Riccardo Selice

High-power microscopy for selecting spermatozoa for ICSI by physiological status

Sperm selection for intracytoplasmic sperm injection (ICSI), based on standard morphology, can fail to select normal cells, and actual methods to evaluate their physiological status do not allow their later use for ICSI. Some authors have demonstrated that sperm selection based on high-magnification morphology is associated with a better ICSI outcome, above all in subjects with severe testicular failure. In this study there was an evaluation of mitochondrial function, chromatin structure and sperm aneuploidies on whole sperm samples from 30 subjects: 10 normozoospermic controls and 20 patients that were severely oligozoospermic due to testicular damage or partial obstruction of the seminal ducts. All severely oligozoospermic patients showed worse mitochondrial function and chromatin status, while sperm aneuploidies were significantly increased only in those subjects with severe testicular damage (P < 0.001). In the latter patients the analysis of a single spermatozoon, performed after morphological selection by high-magnification microscopy, showed significantly better mitochondrial function, chromatin status and aneuploidy rate than observed in unselected cells (all P < 0.001). Interestingly, these parameters were further improved when nuclear vacuoles were lacking. These results suggest a strong relationship between high-magnification morphology and the status of spermatozoa, and they may explain the better results of ICSI obtained using spermatozoa selected by high-magnification microscopy.

Male infertility and androgen receptor gene mutations: clinical features and identification of seven novel mutations.

Objective  Androgens and a functioning androgen receptor (AR) are essential for development and maintenance of the male phenotype and spermatogenesis. Consistent with this, mutations in the AR gene cause a variety of defects related to androgen insensitivity, ranging from complete feminization to phenotypic males with infertility. The aim of his study was to analyse the prevalence of AR gene mutations in male infertility and to clarify the genotype‐phenotype relation.

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.

FSH receptor gene polymorphisms in fertile and infertile Italian men

In women, single nucleotide polymorphisms (SNP) of the FSH receptor (FSHR) gene influence FSH concentrations and the sensitivity of the FSHR to FSH in vivo. In contrast, the significance of FSHR R gene SNP in the male is poorly understood. To this aim, the possible role of three FSHR SNP was evaluated in male infertility. SNP in exon 10 (codon 307 and 680) and in the core promoter region (at position -29) of the FSHR gene were analysed by polymerase chain reaction-restriction fragment length polymorphism technique in 150 men representative of the general population, 107 proven fathers, 92 normozoospermic controls, and 215 infertile patients classified according to sperm parameters (38 azoospermia, 53 severe oligozoospermia, 48 moderate oligozoospermia, and 76 slight oligozoospermia). Reproductive hormones were measured in infertile males and normozoospermic controls. No significant difference was found in allelic variants frequency and genotype distribution between each category of subjects when analysing the FSHR exon 10 SNP alone and in combination with the SNP at position -29. Serum FSH concentrations and other andrological parameters did not differ between subjects with different genotype within each group. The data showed that in the Italian population, FSHR genotypes have no influence on FSH concentrations both in normal and infertile males and do not associate with spermatogenetic impairment.

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 Padovas 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.

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.

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.

The response to FSH treatment in oligozoospermic men depends on FSH receptor gene polymorphisms

In the last years, follice-stimulating hormone (FSH) receptor (FSHR) gene polymorphisms have been studied as potential risk factors for spermatogenetic failure. In this study, we have evaluated the response of FSH treatment in terms of sperm production on the basis of Ala307Thr-Asn680Ser polymorphisms in the FSHR gene in a group of oligozoospermic subjects with hypospermatogenesis and normal FSH levels. Patients were randomized into two groups: 70 treated with recombinant FSH (150 IU thrice per week for 3months) and 35 without treatment. After 3months of treatment, we observed significant increase in total sperm count, sperm concentration, forward motility, percentage of normal morphology forms and total motile sperm. When 70 treated subjects were subdivided based on FSHR genotype, only subjects with at least one serine in position 680 showed a statistically significant increase in these sperm parameters, whereas subjects with homozygote Thr307-Asn680 showed no difference in any seminal parameters evaluated. Non-treated subjects showed no differences in any parameter evaluated. This study suggests that the analysis of this gene represents a valid pharmacogenetic approach to the treatment of male infertility, confirming also the importance of strict criteria for the selection of patients to be treated with FSH.

Paracrine and endocrine roles of insulin-like factor 3

Insulin-like factor 3 (INSL3) is expressed in Leydig cells of the testis and theca cells of the ovary. This peptide affects testicular descent by acting on gubernaculum via its specific receptor leucine-rich repeat-containing G proteincoupled receptor 8 (LGR8). From initial animal data showing the cryptorchid phenotype of Insl3/ Lgr8 mutants, an extensive search for mutations in INSL3 and LGR8 genes was undertaken in human patients with cryptorchidism, and a frequency of mutation of 4–5% has been detected. However, definitive proofs of a causative role for some of these mutations are still lacking. More recent data suggest additional paracrine (in the testis and ovary) and endocrine actions of INSL3 in adults. INSL3 circulates at high concentrations in serum of adult males and its production is dependent on the differentiation effect of LH. Therefore, INSL3 is increasingly used as a specific marker of Leydig cell differentiation and function.

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.