Beyond bone biology: Lessons from team science.

Today, research in biomedicine often requires the knowledge and technologies in diverse fields. Therefore, there is an increasing need for collaborative team science that crosses traditional disciplines. Here, we discuss our own lessons from both interdisciplinary and transdisciplinary teams, which ultimately ushered us to expand our research realm beyond bone biology.

The role of FSH and LH in prostate cancer and cardiometabolic comorbidities.

INTRODUCTION: In this article we advance a potential explanation for the incidence of cardiovascular (CV) and cardiometabolic risk in patients undergoing androgen deprivation therapy (ADT) for prostate cancer. Our conceptual model involves the differential impact of gonadotropin-releasing hormone (GnRH) agonists and antagonists on the follicle-stimulating hormone (FSH) system. MATERIALS AND METHODS: Authors searched online repositories and meeting abstract databases for relevant materials. RESULTS: Mounting evidence links FSH with development and progression of prostate cancer. What is also becoming clear is that the differential effects of GnRH agonists and antagonists on FSH may at least partially explain the differing effects these agents have on CV risk during ADT. While GnRH antagonists immediately suppress FSH, GnRH agonists provoke a transient surge in FSH that may contribute to the higher CV risk observed with these agents. Additionally, recent studies suggest that GnRH antagonists may significantly reduce CV risk compared to GnRH agonists, particularly in men with pre-existing CV disease. CONCLUSIONS: Patients with cardiovascular risk factors who require ADT may benefit from the better control of FSH provided by GnRH antagonists. ADT itself appears to heighten CV risk, and data suggest that FSH may at least partly drive this risk by promoting inflammation, atherosclerosis, insulin resistance, adipocyte rearrangement and plaque instability.

Spexin and a Novel Cichlid-Specific Spexin Paralog Both Inhibit FSH and LH Through a Specific Galanin Receptor (Galr2b) in Tilapia.

Spexin (SPX) is a 14 amino acid peptide hormone that has pleiotropic functions across vertebrates, one of which is involvement in the brain-pituitary-gonad axis of fish. SPX(1) has been identified in each class of vertebrates, and a second SPX (named SPX2) has been found in some non-mammalian species. We have cloned two spexin paralogs, designated as Spx1a and Spx1b, from Nile tilapia (Oreochromis niloticus) that have varying tissue distribution patterns. Spx1b is a novel peptide only identified in cichlid fish, and is more closely related to Spx1 than Spx2 homologs as supported by phylogenetic, synteny, and functional analyses. Kisspeptin, Spx, and galanin (Gal) peptides and their corresponding kiss receptors and Gal receptors (Galrs), respectively, are evolutionarily related. Cloning of six tilapia Galrs (Galr1a, Galr1b, Galr2a, Galr2b, Galr type 1, and Galr type 2) and subsequent in vitro second-messenger reporter assays for Gαs, Gαq, and Gαi suggests that Gal and Spx activate Galr1a/Galr2a and Galr2b, respectively. A decrease in plasma follicle stimulating hormone and luteinizing hormone concentrations was observed with injections of Spx1a or Spx1b in vivo. Additionally, application of Spx1a and Spx1b to pituitary slices decreased the firing rate of LH cells, suggesting that the peptides can act directly at the level of the pituitary. These data collectively suggest an inhibitory mechanism of action against the secretion of gonadotropins for a traditional and a novel spexin paralog in cichlid species.

A pilot study comparing corifollitropin alfa associated with hp-HMG versus high dose rFSH antagonist protocols for ovarian stimulation in poor responders.

Does corifollitropin alfa associated with hp-HMG protocol from the beginning of ovarian stimulation perform better than high dose rFSH alone for ovarian stimulation with GnRH antagonist in poor responders? This retrospective, monocentric, case-control pilot study was conducted in 65 poor responders (Bologna criteria) undergoing 2 consecutive IVF cycles. All patients underwent a first ovarian stimulation cycle with high dose rFSH (≥300 IU/day) alone in antagonist protocol, unfortunately leading to poor ovarian response and no pregnancy. The following cycle was performed with 150 µg of corifollitropin alfa associated with daily injections of hp-HMG from the beginning of the cycle. The primary outcome was the number of mature oocytes retrieved. The secondary outcomes were ovarian stimulation cancellation and embryo transfer rate per initiated cycle. The number of mature oocytes was not significantly different between the 2 groups. However, cycle cancellation rate was significantly lower and the proportion of cycles with embryo transfer was significantly higher with corifollitropin + hp-HMG protocol, leading to an encouraging clinical pregnancy rate of 24.1% per oocyte retrieval. This pilot study based on corifollitropin alfa associated with hp-HMG from the onset of stimulation appears to be promising for ovarian stimulation in poor responders.

Post interferon therapy decreases male fertility through gonadotoxic effect.

Prevalence of hepatitis C virus (HCV) has been seen in more than 15% of Pakistani population. For the treatment of this infection, only two medicines, interferon, and ribavirin were approved in 1998. The concerned physicians evaluate side effects of these two antiviral drugs only during the treatment period. The long-term extra hepatic side effects are being neglected. This retrospective study was conducted with reference to induced infertility in HCV treated 40 male patients from the period 2008-2015. Possible effects of interferon therapy on fertility hormones and seminal parameters were assessed. Level of fertility hormones like serum Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH), and testosterone was measured. For seminal parameters, guidelines from World Health Organization (WHO) were followed. Among forty cases of HCV patients who received interferon, only 14 (35%) have children and 26 (65%) could not conceive (p = 0.0372). After HCV treatment, HCV positive patients showed a significant change in the level of FSH, LH (p<0.05). Especially, it decreased testosterone level (p=0.0096). Similarly, HCV treatment significantly decreased sperm count (p=0.001) and motility (p=0.0005).

Pharmacogenetics of G-protein-coupled receptors variants: FSH receptor and infertility treatment.

Infertility treatment may represent a paradigmatic example of precision medicine. Follicle-stimulating hormone (FSH) has been proposed as a valuable therapeutic option both in males and in females, even if a standardized approach is far to be established. To date, several genetic mutations as well as polymorphisms have been demonstrated to significantly affect the pathophysiology of FSH-FSH receptor (FSHR) interaction, although the underlying molecular mechanisms remain unclear. This review aims to highlight possible aspects of FSH therapy that could benefit from a pharmacogenetic approach, providing an up-to-date overview of the variability of the response to FSH treatment in both sexes. Specific sections are dedicated to the clinical use of FSH in infertility and how FSHR polymorphisms may affect the therapeutic endpoints.

Follistatin during pregnancy and its potential role as an ovarian suppressing agent.

OBJECTIVE: Ovarian quiescence is a common condition during pregnancy. In vitro, follistatin, an antagonist of follicle-stimulating hormone, blocks follicular development at early stages, and its serum levels increase during pregnancy. A possible surrogate biomarker of ovarian arrest during pregnancy is a decrease in anti-mullerian hormone (AMH) levels followed by an increase in these levels on the second day after labor. The purpose of this study was to determine whether follistatin could act as an ovarian-suppressing agent during pregnancy. Follistatin levels and AMH levels were determined at various stages of pregnancy and postpartum. STUDY DESIGN: The follistatin and AMH levels of 69 patients were retrospectively determined with the AMH Gen II ELISA and with the Human Follistatin Quantikine ELISA Kit. For 49 patients, samples were available from various trimesters for cross-sectional analysis; for the other 20, samples were available longitudinally from day one before labor and then daily on days 1 through 4 after labor. Statistical significance was determined with linear regression, the Friedman rank sum test and the Wilcoxon-Nemenyi-McDonald-Thompson post hoc test. RESULTS: The behavior of follistatin levels was exactly opposite that of AMH levels: Follistatin levels increased significantly during pregnancy and on the first day after parturition but declined afterwards, whereas AMH levels decreased significantly during pregnancy and increased after labor. CONCLUSION: Follistatin can induce ovarian arrest during pregnancy.

Rationale for eliminating the hormone-free interval in modern oral contraceptives.

BACKGROUND: Although most low-dose combined oral contraceptives (COCs) include 7-day hormone-free intervals (HFIs), these COCs could incompletely suppress ovarian activity. OBJECTIVES: To review the impact of HFIs on ovarian suppression and tolerability, and evaluate the utility of COCs without traditional 7-day HFIs. SEARCH STRATEGY: PubMed was searched for clinical studies published in English between January 1980 and April 2015 on the impact of HFIs and HFI modifications in COCs. SELECTION CRITERIA: Articles assessing contraceptive efficacy or tolerability as the primary focus were included. DATA COLLECTION AND ANALYSIS: Abstracts of 319 articles were screened. RESULTS: Analysis of the 161 articles selected revealed that suppression of ovarian activity with low-dose COCs with 7-day HFIs is suboptimal. Loss of ovarian suppression during 7-day HFIs is commonly associated with follicular development, and most dominant follicles appear during this period. By contrast, increased ovarian suppression was noted in regimens that shortened or eliminated the HFI, or that substituted low-dose ethinyl estradiol for the HFI. CONCLUSIONS: Extended regimens with modified HFIs may provide greater ovarian suppression with the potential for increased contraceptive effectiveness. Additional research is needed to evaluate whether COC regimens that include 10µg ethinyl estradiol instead of an HFI may improve tolerability.

Dibutyl phthalate induces oxidative stress and impairs spermatogenesis in adult rats.

Phthalates are abundantly produced plasticizers, and dibutyl phthalate (DBP) is the most widely used derivative in various consumer products and medical devices. This study was conducted to further explore the potential testicular toxicity of DBP in adult rats and to elucidate the underlying mechanisms. Adult male albino rats were treated orally with DBP at doses of 0, 200, 400, or 600 mg/kg/day for 15 consecutive days. Testicular weight, sperm count, and motility were significantly decreased. Treatment with DBP decreased serum follicle-stimulating hormone and testosterone levels and testicular lactate dehydrogenase activity. DBP treatment also decreased serum total antioxidant capacity and the activities of the testicular antioxidant enzymes, such as superoxide dismutase, catalase, and glutathione reductase. Further, DBP treatment provoked degeneration with absence of spermatogenesis and sperms and necrosis in some of seminiferous tubules. These results indicated that oxidative stress and subsequent decrease in testosterone secretion were the potential underlying mechanism of DBP-induced testicular toxicity.

Virtual High-Throughput Screening To Identify Novel Activin Antagonists.

Activin belongs to the TGFß superfamily, which is associated with several disease conditions, including cancer-related cachexia, preterm labor with delivery, and osteoporosis. Targeting activin and its related signaling pathways holds promise as a therapeutic approach to these diseases. A small-molecule ligand-binding groove was identified in the interface between the two activin ßA subunits and was used for a virtual high-throughput in silico screening of the ZINC database to identify hits. Thirty-nine compounds without significant toxicity were tested in two well-established activin assays: FSHß transcription and HepG2 cell apoptosis. This screening workflow resulted in two lead compounds: NUCC-474 and NUCC-555. These potential activin antagonists were then shown to inhibit activin A-mediated cell proliferation in ex vivo ovary cultures. In vivo testing showed that our most potent compound (NUCC-555) caused a dose-dependent decrease in FSH levels in ovariectomized mice. The Blitz competition binding assay confirmed target binding of NUCC-555 to the activin A:ActRII that disrupts the activin A:ActRII complex's binding with ALK4-ECD-Fc in a dose-dependent manner. The NUCC-555 also specifically binds to activin A compared with other TGFß superfamily member myostatin (GDF8). These data demonstrate a new in silico-based strategy for identifying small-molecule activin antagonists. Our approach is the first to identify a first-in-class small-molecule antagonist of activin binding to ALK4, which opens a completely new approach to inhibiting the activity of TGFß receptor superfamily members. in addition, the lead compound can serve as a starting point for lead optimization toward the goal of a compound that may be effective in activin-mediated diseases.

Effects of the environmental contaminants DEHP and TCDD on estradiol synthesis and aryl hydrocarbon receptor and peroxisome proliferator-activated receptor signalling in the human granulosa cell line KGN.

Environmental contaminants binding to transcription factors, such as the aryl hydrocarbon receptor (AhR) and the alpha and gamma peroxisome proliferator-activated receptors (PPARs), contribute to adverse effects on the reproductive system. Expressing both the AhR and PPARs, the human granulosa cell line KGN offers the opportunity to investigate the regulatory mechanisms involved in receptor crosstalk, independent of overriding hormonal control. The aim of the present study was to investigate the impact of two environmental contaminants, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, an AhR ligand) and di-(2-ethylhexyl) phthalate (DEHP, a PPAR ligand), on gonadotrophin sensitivity and estrogen synthesis in KGN cells. Accumulation of the DEHP metabolite mono-(2-ethylhexyl) phthalate (MEHP) in DEHP-exposed cells was measured by high-performance liquid chromatography mass spectrometry, thereby demonstrating DEHP metabolism to MEHP by KGN cells. By employing TCDD ( an AhR agonist), rosiglitazone (a PPARgamma agonist) or bezafibrate (a PPARalpha agonist), the presence of a functional AhR and PPAR cascade was confirmed in KGN cells. Cytotoxicity testing revealed no effect on KGN cell proliferation for the concentrations of TCDD and DEHP used in the current study. FSH-stimulated cells were exposed to TCDD, DEHP or a mix of both and estradiol synthesis was measured by enzyme-linked immunosorbent assay and gene expression by quantitative RT-PCR. Exposure decreased estradiol synthesis (TCDD, DEHP, mix) and reduced the mRNA expression of CYP19 aromatase (DEHP, mix) and FSHR (DEHP). DEHP induced the expression of the alpha and gamma PPARs and AhR, an effect which was inhibited by selective PPAR antagonists. Studies in the human granulosa cell line KGN show that the action of endocrine-disrupting chemicals may be due to a direct activation of AhR, for example by TCDD, and by a transactivation via PPARs, for example by DEHP, inducing subsequent transcriptional changes with a broad range of effects on granulosa cell function.

FSH suppression does not affect bone turnover in eugonadal men.

CONTEXT: In vitro and animal studies have reported conflicting results regarding an independent role for FSH in the regulation of bone turnover. OBJECTIVE: Our objective was to test the hypothesis that suppressing serum FSH while holding serum gonadal steroid levels stable in the eugonadal range will affect biochemical markers of bone metabolism in healthy men. PARTICIPANTS, DESIGN, AND SETTING: Eugonadal men aged 20 to 50 years participated in this randomized controlled trial at a tertiary care academic teaching hospital. INTERVENTIONS: Participants received monthly GnRH analog injections to suppress FSH secretion plus daily topical testosterone gel in prespecified doses (intervention group). Controls received matching placebos (control group). Subjects in the intervention group were individually matched with subjects in the control group to ensure that the mean testosterone and estradiol levels (measured every 4 weeks during the 16-week study period) in the 2 groups were similar. MAIN OUTCOME MEASURES: Biochemical markers of bone resorption (serum N-terminal telopeptide and C-terminal telopeptide), bone formation (serum osteocalcin), and FSH were measured at baseline and after 16 weeks of treatment. RESULTS: Serum FSH declined by 2% in the control group and by 60% in the intervention group (P < .0001 for the between-group difference). Despite the substantial suppression of serum FSH in the intervention group, serum N-terminal telopeptide, C-terminal telopeptide, and osteocalcin did not change in the intervention group, nor were any between-group differences observed. CONCLUSION: When gonadal steroid levels are held constant, short-to midterm suppression of FSH does not affect bone turnover in men. FSH does not appear to be a significant regulator of bone metabolism in eugonadal men.

Inhibition of follicle-stimulating hormone-induced preovulatory follicles in rats treated with a nonsteroidal negative allosteric modulator of follicle-stimulating hormone receptor.

We previously described a negative allosteric modulator (NAM) of FSHR (ADX61623) that blocked FSH-induced cAMP and progesterone production but did not block estradiol production. That FSHR NAM did not affect FSH-induced preovulatory follicle development as evidenced by the lack of an effect on the number of FSH-dependent oocytes found in the ampullae following ovulation with hCG. A goal is the development of a nonsteroidal contraceptive. Toward this end, a high-throughput screen using human FSHR identified an additional nonsteroidal small molecule (ADX68692). Although ADX68692 behaved like ADX61623 in inhibiting production of cAMP and progesterone, it also inhibited FSH-induced estradiol in an in vitro rat granulosa primary cell culture bioassay. When immature, noncycling female rats were injected subcutaneously or by oral dosing prior to exogenous FSH administration, it was found that ADX68692 decreased the number of oocytes recovered from the ampullae. The estrous cycles of mature female rats were disrupted by administration by oral gavage of 25 mg/kg and 10 mg/kg ADX68692. In the highest dose tested (25 mg/kg), 55% of animals cohabited with mature males had implantation sites compared to 33% in the 10 mg/kg group and 77% in the control group. A surprising finding was that a structural analog ADX68693, while effectively blocking progesterone production with similar efficacy as ADX68692, did not block estrogen production and despite better oral availability did not decrease the number of oocytes found in the ampullae even when used at 100 mg/kg. These data demonstrate that because of biased antagonism of the FSHR, nonsteroidal contraception requires that both arms of the FSHR steroidogenic pathway must be effectively blocked, particularly estrogen biosynthesis. Thus, a corollary to these findings is that it seems reasonable to propose that the estrogen-dependent diseases such as endometriosis may benefit from inhibition of FSH action at the ovary using the FSHR NAM approach.

Early luteal phase endocrine profile is affected by the mode of triggering final oocyte maturation and the luteal phase support used in recombinant follicle-stimulating hormone-gonadotropin-releasing hormone antagonist in vitro fertilization cycles.

OBJECTIVE: To assess endocrine differences during early luteal phase according to mode of triggering final oocyte maturation with or without luteal phase support (LPS). DESIGN: A prospective randomized study. SETTING: University center for reproductive medicine. PATIENT(S): Four oocyte donors each underwent four consecutive cycles. INTERVENTION(S): To avoid interpatient variation, each donor underwent the same stimulation regimen. However, different modes of triggering final oocyte maturation and LPS were administered: A) 10,000 IU hCG and standard LPS; B) GnRH agonist (GnRHa; 0.2 mg triptorelin), and 35 hours later 1,500 IU hCG, and standard LPS; C) GnRH agonist (0.2 mg triptorelin) and standard LPS; and D) GnRH agonist (0.2 mg triptorelin) without LPS. MAIN OUTCOME MEASURE(S): Blood sampling was performed on the day of ovulation trigger, ovulation trigger + 1 day, and ovum pick-up + 5 days. Serum E2, FSH, LH, and P were measured. RESULT(S): The early luteal phase steroid levels following GnRHa trigger and modified luteal phase support (B) were similar to those seen after hCG trigger (A). However, significant differences were seen between groups A and B compared with C and D, as well as between groups C and D. CONCLUSION(S): Administration of a single bolus of GnRHa effectively induced LH and FSH surges in oocyte donors stimulated with recombinant FSH and cotreated with a GnRH antagonist. However, gonadotropin and steroid levels differed significantly according to the type of luteal phase support used after GnRHa trigger. EUROPEAN COMMUNITY CLINICAL TRIAL SYSTEM (EUDRACT) NUMBER: 2009-009429-26.

Ovotoxic effects of galactose involve attenuation of follicle-stimulating hormone bioactivity and up-regulation of granulosa cell p53 expression.

Clinical evidence suggests an association between galactosaemia and premature ovarian insufficiency (POI); however, the mechanism still remains unresolved. Experimental galactose toxicity in rats produces an array of ovarian dysfunction including ovarian development with deficient follicular reserve and follicular resistance to gonadotrophins that characterize the basic tenets of human POI. The present investigation explores if galactose toxicity in rats attenuates the bioactivity of gonadotrophins or interferes with their receptor competency, and accelerates the rate of follicular atresia. Pregnant rats were fed isocaloric food-pellets supplemented with or without 35% D-galactose from day-3 of gestation and continuing through weaning of the litters. The 35-day old female litters were autopsied. Serum galactose-binding capacity, galactosyltransferase (GalTase) activity, and bioactivity of FSH and LH together with their receptor competency were assessed. Ovarian follicular atresia was evaluated in situ by TUNEL. The in vitro effects of galactose were studied in isolated whole follicles in respect of generation of reactive oxygen species (ROS) and expression of caspase 3, and in isolated granulosa cells in respect of mitochondrial membrane potential, expression of p53, and apoptosis. The rats prenatally exposed to galactose exhibited significantly decreased serum GalTase activity and greater degree of galactose-incorporation capacity of sera proteins. LH biopotency and LH-FSH receptor competency were comparable between the control and study population, but the latter group showed significantly attenuated FSH bioactivity and increased rate of follicular atresia. In culture, galactose increased follicular generation of ROS and expression of caspase 3. In isolated granulosa cells, galactose disrupted mitochondrial membrane potential, stimulated p53 expression, and induced apoptosis in vitro; however co-treatment with either FSH or estradiol significantly prevented galactose-induced granulosa cell p53 expression. We conclude that the ovotoxic effects of galactose involves attenuation of FSH bioactivity that renders the ovary resistant to gonadotrophins leading to increased granulosa cell expression of p53 and follicular atresia.

Decreased bone mineral density in rats rendered follicle-deplete by an ovotoxic chemical correlates with changes in follicle-stimulating hormone and inhibin A.

Bone loss during perimenopause, an estrogen-sufficient period, correlates with elevated serum follicle-stimulating hormone (FSH) and decreased inhibins A and B. Utilizing a recently described ovotoxin-induced animal model of perimenopause characterized by a prolonged estrogen-replete period of elevated FSH, we examined longitudinal changes in bone mineral density (BMD) and their association with FSH. Additionally, serum inhibin levels were assessed to determine whether elevated FSH occurred secondary to decreased ovarian inhibin production and, if so, whether inhibins also correlated with BMD. BMD of the distal femur was assessed using dual-energy X-ray absorptiometry (DXA) over 19 months in Sprague-Dawley rats treated at 1 month with vehicle or 4-vinylcyclohexene diepoxide (VCD, 80 or 160 mg/kg daily). Serum FSH, inhibins A and B, and 17-ß estradiol (E(2)) were assayed and estrus cyclicity was assessed. VCD caused dose-dependent increases in FSH that exceeded values occurring with natural senescence, hastening the onset and prolonging the duration of persistent estrus, an acyclic but E(2)-replete period. VCD decreased serum inhibins A and B, which were inversely correlated with FSH (r(2) = 0.30 and 0.12, respectively). In VCD rats, significant decreases in BMD (5-13%) occurred during periods of increased FSH and decreased inhibins, while BMD was unchanged in controls. In skeletally mature rats, FSH (r(2) = 0.13) and inhibin A (r(2) = 0.15) correlated with BMD, while inhibin B and E(2) did not. Thus, for the first time, both the hormonal milieu of perimenopause and the association of dynamic perimenopausal changes in FSH and inhibin A with decreased BMD have been reproduced in an animal model.

Dynamics and bifurcation of a model for hormonal control of the menstrual cycle with inhibin delay.

A system of 13 ordinary differential equations with 42 parameters is presented to model hormonal regulation of the menstrual cycle. For an excellent fit to clinical data, the model requires a 36 h time delay for the effect of inhibin on the synthesis of follicle stimulating hormone. Biological and mathematical reasons for this delay are discussed. Bifurcations with respect to changes in three important parameters are examined. One parameter represents the level of estradiol adequate for significant synthesis of luteinizing hormone. Bifurcation diagrams with respect to this parameter reveal an interval of parameter values for which a unique stable periodic solution exists and this solution represents a menstrual cycle during which ovulation occurs. The second parameter measures mass transfer between the first two stages of ovarian development and is indicative of healthy follicular growth. The third parameter is the time delay. Changes in the second parameter and the time delay affect the size of the uniqueness interval defined with respect to the first parameter. Saddle-node, transcritical and degenerate Hopf bifurcations are studied.

Feminizing adrenocortical carcinoma with selective suppression of follicle-stimulating hormone secretion and disorganized steroidogenesis: a case report and literature review.

We report a 61-year-old male with gynecomastia, poor libido and erectile dysfunction. Endocrinological studies showed high levels of estradiol and dehydroepiandrosterone sulfate. Although luteinizing hormone (LH) level was within the normal limit, the concentration of follicle-stimulating hormone (FSH) was under the normal limit. Delayed response of LH and poor response of FSH to gonadotropin-releasing hormone administration were detected. Magnetic resonance imaging of the abdomen revealed a left adrenal tumor. Although the surgically-resected tumor was diagnosed as a high grade ACC based on Weiss's criteria of adrenocortical malignancy, no metastasis was detected. Since estrogen levels normalized after resection, feminizing ACC was confirmed. While LH concentration increased slightly after operation, FSH level became transiently elevated over the normal limit, and finally reached the normal range. These data may suggest that FSH was suppressed selectively by hormone produced by ACC different from estrogen.