Sheena E.M. Lewis

Total antioxidant capacity of seminal plasma is different in fertile and infertile men

OBJECTIVE To determine if the total antioxidant capacity of seminal plasma is different in fertile and infertile men. DESIGN An enhanced chemiluminescence assay applied to seminal plasma from groups of fertile and infertile men. SETTING The Assisted Conception Unit, Royal Maternity Hospital, Belfast. SUBJECTS Men of proven fertility whose partners had an ongoing pregnancy resulting from IVF and male partners of couples attending our subfertility clinic. RESULTS Total antioxidant capacity was significantly higher in seminal plasma from fertile men than from that of infertile men with normozoospermic samples that exhibited reactive oxygen species or asthenozoospermic samples with or without reactive oxygen species activity. CONCLUSIONS Seminal plasma from infertile men has lower antioxidant levels than that of fertile men, particularly of patients whose semen have poor sperm motility. The presence of reactive oxygen species activity in sperm of infertile groups also is associated with lower levels of chain-breaking antioxidants in seminal plasma.

Seeds of concern

During the past few decades, worries about environmental threats to human health have centred on the possible induction of cancers. Now risks to the male germ line, both real and potential, are also causing disquiet.

Comparison of individual antioxidants of sperm and seminal plasma in fertile and infertile men

OBJECTIVE To measure individual antioxidants in sperm and seminal plasma from fertile and infertile men to determine if any particular antioxidant is reduced in infertile men. DESIGN Semen samples were prepared by a discontinuous Percoll gradient to separate sperm and seminal plasma, and the antioxidant concentrations of each were assessed. Samples also were screened for phorbol ester-induced reactive oxygen species (ROS) activity. SETTING Departments of Obstetrics and Gynaecology, and Clinical Biochemistry, The Queens University of Belfast, Northern Ireland. PATIENT(S) Fifty-nine male patients attending our infertility center: 18 men whose wives had ongoing pregnancies from IVF with normozoospermic semen profiles, 20 infertile men with normozoospermic and 21 men with asthenozoospermic semen profiles. MAIN OUTCOME MEASURE(S) Ascorbate, urate, sulphydryl groups, tocopherol and carotenoid concentrations were measured in sperm and seminal plasma from fertile and infertile men. RESULT(S) In seminal plasma, ascorbate contributes almost twice as much as urate and thiol levels are about one third of ascorbate. Ascorbate levels in seminal plasma of asthenozoospermic individuals (+ROS) are significantly reduced. In sperm, thiols contributed most and ascorbate only a fraction of the total. CONCLUSION(S) In seminal plasma, ascorbate, urates, and thiols are the major antioxidants present. In contrast, within sperm, this group is the major contributor. In samples exhibiting ROS activity, ascorbate concentrations in the seminal plasma are significantly reduced.

Sperm DNA: organization, protection and vulnerability: from basic science to clinical applications—a position report

This article reports the results of the most recent in a series of EHSRE workshops designed to synthesize the current state of the field in Andrology and provide recommendations for future work (for details see Appendix). Its focus is on methods for detecting sperm DNA damage and potential application of new knowledge about sperm chromatin organization, vulnerability and repair to improve the diagnosis and treatment of clinical infertility associated with that damage. Equally important is the use and reliability of these tests to identify the extent to which environmental contaminants or pharmaceutical agents may contribute to the incidence of sperm DNA damage and male fertility problems. A working group (for workshop details, see Appendix) under the auspices of ESHRE met in May 2009 to assess the current knowledgebase and suggest future basic and clinical research directions. This document presents a synthesis of the working groups understanding of the recent literature and collective discussions on the current state of knowledge of sperm chromatin structure and function during fertilization. It highlights the biological, assay and clinical uncertainties that require further research and ends with a series of 5 key recommendations.

In vitro fertilization and pregnancy rates: the influence of sperm motility and morphology on IVF outcome

OBJECTIVE To determine the relationship between sperm motility and sperm morphology parameters and IVF and pregnancy rates. DESIGN Pre- and postpreparation analysis of semen samples from infertile couples undergoing IVF-ET. SETTING Andrology Laboratory, Royal Maternity Hospital, Belfast, Northern Ireland. PATIENT(S) One hundred fifty couples undergoing IVF-ET treatment at the Regional Fertility Centre. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) The ability of human sperm to achieve IVF and pregnancy was investigated in relation to motility parameters (assessed with computer-aided sperm analysis [Integrated Visual Optical System] and percent normal morphology (determined with the strict criteria). RESULT(S) Significant differences were observed in motility parameters and percent normal morphology in samples that achieved > or =50% fertilization compared with < or =50% fertilization and between samples that achieved a pregnancy compared with those that did not. Significant positive correlations were observed between percent progressive motility, the velocity of sperm movement, and morphology parameters and both IVF and pregnancy. CONCLUSION(S) Both sperm motility parameters and percent normal morphology are significant factors in predicting fertilization and pregnancy rates in IVF.

Cryopreservation of human semen and prepared sperm: effects on motility parameters and DNA integrity

OBJECTIVE To investigate effects of cryopreservation on sperm motility and DNA integrity. DESIGN Pre-cryopreservation and post-cryopreservation analysis of motility and DNA integrity of semen and prepared sperm samples. SETTING A hospital andrology laboratory. PATIENT(S) Forty men attending the Regional Fertility Centre, Belfast, Northern Ireland. INTERVENTION(S) Each sample was divided, and an aliquot was frozen unprepared. Remaining aliquots were prepared by Percoll density centrifugation (95.0:47.5) or direct swim-up procedure and divided into aliquots to allow direct comparison of fresh and frozen semen and prepared sperm (frozen with or without the addition of seminal plasma) from the same ejaculate. Samples were frozen by static-phase vapor cooling and being plunged into liquid nitrogen. Thawing was carried out at room temperature. MAIN OUTCOME MEASURE(S) Sperm DNA integrity was determined using a modified alkaline single cell gel electrophoresis (comet) assay, and motility was determined using computer-assisted semen analysis. RESULT(S) Sperm frozen unprepared in seminal fluid appeared more resistant to freezing damage than frozen prepared sperm. Further improvements can be achieved by selecting out the subpopulation of sperm with best motility and DNA integrity and freezing these sperm in seminal plasma, making this the optimal procedure. CONCLUSION(S) Freezing sperm in seminal plasma improves postthaw motility and DNA integrity.

Clinical significance of sperm DNA damage in assisted reproduction 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.

Antioxidant supplementation in vitro does not improve human sperm motility

OBJECTIVE To determine the effects of supplementation of preparation media with ascorbate and alpha-tocopherol on subsequent sperm motility and reactive oxygen species production. DESIGN Prospective study to analyze postpreparation human sperm motility parameters and reactive oxygen species production following antioxidant supplementation. SETTING Andrology Laboratory, Royal Maternity Hospital, Belfast, Northern Ireland. PATIENT(S) Sixty patients attending the Andrology Laboratory for semen analysis. INTERVENTION(S) Normozoospermic and asthenozoospermic semen samples (n = 10 for each control and antioxidant group) were prepared by Percoll density centrifugation (95%-47.5%) in media supplemented with ascorbate or alpha-tocopherol to different concentrations within physiologic levels. Controls were included that were not exposed to antioxidant. MAIN OUTCOME MEASURE(S) Sperm motility parameters were assessed using computer-assisted semen analysis. The generation of reactive oxygen species was determined using luminol-dependent chemiluminescence. RESULT(S) The production of reactive oxygen species by sperm was reduced by supplementation in vitro with ascorbate and alpha-tocopherol. However, progressive motility, average path velocity, curvilinear velocity, straight-line velocity, and linearity were decreased significantly, with the greatest inhibition observed with the highest concentrations of antioxidants. CONCLUSION(S) Supplementation of preparation media with ascorbate and alpha-tocopherol, either singly or in combination, is not beneficial to sperm motility.

The effects of aging and chronic dietary restriction on whole body growth and protein turnover in the rat

Changes in whole body growth, nucleic acids, and protein turnover have been studied in conjunction with ageing and chronic dietary restriction. Normal developmental changes between weaning and senescence included progressive decreases in the fractional rates of growth, protein synthesis, and protein breakdown; the decline in the synthetic rate correlating with decreases in the ribosomal capacity. Dietary intervention was imposed at weaning and involved pair feeding to 50% of the ad libitum food intake. Although this regime slowed whole body growth by retarding the developmental decline in protein turnover, growth was extended into the second and third years of life. The dietary-induced increase in longevity resulting from a retardation of the ageing process(es) appears therefore to be associated with an enhanced turnover of proteins during the major portion of the life span of dietary restricted rats.

Sperm DNA damage measured by the alkaline Comet assay as an independent predictor of male infertility and in vitro fertilization success

OBJECTIVE To evaluate sperm DNA fragmentation and semen parameters to diagnose male factor infertility and predict pregnancy after IVF. DESIGN Prospective study. SETTING Academic research laboratory. PATIENT(S) Seventy-five couples undergoing IVF and 28 fertile donors. INTERVENTION(S) 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. MAIN OUTCOME MEASURE(S) Semen parameters and sperm DNA fragmentation in semen and DGC sperm compared with fertilization rates, embryo quality, and pregnancy. RESULT(S) 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). CONCLUSION(S) Sperm DNA testing by the alkaline Comet assay is useful for both diagnosis of male factor infertility and prediction of IVF outcome.