FSH, bone, belly and brain.

The collective evidence positions FSH, which increases during the menopausal transition, as an actionable target for bone loss, visceral obesity, and cognitive decline simultaneously. In pursuit of this objective, our group has developed polyclonal and monoclonal antibodies designed to specifically target a computationally defined epitope consisting of 13 amino acids within the receptorbinding domain of human FSHβ. These antibodies have been shown to enhance bone mass in both wild-type and ovariectomized mice by binding to FSHβ, thereby preventing its interaction with FSHR on osteoclasts and osteoblast precursors, resulting in both antiresorptive and anabolic effects ( Zhu et al. 2012a , b Ji et al. 2018 , Gera et al. 2020 ). Moreover, these antibodies have been shown to reduce fat mass and to promote thermogenic adipose tissue ( Liu et al. 2017 , Gera et al. 2020 ), as well as to prevent the onset of AD-like neuropathology and cognitive decline in an array of mouse models ( Xiong et al. 2022 ). These studies led to the development of a panel of 30 humanized FSH blocking antibodies against the human FSHβ epitope, LVYKDPARPKIQK, of which we have selected a lead candidate MS-Hu6 with a binding affinity (K D ) of 7.53 nM, which approaches that of trastuzumab (K D = 5 nM), one of the most successful therapeutic monoclonal antibodies ( Gera et al. 2020 ). When injected into Tg32 mice, in which the FcRn receptor (the receptor that controls antibody clearance) is humanized, MS-Hu6 shows a β phase t ½ of 7.8 days. MS-Hu6 has also shown promising anti-osteoporosis and anti-obesity efficacy and safety in rodent and nonhuman primates ( Gera et al. 2020 , Gera et al. 2022 , Xiong et al. 2022 , Rojekar et al. 2023 , Sant et al. 2022 ). Consistent with a bone- protective effect of blocking FSH selectively, another group generated an anti-FSH vaccine consisting of tandem repeats against the same binding site FSHβ13AA- T-OVA ( Han et al. 2020 ). This vaccine reduced body weight and fat mass in ovariectomized mice with high FSH, in essence, establishing proof of principle that the reduction of bioavailable FSH in the circulation can inhibit fat accrual. Lastly, it is worth noting that FSHR expression is increased in various types of cancers, associated with reproductive and nonreproductive organs, potentially making them a valuable therapeutic target. FSHRs are expressed in prostate ( Ben-Josef et al. 1999 , Mariani et al. 2006 ), ovarian ( Perales-Puchalt et al. 2017 ), endometrial ( Sheng et al. 2022 ), thyroid ( Pawlikowski et al. 2015 ), and head and neck squamous cell cancer ( Olejar et al. 2020 ), as well as in the surrounding tissues including tumor vessels ( Olejar et al. 2020 , Radu et al. 2010 , Siraj et al. 2013 ). Around two-thirds of ovarian cancers express FSHR, and this expression has been associated with a poorer prognosis ( Lenhard et al. 2011 ). Furthermore, exposure of endometrial cancer cell lines to FSH promotes cell proliferation and migration. In in vivo studies using ovariectomized mice with estrogen supplementation, implanted endometrial cancer cells exhibit significant growth in the FSH-treated group ( Sheng et al. 2022 ). A monoclonal anti-FSHR antibody (D2AP11) against the external domain of FSHR has shown promising efficacy in ovarian cancer ( Bordoloi et al. 2022 ). This antibody specifically bound to ovarian cancer tissues of various pathological conditions, such as high- and low-grade serous carcinoma, mucinous adenocarcinoma, clear cell carcinoma, dysgerminoma, endodermal sinus carcinoma and metastatic adenocarcinoma, and showed a dosedependent cytotoxic effect in cancer cell lines in vitro and reduced tumor volume in in vivo mouse models. Collectively, these findings suggest that targeting FSHRs may offer an innovative alternative or complementary therapy to existing cancer treatments.

We documented the expression of the full-length Fshr by qPCR and Sanger sequencing and immunostaining of adipocytes and adipose tissue in mice ( Liu et al. 2017 ). FSHRs were also identified in adipose tissues in other species including boar and humans ( Liu et al. 2015 , Liu et al. 2017 , Han et al. 2021 ). Unlike in ovarian granulosa cells, we find that FSHRs in adipocytes are coupled to Gα i protein and decrease intracellular cAMP ( Liu et al. 2017 ), which subsequently downregulates cAMP-mediated β 3 - adrenergic receptor signaling. As a result, uncoupling protein 1 (UCP1) mediated beiging is downregulated ( Liu et al. 2017 , Cero et al. 2021 ). In addition, FSH induces lipid synthesis by upregulating peroxisome proliferator- activated receptor gamma (PPARγ), CCAAT enhancer binding proteins (C/EBPα), fatty acid synthase (FAS), lipoprotein lipase (LPL), and perilipin through Ca 2+ - dependent signaling ( Liu et al. 2015 ) ( Fig. 1 ). On the other hand, we found that attenuating the action of FSH, genetically in Fshr +/− mice or pharmacologically using our FSH-blocking antibodies in either high-fatdiet-treated or ovariectomized mice, promoted UCP1 expression and mitochondrial biogenesis, resulting in increased energy expenditure and reduced fat mass ( Liu et al. 2017 ). Further evidence comes from animal models that recapitulate primary (high FSH) vs secondary (low FSH) hypogonadism by surgical gonadectomy or GnRH treatment, respectively, where primary hypogonadal animals with elevated FSH show higher subcutaneous and visceral fat mass ( Liu et al. 2015 , Han et al. 2021 ). In addition, a vaccine based on tandem repeats of the FSHβ sequence, which was used to generate our antibody, resulted in low fat mass ( Han et al. 2020 ). Available human data further support the pro-adiposity actions of FSH. It is well established that women during the peri- and postmenopausal periods undergo unfavorable metabolic changes resulting in weight gain, characterized by central redistribution of adipose tissue ( Pollycove et al. 2011 , Abildgaard et al. 2021 ). In the SWAN study that included 2375 perimenopausal women with diverse ethnic backgrounds, FSH levels, which started rising 2–5 years prior to final menstruation, had a remarkably similar trajectory to waist circumference and fat mass during this perimenopausal transition (FSH and fat mass: r = 0.12, CI 0.09–0.16) ( Sowers et al. 2007 ). It is worth re-emphasizing that estrogen levels remained relatively unperturbed during the perimenopausal window ( Randolph et al. 2004 ). Furthermore, the AGES- Reykjavik study shows that elderly women in the highest FSH quartile had higher bone marrow adiposity ( Veldhuis-Vlug et al. 2021 ). Similarly, a population study conducted in China revealed a positive correlation between FSH levels and BMI not only in postmenopausal women but also in older men in whom FSH rises gradually at ~3.5% annually ( Liu et al. 2015 ). This suggests that the impact of FSH may not be limited in women. For example, in men with prostate cancer, surgical orchiectomy (primary hypogonadism, low testosterone, and high FSH) caused greater weight gain and fat mass both in subcutaneous and visceral fat compared with GnRH agonist treatment (secondary hypogonadism, low testosterone, and low FSH) ( Ostergren et al. 2019 ). This interventional study provided compelling evidence that fat mass and body weight can be reduced by lowering serum FSH in humans. A very recent study, an ancillary analysis of the BLADE study, showed that the higher serum FSH levels during therapy with the LH releasing hormone antagonist degarelix in patients with prostate cancer were associated with higher total, limb, and trunk fat mass ( Bergamini et al. 2024 ). The detrimental metabolic effects of FSH may extend beyond adipogenesis and fat redistribution, affecting cardiovascular health both directly and indirectly in both men and women. For example, the Prospective Army Coronary Calcium project examined 126 asymptomatic perimenopausal women and found that serum FSH levels, but not estradiol, were associated with the number of aortic plaques, as measured by contrast-enhanced computed tomography (CT) angiography and carotid ultrasonography (USG) ( Munir et al. 2012 ). Similarly, the SWAN cohort noted that subjects with medium and high FSH levels had significantly thicker carotid intima–media thickness ( El Khoudary et al. 2016 ). Additionally, in men with prostate cancer, androgen deprivation therapy has been linked to increased cardiovascular and metabolic morbidity ( Crawford et al. 2017 ). Pooled data from six phase III prospective randomized trials reveal a lower risk of cardiovascular disease in patients receiving a GnRH antagonist as opposed to GnRH agonists ( Albertsen et al. 2014 ). The latter primarily suppress LH, rather than both FSH and LH. Several mouse studies suggest that FSH promotes atherogenesis via pro-inflammatory signaling. FSHRs have been identified in immune cells, including monocytes and macrophages and their activation promotes TNFα signaling. Furthermore, FSH treatment upregulates IL-1β expression in macrophages, enhances macrophage migration, and accelerates atherosclerosis in atherogenic ApoE - / - mice ( Han et al. 2023 ). The direct role of FSH in endothelial dysfunction and atherogenesis is suggested by its expression in vascular endothelial cells, although the expression was not reported consistently, probably due to low expression levels ( Radu et al. 2010 , Stelmaszewska et al. 2016 , Li et al. 2017 , Tan et al. 2021 , Tedjawirja et al. 2023 ). FSHR activation in human umbilical vein endothelial cells (HUVEC) promoted angiogenesis to a degree comparable with vascular endothelial growth factor (VEGF) and stimulated nitric oxide production ( Stilley et al. 2014b ). Furthermore, FSH upregulated VAV guanine nucleotide exchange factor 3 (VAV3) and laminin alpha 2 (LAMA2), both of which are involved in the cell adhesion and migration ( Tan et al. 2021 ). FSH also enhanced vascular cellular adhesion module-1 (VCAM-1) expression, which regulates leukocyte adhesion and promotes atherosclerosis ( Li et al. 2017 ), together suggesting that FSH might play a crucial role in atherosclerosis and contribute to increased cardiovascular risk of peri- and postmenopausal women and aged men or men with prostate cancer under androgen-deprivation therapy. Finally, a recent study identified the expression of FSHRs in human and mouse pancreatic islet β-cells. Blocking FSH signaling led to impaired glucose tolerance due to decreased insulin secretion, while high FSH levels caused insufficient insulin secretion ( Cheng et al. 2023 ). We first established an effect of FSH on the skeleton using genetically modified mice deficient in Fshb or Fshr as well as by pharmacological intervention using our panel of FSH-blocking antibodies. Haploinsufficient Fshb +/− and Fshr +/− mice, despite having only one functional gene copy, still developed intact uteri and ovaries but displayed a high bone mass ( Sun et al. 2006 ). Subsequent studies by us and others further validated a direct effect of FSH on the skeleton. For instance, injection of FSH into rodents exacerbated bone loss induced by ovariectomy, while the use of an FSH antagonist mitigated this bone loss ( Liu et al. 2010a , b ). FSH induces bone loss by both pro-resorptive and antianabolic actions ( Fig. 2 ). FSH stimulates osteoclast differentiation and activation through a distinct FSHR isoform lacking exon 9 ( Sun et al. 2006 , Robinson et al. 2010 , Sun et al. 2010 , Wang et al. 2015b ). As in adipocytes, FSHRs in CD11b+ osteoclast precursors and osteoclasts are coupled with inhibitory Gα i2 protein. FSHR activation in osteoclasts thus decreases cAMP levels but stimulates ERK1/2 and IκBα phosphorylation, ultimately upregulating osteoclast formation. Consistent with this, the effects of FSH on osteoclastogenesis are attenuated in the absence of Gα i subunit ( Sun et al. 2006 , Zhu et al. 2012a , b ) The pro-osteoclastic FSH effect is also, in part, mediated by cytokines. FSH enhances the expression of RANK ( Cannon et al. 2011 ), as well as IL-1β, TNFα, and IL-6 ( Iqbal et al. 2006 , Cannon et al. 2010 ). Serum FSH levels correlate with circulating cytokines ( Cannon et al. 2010 , Gertz et al. 2010 ). Finally, FSH failed to increase osteoclast formation in mice lacking immunoreceptor tyrosine–based activation motif (ITAM) adapter signaling molecules ( Wu et al. 2007 ). FSH also decreases bone formation by inhibiting osteoblast differentiation from osteoblast precursors ( Zhu et al. 2012a ). Notably, our FSH-blocking antibody increased the number of osteoblast precursors, including mesenchymal stem cells. This increase led to an upregulation of the expression of genes related to osteoblast formation and enhanced bone formation ( Zhu et al. 2012a , Gera et al. 2020 , Robinson et al. 2010 ). Human data supports the independent skeletal actions of FSH. Patients with premature ovarian failure with hypergonadotropic amenorrhea (mean FSH 85 IU/L) showed lower bone mineral density (BMD) in the lumbar spine than patients with hypogonadotropic amenorrhea (mean FSH: 10 IU/L) ( Devleta et al. 2004 ). Likewise, women with functional hypothalamic amenorrhea, wherein both FSH and estrogen are low, show slight to moderate skeletal defects ( Podfigurna-Stopa et al. 2012 ). Furthermore, women harboring an activating FSHR N680 variant display strikingly lower bone mass and high bone resorption markers ( Rendina et al. 2010 , Nenonen et al. 2019 ). Additionally, the FSHR S680 (which transduces lower intracellular signals) and BMP15 IVS1+905A digenic combination has been described as osteoprotective ( Mendoza et al. 2012 ). The SWAN study found a strong correlation between serum levels of FSH and BMD, and, importantly, a rise in serum levels of FSH over 4 years predicted a decline in BMD ( Sowers et al. 2003 ). Several Chinese cohorts similarly found significant associations between high serum levels of FSH and bone resorption markers or BMD, particularly in the highest quartiles of serum FSH ( Xu et al. 2009 , Wu et al. 2010 , Cheung et al. 2011 , Wang et al. 2015a ). The Bone Turnover Range of Normality (BONTURNO) Study showed that women with serum FSH >30 IU/L had higher bone turnover markers than age-matched women with FSH levels of ≤30 IU/L ( Adami et al. 2008 ). Similarly, the NHANES III study and the AGES-Reykjavik Study of Older Adults from Iceland noted an inverse correlation between serum FSH and BMD ( Gallagher et al. 2010 , Veldhuis-Vlug et al. 2021 ).

Utilizing genetically modified mouse models and new genomic technologies, we and others have described independent actions of FSH in various organ systems; this has been a complicated path due to the intricate feedback loops and the dominant effects of sex steroids. Nonetheless, the discovery of the broader physiological role of FSH, recognized traditionally as solely involved in procreation, re-labels FSH as a putative aging hormone, with implications in addressing health consequences in women, including osteoporosis, obesity, and cognitive decline, as well as in men undergoing androgen deprivation therapy. Furthermore, emerging data have suggested that FSH might be a potential therapeutic target for cardiovascular disease and cancer. In all, the exploration of FSH, and other pituitary hormones, hitherto with ascribed unitary functions, highlights the importance of integrative studies to unravel new physiological circuits of medical importance.

Alzheimer’s disease (AD) disproportionately affects women in terms of lifetime risk, rate of progression, and symptom burden, suggesting that the female sex could be a major risk factor for AD, particularly after menopause, which accounts for over 60% of all those affected ( Farrer et al. 1997 , Brookmeyer et al. 1998 , Laws et al. 2016 , Koran et al. 2017 , Fisher et al. 2018 ). This striking sex difference led to the hypothesis that estrogen deficiency might underlie AD in postmenopausal women. However, the relationship between estrogen and AD in postmenopausal women has led to conflicting views. Preclinical studies show that estrogen could potentially yield a neuroprotective effect and mitigate the risk of AD ( Henderson 2006 , Simpkins et al. 2009 , Lan et al. 2015 , Engler-Chiurazzi et al. 2017 ). In ovariectomized rodents, supplementation with estrogen has shown improvements in cognitive function and learning skills ( Gibbs & Gabor 2003 , Daniel et al. 2006 ). Several studies using primates have also shown a cognitive benefit of estrogen ( Voytko 2002 , Lacreuse & Herndon 2003 , Hao et al. 2007 ). For example, young primates began developing attention deficit within 2 weeks of surgical menopause and more significant cognitive impairment after 2 months, which was reversed by estrogen supplements ( Voytko 2002 ). However, human studies have yielded conflicting outcomes. Observational studies and a small-sized clinical trial (20 women) with hormone replacement therapy (HRT) showed fewer progressive symptoms and improved cognition ( Paganini-Hill & Henderson 1996 , Doraiswamy et al. 1997 , Asthana et al. 2001 ). Conversely, several substantial clinical trials failed to establish a cognitive advantage from HRT ( Henderson et al. 2000 , Mulnard et al. 2000 , Wang et al. 2000 , Zandi et al. 2002 ). The Women’s Health Initiative Memory study recognized as one of the largest randomized, double-blind, placebo- controlled trial, conducted with community-dwelling women aged 65–79 years not only failed to demonstrate a protective effect but also revealed an increased risk of dementia and mild cognitive impairment ( Shumaker et al. 2004 , Espeland et al. 2004 ). Consequently, there remains a debate surrounding the potential of HRT, specifically estrogen, to alleviate AD symptoms. This raises the question of whether the increase in FSH levels during the perimenopausal transition and thereafter contributes to cognitive decline. Several large observational studies seem to make this hypothesis plausible. Longitudinal studies such as the SWAN and the Penn Ovarian Aging Study have shown a decline in cognitive performance specifically in cognitive processing speed, verbal encoding, and verbal episodic memory during perimenopause, independently of anxiety, depressive or stress symptoms; at this time, FSH starts rising prior to the decline of estrogen ( Greendale et al. 2009 , Epperson et al. 2013 , Greendale et al. 2020 ). Furthermore, FSH levels were positively correlated with the risk of dementia in men ( Bowen et al. 2000 , Li et al. 2020 ) and women ( Short et al. 2001 ). Additionally, a polymorphism in the FSHR (FSHR A307,S680/A307,S680 ) was linked to a lower risk of AD in women (OR = 0.36; 95% CI: 0.15–0.85) ( Corbo et al. 2011 ). We recently documented the presence of FSH receptors in multiple regions of the mouse and human brain, importantly, the granular layer of the dentate gyrus of the hippocampus and entorhinal cortex, which participate in learning behaviors and memory ( Ryu et al. 2022 ). We reported that intraperitoneal FSH injection into female AD-prone 3xTg mice induced the activation of the δ-secretase arginine endopeptidase (AEP) that caused the cleavage of amyloid precursor protein (APP) and TAU, resulting in Aβ plaque and neurofibrillary tangle deposition and cognitive decline ( Fig. 3 ). The response to FSH was attenuated in the C/EBPβ haploinsufficient mice, suggesting FSH-induced AD pathology was mediated, at least in part, by the transcription factor C/EBPβ ( Xiong et al. 2022 ). FSH treatment of human neuroblastoma SH-SY5Y cells and primary rat cortical neurons thus increased C/EBPβ and AEP expression in a time-dependent manner ( Xiong et al. 2022 ). Consistent with the hypothesis that FSH promotes AD-like pathology and cognitive decline, downregulation of the Fshr in the hippocampus by stereotactic si Fshr injections or our FSH-blocking antibody attenuated cognitive decline and neuropathology in the 3xTg mouse ( Xiong et al. 2022 ).

Two gonadotropins secreted by the anterior pituitary gland, namely, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), along with thyroidstimulating hormone (TSH), as well as their respective receptors, exhibit significant structural similarities. These similarities can be traced back to their evolutionary origins from thyrostimulin, an ancestral glycoprotein that later evloved into several glycoprotein hormones in vertebrates ( Kleinau & Krause 2009 ). Both the luteinizing hormone/choriogonadotropin receptor (LHCGR) and FSH receptor (FSHR) are expressed in the gonads of fish, with higher-order species maintaining a high level mainly of gonadal expression. Additionally, in fish, multiple forms of the FSHR have been identified, some of which can bind both FSH and LH ( Bogerd et al. 2005 , Kobayashi et al. 2008 ). In addition to fish, FSH has been identified in a wide range of species, including mammals, birds, reptiles, and amphibians ( Borrelli et al. 2002 , Kobayashi & Andersen 2008 , Ubuka et al. 2013 ). The effects of pituitary glycoprotein hormones can overlap, both within the same species and across different species. For instance, the TSH receptor (TSHR) in striped bass shares homology with mammalian TSH, LH, and FSH receptors, as well as gonadotropin receptors in salmon and catfish ( Kumar et al. 2000 ). It can be activated by bovine TSH and striped bass LH ( Kumar et al. 2000 ). In all, these observations imply that pituitary hormones, ancient molecules that have evolved to serve diverse somatic and sensory functions may not have singular actions in mammalian physiology. We provided the first evidence for an extra-thyroidal function of mammalian TSH by showing that haploinsufficiency of the Tshr in mice results in a low bone mass without affecting thyroid development and thyroid hormone secretion ( Abe et al. 2003 ). This study laid a firm foundation for over two decades of work that led to the discovery of diverse functions of both anterior and posterior pituitary hormones in mammals. A primary role for FSH in reproduction was shown in mammals when FSH isolated from sheep grew the size of ovarian follicles in hypophysectomized rats ( Li et al. 1949 ). Similarly, partially purified FSH obtained from human pituitaries was administered to amenorrheic women leading to elevated urinary estrogen levels and enlargement of the uterine cavity and ovaries ( Gemzell et al. 1958 ). With the development of sensitive and specific assays for FSH, the intricate relationship between FSH and estrogen during menstrual cycles and peri-menopause was demonstrated ( Odell et al. 1968 ). FSH exerts its action on gonads by binding to G protein- coupled receptor (GPCR) that is coupled with a stimulatory Gα s protein. This, in turn, activates adenylate cyclase, resulting in an increase in cAMP and protein kinase A (PKA) activation, which leads to the upregulation of several key factors, including cAMP regulatory elementbinding protein (CREB), p38 MAP kinase, p70-S6 kinase, phosphoinositide-3 kinase (PI3K), PKB/Akt, and FOXO1. Through these pathways, FSH regulates the maturation of Graafian follicles, promotes granulosa cell proliferation, enhances estrogen, and, in males, plays a role in spermatogenesis in Sertoli cells ( Lizneva et al. 2019 ). Within the past two decades, an emerging body of evidence has revealed that the expression of FSHR extends beyond the gonads and is found in various extragonadal tissues in mammals. FSHR has been identified in nongonadal reproductive tracts, blood vessels, endothelial cells, the liver, skeletal tissue, bone cells, adipose tissue, and the brain ( Table 1 ). These discoveries have raised the question of whether FSH exerts extragonadal effects independently of estrogen and whether it holds pathophysiological significance, particularly in the context of metabolic, skeletal, and cognitive changes during the perimenopausal transition. This is particularly relevant considering the selective rise of FSH preceding the loss of estrogen that was shown in the Study of Women’s Health Across the Nation (SWAN) ( Randolph et al. 2004 ).