DNA strand breaks in human spermatozoa: a possible factor, to be considered in couples suffering from unexplained infertility.

BACKGROUND: The aim of this study was to assess the presence of DNA strand breaks and sperm cell morphology in men suffering from unexplained infertility, and to compare these results with normal fertile and oligozoospermic men. METHODS: One fresh sperm sample from proven fertile sperm donors (n=20) and from infertile men with oligozoospermia, (<20 x 10(6)/ml sperm cells) (n=74), and men suffering from unexplained infertility (>20 x 10(6)/ml sperm cells) (n=39) who delivered two sperm samples with 24 hours interval, were tested for the presence of DNA strand breaks in the spermatozoa by direct immunoperoxidase detection of digoxigenin-labeled genomic DNA. Correlations to other sperm parameters, sperm cell counts, motility, activation and Krüger's strict criteria were performed. RESULTS: DNA strand breaks in sperm cell nuclei were found significantly more often in sperm samples from men suffering from unexplained infertility compared to those from normal fertile men, and significantly more rarely compared with sperm samples from men with oligozoospermia. The percentages of normal spermatozoa (Krüger's strict criteria) were significantly lower in samples from men suffering from unexplained infertility compared to those of normal fertile men, but significantly higher compared to those of men with oligozoospermia. No difference was found between first and second day samples used for insemination, as regards DNA strand breaks, sperm cell morphology, total number of motile sperm cells, activation and motility degree. CONCLUSION: The present data suggest that a subgroup of men suffering from unexplained infertility have DNA strand breaks in their sperm cell DNA. This group might suffer from the same malfunction as many men with oligozoospermia, however, their apoptotic activated sites in the testis are different. Delivery of sperm samples with 24 hours interval does not affect any sperm cell counts, CASA, DNA strand breaks or morphology findings in sperm samples from men suffering from unexplained infertility.     

The role of DNA strand breaks in human spermatozoa used for IVF and ICSI

BACKGROUND: The objective of this study was to determine the incidence of spermatozoa with DNA strand breaks in four clinically different groups of infertile couples, and to correlate DNA damage with other semen analysis parameters, as well as fertilization rates and IVF outcome. METHODS: One group consisted of 75 men where the female partners had a tubal obstruction, Group A. Fifty sperm samples were collected from men in unexplained infertile couples, Group B. Fifty men with oligozoospermia and IVF made up Group C. Finally, 61 men with oligozoospermia and where ICSI was performed made up Group D. Sperm samples were assessed according to the WHO manual and for the presence of DNA strand breaks in spermatozoa. The study was blinded for the technician involved in the assessment of DNA strand breaks. IVF was carried out according to a long down regulation protocol using GnRH, FSH and hCG. Embryos were transferred on day 2 after fertilization with a maximum of three embryos. RESULTS: This study demonstrated a negative correlation between the proportion of spermatozoa having DNA strand breaks and the proportion of oocytes fertilized after IVF (p<0.01). Furthermore, the number of spermatozoa with DNA strand breaks was important for the pregnancy rate in the group of unexplained infertile couples. After ICSI no association was found between spermatozoa with DNA strand breaks and fertilization rates (p>0.05). CONCLUSION: DNA strand breaks in human spermatozoa impairs fertilization in both unexplained infertile couples and those with oligozoospermia and IVF. However, after ICSI, this impact of DNA strand breaks were not seen. This creates a specific indication and treatment for this new diagnosed group of otherwise unexplained infertile men.     

Sperm morphology and IVF: embryo quality in relation to sperm morphology following the WHO and Krüger's strict criteria.

BACKGROUND: The aim of this study was to examine the correlation between sperm morphology and embryo quality/IVF outcome. METHODS: The implication of sperm morphology assessment before an IVF cycle was evaluated. A total of 100 IVF couples where the female partner had either tubal factor (n=50) or unexplained infertility (n= 50) entered a prospective study, and sperm samples for the actual cycle were assessed according to the strict criteria and WHO criteria. The study was blinded for the technician involved in sperm morphology analyzing. IVF was carried out according to a long down regulation protocol using GnRH/FSH/hCG and ova were inseminated with 200,000 spermatozoa/ml. Embryos were transferred on day 2 post fertilization in a maximum of three embryos. RESULTS: No significant differences were found between the groups regarding age of the female partner (mean=34.3), no. oocytes retrieved (mean=8.5), fertilization (66.5%), pregnancies (pos. S-hCG/transfer 39.6%) or 'Take home baby rate' (birth rate/transfer 30.0%). As to the score of Krüger's strict criteria and the WHO criteria, we found no correlation between this score and cleavage rate, embryo development or pregnancies. The WHO criteria were found to be a better predictor for fertilization rate than the Krüger's criteria (p<0.002). CONCLUSION: The strict criteria or sperm evaluation according to WHO have no better predictive value for the outcome of routine IVF.     

Comparison between a two-layer discontinuous Percoll gradient and swim-up for sperm preparation on normal and abnormal semen samples

Purpose This work was to compare the effects of Percoll gradient and swim-up treatments for sperm preparation on the percentage of progressive motility, recovery of motile sperm, removal of debris, percentage of normal forms according to strict criteria, and movement characteristics of sperm using computer-assisted velocity analysis.Results In total, 50 semen samples from 50 patients were tested and divided into two groups: a normal group (n=27) with normal parameters and an abnormal group (n=23) with abnormal parameters. The results in both the normal and the abnormal groups revealed that the sperm concentration in the Percoll samples was significantly greater than that in the swim-up samples. Although the percentage of progressive motility was greater in the swim-up samples than in the Percoll samples, the number of motile sperm, reflecting the percentage of motile sperm recovery, was still greater in the Percoll samples. The debris of semen were equally removed by both methods and the percentage of normal forms was also similar in the samples treated according to these two procedures. Both curvilinear velocity (VCL) and straight-line velocity (VSL) of sperm were significantly greater in the swim-up samples than in the Percoll samples. Sperm from the swim-up procedure also showed a greater mean amplitude of lateral head displacement than that from the Percoll gradient procedure, but the distinction was insignificant.Conclusion The Percoll gradient technique, by recovering more motile sperm, may be applied to prepare oligospermic samples. The swim-up method may become the standard choice to prepare normal semen which could obtain sufficiently motile sperm, due to its simplicity and recovered sperm with superior motility.     

The significance of sperm nuclear DNA strand breaks on reproductive outcome

PURPOSE OF REVIEW: A growing body of evidence indicates that ejaculated spermatozoa from men being treated with intracytoplasmic sperm injection contain nuclear abnormalities. Many of these nuclear anomalies manifest themselves as breaks in the sperm nuclear DNA. This review examines the mechanisms involved in generating DNA strand breaks during spermatogenesis in the human, the main techniques used to assess the sperm nucleus and the evidence, in relation to assisted reproduction, showing that sperm nuclear DNA strand breaks may impact on reproductive outcome. RECENT FINDINGS: Techniques such as the TUNEL assay and the sperm chromatin structure assay both show increased levels of DNA abnormalities in spermatozoa from men who have poor semen parameters. The reproductive parameters affected by an increased presence of DNA abnormalities in ejaculated spermatozoa include fertilization, blastocyst development, and pregnancy rates. SUMMARY: There is accumulating evidence linking sperm nuclear DNA anomalies to poor reproductive outcome in relation to assisted reproduction technologies. The tests currently available only provide an inkling of the impact of sperm nuclear DNA abnormalities on reproductive outcomes. Although the impact an abnormal paternal genome may have on reproductive outcome is unquestionably less than that of its female counterpart, it cannot be ignored.     

DNA strand breaks in human spermatozoa: correlation with fertilization in vitro in oligozoospermic men and in men with unexplained infertility

BACKGROUND: The purpose of this study was to examine the correlation between DNA strand breaks in human spermatozoa and semen quality, fertilization rate and IVF outcome. METHODS: A total of 50 men suffering from unexplained infertility and 50 men with oligozoospermia undergoing IVF treatment entered a prospective study. Sperm samples were assessed according to the WHO manual and for the presence of DNA strand breaks in spermatozoa. The study was blinded for the technician involved in assessment of DNA strand breaks. IVF was carried out according to a long down regulation protocol using GnRH, FSH and hCG. The ova were inseminated with 200,000 spermatozoa/ml. Embryos were transferred on day 2 after fertilization with a maximum of three embryos. RESULTS: This study demonstrates a negative correlation between the proportion of spermatozoa having DNA strand breaks and the proportion of oocytes fertilized after IVF. CONCLUSION: The number of human spermatozoa with DNA strand breaks is a good predictor for fertilization rate in couples suffering from unexplained infertility and undergoing IVF treatment.     

Morphology of spermatozoa used in IVF and ICSI from oligozoospermic men

The purpose of this study was to determine morphology in human spermatozoa from men with oligozoospermia and correlate with the fertilization rate, embryo score and pregnancy rate after IVF and intracytoplasmic sperm injection (ICSI) respectively. The study group consisted of 125 couples where the male partner suffered from oligozoospermia. Fifty of these had IVF (group A). Seventy-five couples in whom ICSI had been performed made up group B. Sperm samples were assessed according to the WHO manual. For each male, morphology of spermatozoa was judged according to Krüger's strict criteria, WHO criteria and the teratozoospermia index (TZI). Oocyte monitoring was carried out according to a long down-regulation protocol using gonadotrophin-releasing hormone, recombinant FSH and human chorionic gonadotrophin. Embryos were transferred on day 2 after fertilization, with a maximum of three embryos. This study demonstrated no correlation between any of the morphological assessments of spermatozoa and the fertilization rate, embryo score and pregnancy rate, either after IVF or ICSI. Morphology in human spermatozoa according to Krüger's strict criteria, WHO criteria and the TZI had no predictive value for the outcome after either IVF or ICSI.     

Flow cytometric comparison between swim-up and Percoll gradient techniques for the separation of frozen-thawed human spermatozoa.

Viable human spermatozoa were recovered from cryopreservation by either swim-up or Percoll density gradient techniques and their cell-surface oligosaccharide components were compared to those of fresh sperm. Sperm were labeled with FITC-Con A and analyzed by fluorescence microscopy and flow cytometry. By fluorescence microscopy, fresh sperm were uniformly labeled in the neck region alone. Frozen-thawed sperm, obtained by either swim-up or Percoll density gradients, showed two patterns of staining: one sperm population was stained in the neck region only, whilst another showed staining in both the neck and acrosome regions. No difference between sperm recovered by the two techniques was apparent. Flow cytometry profiles of fresh sperm revealed a single peak of fluorescence, whereas cryopreserved sperm gave two peaks of fluorescence. Samples recovered by Percoll density gradient contained significantly more sperm with the same fluorescence pattern as that observed for fresh sperm than did those recovered by swim-up. Cryopreservation alters the cell-surface oligosaccharide components of spermatozoa. Recovery of cryopreserved sperm by Percoll density gradient yields a greater proportion of sperm which resemble fresh sperm than does swim-up.     

Semen manipulation: improved sperm recovery and function with a two-layer Percoll gradient

The authors compared a simple, two-layer Percoll density gradient technique with the swim-up technique for semen preparation in 128 men. In samples from normospermic (n = 55), oligospermic (n = 26), and asthenospermic (n = 29) men, the Percoll technique significantly improved yield, percent motility, and absolute number of motile sperm recovered, but in samples from oligoasthenospermic men (n = 18), only percent motility was improved. The Percoll density gradient also selected sperm with markedly improved function as assessed by both the sperm penetration assay and the fertility index. In 37 samples negative on the sperm penetration assay when processed with the swim-up technique, 19 (51%) became positive when processed with the Percoll technique. The Percoll density gradient is an improved method for semen manipulation as it allows greater recovery of sperm with higher motility and improved sperm function.     

Clinical significance of sperm DNA damage threshold value in the assessment of male infertility

Sperm DNA integrity is a prerequisite for normal spermatozoal function. The aim of the study was to evaluate the role of sperm chromatin damage, its cut-off level and its effect on sperm parameters in men with idiopathic infertility by analyzing 100 idiopathic infertile men and 50 fertile controls. Semen samples were analyzed as per WHO 1999 guidelines and sperm chromatin structure assay (SCSA) was applied to measure DNA fragmentation index (DFI) in sperm. The mean DFI of infertile men (35.75) was significantly (P < .0001) higher as compared to controls (26.22). The threshold level of 30.28% was obtained as cut-off value to discriminate infertile men from fertile controls. Sperm count, forward motility, and normal morphology found to be negatively associated with DFI in overall study subjects. Infertile men with severe oligozoospermia had higher mean DFI (40.01 ± 11.31) than infertile men with oligozoospermia (35.11 ± 10.05) and normal sperm count (33.99 ± 9.96). Moreover 64% of infertile men have DFI > 30 against 6% of fertile controls (P < .0001). Higher sperm DNA fragmentation may be the underlying cause for poor semen quality in idiopathic infertile men and the threshold value of 30.28% is a clear discriminator to distinguish infertile men from fertile men of Indian population. Thus, DFI is a good prognostic marker as cases with higher sperm DFI may have poor success rate even after assisted conception and may experience recurrent pregnancy loss (RPL) and should be counseled accordingly.     

Effects of four methods of sperm preparation on the motile concentration,morphology, and acrosome status of recovered sperm from normal semen samples

Purpose This prospective study compared the effects of four sperm preparation methods on semen samples with normal sperm parameters. The samples were obtained from husbands of infertile couples being evaluated at a fertility center in New York City. Twenty-nine of 51 men in the study (56.9%) had fathered a child within the past 2–5 years. Specifically, the study compared the effects of simple wash (unselective) and three selective methods—conventional swim-up, three-gradient Percoll separation, and swim-up from the Percoll-separated fraction—on the concentration, percentage motile, percentage normal (oval) morphology, percentage intact acrosomes, percentage motile sperm recovery, and motile sperm concentration in the processed samples. The same parameters were measured in the untreated ejaculate, resulting in five data sets. These data were analyzed by both comparison across the five sets and pairwise multiple comparisons between sets.Results The study showed that of the selective methods, conventional swim-up and Percoll separation yielded comparably optimal percentages motile sperm recovery and motile sperm concentrations.Conclusion However, swim-up yielded a significantly higher percentage oval and percentage motile sperm than Percoll, while Percoll had a significantly higher percentage acrosome-intact. Swim-up after Percoll separation yielded the highest percentage motility and percentage oval but had the lowest motile sperm concentration with the lowest percentage acrosome-intact.This study was undertaken on patients undergoing fertility evaluation and treatment at the Brandeis Fertility Center. There was no outside support in the form of financial aid, grants, or equipment.     

Assessment of DNA fragmentation of spermatozoa that were surgically retrieved from men with obstructive azoospermia

OBJECTIVE: To determine the degree of DNA fragmentation in spermatozoa of men with obstructive azoospermia or anejaculation compared with that of ejaculated spermatozoa from fertile donors. DESIGN: Observational study. SETTING; University Medical Center St. Radboud, Nijmegen. The Netherlands. PATIENT(S): Forty-one patients with obstructive azoospermia or anejaculation and 10 fertile donors. MAIN OUTCOME MEASURE(S): Sperm samples were obtained surgically from the epididymis or testis of men with azoospermia or anejeculation and by ejaculation in fertile patients. DNA fragmentation was analyzed in the total sample and in a motile fraction that was isolated as in routine ICSI procedures. DNA breaks were measured by using the TdT-mediated dUTP nick-end labeling assay. RESULT(S): A higher percentage of cells with DNA breaks was found in men with obstructive azoospermia or anejaculation compared with donors (mean, 18.9% vs. 6.2%). A significant lower degree of DNA fragmentation was observed in the motile fraction from patients compared with donors (0.4% vs. 0.6%). CONCLUSION(S): High percentages of cells with DNA damage were found in sperm samples from men with obstructive azoospermia or anejaculation, but a very low frequency of damage to the DNA was observed in the motile fraction. In an ICSI setting, the use of motile sperm retrieved from epididymis or testis of men with obstructive azoospermia does not seem to pose a higher genetic risk to the progeny than does use of motile ejaculated sperm.     

In vitro reconstruction of inflammatory reaction in human semen: effect on sperm DNA fragmentation

The study was aimed at evaluating an in vitro induction of DNA damage in three sperm subpopulations exposed to selected inflammatory mediators, such as leukocytes, two combinations of pro-inflammatory cytokines (interleukin [IL]-6 + IL-8 and IL-12 + IL-18) and two bacterial strains (Escherichia coli and Bacteroides ureolyticus). Semen samples from normozoospermic volunteers were differentiated by swim-up (swim-up fraction) and Percoll gradient procedures (90% and 47% Percoll fractions). Leukocytes were isolated from the whole heparinized blood using the density gradient centrifugation technique. DNA fragmentation in sperm fractions was evaluated using flow cytometry with TUNEL labeling and Comet assay. Out of the inflammatory factors tested, bacteria were found to have a greatest toxic effect on sperm DNA, especially in fractions isolated by Percoll gradient, compared with untreated cells (P < 0.05). The results indicate that inflammatory mediators can be a direct cause of DNA fragmentation in ejaculated spermatozoa, which can ultimately lead to limited fertilizing abilities of the germ cells. In contrast to the swim-up technique, the selection of spermatozoa by gradient procedures increases the vulnerability of mature spermatozoa to the harmful effects of infectious agents on DNA integrity. This observation may have some meaning for recommendations concerning laboratory techniques used in assisted reproductive therapy.     

Simultaneous swim-up/swim-down of sperm in assisted reproduction procedures

Purpose High-quality motile human spermatozoa were obtained following treatment of semen by simultaneous swim-up into medium and swim-down into an isotonic 40% Percoll solution.Results This procedure was significantly better than the swim-up method and comparable to discontinuous Percoll gradient centrifugation. Recovery rates of motile sperm were 35% for swim-up, 65% for Percoll gradient centrifugation, and 73% for swim-up/swim-down.Conclusion The swim-down sperm was inferior to the swim-up sperm in its upward migration capacity but superior in morphology. Spermatozoa obtained by the swim-up/swim-down procedure demonstrated fertilizing ability in IVF, and clinical pregnancies were established. The simultaneous swim-up/swim-down procedure offers an alternative efficient method of simple separation of high-quality motile sperm for various assisted reproduction techniques.     

Potential adverse effect of semen processing on human sperm deoxyribonucleic acid integrity.

OBJECTIVE: To examine the effect of standard Percoll density-gradient centrifugation on human sperm DNA denaturation. DESIGN: Prospective, observational study. SETTING: University-based infertility clinic. PATIENT(S): Twenty-five nonazoospermic men. INTERVENTION(S): Semen samples (n = 25) were obtained from consecutively seen nonazoospermic men presenting for infertility evaluation. Samples were processed by two-layer and four-layer Percoll density gradients. Sperm motility and sperm chromatin structure (evaluated by flow cytometry analysis of acridine orange-treated spermatozoa) were monitored before and after semen processing. Sperm chromatin integrity was expressed as the percentage of spermatozoa that demonstrated denatured DNA. MAIN OUTCOME MEASURE(S): Sperm motility and DNA integrity. RESULT(S): Mean sperm motility improved significantly after processing with two-layer and four-layer Percoll gradients compared with whole semen (54% and 57% motility versus 44% motility, respectively). In contrast, the percentage of sperm with denatured DNA increased after processing with two-layer and four-layer Percoll gradients compared with whole semen (34% and 32% versus 18%, respectively). CONCLUSION(S): Our data demonstrate that the improvement seen in sperm motility after Percoll processing is not associated with a similar improvement in sperm DNA integrity. These data suggest that we reexamine current sperm processing techniques to minimize sperm DNA damage and the potential transmission of genetic mutations in assisted reproductive cycles.     

Human sperm endothelial nitric oxide synthase expression: correlation with sperm motility

Objective: To characterize the pattern of endothelial nitric oxide synthase (eNOS) expression on human spermatozoa and to determine whether sperm eNOS expression correlates with sperm function.

Design: Prospective, observational study.

Setting: University infertility clinic.

Patient(s): Twelve nonazoospermic infertile men.

Intervention(s): Semen samples (n = 12) obtained from nonazoospermic infertile men were fractionated on discontinuous Percoll gradients. Endothelial nitric oxide synthase staining on spermatozoa was correlated with sperm motility in Percoll gradient–fractionated spermatozoa. Endothelial nitric oxide synthase protein was detected with the use of a previously characterized monoclonal antibody. Control slides were incubated with preabsorbed antibody or mouse immunoglobulin G.

Main Outcome Measure(s): Localization of eNOS on human spermatozoa and correlation between the pattern of sperm eNOS expression and sperm motility.

Result(s): Morphologically normal spermatozoa exhibited postacrosomal and equatorial eNOS immunostaining. However, abnormally shaped spermatozoa often exhibited aberrant staining (in the midpiece and/or head region). A significant negative correlation was observed between the percentage of sperm with aberrant eNOS immunostaining and the percentage of motile sperm (r = −.46).

Conclusion(s): The specific localization of eNOS to human spermatozoa suggests that nitric oxide may be involved in normal sperm physiology. However, aberrant patterns of sperm eNOS expression are associated with decreased sperm motility, possibly through the generation of excessive cytotoxic oxidants.     

Easy sperm processing technique allowing exclusive accumulation and later usage of DNA-strandbreak-free spermatozoa

Sperm DNA fragmentation is increased in poor-quality semen samples and correlates with failed fertilization, impaired preimplantation development and reduced pregnancy outcome. Common sperm preparation techniques may reduce the percentage of strandbreak-positive spermatozoa, but, to date, there is no reliable approach to exclusively accumulate strandbreak-free spermatozoa. To analyse the efficiency of special sperm selection chambers (Zech-selectors made of glass or polyethylene) in terms of strandbreak reduction, 39 subfertile men were recruited and three probes (native, density gradient and Zech-selector) were used to check for strand breaks using the sperm chromatin dispersion test. The mean percentage of affected spermatozoa in the ejaculate was 15.8 ± 7.8% (range 5.0–42.1%). Density gradient did not significantly improve the quality of spermatozoa selected(14.2 ± 7.0%). However, glass chambers completely removed 90% spermatozoa showing strand breaks and polyethylene chambers removed 76%. Both types of Zech-selectors were equivalent in their efficiency, significantly reduced DNA damage (P < 0.001) and,with respect to this, performed better than density gradient centrifugation (P < 0.001). As far as is known, this is the first report ona sperm preparation technique concentrating spermatozoa unaffected in terms of DNA damage. The special chambers most probably select for sperm motility and/or maturity.     

Analysis of aneuploidy in mini-percoll gradient centrifuged human sperm for intracytoplasmic sperm injection (ICSI) using fluorescence in situ hybridization

AIM: To study the aneuploidy rates of chromosomes 13, 18, 21, X and Y in Percoll gradient centrifuged sperm from infertile patients with male infertility factor treated by intracytoplasmic sperm injection (CSI) compared with healthy fertile donors and infertile patients with normal semen parameters. METHODS: This case-controlled study was conducted in a university hospital. Semen samples were obtained from three healthy fertile donors, eight infertile patients with normal semen parameters, and 18 infertile patients with male infertility factor. All samples were subjected to mini-Percoll gradient centrifugation before being processed through fluorescent in situ hybridization. The incidences of aneuploidy were compared using Chi-squared test. RESULTS AND CONCLUSIONS: A total of 64949 spermatozoa were analyzed. The disomy rates for chromosomes 13, 18, 21, and X or Y of sperm from patients with male infertility factor were 0.21%, 0.37%, 0.36% and 0.63%, respectively, whereas the diploidy rate was 0.17-0.23%. These incidences were higher than those from men with normal semen parameters. The result suggested that the embryos of patients with male infertility factor treated by ICSI are at increased risk of chromosome abnormalities.     

Clinical relevance of sperm DNA damage in assisted reproduction

Many studies have shown how a 'paternal effect' can cause repeated assisted reproduction failures. In particular, with increasing experience of intracytoplasmic sperm injection (ICSI), it became evident that spermatozoa from some patients repeatedly fail to form viable embryos, although they can fertilize the oocyte and trigger early preimplantation development. Many authors have shown how this paternal effect can be traced back to anomalies in sperm chromatin organization: the spermatozoa of subfertile men are characterized by numerical abnormalities in spermatozoal chromosome content, Y chromosome microdeletions, alterations in the epigenetic regulation of paternal genome and non-specific DNA strand breaks. In particular, pathologically increased sperm DNA fragmentation is one of the main paternal-derived causes of repeated assisted reproduction failures in the ICSI era. The intention of this review is to describe nuclear sperm DNA damage, with emphasis on its clinical significance and its relationship with male infertility. Assessment of sperm DNA damage appears to be a potential tool for evaluating semen samples prior to their use in assisted reproduction, helping to select spermatozoa with intact DNA or with the least amount of DNA damage for use in assisted conception.     

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.