Injection of luteinizing hormone or human chorionic gonadotropin increases calcium excretion and serum PTH in males.

Animal and human studies have suggested that sex steroids have calciotropic actions, and it has been proposed that follicle-stimulating hormone (FSH) may exert direct effects on bone. Here, we demonstrate the expression of the receptor for Luteinizing hormone (LH) and human choriogonadotropin (hCG), LHCGR, in human kidney tissue, suggesting a potential influence on calcium homeostasis. To investigate the role of LHCGR agonist on calcium homeostasis in vivo, we conducted studies in male mice and human subjects. Male mice were treated with luteinizing hormone (LH), and human extrapolation was achieved by injecting 5000 IU hCG once to healthy men or men with hypergonadotropic or hypogonadotropic hypogonadism. In mice, LH treatment significantly increased urinary calcium excretion and induced a secondary increase in serum parathyroid hormone (PTH). Similarly, hCG treatment in healthy men led to a significant increase in urinary calcium excretion, serum PTH levels, and 1,25 (OH)2D3, while calcitonin, and albumin levels were reduced, possibly to avoid development of persistent hypocalcemia. Still, the rapid initial decline in ionized calcium coincided with a significant prolongation of the cardiac QTc-interval that normalized over time. The observed effects may be attributed to LH/hCG-receptor (LHCGR) activation, considering the presence of LHCGR expression in human kidney tissue, and the increase in sex steroids occurred several hours after the changes in calcium homeostasis. Our translational study shed light on the intricate relationship between gonadotropins, sex hormones and calcium, suggesting that LHCGR may be influencing calcium homeostasis directly or indirectly.

Denosumab stimulates spermatogenesis in infertile men with preserved Sertoli cell capacity.

Sperm production depends on proper Sertoli-germ cell interaction, and we hypothesized that receptor activator of nuclear factor κB ligand (RANKL) activity in Sertoli cells may influence spermatogenesis. Treatment with the RANKL inhibitor denosumab, normally used to treat osteoporosis, increased testicular weight, inhibin B, and germ cell proliferation in ex vivo testis cultures and in vivo in a humanized RANKL mouse. The effect on germ cell proliferation was positively associated with baseline serum concentrations of anti-müllerian hormone (AMH). In accordance, denosumab increased germ cell proliferation in ex vivo human testis cultures with low/moderate but not severe impairment of Sertoli cell function. In a placebo-controlled randomized clinical trial, denosumab had no effect on semen quality but increased sperm concentration in a subgroup of infertile men with serum AMH ≥38 pmol/L at baseline. In conclusion, high serum AMH may increase the probability of a beneficial response to denosumab treatment in infertile men, thus suggesting a possible venue for precision medicine in male infertility.

Effect of a single-dose denosumab on semen quality in infertile men (the FITMI study): study protocol for a randomized controlled trial.

BACKGROUND: Infertility is a common problem globally and impaired semen quality is responsible for up to 40% of all cases. Almost all infertile couples are treated with either insemination or assisted reproductive techniques (ARTs) independent of the etiology of infertility because no medical treatment exists. Denosumab is an antibody that blocks RANKL signaling and inhibition of testicular RANKL signaling has been suggested to improve semen quality in a pilot study. This RCT aims to assess whether treatment with denosumab can improve spermatogenesis in infertile men selected by serum AMH as a positive predictive biomarker. This paper describes the design of the study. METHODS/DESIGN: FITMI is a sponsor-investigator-initiated, double-blinded, placebo-controlled 1:1, single-center, randomized clinical trial. Subjects will be randomized to receive either a single-dose denosumab 60 mg subcutaneous injection or placebo. The study will be carried out at the Department of Growth and Reproduction, Copenhagen University Hospital, Rigshospitalet, Copenhagen. The primary outcome of the study is defined as the difference in sperm concentration (millions pr. mL) one spermatogenesis (80 days) after inclusion. DISCUSSION: We describe a protocol for a planned RCT aimed at evaluating whether treatment with denosumab can improve the semen quality in infertile men selected by using serum AMH as a positive predictive biomarker. The results will provide evidence crucial for future treatment in a patient group where there is a huge unmet need. TRIAL REGISTRATION: Clinical Trials.gov NCT05212337 . Registered on 14 January 2022. EudraCT 2021-003,451-42. Registered on 23 June 2021. Ethical committee H-21040145. Registered on 23 December 2021.

Possible Relevance of Soluble Luteinizing Hormone Receptor during Development and Adulthood in Boys and Men.

Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) are agonists for the luteinizing hormone receptor (LHCGR) which regulates male reproductive function. LHCGR may be released into body fluids. We wish to determine whether soluble LHCGR is a marker for gonadal function. Cross-sectional, longitudinal, and intervention studies on 195 healthy boys and men and 396 men with infertility, anorchia, or Klinefelter Syndrome (KS) were used to correlate LHCGR measured in serum, seminal fluid, urine, and hepatic/renal artery and vein with gonadal function. LHCGR was determined in fluids from in vitro and in vivo models of human testicular tissue and cell lines, xenograft mouse models, and human fetal kidney and adrenal glands. Western blot showed LHCGR fragments in serum and gonadal tissue of similar size using three different antibodies. The LHCGR-ELISA had no species cross-reactivity or unspecific reaction in mouse serum even after human xenografting. Instead, sLHCGR was released into the media after the culture of a human fetal kidney and adrenal glands. Serum sLHCGR decreased markedly during puberty in healthy boys (p = 0.0001). In healthy men, serum sLHCGR was inversely associated with the Inhibin B/FSH ratio (ß -0.004, p = 0.027). In infertile men, seminal fluid sLHCGR was inversely associated with serum FSH (ß 0.006, p = 0.009), sperm concentration (ß -3.5, p = 0.003) and total sperm count (ß -3.2, p = 0.007). The injection of hCG lowered sLHCGR in serum and urine of healthy men (p < 0.01). In conclusion, sLHCGR is released into body-fluids and linked with pubertal development and gonadal function. Circulating sLHCGR in anorchid men suggests that sLHCGR in serum may originate from and possibly exert actions in non-gonadal tissues. (ClinicalTrials: NTC01411527, NCT01304927, NCT03418896).

RANKL regulates male reproductive function.

Infertile men have few treatment options. Here, we demonstrate that the transmembrane receptor activator of NF-kB ligand (RANKL) signaling system is active in mouse and human testis. RANKL is highly expressed in Sertoli cells and signals through RANK, expressed in most germ cells, whereas the RANKL-inhibitor osteoprotegerin (OPG) is expressed in germ and peritubular cells. OPG treatment increases wild-type mouse sperm counts, and mice with global or Sertoli-specific genetic suppression of Rankl have increased male fertility and sperm counts. Moreover, RANKL levels in seminal fluid are high and distinguishes normal from infertile men with higher specificity than total sperm count. In infertile men, one dose of Denosumab decreases RANKL seminal fluid concentration and increases serum Inhibin-B and anti-Müllerian-hormone levels, but semen quality only in a subgroup. This translational study suggests that RANKL is a regulator of male reproductive function, however, predictive biomarkers for treatment-outcome requires further investigation in placebo-controlled studies.

Vitamin D and sex steroid production in men with normal or impaired Leydig cell function.

Production of testosterone is under tight control by human chorion gonadotropin (hCG) during fetal life and luteinizing hormone (LH) in adulthood. Several animal and human studies have linked vitamin D status with sex steroid production although it is not clear whether there exist a direct or indirect involvement in androgen production. Few studies have investigated this crosslink in young healthy men and putative direct or synergistic effect of activated vitamin D (1,25(OH)2D3) and LH/hCG on sex steroid production in vitro. Here, we present cross-sectional data from 300 young men and 41 hCG-stimulated men with impaired Leydig cell function combined with data from an ex vivo culture of human testicular tissue exposed to 1,25(OH)2D3 alone or in combination with hCG. Serum 25-OHD was positively associated with SHBG (ß:0.002; p = 0.023) and testosterone/estradiol-ratio (ß:0.001; p = 0.039), and inversely associated with free testosterone (%) (free testosterone/total testosterone) (ß:-0.002; p = 0.016) in young men. Vitamin D deficient men had higher total and free estradiol concentrations than men with higher vitamin D status (19% and 18%, respectively; p < 0.01). Interestingly, men with impaired Leydig cell function and vitamin D deficiency had a significantly lower hCG-mediated increase in total and free testosterone compared with vitamin D sufficient men (p < 0.05). Accordingly, testicular tissue exposed to 100 nM 1,25(OH)2D3 had a 15% higher testosterone release into the media compared with vehicle treated specimens (p = 0.030). In conclusion, vitamin D deficiency is associated with lower testosterone/estradiol ratio in young men and lower Leydig cell sensitivity after hCG-stimulation in men with impaired gonadal function. The significant effect of 1,25(OH)2D3 on testosterone production in a human testis model supports that the stimulatory effect at least in part may be direct. Larger placebo-controlled studies are needed to determine whether vitamin D supplementation can influence testosterone production.

Possible link between FSH and RANKL release from adipocytes in men with impaired gonadal function including Klinefelter syndrome.

INTRODUCTION: The FSH receptor (FSHR) has been found to be expressed in human bone cells and bone marrow-adipocytes, and highly-debated mouse studies have suggested extra-gonadal effects of gonadotropins on glucose, adipocyte and bone homeostasis. These putative effects could be direct or indirectly mediated by endocrine factors released from bone-cells or adipocytes. Here, we investigated whether gonadotropins are linked with glucose- and lipid-metabolism in hypergonadotropic men. METHODS: Single centre, cross-sectional study of 307 men with idiopathic infertility and 28 men with Klinefelter syndrome (KS). OUTCOME: associations between serum LH and FSH with soluble-RANKL (sRANKL), osteoprotegerin (OPG), osteocalcin, fasting glucose and insulin, sex steroids, and body composition. Expression of FSHR was studied in human-derived adipocyte-cell-models (hMADS, TERT-hWA) and FSH stimulation of RANKL expression and secretion in hMADS in vitro. RESULTS: Serum FSH was not directly linked with glucose- and lipid-metabolism. However, FSH was inversely associated with sRANKL in both infertile men and KS men (p = .023 and p = .012). Infertile men with elevated FSH (>11 U/L) had significantly lower sRANKL (p = .015). sRANKL was positively associated with fat percentage, fasting insulin, and glucose (all p < .05). Men with prediabetes had higher sRANKL (p = .021), but lower testosterone (p < .0001) and Inhibin B (p = .005). The FSHR was expressed in the investigated human derived adipocytes, and 3-6 h treatment with FSH markedly increased RANKL release (p < .05). CONCLUSION: KS and infertile men with prediabetes have low Inhibin B, and testosterone but elevated RANKL compared with non-prediabetic men despite comparable levels of serum gonadotropins. Serum FSH and sRANKL was inversely associated in both infertile and KS men, but the increased release of RANKL from FSH treated adipocytes suggest a direct effect of FSH on RANKL production in some tissues. Further studies are required to clarify whether FSH targets RANKL in the skeleton. ClinicalTrial_ID:NCT01304927.

Germ Cell Neoplasia in Situ and Preserved Fertility Despite Suppressed Gonadotropins in a Patient With Testotoxicosis.

Context: Testotoxicosis is an autosomal-dominant, male-limited disorder. Activating mutations in the luteinizing hormone receptor gene (LHCGR) cause high autonomous testosterone secretion, resulting in early-onset peripheral precocious puberty. Little is known about long-term consequences of testotoxicosis. Case Description: We present a rare case of a patient followed for 25 years with two remarkable outcomes: preserved fertility and germ cell neoplasia in situ (GCNIS). He presented with precocious puberty at 10 months of age and was diagnosed with testotoxicosis due to a de novo heterozygous Asp578Tyr mutation in LHCGR. Testicular biopsy in childhood showed Leydig cell hyperplasia with altered cell maturation. From infancy throughout adulthood, elevated testosterone and estradiol, low inhibin B and anti-Müllerian hormone, and completely suppressed follicle-stimulating hormone and luteinizing hormone were noted. Height acceleration and advanced bone age resulted in a reduced final height. Semen analysis revealed ongoing spermatogenesis, and the patient fathered a child by natural conception. Ketoconazole treatment decreased circulating testosterone in childhood, supported by experimental suppression of testosterone production in his adult testis tissue cultured ex vivo. At 25 years of age, ultrasound revealed a testicular tumor, identified as a Leydig cell adenoma, but unexpectedly with GCNIS present in adjacent seminiferous tubules. Conclusion: The case illustrates that absence of gonadotropins but high intratesticular testosterone concentration is sufficient for spermatogenesis and to allow fatherhood. Our study is also the first description, to our knowledge, of GCNIS in a patient with testotoxicosis. We recommend regular clinical examination and ultrasonic evaluation of the testes in these patients due to potential increased risk of malignancy.