®, 24 ng/ml Bemfola®) by real-time PCR. Each value was normalized over the RPS7 gene expression (means ± SEM; n = 4). Unstimulated cells served as control and are indicated as a dotted line. (\*= significant vs. Bemfola®, Kruskal Wallis test; p < 0.05).

TABLE 6 | FSH EC50 values (ng/ml) in inducing 8 h- and 24 h-progesterone and estradiol production induced by reference and biosimilar follitropin alfa preparations (means±SEM; n = 5) in human primary granulosa cells.

aKruskal-Wallis test.

Ca2+ increase (Kruskal-Wallis test; p ≥ 0.05; n = 3). Maximal levels of intracellular Ca2+ were achieved under thapsigargin treatment, which served as a positive control and induced an about 600-fold greater increase compared to the basal level (Kruskal-Wallis test; p < 0.05; n = 3).

#### DISCUSSION

We compared the biochemical profiles and hormone-induced cell responses of the reference follitropin alfa (Gonal-f R ) and two biosimilars, Ovaleap R and Bemfola R , in vitro, revealing

overall comparable hormone-induced intracellular signaling and steroidogenesis. Only the originator follitropin alfa induced hormone-specific pattern of CREB phosphorylation and, at supra-physiological concentrations (62), intracellular Ca2+ increase to transfected, FSHR-expressing cell lines.

(n = 3). Area under the curve (AUC) values were calculated and differences

were considered for p < 0.05 (Kruskal Wallis test).

Several gonadotropin formulations are commercially available, differing by source, purification process, and purity. Clinicians choose freely what preparation or combination of preparations will be administered to women undergoing ART (35). These preparations may differ in oligosaccharide content and number of branches attached to the protein backbone (63), depending on the glycosyltransferases equipment of the source cell. Gonal-f R is expressed by Chinese hamster ovary (CHO) cell lines (64), ensuring high bioactivity and batch-to-batch consistency (65, 66). Bemfola R is produced by a pre-adapted dihydrofolate reductase deficient CHO (CHO DHFR-) host cell line (67) and has demonstrated similar efficacy and safety in vivo as compared to the reference follitropin alfa, in a multi-center phase 3 study (68). Ovaleap R is also produced by a CHO-derived cell line after adaptation to serum free conditions (69) and has been demonstrated to be similar to follitropin alfa in vivo in a phase 3 clinical study (70).

Gonal-f R and Ovaleap R share two similar Western blotting patterns under denaturing and reducing conditions, likely due to specifically glycosylated FSH β-subunits (36, 37), while Bemfola R featured a ∼23 instead of 20 KDa band according to its specific glycosylation pattern detected by mass spectrometry (36). Most of these signals were confirmed by silver staining, except for the absence of the 15 KDa band, likely due to sub-optimal sensitivity of the method (54). Analysis of native proteins contained in Gonal-f R and Ovaleap R batches, which were obtained by omitting treatment of samples by 100◦Cheating and 2-mercaptoethanol reduction, revealed a single 37 Riccetti et al. FSH Biosimilars in vitro

KDa band consistent with the FSH heterodimer (71), while Bemfola R resulted in slightly higher apparent molecular weight. On the other hand, lectin assay revealed higher DSA signal in Bemfola R than Gonal-f R , likely due to different multiantennary complex structures on N-glycans demonstrated by glycopeptide mapping (36), and suggesting Bemfola R -specific glycosylation patterns. Lower ricin binding to Ovaleap R than to Bemfola R and Gonal-f R indicated a different content of Galβ1-4GlcNAc molecules (72).