High incidence of imperforate vagina in ADGRA3-deficient mice.

BACKGROUND: Ten percent of the female population suffers from congenital abnormalities of the vagina, uterus, or oviducts, with severe consequences for reproductive and psychological health. Yet, the underlying causes of most of these malformations remain largely unknown. ADGRA3 (GPR125) is involved in WNT signaling and planar cell polarity, mechanisms vital to female reproductive tract development. Although ADGRA3 is a well-established spermatogonial stem cell marker, its role within the female urogenital system remains unclear. RESULTS: In this study, we found Adgra3 to be expressed throughout the murine female urogenital system, with higher expression pre-puberty than after sexual maturation. We generated a global Adgra3-/- mouse line and observed imperforate vagina in 44% of Adgra3-/- females, resulting in distension of the reproductive tract and infertility. Ovarian morphology, plasma estradiol, ovarian Cyp19a1, and vaginal estrogen receptor α (Esr1) expression were unaffected. However, compared to controls, a significantly lower bone mineral density was found in Adgra3-/- mice. Whereas vaginal opening in mice is an estrogen-dependent process, 17ß-estradiol treatment failed to induce vaginal canalization in Adgra3-/- mice. Furthermore, a marked reduction in vaginal and ovarian progesterone receptor expression was observed concomitant with an upregulation of apoptotic regulators Bcl2, Bid, and Bmf in adult Adgra3-/- females with a closed vagina. CONCLUSIONS: Our collective results shed new insights into the complex mechanisms by which the adhesion receptor ADGRA3 regulates distal vaginal tissue remodeling during vaginal canalization via altered sex hormone responsiveness and balance in apoptotic regulators. This highlights the potential of ADGRA3 as a target in diagnostic screening and/or therapy for obstructive vaginal malformations in humans.

Low-serum antimüllerian hormone is linked with poor semen quality in infertile men screened for participation in a randomized controlled trial.

OBJECTIVE: To investigate possible associations between serum antimüllerian hormone (AMH) concentration and semen quality in infertile men. Studies investigating the associations between serum AMH concentration and semen quality in infertile men have shown conflicting results. DESIGN: Infertile men were included during screening for participation in the First in Treating Male Infertility Study, a double-blinded, placebo-controlled, 1:1, single-center randomized controlled trial. SETTING: Not applicable. PATIENTS: At the screening visit, 400 participants produced a semen sample and had their serum analyzed for AMH concentration. INTERVENTION: Not applicable. MAIN OUTCOME MEASURES: Serum AMH concentration and semen quality. RESULTS: All men were stratified according to serum AMH concentrations in quartiles (Q1-Q4). Men in the lowest quartile had a lower sperm concentration (1 × 106/mL) (Q1: 8.0 vs. Q2: 10.4 vs. Q3: 11.0 vs. Q4: 13.0), total sperm count (1 × 106) (Q1: 29.1 vs. Q2: 38.2 vs. Q3: 44.4 vs. Q4: 55.7), sperm motility (%) (Q1: 41 vs. Q2: 57 vs. Q3: 50 vs. Q4: 53), and progressive sperm motility (%) (Q1: 31 vs. Q2: 44 vs. Q3: 35 vs. Q4: 40) compared with the other quartiles. Moreover, men with a sperm concentration <2 million/mL had a lower serum AMH concentration compared with men having 2-16 × 106 /mL and >16 × 106/mL (31 pmol/L vs. 38 pmol/L vs. 43 pmol/L, respectively). In accordance, men with sperm motility <20% had a lower serum AMH concentration compared with men with sperm motility 20%-42%, and >42% (31 pmol/L vs. 43 pmol/L. vs. 39 pmol/L, respectively). CONCLUSION: This study shows that low serum AMH concentration is associated with poor semen quality in infertile men, which implies that serum AMH concentration may have clinical value during the evaluation of male infertility. CLINICAL TRIAL REGISTRATION NUMBER: NCT05212337.

Cancer risk in patients treated with denosumab compared with alendronate: A population-based cohort study.

BACKGROUND: Antiresorptive treatment is currently used in millions of patients with osteoporosis and cancer worldwide. Early studies of denosumab suggested a small signal in ovarian cancer incidence and emerging data suggest that denosumab stimulates germ cell proliferation in the gonads. This study aims to determine the association between the use of denosumab and the risk of reproductive cancers compared with the use of alendronate. RESEARCH DESIGN AND METHODS: Using a cohort study design, we used the Danish nationwide registries to identify a population of subjects ≥50 years of age during 2010-2017 who started denosumab after being on alendronate treatment for at least six months. The cohort was matched 1:2 with patients who had been treated with alendronate alone for at least six months. The risk of reproductive cancers and the risk difference between groups were estimated using the Longitudinal Targeted Maximum Likelihood Estimation (L-TMLE) method. RESULTS: We identified 6054 Danish individuals who underwent treatment with denosumab. These individuals were matched with 12,108 receiving alendronate. The absolute risk of reproductive cancer was 1.05 % (95 % CI 0.75-1.34) after three years for denosumab users and was not different 0.03 % (-0.34-0.39) than for alendronate users. In supplemental analyses, there was no increased risk of non-reproductive cancers associated with the use of denosumab (risk difference of 0.54 % (-0.41-1.19). Analysis comparing denosumab users with the general population gave similar results. CONCLUSION: There was no difference in the risk of cancer following treatment with denosumab compared to treatment with alendronate assessed after a short follow-up of 3 years.

High-dose vitamin D3 supplementation shows no beneficial effects on white blood cell counts, acute phase reactants, or frequency of respiratory infections.

BACKGROUND: Vitamin D has been suggested to influence the immune system, and vitamin D metabolites and the vitamin D receptor (VDR) are generated and expressed in white blood cells (WBC). Moreover, vitamin D status has been associated with incidence and prognosis of some respiratory tract infections (RTI). Therefore, we investigated the effect of vitamin D3 supplementation on WBC, acute phase reactants (APR), and the risk of developing RTIs. METHODS: A double-blinded, randomized, placebo-controlled clinical trial of 307 infertile men with multiple secondary immunological endpoints. The vitamin D3 group (n = 151) initially received 300,000 IU (7,500 µg) cholecalciferol once - followed by 1,400 IU (35 µg) daily for 150 days. The placebo group (n = 156) did not receive active ingredients. RESULTS: At baseline, stratification into clinically relevant groups of vitamin D status (< 25; 25-50; 50-75; >75 nmol/L), showed an inverse association with total leucocyte concentrations (7.0 vs. 6.0 vs. 6.0 vs. 5.5 (109/L); p = 0.007), lymphocytes (2.4 vs. 2.1 vs. 2.0 vs. 2.0 (109/L); p = 0.048), CRP (2.0 vs. 1.7 vs. 1.2 vs. 1.2 (mg/L); p = 0.037), and orosomucoid (0.82 vs. 0.77 vs. 0.76 vs. 0.70 (g/L); p = 0.015). After 150 days, no differences were detected in WBC counts or APRs between the vitamin D3 and the placebo group. However, vitamin D3 treated men had a higher prevalence of self-reported RTIs compared with the placebo group (55% vs. 39%; p = 0.005). CONCLUSIONS: High-dose vitamin D3 supplementation did not alter WBCs or APRs, but a higher prevalence of respiratory infections was observed in the vitamin D3 group. Serum 25(OH)D3 was negatively correlated with most WBCs, indicating that vitamin D status may be linked with inflammation and WBC turnover, but not an important determinant of developing RTIs. TRIAL REGISTRATION: NCT01304927 (ClinicalTrials.gov). Registered February 20, 2011.

Effects of vitamin D on sex steroids, luteinizing hormone, and testosterone to luteinizing hormone ratio in 307 infertile men.

OBJECTIVE: Vitamin D status has been associated with sex steroid production. The question is whether vitamin D supplementation has an impact on sex steroid production in infertile men with vitamin D insufficiency? DESIGN: A single-center, double-blinded, randomized clinical trial. Differences in sex steroids and reproductive hormones were predefined secondary outcomes, vitamin D status at baseline was a predefined subgroup and the primary outcome was differences in semen quality. METHODS: A total of 307 infertile men were included and randomized 1:1 to active or placebo treatment for 150 days. Men in the active group initially received an oral bolus of 300,000 IU cholecalciferol, followed by daily supplementation with 1400 IU cholecalciferol and 500 mg calcium. RESULTS: After intervention, no differences were found in serum concentrations of sex steroids, luteinizing hormone, testosterone/luteinizing hormone ratio or SHBG between the vitamin D and placebo group. However, in a predefined subgroup analysis of men with serum 25OHD ≤ 50 nmol/L, men treated with vitamin D had a significantly higher testosterone/luteinizing hormone ratio [4.2 (3.8-4.4) vs. 3.7 (3.4-4.0); p = 0.033] compared with placebo treatment. In men with vitamin D deficiency, the difference between groups was larger but not significant due to few men with serum 25OHD < 25 nmol/L. CONCLUSION: Vitamin D + calcium supplementation did not alter sex steroid production in infertile men. However, vitamin D insufficient men treated with vitamin D supplementation had a significantly higher testosterone/LH ratio compared with placebo-treated men, suggesting that optimal Leydig cell function are dependent on adequate vitamin D status.

High-dose cholecalciferol supplementation to obese infertile men is sufficient to reach adequate vitamin D status.

Obesity is associated with low vitamin D status, and the optimal supplement and dosage of cholecalciferol (vitamin D3) or calcidiol (25OHD) for individuals with obesity have been debated. We aimed to determine the effect of high-dose vitamin D3 supplementation on achieving adequate vitamin D levels among infertile men with normal weight v. obesity. Here, we present secondary end points from a single-centre, double-blinded, randomised clinical trial, comprising 307 infertile men randomised to active or placebo treatment for 150 days. Men in the active group initially received an oral bolus of 300 000 mg of vitamin D3, followed by daily supplementation with 1400 mg of vitamin D3 and 500 mg of calcium. Baseline BMI was listed as a predefined subgroup. At baseline, serum 25OHD was significantly higher in men with normal weight (BMI < 25 kg/m2) compared with men with overweight (BMI 25-30 kg/m2) and obesity (BMI > 30 kg/m2) (48 nmol/l v. 45 nmol/l and 39 nmol/l, respectively; P = 0·024). After the intervention, men with normal weight, overweight and obesity treated with vitamin D3 had a significantly higher serum 25OHD compared with corresponding placebo-treated men (BMI < 25 kg/m2: 92 nmol/l v. 53 nmol/l, BMI = 25-30 kg/m2: 87 nmol/l v. 49 nmol/l and BMI > 30 kg/m2: 85 nmol/l v. 48 nmol/l; P < 0·001 for all, respectively). In conclusion, we show that high-dose vitamin D3 supplementation to infertile men with obesity and low vitamin D status is sufficient to achieve adequate serum 25OHD levels.

Calcium and vitamin D homoeostasis in male fertility.

Calcium and vitamin D have well-established roles in maintaining calcium balance and bone health. Decades of research in human subjects and animals have revealed that calcium and vitamin D also have effects on many other organs including male reproductive organs. The presence of calcium-sensing receptor, vitamin D receptor, vitamin D activating and inactivating enzymes and calcium channels in the testes, male reproductive tract and human spermatozoa suggests that vitamin D and calcium may modify male reproductive function. Functional animal models have shown that vitamin D deficiency in male rodents leads to a decrease in successful mating and fewer pregnancies, often caused by impaired sperm motility and poor sperm morphology. Human studies have to a lesser extent validated these findings; however, newer studies suggest a positive effect of vitamin D supplementation on semen quality in cases with vitamin D deficiency, which highlights the need for initiatives to prevent vitamin D deficiency. Calcium channels in male reproductive organs and spermatozoa contribute to the regulation of sperm motility and capacitation, both essential for successful fertilisation, which supports a need to avoid calcium deficiency. Studies have demonstrated that vitamin D, as a regulator of calcium homoeostasis, influences calcium influx in the testis and spermatozoa. Emerging evidence suggests a potential link between vitamin D deficiency and male infertility, although further investigation is needed to establish a definitive causal relationship. Understanding the interplay between vitamin D, calcium and male reproductive health may open new avenues for improving fertility outcomes in men.

Low serum anti-Müllerian hormone is associated with semen quality in infertile men and not influenced by vitamin D supplementation.

BACKGROUND: Anti-Müllerian hormone (AMH) is released by testicular Sertoli cells and of great importance during fetal male sexual development, but less is known about the role of circulating AMH during adulthood. In vitro studies have shown that vitamin D may induce AMH transcription, but a controlled trial investigating the possible effect of vitamin D on serum AMH has not been conducted in men. METHODS: A single-center, double-blinded, randomized placebo-controlled clinical trial (NCT01304927) conducted in Copenhagen, Denmark. A total of 307 infertile men were included and randomly assigned (1:1) to a single dose of 300,000 IU cholecalciferol followed by 1400 IU cholecalciferol + 500 mg of calcium daily (n = 151) or placebo (n = 156) for 150 days. Difference in serum AMH was a predefined secondary endpoint. Explorative outcomes were associations between serum AMH and gonadal function in infertile men. The primary endpoint was difference in semen quality and has previously been published. RESULTS: Infertile men in the lowest AMH tertile had significantly lower sperm concentration (∆T3-1 16 mill/mL (228%); P < 0.001), sperm count (∆T3-1 55 million (262%); P < 0.001), motile sperm count (∆T3-1 28 million (255%); P < 0.001), progressive motile sperm count (∆T3-1 18 million (300%); P < 0.001), testis size (∆T3-1 2.7 mL (16%); P < 0.001), serum inhibin B (∆T3-1 72 pg/mL (59%); P < 0.001), inhibin B/FSH ratio (∆T3-1 48 (145%); P < 0.001), and higher FSH (∆T3-1 2.6 (38%); P < 0.001) than the tertile of infertile men with highest serum AMH. Vitamin D supplementation had no effect on serum AMH compared with placebo treatment. CONCLUSIONS: In infertile men, low serum AMH is associated with severely impaired gonadal function illustrated by poor semen quality and lower testosterone/LH ratio. Serum AMH in infertile men was not influenced by vitamin D supplementation.

Loss of Adgra3 causes obstructive azoospermia with high penetrance in male mice.

The adhesion receptor ADGRA3 (GPR125) is a known spermatogonial stem cell marker, but its impact on male reproduction and fertility has not been examined. Using a mouse model lacking Adgra3 (Adgra3-/- ), we show that 55% of the male mice are infertile from puberty despite having normal spermatogenesis and epididymal sperm count. Instead, male mice lacking Adgra3 exhibited decreased estrogen receptor alpha expression and transient dilation of the epididymis. Combined with an increased estradiol production, this indicates a post-pubertal hormonal imbalance and fluid retention. Dye injection revealed a blockage between the ejaculatory duct and the urethra, which is rare in mice suffering from infertility, thereby mimicking the etiologies of obstructive azoospermia found in human male infertility. To summarize, male reproductive tract development is dependent on ADGRA3 function that in concert with estrogen signaling may influence fluid handling during sperm maturation and storage.

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.

RANKL regulates testicular cancer growth and Denosumab treatment has suppressive effects on GCNIS and advanced seminoma.

BACKGROUND: Testicular germ cell tumours (TGCTs) have a high sensitivity to chemotherapy and a high cure rate, although with serious adverse effects. In the search for tumour suppressive drugs, the RANKL inhibitor Denosumab, used to treat osteoporosis, came up as a candidate since RANKL signalling was recently identified in the testis. METHODS: Expression of RANKL, RANK and OPG, and the effects of RANKL inhibition were investigated in human TGCTs, TGCT-derived cell-lines, and TGCT-xenograft models. Serum RANKL was measured in TGCT-patients. RESULTS: RANKL, RANK, and OPG were expressed in germ cell neoplasia in situ (GCNIS), TGCTs, and TGCT-derived cell lines. RANKL-inhibition reduced proliferation of seminoma-derived TCam-2 cells, but had no effect on embryonal carcinoma-derived NTera2 cells. Pretreatment with Denosumab did not augment the effect of cisplatin in vitro. However, inhibition of RANKL in vivo reduced tumour growth exclusively in the TCam-2-xenograft model and Denosumab-treatment decreased proliferation in human GCNIS cultures. In TGCT-patients serum RANKL had no prognostic value. CONCLUSIONS: This study shows that the RANKL signalling system is expressed in GCNIS and seminoma where RANKL inhibition suppresses tumour growth in vitro and in vivo. Future studies are needed to determine whether RANKL is important for the malignant transformation or transition from GCNIS to invasive tumours.

Vitamin D Supplementation Improves Fasting Insulin Levels and HDL Cholesterol in Infertile Men.

CONTEXT: Vitamin D has been linked with glucose and lipid metabolism. Men with impaired gonadal function have a higher risk of metabolic syndrome and mortality, and vitamin D status may be a reversible modulator. OBJECTIVE: This work aimed to determine the effect of daily vitamin D and calcium supplementation for 150 days on glucose and lipid homeostasis in infertile men. METHODS: A single-center, double-blinded, randomized clinical trial (NCT01304927) was conducted. A total of 307 infertile men were randomly assigned (1:1) to a single dose of 300 000 IU cholecalciferol followed by 1400 IU cholecalciferol + 500 mg of calcium daily (n = 151) or placebo (n = 156) for 150 days. Reported metabolic parameters including fasting plasma glucose, glycated hemoglobin A1c, fasting serum insulin, homeostatic model assessment of insulin resistance (HOMA-IR), fasting plasma cholesterols, and triglycerides were secondary end points. The primary end point semen quality has previously been reported. RESULTS: Men receiving vitamin D supplementation improved their vitamin D status, whereas vitamin D status was aggravated in the placebo group characterized by higher serum parathyroid hormone. At the end of the trial, men receiving vitamin D supplementation had 13% lower fasting serum insulin concentrations compared with the placebo-treated group (65 vs 74 pmol/L, P = .018) and 19% lower HOMA-IR (2.2 vs 2.7, P = .025). Moreover, men in the vitamin D group had higher high-density lipoprotein (HDL) cholesterol levels (1.38 vs 1.32 mmol/L, P = .008) compared with the placebo group. CONCLUSION: High-dose vitamin D supplementation has beneficial effects on glucose homeostasis and HDL cholesterol levels in infertile men.

Treatment options for hypercalcemia after cosmetic oil injections: Lessons from human tissue cultures and a pilot intervention study.

OBJECTIVE: Granuloma formation following self-administered cosmetic oil injections can lead to severe hypercalcemia and renal calcifications due to extra-renal vitamin D activation. This translational study aims to identify Prednisolone sparing therapeutics for hypercalcemia after development of granulomatous disease secondary to paraffin oil injections. MATERIALS AND METHODS: Granuloma tissue isolated from five men were cultured ex vivo and treated with selected drugs to block generation of activated vitamin D (1,25(OH)2D3). In a retrospective study, we included data before and during different treatments of 21 men with paraffin oil induced granulomatous hypercalcemia (46 treatment courses) where serum calcium, parathyroid hormone, vitamin D metabolites, creatinine and inflammatory markers were measured. RESULTS: Addition of Ketoconazole or Ciclosporin to granuloma tissue ex vivo culture, significantly suppressed production of 1,25(OH)2D3 after 48 h (both p < 0.05). Prednisolone was the first treatment option in most men and lowered serum levels of ionized calcium after 1, 2, 3 and 6 months compared with baseline (p < 0.05). Ketoconazole or Hydroxychloroquine had no significant effect on serum calcium levels and were unable to reduce the concomitant daily Prednisolone doses (p > 0.05). Azathioprine did not reduce calcium levels. However, addition of Tacrolimus to Prednisolone treatment enabled a reduction in Prednisolone dose after 3 months (p = 0.014), but with no additional effect on calcium homeostasis. CONCLUSION: This study verifies that Prednisolone is an effective treatment and suggests that calcineurin inhibitors may be used as Prednisolone sparing treatment for paraffin oil-induced granulomatous hypercalcemia. Randomized clinical trials are needed to determine clinical efficacy.

Prediabetes Defined by First Measured HbA1c Predicts Higher Cardiovascular Risk Compared With HbA1c in the Diabetes Range: A Cohort Study of Nationwide Registries.

OBJECTIVE: To assess the risk of major adverse cardiovascular events (MACE), all-cause mortality, and initiation of medical treatment in subjects with prediabetes according to first-time measured HbA1c. RESEARCH DESIGN AND METHODS: Through registry databases, we identified 326,305 Danish patients with a first HbA1c between 40 and 51 mmol/mol (5.8-6.8%) from 2011 to 2017. After exclusion of patients with prior disease, 84,678 patients were followed 12 months after first HbA1c measurement. Cox regression models were used to estimate hazard ratios (HRs) of MACE and standardized absolute risks. Cumulative incidences were used to analyze initiation of glucose-lowering, antihypertensive, cholesterol-lowering, and antithrombotic medication. RESULTS: The 12-month risk of MACE and all-cause mortality increased gradually with increasing HbA1c until 47 mmol/mol (6.5%). In comparisons of subjects with HbA1c 40-41 mmol/mol (5.8-5.9%), subjects with HbA1c 46-47 mmol/mol (6.4-6.5%) had a 0.79% (95% CI 0.33-1.24) higher standardized absolute risk and an HR of 2.21 (95% CI 1.67-2.92) of MACE. Patients with HbA1c 48-49 mmol/mol (6.5-6.6%) had a 0.09% (95% CI -0.35 to 0.52) lower absolute risk and an HR of 1.33 (95% CI 0.87-2.05) of MACE. Initiation of medication was significantly lower among patients with HbA1c of 46-47 mmol/mol (6.4-6.5%) than among patients with HbA1c of 48-49 mmol/mol (6.5-6.6%). CONCLUSIONS: In the Danish population screened for diabetes with HbA1c, the highest risk of MACE and all-cause mortality was found in subjects with HbA1c just below the diagnostic threshold for diabetes. Our results highlight the need for increased focus on the treatment of cardiovascular risk factors for subjects with prediabetes.

Calcium transport in male reproduction is possibly influenced by vitamin D and CaSR.

Vitamin D is important for gonadal function in rodents, and improvement of vitamin D status in men with low sperm counts increases live birth rate. Vitamin D is a regulator of transcellular calcium transport in the intestine and kidney and may influence the dramatic changes in the luminal calcium concentration in epididymis. Here, we show spatial expression in the male reproductive tract of vitamin D receptor (VDR)-regulated factors involved in calcium transport: transient receptor potential vanilloid 5/6 , sodium/calcium exchanger 1, plasma membrane calcium ATPase 1, calbindin D9k, calcium-sensing receptor (CaSR), and parathyroid hormone-related peptide (PTHrP) in mouse and human testis and epididymis. Testicular Casr expression was lower in Vdr ablated mice compared with controls. Moreover, expression levels of Casr and Pthrp were strongly correlated in both testis and epididymis and Pthrp was suppressed by 1,25(OH)2D3 in a spermatogonial cell line. The expression of CaSR in epididymis may be of greater importance than in the gonad in mice as germ cell-specific Casr deficient mice had no major reproductive phenotype, and coincubation with a CaSR-agonist had no effect on human sperm-oocyte binding. In humans, seminal calcium concentration between 5 and 10 mM was associated with a higher fraction of motile and morphologically normal sperm cells, and the seminal calcium concentration was not associated with serum calcium levels. In conclusion, VDR regulates CaSR and PTHrP, and both factors may be involved in the regulation of calcium transport in the male reproductive tract with possible implications for sperm function and storage.

Severe hypocalcemia due to hypoparathyroidism associated with HIV: A case report.

Calcemia is not routinely determined among people living with human immunodeficiency virus (HIV). In people living with HIV, the most frequent electrolyte disturbance is hyponatremia and since symptoms of hypocalcemia often are unspecific, calcium is typically measured with some delay. Hypocalcemia in people living with HIV is mainly due to indirect causes such as vitamin D deficiency, renal failure, or drug related. However, in rare cases direct viral involvement of the parathyroid glands has been reported. We present a case of a 67-year-old male living with HIV who presented at an emergency department with symptomatic severe hypocalcemia, without any previous history of neck surgery, radiation therapy or large infections in the head and neck area. At the time of admission serum concentrations were for ionized calcium 0.98 mmol/L (ref. 1.18-1.32 mmol/L) and PTH 1.3 mmol/L (ref. 2.0-8.5 pmol/L). Vitamin D status was sufficient with 25OHD at 73 nmol/L to 112 nmol/L (ref. 60-160 nmol/L) from 2016 through 2019. The patient was diagnosed with primary hypoparathyroidism and was treated with Alphacalcidol 0,5 µg × 1/daily, calcium 500 mg × 4 the first day followed by 400 mg × 2 and magnesium 360 mg × 3, which induced rapid clinical recovery with dissolvement of muscular pain and biochemical improvement. This case study suggests that further studies are needed to investigate the added value of routine monitoring for hypocalcemia as part of clinical follow-up of people living with HIV.

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.

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).

Hypercalcemia After Cosmetic Oil Injections: Unraveling Etiology, Pathogenesis, and Severity.

Intramuscular injections of paraffin oil can cause foreign body granuloma formation and hypercalcemia. Macrophages with the ability to produce high levels of 1,25(OH)2 D3 may induce the mineral disturbance, but no major series of patients have been published to date. Here, medical history, physical evaluation, biochemical, and urinary analysis for calcium homeostasis were obtained from 88 males, who 6 years previously had injected paraffin or synthol oil into skeletal muscle. Moreover, granuloma tissue from three men was cultured for 48 hours ex vivo to determine 1,25(OH)2 D3 production supported by qPCR and immunohistochemistry of vitamin D metabolism and immune cell populations after treatment with 14 different drugs. The 88 men were stratified into men with hypercalcemia (34%), whereas normocalcemic men were separated into men with either normal (42%) or suppressed parathyroid hormone (PTH) (24%). All men had high calcium excretion, and nephrolithiasis was found in 48% of hypercalcemic men, 22% of normocalcemic men with normal PTH, and 47% of normocalcemic men with suppressed PTH. Risk factors for developing hypercalcemia were oil volume injected, injection of heated oil, high serum interleukin-2 receptor levels, and high urine calcium. High 1,25(OH)2 D3 /25OHD ratio, calcium excretion, and low PTH was associated with nephrolithiasis. The vitamin D activating enzyme CYP27B1 was markedly expressed in granuloma tissue, and 1,25(OH)2 D3 was released in concentrations corresponding to 40% to 50% of the production by human kidney specimens. Dexamethasone, ketoconazole, and ciclosporin significantly suppressed granulomatous production of 1,25(OH)2 D3 . In conclusion, this study shows that injection of large oil volumes alters calcium homeostasis and increases the risk of nephrolithiasis. Hypercalciuria is an early sign of disease, and high granulomatous 1,25(OH)2 D3 production is part of the cause. Prospective clinical trials are needed to determine if ciclosporin, ketoconazole, or other drugs can be used as prednisolone-sparing treatment. © 2020 American Society for Bone and Mineral Research (ASBMR).

The Calcium-Sensing Receptor Is Essential for Calcium and Bicarbonate Sensitivity in Human Spermatozoa.

CONTEXT: The calcium-sensing receptor (CaSR) is essential to maintain a stable calcium concentration in serum. Spermatozoa are exposed to immense changes in concentrations of CaSR ligands such as calcium, magnesium, and spermine during epididymal maturation, in the ejaculate, and in the female reproductive environment. However, the role of CaSR in human spermatozoa is unknown. OBJECTIVE: This work aimed to investigate the role of CaSR in human spermatozoa. METHODS: We identified CaSR in human spermatozoa and characterized the response to CaSR agonists on intracellular calcium, acrosome reaction, and 3',5'-cyclic adenosine 5'-monophosphate (cAMP) in spermatozoa from men with either loss-of-function or gain-of-function mutations in CASR and healthy donors. RESULTS: CaSR is expressed in human spermatozoa and is essential for sensing extracellular free ionized calcium (Ca2+) and Mg2+. Activators of CaSR augmented the effect of sperm-activating signals such as the response to HCO3- and the acrosome reaction, whereas spermatozoa from men with a loss-of-function mutation in CASR had a diminished response to HCO3-, lower progesterone-mediated calcium influx, and were less likely to undergo the acrosome reaction in response to progesterone or Ca2+. CaSR activation increased cAMP through soluble adenylyl cyclase (sAC) activity and increased calcium influx through CatSper. Moreover, external Ca2+ or Mg2+ was indispensable for HCO3- activation of sAC. Two male patients with a CASR loss-of-function mutation in exon 3 presented with normal sperm counts and motility, whereas a patient with a loss-of-function mutation in exon 7 had low sperm count, motility, and morphology. CONCLUSION: CaSR is important for the sensing of Ca2+, Mg2+, and HCO3- in spermatozoa, and loss-of-function may impair male sperm function.