A cryptozoospermic infertile male with Y chromosome AZFc microdeletion and low FSH levels due to a simultaneous polymorphism in the FSHB gene: a case report.

Genetic causes account for 10-15% of male factor infertility, making the genetic investigation an essential and useful tool, mainly in azoospermic and severely oligozoospermic men. In these patients, the most frequent findings are chromosomal abnormalities and Y chromosome long arm microdeletions, which cause a primary severe spermatogenic impairment with classically increased levels of FSH. On the other hand, polymorphisms in the FSH receptor (FSHR) and FSH beta chain (FSHB) genes have been associated with different FSH plasma levels, due to variations in the receptor sensitivity (FSHR) or in the production of FSH from the pituitary gland (FSHB). Here, we describe an unusual patient with a combined genetic alteration (classic AZFc deletion of the Y chromosome and TT homozygosity for the -211G>T polymorphism in the FSHB gene (rs10835638)), presenting with cryptozoospermia, severe hypospermatogenesis, and normal LH and testosterone plasma concentrations, but low FSH levels. The patient partially benefitted from treatment with FSH (150 IU three times/week for 6 months) which allowed him to cryopreserve enough motile spermatozoa to be used for intracytoplasmic sperm injection. According to our knowledge, this is the first report of an infertile man with AZFc microdeletion with low FSH plasma concentrations related to homozygosity for the -211G>T polymorphism in the FSHB gene.

Osteoporosis and bone metabolism in patients with Klinefelter syndrome.

Low bone mass is common in men with Klinefelter syndrome (KS), with a prevalence of 6-15% of osteoporosis and of 25-48% of osteopenia. Reduced bone mass has been described since adolescence and it might be related to both reduced bone formation and higher bone resorption. Although reduced testosterone levels are clearly involved in the pathogenesis, this relation is not always evident. Importantly, fracture risk is increased independently from bone mineral density (BMD) and testosterone levels. Here we discuss the pathogenesis of osteoporosis in patients with KS, with a particular focus on the role of testosterone and testis function. In fact, other hormonal mechanisms, such as global Leydig cell dysfunction, causing reduced insulin-like factor 3 and 25-OH vitamin D levels, and high follicle-stimulating hormone and estradiol levels, might be involved. Furthermore, genetic aspects related to the supernumerary X chromosome might be involved, as well as androgen receptor expression and function. Notably, body composition, skeletal mass and strength, and age at diagnosis are other important aspects. Although dual-energy x-ray absorptiometry is recommended in the clinical workflow for patients with KS to measure BMD, recent evidence suggests that alterations in the microarchitecture of the bones and vertebral fractures might be present even in subjects with normal BMD. Therefore, analysis of trabecular bone score, high-resolution peripheral quantitative computed tomography and vertebral morphometry seem promising tools to better estimate the fracture risk of patients with KS. This review also summarizes the evidence on the best available treatments for osteoporosis in men with KS, with or without hypogonadism.

Estradiol-Testosterone Imbalance Is Associated with Erectile Dysfunction in Patients with Klinefelter Syndrome.

Erectile dysfunction (ED) is a frequent sexual disorder in adult men. Klinefelter syndrome (KS) is the most common sex chromosomal disorder and a frequent cause of male hypogonadism. Psychological and cognitive aspects are quite typical in KS and have been linked to ED, while the role of testosterone (T) levels in sexual function of KS subjects has not been fully elucidated. The purpose of the present study is to investigate the role of hormonal disturbances in erectile function of subjects with KS. We conducted a retrospective study involving 52 Klinefelter patients newly diagnosed who never received androgen replacing therapy. All the subjects underwent medical history, accurate physical examination, and blood tests. The International Index of Erectile Function questionnaire (IIEF-EF) score correlated negatively with estradiol/testosterone ratio (E2/T); this correlation remained statistically significant after correction for age (ρ -0.320 p = 0.018). A multiple linear regression analysis identified age and E2/T as the main predictors of IIEF-EF score (R2 0.169 F = 3.848 p = 0.008). Our findings corroborate previous KS data obtained in the general population showing an association between higher E2/T ratio and impaired erectile function. Larger studies are required to better elucidate the pathophysiology of ED in patients with KS.

Sperm Count and Hypogonadism as Markers of General Male Health.

BACKGROUND: Some evidence suggests that infertile men, who are at increased risk for hypogonadism, metabolic derangements, and osteoporosis, have higher long-term morbidity and mortality than controls, but data are scarce and not conclusive. OBJECTIVE: We tested whether semen quality and reproductive function could represent a marker of general male health. DESIGN, SETTING, AND PARTICIPANTS: A retrospective study of 5177 individuals from a prospectively collected database of 11516 males of infertile couples who had semen analysis in a tertiary university center. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Of them, 5177 had all data for reproductive hormones, testis ultrasound, and biochemical determinations for glucose and lipid metabolism. Hypogonadism was defined as testosterone <10.5nmol/l and/or luteinizing hormone >9.4 IU/l. Individuals with a total sperm count of <10 million had genetic testing (karyotype, Y chromosome microdeletions, and CFTR gene mutations) and those with hypogonadism underwent dual-energy x-ray absorptiometry for bone mineral density. Descriptive statistics and odds ratio (OR) calculation were used. RESULTS AND LIMITATIONS: Men with a low sperm count (<39 million/ejaculate) are at a high risk of hypogonadism (OR 12.2, 95% confidence interval [CI] 10.2-14.6) and have higher body mass index, waist circumference, systolic pressure, low-density lipoprotein cholesterol, triglycerides, and homeostatic model assessment (HOMA) index; lower high-density lipoprotein cholesterol; and a higher prevalence of metabolic syndrome (OR 1.246, 95 CI 1.005-1.545). All data are worse in men with hypogonadism, but a low sperm count per se is associated with a poor metabolic parameter. Men with hypogonadism have lower bone mineral density and 51% prevalence of osteoporosis/osteopenia. Longitudinal studies are necessary to support these data. CONCLUSIONS: This is the largest study with comprehensive evaluation of semen quality and reproductive function, etiology and risk factor determination, and metabolic, cardiovascular, and osteoporosis risk assessment, performed in men referred for fertility evaluation. A low sperm count is associated with poorer metabolic, cardiovascular, and bone health. Hypogonadism is mainly involved in this association, but a low sperm count in itself is a marker of general health. PATIENT SUMMARY: This large study evaluated semen quality, reproductive function, and metabolic risk in men referred for fertility evaluation, and showed that a man's semen count is a marker of his general health. Men with low sperm counts are more likely than those with normal sperm counts to have greater body fat, higher blood pressure, higher "bad" (low-density lipoprotein) cholesterol and triglycerides, and lower "good" (high-density lipoprotein) cholesterol. They also have a higher frequency of metabolic syndrome and insulin resistance, a condition that can lead to diabetes. Men with low sperm counts had a 12-fold increased risk of hypogonadism or low testosterone levels, and half of them had osteoporosis or low bone mass. Fertility evaluation gives men the unique opportunity for health assessment and disease prevention.

Negative Association Between Sclerostin and INSL3 in Isolated Human Osteocytes and in Klinefelter Syndrome: New Hints for Testis-Bone Crosstalk.

Context: The regulation of bone mass by the testis is a well-recognized mechanism, but the role of Leydig-specific marker insulin-like 3 peptide (INSL3) on the most abundant bone cell population, osteocytes, is unknown. In this study, we aimed to investigate the relationship between INSL3 and sclerostin, an osteocyte-specific protein that negatively regulates bone formation. Design: Serum sclerostin and INSL3 levels were evaluated in Klinefelter syndrome (KS) and healthy controls. In vitro effect of INSL3 on sclerostin production was evaluated in human cultured osteocytes. Patients: A total of 103 KS patients and 60 age- and sex-matched controls were recruited. Main Outcome Measures: Serum sclerostin and INSL3 levels were assessed by enzyme-linked immunosorbent assay. Osteocytes were isolated by fluorescence-assisted cell sorting. Sclerostin expression was evaluated by western blot, immunofluorescence, and reverse transcription polymerase chain reaction. Measurement of bone mineral density was done by dual-energy X-ray absorptiometry at lumbar spine (L1-L4) and femoral neck. Results: Sclerostin levels were significantly increased in KS subjects, and negatively correlated with INSL3 levels in both cohorts and with bone mineral density in the KS group. Stimulation of cultured osteocytes with INSL3 at 10-7 M significantly decreased both sclerostin messenger RNA and protein expression. Conclusions: We report a negative association between the testicular hormone INSL3 and the osteocytic negative regulator of bone formation, sclerostin. We further explored this association in vitro and showed that INSL3 was able to reduce sclerostin expression. These results add further knowledge on the emerging role of sclerostin as a therapeutic target for osteoporosis treatment.

Novel insights on testicular volume and testosterone replacement therapy in Klinefelter patients undergoing testicular sperm extraction. A retrospective clinical study.

STUDY QUESTION: To investigate whether sperm recovery is related to clinical features, hormone parameters and testosterone replacement therapy (TRT) in patients with Klinefelter syndrome (KS). SUMMARY ANSWER: This study provides three interesting insights: (i) the probability to retrieve sperm is not related to testicular volume; (ii) TRT does not affect sperm retrieval rate (SRR); and (iii) reduced levels of LH and FSH represent a negative predictor of sperm retrieval in patients with TRT. WHAT IS KNOWN ALREADY: Classical KS shows a karyotype with one extra X chromosome in all of somatic cells and clinical manifestations characterized by hypergonadotropic hypogonadism and infertility. STUDY DESIGN, SIZE AND DURATION: We performed a retrospective cohort study. Data from 111 consecutive KS azoospermic patients undergoing testicular sperm extraction (TESE) were collected from 2005 to 2016. PARTICIPANTS/MATERIALS, SETTING AND METHODS: Data on anthropometric parameters, reproductive hormones and testicular volumes were collected. SRR was related to clinical characteristics and compared between TRT and untreated patients. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 38 patients (34.2%) had successful sperm recovery. The comparison of clinical characteristics did not differ between patients with and without sperm recovery. Sperm retrieval was successful also in subjects with smaller testes. The comparison of SRR in patients with or without TRT was not different (33.3% vs 34.6%). In TRT group, LH and FSH levels were significantly lower in subjects with no sperm retrieval (P values, respectively, <.05 and <.001). LIMITATIONS AND REASONS FOR CAUTION: Well-designed controlled studies are necessary to confirm these data aimed to set the best therapeutic approach for fertility management in hypogonadal patients with nonmosaic KS. WIDER IMPLICATIONS OF THE FINDINGS: Age at TESE, anthropometric measures, testis volume, sex hormones levels and semen parameters are not predictive parameters of SRR. Among TRT patients, reduced gonadotropin is related to failure in sperm retrieval.

Is there any clinical relevant difference between non mosaic Klinefelter Syndrome patients with or without Androgen Receptor variations?

Klinefelter Syndrome (KS) is the most common chromosomal disorder in men leading to non-obstructive azoospermia. Spermatozoa can be found by TESE in about 50% of adults with KS despite severe testicular degeneration. We evaluated AR variations and polymorphism length in 135 non-mosaic KS patients, aimed to find possible correlation with clinical features, sex hormones and sperm retrieval. Among 135 KS patients we found AR variations in eight subjects (5.9%). All variations but one caused a single amino acid substitution. Four variations P392S, Q58L, L548F, A475V found in six patients had been previously described to be associated with different degrees of androgen insensitivity. Moreover we observed in two patients Y359F and D732D novel variations representing respectively a missense variation and a synonymous variation not leading to amino acid substitution. All the Klinefelter patients with AR gene variations were azoospermic. Spermatozoa were retrieved with TESE for two men (40%), sperm retrieval was unsuccessful in other 3 patients. This is the only study reporting AR variations in KS patients. Relevant clinical differences not emerged between AR mutated and not AR mutated KS patients, but does each variation play an important role in the trasmission to the offspring obtained by ART in this patients?

Impact of Bep or Carboplatin Chemotherapy on Testicular Function and Sperm Nucleus of Subjects with Testicular Germ Cell Tumor.

Young males have testicular germ cells tumors (TGCT) as the most common malignancy and its incidence is increasing in several countries. Besides unilateral orchiectomy (UO), the treatment of TGCT may include surveillance, radiotherapy, or chemotherapy (CT), basing on tumor histology and stage of disease. It is well known that both radio and CT may have negative effects on testicular function, affecting spermatogenesis, and sex hormones. Many reports investigated these aspects in patients treated with bleomycin, etoposide, and cisplatin (BEP), after UO. In contrast no data are available on the side effects of carboplatin treatment in these patients. We included in this study 212 consecutive subjects who undergone to sperm banking at our Andrology and Human Reproduction Unit after UO for TGCT. Hundred subjects were further treated with one or more BEP cycles (BEP-group), 54 with carboplatin (CARB group), and 58 were just surveilled (S-group). All patients were evaluated for seminal parameters, sperm aneuploidy, sperm DNA, sex hormones, volume of the residual testis at baseline (T0) and after 12 (T1) and 24 months (T2) from UO or end of CT. Seminal parameters, sperm aneuploidies, DNA status, gonadic hormones, and testicular volume at baseline were not different between groups. At T1, we observed a significant reduction of sperm concentration and sperm count in the BEP group versus baseline and versus both Carb and S-group. A significant increase of sperm aneuploidies was present at T1 in the BEP group. Similarly, the same group at 1 had altered sperm DNA integrity and fragmentation compared with baseline, S-group and Carb group. These alterations were persistent after 2 years from the end of BEP treatment. Despite a slight improvement at T2, the BEP group had still higher percentages of sperm aneuploidies than other groups. No impairment of sperm aneuploidies and DNA status were observed in the Carb group both after 1 and 2 years from the end of treatment. Despite preliminary, these data demonstrate that in selected patients with TGCTs CT with carboplatin represents a therapeutic option that that seems to not affect sex hormones, spermatogenesis, and sperm nucleus.

Spermatid count as a predictor of response to FSH therapy.

This study evaluated the predictive power of spermatid count and cytology for assisted reproduction outcome after FSH therapy. A total of 174 men with severe oligozoospermia and normal plasma FSH concentration underwent semen analysis including spermatid count, TUNEL test, FISH analysis for sperm aneuploidies and testicular fine-needle aspiration cytology. Ninety-two men with hypospermatogenesis received FSH therapy for 3 months and 82 patients with maturative disturbance or partial obstruction served as controls. Semen was analysed at baseline, after FSH therapy and after 3- and 9-month follow up, and pregnancies were recorded. Subjects not reaching pregnancy at 3-month follow up were recommended assisted reproduction treatment. Spermatid count was related to testicular cytology: spermatid concentrations <0.01, 0.01-0.3 and >0.3 × 10(6)/ml were predictive of partial obstruction, hypospermatogenesis and maturative disturbance. FSH therapy patients showed increases in sperm number and motility (both P < 0.001), allowing some couples to undergo intrauterine insemination instead of IVF. Cumulative pregnancy rate after 12 months was higher with FSH therapy (44.6%) than without (22.0%; P = 0.002). FSH therapy improved pregnancy rate and sometimes allowed less invasive assisted reproduction treatment in well-selected patients. Spermatid count could represent a new parameter to predict response to FSH therapy. One-hundred seventy-four patients with severe reduction of sperm count and normal sex hormones plasma levels underwent semen analysis with spermatid count, and testicular fine needle aspiration cytologiy (FNAC). Ninety-two men infertile men with reduced sperm production (hypospermatogenesis) were treated with highly purified urofollitropin and 82 patients with sperm maturative defects or partial obstruction of the seminal tract served as controls. After treatment and after the following 3 and 9 months all subjects performed a new semen analysis and pregnancies were recorded. Subjects who had not reached spontaneous pregnancy were suggested to undergo assisted reproductive techniques (ARTs). Spermatid count was strongly related to testicular cytology: spermatid concentrations were predictive of partial obstruction, hypospermatogenesis and maturative disturbance respectively. Treated patients showed significant increase in sperm number and motility allowing some couples to undergo easier and less invasive assisted reproductive techniques. The number of pregnancies was significantly higher among treated (44.6%) than untreated couples (22.0%). Our data confirmed that FSH treatment can induce a significant improvemet of pergnancy rate and sometimes allows less invasive ARTs use in well selected severe oligozoospermic patients. Moreover, we suggest that spermatid count can be useful to define tubular status and could represent a new parameter to predict response to FSH therapy.

Low serum testosterone as a new risk factor for chronic rejection in heart transplanted men.

BACKGROUND: Among the various complications of heart transplantation (HTx), the vasculopathy of the allograft (CAV), a phenomenon of chronic rejection, is still a serious problem. Recently, the literature has shown that low testosterone levels in men are associated with cardiovascular disease. In this study, we evaluated the influence of testosterone plasma levels on CAV development. METHODS: We studied, with a prospective observational study, all consecutive male HTx patients evaluated from May 2010 to June 2011 at our center. All subjects underwent accurate medical history collection, physical examination, biochemical blood tests, hormone levels, transthoracic Doppler echocardiography, coronary flow velocity reserve assessment, and coronary angiogram. RESULTS: HTx subjects with CAV had significant lower total testosterone plasma levels (12.9±3.9 vs. 15.8±5.8 nmol/L), free testosterone (0.26±0.07 vs. 0.31±0.08 nmol/L), and coronary flow velocity reserve (2.35±0.60 vs. 2.81±0.78 s) with respect to No-CAV patients. Considering the patients as a whole group, a significant negative relation was found between free and total testosterone plasma levels and some cardiovascular risk factors (cholesterol and fasting blood glucose). A significant linear inverse relation was found between total and free testosterone plasma levels and CAV grading. Only free testosterone plasma levels were independent predictors for CAV. CONCLUSIONS: We showed for the first time the influence of testosterone plasma levels on CAV development: indirectly increasing traditional risk factors and directly with a probable influence on alloimmune response.

Erectile dysfunction, penile atherosclerosis, and coronary artery vasculopathy in heart transplant recipients.

INTRODUCTION: Vascular erectile dysfunction (ED) is the expression of a systemic vascular disease and in particular of endothelial dysfunction. Dysfunctional endothelium plays also a significant role in the onset and progression of coronary artery vasculopathy (CAV). AIM: This pilot study was designed to evaluate the prevalence and pathogenesis of ED and its correlation with CAV in heart transplanted male. METHODS: A total of 77 male heart transplanted patients (HTx) evaluated in our center (mean age 61.6 + 10.6 years) were enrolled in the study. MAIN OUTCOME MEASURES: All subjects underwent accurate medical history collection, including lifestyle (cigarette smoking, dietary and sedentary habits, drug intake, and erectile function before cardiac transplantation), physical examination (body mass index and arterial pressure), biochemical blood tests (fasting glucose, total cholesterol, high-density lipoprotein cholesterol, and triglycerides), and hormones (prolactin, luteinizing hormone and total testosterone). Furthermore, they were studied with penile, carotid, femoral echo-color Doppler ultrasonography and coronary angiogram. RESULTS: Incidence of ED was 24% before HTx and increased up to 65% after. Postischemic cardiomiopathy was an indication to HTx in ED group more frequently than in patients without ED (No-ED group) (45.1% vs. 20%). ED patients showed a lower peak systolic velocity, a higher cavernosal intima-media thickness (IMT), a higher prevalence of cavernosal plaques (26.7% vs. 5.2%, P < 0.05), peripheral vascular disease (60.87% vs. 26.1%, P < 0.05) and CAV (45.8% vs. 25.8%, P < 0.05) with respect to No-ED patients. Coronary flow reserve was significantly reduced in ED vs. No-ED patients (2.43 + 0.7 vs. 2.9 + 0.8, P < 0.04). Finally, cavernous plaque and testosterone plasma levels were statistically associated with CAV. CONCLUSIONS: We showed that ED is a frequent disease in HTx patients, more common when the original pathology is postischemic cardiomiopathy and associated with higher prevalence of cavernous plaques and CAV. Its evaluation should be integral to an HTx rehab program.

Testicular function and bone metabolism--beyond testosterone.

Findings in the past few years have advanced understanding of the crosstalk between testis and bone and could contribute to defining an improved clinical approach to the biochemical diagnosis and therapeutic management of hypogonadism and male osteoporosis. This Review focuses on the Leydig cells of the testis. Other than being responsible for steroidogenesis and production of testosterone, the function of these cells is fundamental to bone health in at least two other ways: Leydig cells produce insulin-like 3 (INSL3), which has a role in osteoblast function, and they contribute to 25-hydroxylation of vitamin D. Impairment of testicular function leads to low levels of testosterone, INSL3 and 25-hydroxyvitamin D and consequently to an increased risk of osteopenia and osteoporosis.

Effect of relaxin on human sperm functions.

Relaxin is a circulating hormone with functions in pregnancy, parturition, and other aspects of female reproduction. It is also secreted from the prostate gland into the seminal fluid; however, the role of relaxin in male reproduction is debated. Studies conducted in the past have suggested possible actions on human spermatozoa, but the data were contrasting. Here, we show that the relaxin receptor RXFP1 (Relaxin Family Peptide Receptor 1) is expressed in human spermatozoa, and it mainly localizes in the astrodome. In vitro studies on human sperm demonstrated that this hormone attenuates the natural decline in sperm motility and maintains higher mitochondrial activity and lower apoptosis level. Furthermore, relaxin induced an increase in sperm hyperactivation, intracellular calcium and cAMP, and acrosome reaction. These effects were abolished by the use of the specific anti-RXFP1 antibody. Relaxin concentrations were low in the blood (x ± SD, 0.16 ± 0.03 nM) and very high in the seminal plasma (x ± SD, 10.3 ± 4.0 nM), confirming its secretion mainly by the prostate. Taken together, these data demonstrate that relaxin influences positively many sperm functions linked to fertilizing ability, and it preserves sperm functionality, with possible practical value in assisted reproduction techniques.

Toward a pharmacogenetic approach to male infertility: polymorphism of follicle-stimulating hormone beta-subunit promoter.

OBJECTIVE: To verify in another population (Italians) whether a single-nucleotide polymorphism in the FSHB gene promoter previously associated with serum FSH levels in Estonians is indeed associated with sperm count and FSH plasma levels, and especially to verify whether it could be a pharmacogenetic tool for the treatment of male infertility with FSH. DESIGN: Cross-sectional and prospective study. SETTING: Infertility center at a university hospital. PATIENT(S): Five hundred fourteen subjects with nonobstructive azoospermia and oligozoospermia and 248 subjects with normozoospermia. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Semen parameters, reproductive hormone levels, and FSHB -211 G/T polymorphism (rs10835638). RESULT(S): FSHB -211 TT genotype was associated with significantly lower FSH levels (mean ± SD: 3.3 ± 2.5 IU/L vs. 9.1 ± 8.9 IU/L in GG homozygotes). TT homozygotes were seen in 25% of subjects with azoo-oligozoospermia and low FSH levels (≤1.5 IU/L). We did not observe this genotype in men with high FSH levels (>8 IU/L) or in men with normozoospermia. Treatment with FSH induced a significantly higher improvement in sperm count and quality in TT homozygotes regarding carriers of the G allele. CONCLUSION(S): FSHB -211 TT genotype might represent a novel treatable form of male infertility characterized by severe spermatogenic impairment and low or inappropriately normal FSH plasma levels. This genetic marker could represent a valid pharmacogenetic approach for identification of potential responders to FSH treatment.

Metabolic syndrome and erectile dysfunction: the ultrasound evaluation of cavernosal atherosclerosis.

OBJECTIVE: To study the relation between metabolic syndrome (MS), cavernosal morphological vasculopathy, and peripheral vascular alterations (carotid and femoral wall) in patients with erectile dysfunction. RESEARCH DESIGN AND METHODS: A total of 207 patients and 50 control subjects were evaluated for cardiovascular risk factors, physical examination, reproductive hormones, ultrasound analysis of cavernosal, carotid and femoral arteries (intima-media thickness), and cavernosal flow measurement (peak systolic velocity). RESULTS: A total of 28% of patients had MS, and they presented with a high prevalence of cavernosal alterations (70.3%) and systemic vascular impairment (59.3%), whereas patients with cavernosal alterations (44%) showed the higher prevalence of MS (48.9%). The number of MS components was related to the prevalence of penile vasculopathy. However, multivariate analysis showed that MS is not an independent predictor for cavernosal vasculopathy. CONCLUSIONS: Patients with cavernosal vasculopathy have an increased cardiometabolic risk, and screening for MS components might identify individuals with a higher risk for cavernosal and systemic atherosclerosis.

Bone mass in subjects with Klinefelter syndrome: role of testosterone levels and androgen receptor gene CAG polymorphism.

CONTEXT: Klinefelter syndrome (KS) is a chromosomal alteration characterized by supernumerary X-chromosome(s), primary hypogonadism, decreased pubertal peak bone mineral density (BMD), and accelerated bone loss during adulthood. Decreased bone mass has been traditionally related to low testosterone levels. However, testosterone replacement therapy does not necessarily increase bone mass in these patients, and low BMD can be observed also in patients with normal testosterone levels. The androgen receptor (AR) gene CAG polymorphism seems to modulate the sensitivity to testosterone and previous studies have related it to some clinical aspects of KS, to include BMD, gynecomastia, testes and prostate volume, and hemoglobin concentration. OBJECTIVE: To analyze the relation between bone mass, testosterone, and AR CAG polymorphism in men with KS. DESIGN: Cross-sectional cohort study. SETTING: University department. PATIENTS: One hundred twelve consecutive treatment-naïve 47,XXY Klinefelter patients (mean age 33.5 ± 4.7 yr) and 51 age-matched normal male controls. MAIN OUTCOME MEASURES: Dual-energy x-ray absorptiometry, CAG repeat length polymorphism, X-chromosome inactivation, and testosterone levels. RESULTS: Forty-nine of 112 KS subjects (42.5%) had low bone mass (osteopenia or osteoporosis). Lumbar and/or femoral T-scores were lower in KS patients compared with controls. No significant relationship was observed between testosterone levels and bone parameters, and the prevalence of osteopenia/osteoporosis was similar in subjects with normal and low testosterone levels (43.7% and 40.5%, respectively). The mean CAG repeat length calculated after X-chromosome inactivation analysis showed no differences between patients with normal and low bone mass. CONCLUSIONS: Testosterone levels and AR CAG polymorphism are not associated with bone mass phenotype in KS.

Bone mineral density and testicular failure: evidence for a role of vitamin D 25-hydroxylase in human testis.

WORKING HYPOTHESIS: Mutations in the CYP2R1 gene, highly expressed in the testis and encoding vitamin D 25-hydroxylase, result in a vitamin D deficiency and a defective calcium homeostasis leading to rickets. OBJECTIVE: Our aim was to investigate CYP2R1 expression in pathological testis samples and relate this to vitamin D metabolism in testiculopathic patients. DESIGN, PATIENTS, SETTING: Testis samples for in vitro study and 98 young men were transversally evaluated at Padova's Center for Male Gamete Cryopreservation. METHODS: CYP2R1 mRNA expression and protein production were evaluated by quantitative RT-PCR, Western blot analysis, and immunofluorescence. Hormonal and bone-marker levels, and bone densitometry by dual-energy x-ray absorptiometry, were determined in patients with Sertoli-cell-only syndrome and severe hypospermatogenesis. RESULTS: We found a lower gene and protein expression of CYP2R1 in samples with hypospermatogenesis and Sertoli-cell-only syndrome (P < 0.05) and a colocalization with INSL-3, a Leydig cell marker, at immunofluorescence. In all testiculopathic patients 25-hydroxyvitamin D levels were significantly lower and PTH levels higher compared to controls (P < 0.05). Furthermore, testiculopathic patients showed osteopenia and osteoporosis despite normal testosterone levels compared with controls both with increased bone-marker levels and altered dual-energy x-ray absorptiometry in the femoral neck and lumbar spine (for all parameters, P < 0.05). CONCLUSIONS: Our data show an association between testiculopathy and alteration of the bone status, despite unvaried androgen and estrogen levels and no other evident cause of vitamin D reduction. Further studies in larger cohorts are needed to confirm our results.

Association of testicular germ cell tumor with polymorphisms in estrogen receptor and steroid metabolism genes.

It is generally assumed that the development of testicular germ cell tumor (TGCT) is under endocrine control. In particular, unbalanced androgen/estrogen levels and/or activity are believed to represent the key events for TGCT development and progression. Furthermore, recent evidence has suggested a strong genetic component for TGCT. In this study, we analyzed whether a genetic variation in estrogen receptor (ESR) genes and steroid hormone metabolism genes is associated with TGCT. We genotyped for 17 polymorphic markers in 11 genes in 234 TGCT cases and 218 controls: ESR (ESR1 and ESR2); CYP19A1 (aromatase); 17beta-hydroxysteroid dehydrogenase types 1 and 4 (HSD17B1 and HSD17B4) dehydrogenases that convert potent androgens and estrogens to weak hormones; cytochrome P450 hydroxylating enzymes CYP1A1, CYP1A2, and CYP1B1; and the metabolic enzymes COMT, SULT1A1, and SULT1E1. We observed a significant association of rs11205 in HSD17B4 with TGCT. TGCT risk was increased twofold per copy of the minor A allele at this locus (odds ratios (OR)=2.273, 95% confidence interval (CI)=1.737-2.973). Homozygous carriage of the minor A allele was associated with an over fourfold increased risk of TGCT (OR=4.561, 95% CI=2.615-7.955) compared with homozygous carriage of the major G allele. The risk was increased both for seminoma (OR=5.327, 95% CI=2.857-9.931) and for nonseminoma (OR=3.222, 95% CI=1.471-7.059). We found for the first time an association of polymorphisms in HSD17B4 gene with TGCT. Our findings expand the current knowledge on the role of genetic contribution in testicular cancer susceptibility, and support the hypothesis that variations in hormone metabolism genes might change the hormonal environment implicated in testicular carcinogenesis.

Mutations in INSL3 and RXFP2 genes in cryptorchid boys.

Mutations in the INSL3 and RXFP2 genes have been associated with human cryptorchidism but with contrasting data. We analyzed the frequency of mutations in these genes in 600 newborns with cryptorchidism (396 unilateral and 204 bilateral) and 300 noncryptorchid subjects. We found five RXFP2 mutations in five bilateral cryptorchid boys, one INSL3 mutation in a unilateral cryptorchid boy, and one INSL3 mutation in a boy with unilateral cryptorchidism at birth and spontaneous descent during the first month of life. Overall, the frequency of INSL3 and RXFP2 mutations was therefore 7/600 at birth (1.2%) and 7/303 (2.3%) in persistent cryptorchid boys, with a higher prevalence of bilateral forms (5/120, 4.2%). No mutations were found in controls. This study confirmed the association between INSL3 and RXFP2 gene mutations and human cryptorchidism.