Mutational Screening of Androgen Receptor Gene in 8224 Men of Infertile Couples.

CONTEXT: Mutations in the androgen receptor (AR) gene might be associated with infertility mainly because they cause various degrees of androgen insensitivity. OBJECTIVE: The aim of the study was to evaluate the frequency and type of AR variants in a large cohort of infertile males. METHODS: A total of 8224 males of Italian idiopathic infertile couples were referred to the University Hospital of Padova. The main outcome measures were mutational screening of AR, computational, and functional analyses. RESULTS: We found 131 patients (1.6%) harboring 45 variants in AR gene, of which 18 were novel missense AR variants. Patients with AR gene variants had lower sperm count (P = .048), higher testosterone (T) concentration (P < .0001), and higher androgen sensitivity index (ASI) (luteinizing hormone × T, P < .001) than patients without variants. Statistical analyses found T ≥ 15.38 nmol/L and ASI ≥ 180 IU × nmol/L2 as the threshold values to discriminate with good accuracy patients with AR variants. Patients with oligozoospermia and T ≥ 15.38 nmol/L had a 9-fold increased risk of harboring mutations compared with patients with normal sperm count and T < 15.38 nmol/L (odds ratio 9.29, 95% CI 5.07-17.02). Using computational and functional approaches, we identified 2 novel variants, L595P and L791I, as potentially pathogenic. CONCLUSION: This is the largest study screening AR gene variants in men of idiopathic infertile couples. We found that the prevalence of variants increased to 3.4% in oligozoospermic subjects with T ≥ 15.38 nmol/L. Conversely, more than 80% of men with AR gene variants had low sperm count and high T levels. Based on our findings, we suggest AR sequencing as a routine genetic test in cases of idiopathic oligozoospermia with T ≥ 15.38 nmol/L.

Human Papillomavirus Prophylactic Vaccination improves reproductive outcome in infertile patients with HPV semen infection: a retrospective study.

In this study we aimed to evaluate the effect on reproductive outcome of HPV vaccination in male subjects of infertile couples with HPV semen infection. In this single-center study, we retrospectively enrolled 151 infertile couples with detection of HPV in semen, attending our Hospital Unit of Andrology between January 2013 and June 2015, counseled to receive adjuvant HPV vaccination. Seventy-nine accepted vaccination (vaccine group) whilst 72 did not (control group). Our protocol of follow-up, aimed to evaluate HPV viral clearance, consisted in semen analysis, INNO-LiPA and FISH for HPV in semen cells after 6 and 12 months from basal evaluation. Spontaneous pregnancies, miscarriages and live births were recorded. Progressive sperm motility and anti-sperm antibodies were improved in the vaccine group at both time points (p < 0,05 vs control arm). Forty-one pregnancies, 11 in the control group and 30 in the vaccine group, were recorded (respectively 15% and 38,9%, p < 0,05) and resulted into 4 deliveries and 7 miscarriages (control group) and 29 deliveries and one miscarriage (vaccine group, p < 0,05 vs control group). HPV detection on sperms was predictive of negative pregnancy outcome. Adjuvant vaccination associated with enhanced HPV healing in semen cells and increased rate of natural pregnancies and live births.

FSH treatment in infertile males candidate to assisted reproduction improved sperm DNA fragmentation and pregnancy rate.

The purpose of this study is to evaluate whether follicle-stimulating hormone treatment improves sperm DNA parameters and pregnancy outcome in infertile male candidates to in-vitro fertilization.Observational study in 166 infertile male partners of couples undergoing in-vitro fertilization. Eighty-four patients were receiving follicle-stimulating hormone treatment (cases) and 82 refused treatment (controls). Semen parameters, sexual hormones, and sperm nucleus (fluorescence in-situ hybridization, acridine orange, TUNEL, and γH2AX) were evaluated at baseline (T0) and after 3 months (T1), when all subjects underwent assisted reproduction techniques. Statistical analysis was performed by analysis of variance.Compared to baseline, cases showed significant improvements in seminal parameters and DNA fragmentation indexes after follicle-stimulating hormone therapy (all P < 0.05), whereas no changes were observed in controls. Within cases, follicle-stimulating hormone treatment allowed to perform intrauterine insemination in 35 patients with a pregnancy rate of 23.2 %. Intracytoplasmic sperm injection was performed in all controls and in 49 patients from cases, with pregnancy rates of 23.2 and 40.8 %, respectively (P < 0.05). After 3 months (T0 vs. T1) of follicle-stimulating hormone therapy, cases with positive outcome had reduced DNA fragmentation index and lower double strand breaks (P < 0.05 and P < 0.001 vs. negative outcome, respectively).In this observational study, we showed that follicle-stimulating hormone treatment improves sperm DNA fragmentation, which in turn leads to increased pregnancy rates in infertile males undergoing in-vitro fertilization. In particular, double strand breaks (measured with γH2AX test) emerged as the most sensible parameter to follicle-stimulating hormone treatment in predicting reproductive outcome.

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.

Spontaneous fertility and in vitro fertilization outcome: new evidence of human papillomavirus sperm infection.

OBJECTIVE: To evaluate the reproductive outcome of infertile couples undergoing assisted reproduction techniques (ART) with or without human papillomavirus (HPV) semen infection. DESIGN: Cross-sectional clinical study. SETTING: Units of andrology, reproductive medicine, and gynecology. PATIENT(S): A total of 226 infertile couples. INTERVENTION(S): Male partners were evaluated by means of fluorescence in situ hybridization (FISH) for HPV on semen. After a diagnostic period, female partners underwent intrauterine insemination (IUI) or intracytoplasmic sperm injection (ICSI). MAIN OUTCOME MEASURE(S): Seminal parameters and FISH analysis for HPV in sperm head. Spontaneous or assisted pregnancies, live births, and miscarriages were recorded. Statistical analysis included unpaired Student t test and chi-square test. RESULT(S): Fifty-four male partners (23.9%) had HPV semen infection confined to sperm, confined to exfoliated cells, or in both cells. During the diagnostic period, noninfected couples showed spontaneous pregnancies. IUI and ICSI treatments were performed in, respectively, 60 and 98 noninfected and in 21 and 33 infected couples, with 38.4% and 14.2% cumulative pregnancy rates, respectively. The follow-up of pregnancies showed a higher miscarriage rate in infected couples (62.5% vs. 16.7%). Ongoing pregnancies of the latter group were characterized by HPV infection confined to exfoliated cells. CONCLUSION(S): A reduction in natural and assisted cumulative pregnancy rate and an increase in miscarriage rate are related to the presence of HPV at sperm level. Although the exact mechanism by which sperm infection is able to impair fertility remains unclear, this aspect is worthy of further investigations. If confirmed, these results could change the clinical and diagnostic approach to infertile couples.

Molecular karyotyping of human single sperm by array- comparative genomic hybridization.

No valid method is currently available to analyze the entire genome of sperm, including aneuploidies and structural chromosomal alterations. Here we describe the optimization and application of array-Comparative Genomic Hybridization (aCGH) on single human sperm. The aCGH procedure involves screening of the entire chromosome complement by DNA microarray allowing having a molecular karyotype, and it is currently used in research and in diagnostic clinical practice (prenatal diagnosis, pre-implantation genetic diagnosis), but it has never been applied on sperm. DNA from single human sperm isolated by micromanipulator was extracted, decondensed and amplified by whole-genome amplification (WGA) and then labeled, hybridized to BAC array, and scanned by microarray scanner. Application of this protocol to 129 single sperm from normozoospermic donors identified 7.8% of sperm with different genetic anomalies, including aneuploidies and gains and losses in different chromosomes (unbalanced sperm). On the contrary, of 130 single sperm from men affected by Hodgkin lymphoma at the end of three months of chemotherapy cycles 23.8% were unbalanced. Validation of the method also included analysis of 43 sperm from a man with a balanced translocation [46,XY,t(2;12)(p11.2;q24.31)], which showed gains and losses corresponding to the regions involved in the translocation in 18.6% of sperm and alterations in other chromosomes in 16.3% of sperm. Future application of this method might give important information on the biology and pathophysiology of spermatogenesis and sperm chromosome aberrations in normal subjects and in patients at higher risk of producing unbalanced sperm, such as infertile men, carriers of karyotype anomalies, men with advanced age, subjects treated with chemotherapy, and partners of couples with repeated miscarriage and repeated failure during assisted reproduction techniques.