Male infertility is a major cause of problems for many couples in conceiving a child. Recently, lifestyle pastimes such as alcohol, tobacco and marijuana have been shown to have further negative effects on male reproduction. The endocannabinoid system (ECS), mainly through the action of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) at cannabinoid (CB(1), CB(2)) and vanilloid (TRPV1) receptors, plays a crucial role in controlling functionality of sperm, with a clear impact on male reproductive potential. Here, sperm from fertile and infertile men were used to investigate content (through LC-ESI-MS), mRNA (through quantitative RT-PCR), protein (through Western Blotting and ELISA) expression, and functionality (through activity and binding assays) of the main metabolic enzymes of AEA and 2-AG (NAPE-PLD and FAAH, for AEA; DAGL and MAGL for 2-AG), as well as of their binding receptors CB(1), CB(2) and TRPV1. Our findings show a marked reduction of AEA and 2-AG content in infertile seminal plasma, paralleled by increased degradation: biosynthesis ratios of both substances in sperm from infertile versus fertile men. In addition, TRPV1 binding was detected in fertile sperm but was undetectable in infertile sperm, whereas that of CB(1) and CB(2) receptors was not statistically different in the two groups. In conclusion, this study identified unprecedented alterations of the ECS in infertile sperm, that might impact on capacitation and acrosome reaction, and hence fertilization outcomes. These alterations might also point to new biomarkers to determine male reproductive defects, and identify distinct ECS elements as novel targets for therapeutic exploitation of ECS-oriented drugs to treat male fertility problems.

To evaluate sperm DNA fragmentation and semen parameters to diagnose male factor infertility and predict pregnancy after IVF.Prospective study.Academic research laboratory.Seventy-five couples undergoing IVF and 28 fertile donors.Sperm DNA fragmentation was measured by the alkaline Comet assay in semen and sperm after density gradient centrifugation (DGC). Binary logistic regression was used to analyze odds ratios (OR) and relative risks (RR) for IVF outcomes.Semen parameters and sperm DNA fragmentation in semen and DGC sperm compared with fertilization rates, embryo quality, and pregnancy.Men with sperm DNA fragmentation at more than a diagnostic threshold of 25% had a high risk of infertility (OR: 117.33, 95% confidence interval [CI]: 12.72-2,731.84, RR: 8.75). Fertilization rates and embryo quality decreased as sperm DNA fragmentation increased in semen and DGC sperm. The risk of failure to achieve a pregnancy increased when sperm DNA fragmentation exceeded a prognostic threshold value of 52% for semen (OR: 76.00, CI: 8.69-1,714.44, RR: 4.75) and 42% for DGC sperm (OR: 24.18, CI: 2.89-522.34, RR: 2.16).Sperm DNA testing by the alkaline Comet assay is useful for both diagnosis of male factor infertility and prediction of IVF outcome.

BACKGROUND:Sperm DNA damage shows great promise as a biomarker of infertility. The study aim is to determine the usefulness of DNA fragmentation (DF), including modified bases (MB), to predict assisted reproduction treatment (ART) outcomes.METHODS:DF in 360 couples (230 IVF and 130 ICSI) was measured by the alkaline Comet assay in semen and in sperm following density gradient centrifugation (DGC) and compared with fertilization rate (FR), embryo cumulative scores (ECS(1)) for the total number of embryos/treatment, embryos transferred (ECS(2)), clinical pregnancy (CP) and spontaneous pregnancy loss. MB were also measured using formamidopyrimidine DNA glycosylase to convert them into strand breaks.RESULTS:In IVF, FR and ECS decreased as DF increased in both semen and DGC sperm, and couples who failed to achieve a CP had higher DF than successful couples (+12.2% semen, P = 0.004; +9.9% DGC sperm, P = 0.010). When MB were added to existing strand breaks, total DF was markedly higher (+17.1% semen, P = 0.009 and +13.8% DGC sperm, P = 0.045). DF was not associated with FR, ECS or CP in either semen or DGC sperm following ISCI. In contrast, by including MB, there was significantly more DNA damage (+16.8% semen, P = 0.008 and +15.5% DGC sperm, P = 0.024) in the group who did not achieve CP.CONCLUSIONS:DF can predict ART outcome for IVF. Converting MB into further DNA strand breaks increased the test sensitivity, giving negative correlations between DF and CP for ICSI as well as IVF.

DNA fragmentation in testicular sperm from men with obstructive azoospermia is increased by 4-hour and 24-hour incubations and after cryopreservation with the effect is intensified by post-thaw incubation. Testicular sperm for use in intracytoplasmic sperm injection (ICSI) should be injected without delay.