Human papillomavirus in spermatozoa is efficiently removed by washing: a suitable approach for assisted reproduction.

RESEARCH QUESTION: Is it possible, by sperm-washing spermatozoa from clinically HPV-positive men, to obtain spermatozoa free of human papillomavirus (HPV) to be employed in assisted reproduction? DESIGN: This was an observational study performed on HPV-positive men. Freshly ejaculated semen was collected and readily processed by gradient separation followed by swim-up from the washed pellet. The resulting fractions were seminal plasma, cell pellet, round cells, non-motile spermatozoa and motile spermatozoa. All fractions were then tested for the presence of HPV DNA. RESULTS: Of the 15 clinically HPV-positive subjects, 67% were positive in at least one of the seminal fractions. If any postivity was detected, the plasma was always HPV positive. No consistent pattern was observed throughout different samples in the cell pellet, round cell and non-motile spermatozoa fractions. However, after the sperm-wash procedure, the fraction of motile spermatozoa was never found to be HPV-positive. CONCLUSIONS: The sperm-washing technique, which was previously successfully used to remove human immunodeficiency virus, can efficiently remove HPV from spermatozoa. However, the present study was conducted on a small population so a larger follow-up study is recommended. HPV screening should be performed in sperm samples and, upon HPV positivity, sperm-washing should be considered before assisted reproduction techniques are used.

Effects of α-lipoic acid and myo-inositol supplementation on the oocyte environment of infertile obese women: A preliminary study.

Obesity is becoming pandemic and is associated with impaired reproductive potential. Oxidative stress, low-grade chronic inflammation and mitochondrial dysfunctions, which characterize obesity, strongly affect oocyte environment and function. Supplementation with antioxidant and anti-inflammatory compounds has been suggested to improve fertility. Here we evaluated the effect of α-lipoic acid and myo-inositol supplementation on the oocyte environment of infertile obese women. Nineteen normal-weight and twenty-three obese women, infertile for non-ovarian reasons, were recruited. For two months before ovarian stimulation, all women received 400 µg/die folic acid, whereas 15 obese were additionally supplemented with 800 mg α-lipoic acid, 2 g myo-inositol/die. Antioxidant capacity was measured in follicular fluid by enzymatic assay; mitochondrial DNA (mtDNA) content and mRNA levels of two respiratory chain subunits were analyzed in granulosa cells by Real-time PCR. Pregnancy rate was similar between normal-weight and treated obese, and lower in untreated obese patients. Supplemented women showed significantly higher antioxidant levels in follicular fluid compared to the two groups taking only folic acid. Conversely, granulosa cells mtDNA content was decreased in treated and higher in untreated obese patients compared to normal-weight women, suggesting mtDNA increases to compensate for oxidative-stress damages. Reduced expression of respiratory subunits in untreated obese may confirm mitochondria impairment. Interestingly, mtDNA levels inversely correlated to both total and metaphase II oocyte number. In this preliminary study, combined supplementation of α-lipoic acid and myo-inositol in infertile obese women was associated with amelioration in the oxidative status of the oocyte environment, possibly contributing to a higher pregnancy rate.

The antifibrogenic effect of hepatocyte growth factor (HGF) on renal tubular (HK-2) cells is dependent on cell growth.

Although several reports suggest an antifibrogenic effect of hepatocyte growth factor (HGF), an increased deposition of matrix induced by HGF has also been reported. These conflicting effects could result from a diverse proliferative state of the target cells. Aim of the present study was to evaluate HGF effects on growth arrested (quiescent) and actively proliferating renal tubular epithelial (HK-2) cells. HK-2 cells were cultured in RPMI medium either on agarose gel or on plastic surface in order to inhibit or to allow cell proliferation. Cells were incubated with RPMI containing HGF (50 ng/ml) for 24 h at 37 degrees C. Untreated HK-2 were used as control. After 24 h of incubation, cells were counted by Coulter counter. (alpha2)IV collagen, transforming growth factor-beta (TGF-beta), Tissue inhibitor of metalloproteases (TIMP1 and 2) mRNA levels were determined by RT-PCR. The production of type IV collagen, c-met, proliferating cell nuclear antigen (PCNA), and SnoN, a transcriptional Smad corepressor and thus a TGF-beta inhibitor, was evaluated by ELISA or western blotting. MMP-9 and 2 gelatinolytic activity was studied by zymography. Treatment with HGF did not increase HK-2 cell number and PCNA synthesis when the cells were grown on agarose as it did for cells grown on plastic surface. HGF increased (alpha2)IV collagen in proliferating cells whereas it reduced (alpha2)IV collagen and c-met synthesis in growth arrested cells. HGF treatment increased TGF-beta and TIMP-2 in proliferating cells while reduced TIMP-1 mRNA levels of quiescent cells. Furthermore, production of the co repressor SnoN was significantly decreased by HGF in proliferating cells. Quiescent and proliferating HK-2 showed a different pattern of metalloproteases activity with a prevalence of MMP2 in quiescent and MMP9 in proliferating cells. In summary, HGF showed opposite effects on growth arrested and proliferating HK-2 cells favouring matrix deposition in the latter with increasing expression of collagen, TIMP-1 and TGF-beta. Our results demonstrate that the proliferative state of target cells may influence the effects of HGF on extracellular matrix turnover in HK-2 cells.

Effects of highly active antiretroviral therapy on semen parameters of a cohort of 770 HIV-1 infected men.

BACKGROUND: HIV-1 infected patients show impaired semen parameters. Currently, it is not clear whether HIV-1 infection itself or antiretroviral therapy have an effect on semen parameters. We aim evaluate semen quality in a large cohort of fertile HIV-1 infected men under stable highly active antiretroviral therapy (HAART) and to assess the effect of HAART type and duration on semen parameters. MATERIALS AND METHODS: Between January 2010 and June 2014, we enrolled in a retrospective case-control study 770 HIV-1 patients under stable HAART asking a reproductive counselling with their HIV negative partner. Co-infections with HBV or HCV, genital tract infections and known causes of infertility represented exclusion criteria. Semen samples were analysed and compared with the WHO reference values. A multivariate analysis including HAART type and duration, age, viral load and CD4 count, was performed on 600 patients out of 770. RESULTS: The median values of all semen parameters were significantly lower among HIV-1 infected patients compared to the WHO reference group, with a significant proportion of patients having values below the 5th percentile of the WHO reference value. In a multivariate analysis, only age and viral load negatively impacted progressive motility (ß -0.3 (95% CI: -0.5; -0.0) %, p<0.05) and semen morphology (ß -0.00 (95% CI: -0.00; -0.00) %, p≤0.01), while no associations were detected as regards HAART type and duration. CONCLUSIONS: HIV-1 infected patients showed a significant impairment of semen parameters compared to the reference values. HAART type and duration showed no associations with semen quality. Further research is needed to investigate implications for clinical care of HIV infected men desiring a child.

Effects of Antiretroviral Therapy on Sperm DNA Integrity of HIV-1-Infected Men.

HIV-1-affected couples' desire to have children and free sexual intercourses with the use of pre-exposure prophylaxis for the negative partner has emerged as an alternative option to assisted reproduction in aviremic patients under highly active antiretroviral therapy (HAART). It is already known that sperm quality may be impaired in HIV-infected men. The underlying physiopathological mechanism is still debated. The aim of this study was to evaluate the effects of HAART on sperm DNA fragmentation, comparing HIV-1-infected patients taking HAART versus naïve HIV-1-infected patients. This is a prospective case-control study. Sperm nuclear DNA fragmentation rate was evaluated by the sperm chromatin dispersion test in 77 HIV-infected men: 53 HIV-1 patients receiving HAART (Group 1) versus 24 naïve HIV-1 patients not receiving HAART (Group 2). Complete semen analysis was performed according to WHO 2010 recommendations. Patients with HBV infection or HCV infection coinfections and genital tract infections wre excluded. All the patients did not present any clinical signs of their disease. Seminal parameters were examined in the two groups, showing no significant differences. Increased sperm DNA fragmentation > 30% was demonstrated in 67.9% of patients in Group 1 and 37.5% of patients in Group 2, respectively ( p = .02). A positive but nonsignificant trend toward increased fragmentation was reported with advancing patients' age. In conclusion, sperm nuclear fragmentation rate is increased in HIV-1-infected patients taking HAART compared to HIV-1 patients not receiving HAART.

Should HCV discordant couples with a seropositive male partner be treated with assisted reproduction techniques (ART)?

OBJECTIVE: The debate on HCV discordant couples requiring assisted reproduction is still open today, and specific guidelines have not yet been established on whether or not physicians should treat HCV discordant couples who require ART. We studied the results of our reproductive assistance with sperm washing in HCV discordant couples, all treated in a single center, including the serological status of mothers and babies, and the outcome of the pregnancies. STUDY DESIGN: Prospective study conducted between January 2008 and December 2010 in our Reproductive Center in Sacco Hospital, University of Milan. Thirty-five HCV serodiscordant infertile couples with an HCV viremic positive male partner were enrolled. All of them completed the immuno-virological and fertility triage, and were treated according to our clinical protocols. RESULTS: Forty-seven superovulation and IUI and 38 second-level ART procedures are reported. The pregnancy rates for IUI and ICSI are similar to those reported by the Italian ART register. All the 85 sperm samples were treated with sperm washing technique to reduce HCV in semen and the possible risk of transmission. We did not observe any preterm delivery or negative perinatal outcome. No mothers or babies are infected by HCV. CONCLUSION: This is the biggest prospective study conducted in a single center involving HCV discordant infertile couples in an ART program. Although sexual transmission of HCV is very low, in subfertile or infertile couples sperm washing should be used to treat HCV positive semen before ART. We suggest that it is not necessary to perform nested PCR to detect HCV RNA in the final swim-up. Since the presence of HCV in semen implies a possible risk of nosocomial contamination, safety regulations must be strictly applied in assisted reproduction laboratories.

Reproductive assistance in HIV-1 discordant couples.

PURPOSE OF REVIEW: The total number of people living with HIV has reached 39.5 million. In developed countries, the major risk for HIV transmission is by heterosexual intercourse. The general condition and life expectancy of many patients with HIV infection is very good, and three-quarter of these individuals are in their reproductive years. For these reasons a large number of young couples are expected to make future plans to have children. Assisted reproduction in HIV-1 discordant couples has become important for clinicians in preventing HIV-1 transmission. RECENT FINDINGS: The main theme covered in this article is epidemiology of HIV-1 in the world, the biology of the virus present in the sperm, the recently published guidelines from the American Society for Reproductive Medicine and ethics recommendations concerning assisted reproduction in people infected by virus, and all clinical aspects and scientific data published that have been modified to allow assisted reproduction in HIV-serodiscordant couples. SUMMARY: To evaluate in clinical practice the safety of HIV-1 assisted reproduction with sperm washing and the outcome of assisted reproductive technologies offered to HIV-1 serodiscordant couples counselled for the prevention of HIV transmission.

Hepatocyte growth factor (HGF) modulates matrix turnover in human glomeruli.

BACKGROUND: The imbalance between synthesis and degradation of mesangial matrix causes glomerulosclerosis and leads to renal failure. Hepatocyte growth factor (HGF) has been shown to reduce the progression in murine models of chronic renal failure. The present study evaluated the effect of HGF on the extracellular matrix turnover and on c-met receptor in human glomeruli. METHODS: Human glomeruli microdissected from donor kidney biopsies before transplantation were incubated with culture media containing HGF (50 ng/mL). After 24 and 48 hours, the expression of c-met, (alpha2) IV collagen, transforming growth factor-beta (TGF-beta), metalloprotease (MMP) 2 and 9 and of the inhibitor of MMP-2, tissue inhibitors of metalloprotease-1 (TIMP-1), was evaluated by polymerase chain reaction (PCR). beta-actin was used as housekeeping gene. The production of collagen type IV and TGF-beta was evaluated by enzyme-linked immunosorbent assay (ELISA) and Western blotting and the activity of MMP by zymography. RESULTS: (alpha2) IV collagen, TGF-beta, and TIMP-1 mRNA levels were markedly decreased in glomeruli treated with HGF at 24 and 48 hours. The expression of c-met was up-regulated by HGF treatment. HGF reduced the production of collagen type IV and TGF-beta. MMP-2 but not MMP-9 mRNA level was increased in HGF-treated glomeruli, although the gelatinolytic activity of the supernatant was not changed. By light microscopic examination kidney biopsies neither showed glomerular hypercellularity nor mesangial expansion. CONCLUSION: HGF reduced expression and synthesis of TGF-beta and collagen type IV and increased MMP-2 mRNA level in normal human glomeruli. These results suggest an antifibrotic effect of HGF on glomerular cells and may explain its beneficial role in glomerulosclerosis.