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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., (Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To determine the effects of sildenafil citrate, a cyclic monophosphate-specific type 5 phosphodiesterase inhibitor known to affect sperm function, on fertilization and early embryo cleavage., Design: This acute mammal study included male and female mice assigned randomly, the females sacrificed after mating and their oocytes/embryos evaluated at four time periods after treatment., Setting: Academic research environment., Animal(s): Male and female CBAB(6) mice., Intervention(s): Female mice were injected intraperitoneally with 5 IU gonadotropin (hCG) to stimulate follicular growth and induce ovulation. They were each caged with a male that had been gavaged with sildenafil citrate (0.06 mg/0.05 mL) and allowed to mate. After 12, 36, 60, and 84 h, females were killed, their oviducts were dissected out, and retrieved embryos were assessed for blastomere number and quality., Main Outcome Measure(s): Fertilization rates and numbers of embryos were evaluated after treatment., Result(s): Fertilization rates (day 1) were markedly reduced (-33%) in matings where the male had taken sildenafil citrate. Over days 2-4, the numbers of embryos developing in the treated group were significantly fewer than in the control group. There was also a trend for impaired cleavage rates within those embryos, although this did not reach significance., Conclusion(s): The impairments to fertility caused by sildenafil citrate have important implications for infertility centers and for couples who are using this drug precoitally while attempting to conceive.

Objective: To determine whether sildenafil citrate, a cyclic monophosphate-specific type 5 phosphodiesterase inhibitor, influences sperm motility or the acrosome reaction., Design: Laboratory analysis of sperm motility after exposure to sildenafil citrate using computer-assisted semen analysis and acrosome reaction by fluorescein isothiocyanate-labeled peanut agglutinin staining., Setting: An assisted reproductive technology (ART) unit., Patient(s): Fifty-seven male patients., Intervention(s): Sperm were divided into 90% (those with the best fertilizing potential used in assisted conception) and 45% (the poorer population) fractions by density centrifugation and incubated with sildenafil citrate (0.67 muM) at 37 degrees C for up to 180 minutes., Main Outcome Measure(s): Both the number and velocity of progressively motile sperm were significantly increased by sildenafil citrate between 15 and 135 minutes. Furthermore, samples revealed that these effects were consistent in the 90% and 45% populations of sperm. In both populations, sildenafil also caused a significant increase in the proportion of acrosome-reacted sperm-22.1% compared with 11.8% in the control group of the good quality fraction and 16.6% compared with 9.4% in the control group of the poorer quality fraction., Conclusion(s): The use of sildenafil citrate may adversely affect male fertility.

Objective: To compare sperm yields, apoptotic indices, and sperm DNA fragmentation from vasectomized men and fertile men undergoing vasectomy., Design: Testicular biopsies from vasectomized (n = 26) and fertile men (n = 46), were milked to calculate sperm/gram and also formalin-fixed to determine the numbers of developing sperm and incidence and intensities of testicular FasL, Fas, Bax, and Bcl-2. Testicular sperm DNA fragmentation was assessed using the alkaline Comet assay., Setting: An ART unit., Patient(s): Twenty-six men attending for intracytoplasmic sperm injection (ICSI) and 46 men attending for vasectomies., Main Outcome Measure(s): Spermatocyte, spermatid and sperm yields, Fas, FasL, and Bax staining., Result(s): Sperm yields from men vasectomized >5 years previously were markedly reduced compared to fertile men. Increased intensities of FasL and Bax staining were observed in the seminiferous tubules of vasectomy men. FasL positivity (percentage) also increased in Sertoli cells, and both FasL and Fas positivity (percentage) increased in primary spermatocytes and round spermatids of vasectomized men. Sperm DNA fragmentation, an end point marker of apoptosis, increased significantly in vasectomized men compared to fertile men., Conclusion(s): Reduced sperm yields after vasectomy are associated with increased apoptosis through the Fas-FasL and Bax pathways. Sperm after vasectomy displayed increased DNA fragmentation.

Objective: To investigate effects of delta-9-tetrahydrocannabinol (THC) on human sperm function in vitro., Design: Laboratory analysis of sperm motility after exposure to THC using computer-assisted semen analysis and acrosome reaction by fluoroscein isothiocyanate-labeled peanut agglutinin staining., Setting: An assisted reproductive technology unit., Patient(s): Seventy-eight male patients., Intervention(s): Sperm were divided into 90% (the best fertilizing potential used in assisted conception) and 45% (the poorer subpopulation) fractions by density centrifugation and incubated with THC at concentrations equivalent to therapeutic (0.032 microM) and recreational (0.32 and 4.8 microM) plasma levels at 37 degrees C for 3 h., Main Outcome Measure(s): Sperm motility and spontaneous and induced acrosome reactions., Result(s): Percentage progressive motility was decreased dose dependently in the 90% fraction (by 2%-21%; P<.05; P<.001). The 45% fraction showed a greater decrease in percentage progressive motility (by 28% at 0.032 microM; 56% at 4.8 microM; P=.004 and P=.01 res). Straight line velocity and the average path velocity also were reduced (by 10%, in the 90% LAYER) in both fractions. Spontaneous acrosome reactions were reduced in the 90% (17% at 0.032 microM, 35% at 4.8 microM P=.004 and P<.001 resp) and more markedly in the 45% fractions (17%-35%; P<.001). When the acrosome reaction was artificially induced (90% fraction) by A23187, THC (4.8 microM) resulted in a 57% inhibition (P<.001)., Conclusion(s): The use of THC as a recreational drug may adversely affect male fertility.

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.

Objective: To determine the incidence of Fas positivity and DNA double-strand breaks (DSB) as indicators of early- and late-stage apoptosis in ejaculated sperm., Design: Fas positivity was assessed by flow cytometry and DSB by the neutral Comet assay., Setting: Andrology Laboratory, Royal Maternity Hospital, Belfast, Northern Ireland, United Kingdom., Patient(s) and Intervention(s): Forty-five infertile men undergoing infertility investigations and 10 fertile men undergoing vasectomies., Main Outcome Measure(s): Percentage Fas-positive cells, percentage DNA fragmentation, olive tail moment., Result(s): The apoptotic marker Fas was detected in ejaculated sperm, with a higher incidence of Fas positivity in teratozoospermic and asthenozoospermic than in normozospermic semen. No Fas positivity was observed in fertile men's sperm. Deoxyribonucleic acid fragmentation (DSB) was greater in infertile than in fertile men's sperm and also greater in sperm in semen than in sperm prepared for assisted conception. There was an inverse relationship between DSB and both sperm concentration and motility. There was no relationship between Fas positivity and DNA damage., Conclusion(s): Fas was expressed in sperm of infertile men. In contrast, DNA fragmentation was observed in all sperm of fertile and infertile men and correlated with inadequate concentration and motility, which suggests that sperm DSB are ubiquitous and are not solely associated with apoptosis.

Objective: To determine the mitochondrial DNA and nuclear DNA fragmentation of sperm populations separated by using discontinuous density gradient., Design: Analysis of mitochondrial and nuclear DNA status of sperm from high and low density layers., Setting: Regional fertility center., Patient(s): Twenty-eight men who presented for an initial infertility investigation., Main Outcome Measure(s): Semen was prepared by using discontinuous density gradient (90.0%:45.0%) and subjected to a modified long polymerase chain reaction to assess mitochondrial DNA deletions and to a modified single-cell alkaline gel electrophoresis assay to determine nuclear DNA fragmentation., Result(s): The high-density fraction displayed significantly more wild-type mitochondrial DNA (75% of samples) than did the low-density fraction (25% of samples). In the high-density fraction, the incidence of single deletions was higher than that of double or multiple deletions, and the deletions were predominantly small. A strong correlation was observed between nuclear DNA fragmentation and the number and size of mitochondrial DNA deletions., Conclusion(s): Density centrifugation isolates a population of sperm with high-quality mitochondrial DNA and nuclear DNA.

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.

Objective: To assess the effect of Trucut needle biopsy on the ultrasound appearances of the testis in obstructive and nonobstructive azoospermia to test serum samples for antisperm antibodies and gonadotropin and testosterone levels., Design: Prospective case analysis., Setting: IVF unit., Patient(s): Sixteen subjects with obstructive azoospermia had postbiopsy ultrasound scans, 18 had assessment of hormone profiles, and 20 had evaluation of antisperm antibodies., Intervention(s): Trucut needle testicular biopsies under local anesthetic., Main Outcome Measure(s): Postbiopsy testicular ultrasound, the presence of serum antisperm antibodies, and follicle stimulating and luteinizing hormone and testosterone levels., Result(s): There were no postbiopsy hematomas or scars, antisperm antibodies did not develop, and pituitary gonadotropins did not rise nor testosterone levels fall., Conclusion(s): Trucut needle testicular biopsy in men with obstructive azoospermia is not associated with defects of parenchymal structure or function.

Objective: To assess the effects of two methods of freezing on testicular sperm DNA from subjects with obstructive azoospermia and to compare these with samples of fresh and freeze-thawed testicular sperm from fertile men., Design: The Comet assay was used to determine the percentage of undamaged DNA in fresh testicular sperm, testicular sperm freeze-thawed in suspension and in a biopsy sample (men with obstructive azoospermia), and in fresh and freeze-thawed testicular sperm (fertile men)., Setting: The Regional Fertility Center, Royal Maternity Hospital, Belfast, Northern Ireland, United Kingdom., Patient(s): Twelve males with obstructive azoospermia (normal testicular volume and hormone profiles) and nine fertile control subjects., Intervention(s): Trucut needle testicular biopsy under local anesthetic., Main Outcome Measure(s): Measurement of the percentage of undamaged DNA in fresh and freeze-thawed testicular sperm using the Comet assay., Result(s): No significant difference was found between the percentage of undamaged DNA of fresh testicular sperm and of both types of freeze-thawed testicular sperm. There was also no significant difference between the percentage of undamaged DNA in fresh or freeze-thawed testicular sperm from controls. Control ejaculated sperm DNA was significantly more damaged than testicular sperm DNA from control men., Conclusion(s): Cryopreservation of testicular sperm does not increase baseline levels of DNA damage.

Objective: To assess the sperm yield and patient acceptability of Trucut needle testicular biopsy followed by seminiferous tubule milking., Design: Prospective case analysis., Setting: The Regional Fertility Center, Royal Maternity Hospital, Belfast, Northern Ireland, United Kingdom., Patient(s): Forty-one males with obstructive azoospermia (normal testicular volume and FSH and LH levels)., Intervention(s): Trucut needle testicular biopsies under local anesthetic with milking of the seminiferous tubules., Main Outcome Measure(s): Quantitation of sperm retrieved per biopsy core and patient follow-up by questionnaire., Result(s): A mean of 105,634 sperm (range, 5,000-427,800) were retrieved, and the mean biopsy weight was 9.17 mg. Twenty-six subjects found the biopsy painless and 15 were pain-free after biopsy., Conclusion(s): The Trucut needle can be used in combination with seminiferous tubule milking to obtain large numbers of sperm in men with obstructive azoospermia.

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.

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.

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 Queen's 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.

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.

Objective: To determine if pentoxifylline has an in vitro effect on human sperm motility in subjects with normal semen analysis., Design: Prospective, longitudinal study of the effect of pentoxifylline on normospermic semen samples using computerized analysis., Setting: The Assisted Conception Unit, Royal Maternity Hospital, Belfast., Subjects: Eight healthy male volunteers of proven fertility within 2 years of study and whose general health was unchanged in the intervening period., Results: Pentoxifylline did not consistently increase the number of progressively motile sperm or their straight line velocity (VSL). In marked contrast, the curvilinear velocity (VCL) was increased significantly at all times, up to and including 240 minutes of exposure to the drug. This resulted in a substantial reduction in the linearity (VSL/VCL) of sperm progression. The most dramatic effect that pentoxifylline had on sperm movement was to increase the amplitude of lateral head displacement (ALH) over the same time interval. This change was accompanied by an increase in the beat cross frequency., Conclusions: The addition of pentoxifylline to prepared sperm samples from normospermic men does not increase the number of progressively motile sperm nor the progressive velocity. However, it does significantly alter the quality of certain characteristics of already motile sperm.