An algorithm to predict pregnancy in assisted reproduction

BACKGROUND: Male fertility potential cannot be measured by conventional parameters for the assisted reproduction technique; ICSI. This study determines the relationship between testicular and ejaculated sperm mitochondrial (mt) DNA deletions, nuclear (n) DNA fragmentation, and fertilization and pregnancy rates in ICSI. METHODS: Ejaculated sperm were obtained from 77 men and testicular sperm from 28 men with obstructive azoospermia undergoing ICSI. Testicular sperm were retrieved using a Trucut needle. mtDNA was analysed using a long PCR. The alkaline Comet assay determined nDNA fragmentation. RESULTS: Of subjects who achieved a pregnancy (50%) using testicular sperm, only 26% had partners' sperm with wild-type (WT) mtDNA. Of pregnant subjects (38%) using ejaculated sperm, only 8% had partner sperm with WT mtDNA. In each, the successful group had less mtDNA deletions and less nDNA fragmentation. There were inverse relationships between pregnancy and mtDNA deletion numbers, size and nDNA fragmentation for both testicular and ejaculated sperm. No relationships were observed with fertilization rates. An algorithm for the prediction of pregnancy is presented based on the quality of sperm nDNA and mtDNA. CONCLUSION: In both testicular and ejaculated sperm, mtDNA deletions and nDNA fragmentation are closely associated with pregnancy in ICSI.     

Mitochondrial DNA deletions and nuclear DNA fragmentation in testicular and epididymal human sperm

BACKGROUND: There are still concerns about the safety of intracytoplasmic sperm injection (ICSI) due to its brief clinical record and lack of animal testing. Testicular and epididymal sperm are now used routinely for ICSI in patients with obstructive azoospermia. The use of such immature sperm compounds fears, since little is known of their mitochondrial and nuclear DNA quality. METHODS: A modified long polymerase chain reaction (LPCR) was employed to study mitochondrial DNA (mtDNA) and a modified alkaline Comet assay to determine nuclear DNA (nDNA) fragmentation in testicular and epididymal sperm from men with obstructive azoospermia (n = 25) attending the Regional Fertility Centre. RESULTS: Testicular sperm displayed significantly more wild-type mtDNA (45% of patients) than epididymal sperm (16% of patients). They also had a lower incidence of multiple deletions and smaller mtDNA fragments. Epididymal sperm harboured more large-scale deletions (P < 0.05). There was a strong correlation between nuclear DNA fragmentation, the number of mtDNA deletions (r = 0.48, r = 0.50, P < 0.001) and their size (r = 0.58, r = 0.60, P < 0.001) in both epididymal and testicular sperm. CONCLUSION: This study suggests that mtDNA and nDNA of testicular sperm have fewer mutations and fragmentation than epididymal sperm and should be used in preference for ICSI in clinical treatment.     

A comparison of DNA damage in testicular and proximal epididymal spermatozoa in obstructive azoospermia.

Testicular and epididymal spermatozoa are used routinely for intracytoplasmic sperm injection (ICSI) to treat men with obstructive azoospermia. Little is known of the effects of obstruction and stasis on the DNA of these spermatozoa, particularly in the epididymis where spermatozoa have been retained for long periods. Surgical epididymal aspiration for ICSI could provide spermatozoa that are senescent or dying. Using the Comet assay, the percentage of undamaged DNA of testicular spermatozoa from 20 men with obstructive azoospermia was significantly better (83.0 +/- 1. 2%) than from proximal epididymal spermatozoa (75.4 +/- 2.3%; P < 0. 05). There was no difference between the percentage of undamaged DNA of testicular spermatozoa from 39 men with obstructive azoospermia (84.0 +/- 0.9) or from 10 fertile men at vasectomy (86.8 +/- 1.8) or from ejaculated spermatozoa from five of the controls (78.9 +/- 3.9; P > 0.05). In nine subjects, a second biopsy was carried out 6 months later. There was no significant difference in undamaged DNA on these two occasions (83.5 +/- 5.6 and 84.1 +/- 4.2; P > 0.05). This confirms the reproducibility of the Comet assay for non-ejaculated spermatozoa. Our data suggest that testicular sperm DNA appears to be significantly less damaged than epididymal sperm DNA, and so testicular spermatozoa should be used in preference for ICSI to treat men with obstructive azoospermia.     

Chromosome analysis of epididymal and testicular sperm in azoospermic patients undergoing ICSI

BACKGROUND: Although ICSI provides a way of treating azoospermic men, concern has been raised about the potential risk for transmission of genetic abnormalities to the offspring. We quantified the incidence of chromosomal abnormalities in epididymal and testicular sperm retrieved from azoospermic patients undergoing ICSI. METHODS: Individual testicular sperm were collected from testicular biopsies with an ICSI pipette, and epididymal sperm were retrieved by microsurgical epididymal sperm aspiration. Samples were processed by fluorescent in-situ hybridization (FISH) for chromosomes 18, 21, X and Y and the results compared with those from normal ejaculated samples. RESULTS: The overall aneuploidy rate of 11.4% in men with non-obstructive azoospermia was significantly higher (P = 0.0001) than the 1.8% detected in epididymal sperm from men with obstructive azoospermia and also the 1.5% found in ejaculated sperm. No significant difference was found between the epididymal and ejaculated samples. When the chromosomal abnormalities were analysed, gonosomal disomy was the most recurrent abnormality in both obstructive and non-obstructive azoospermic patients, while autosomal disomy was the most frequent in ejaculated sperm. CONCLUSIONS: Sperm of non-obstructive azoospermic men had a higher incidence of chromosomal abnormalities, of which sex chromosome aneuploidy was the most predominant. Genetic counselling should be offered to all couples considering infertility treatment by ICSI with testicular sperm.     

Analysis of chromosomal abnormalities in testicular and epididymal spermatozoa from azoospermic ICSI patients by fluorescence in-situ hybridization

BACKGROUND: An increased incidence of numerical chromosomal abnormalities has been reported in the ejaculated spermatozoa of infertile patients. However, there are few cytogenetic studies of testicular and epididymal spermatozoa, and their results are still controversial. METHODS: Fluorescence in-situ hybridization (FISH) analysis of chromosomes 13, 18, 21, X and Y was performed on seven testicular samples and two epididymal samples from patients with obstructive azoospermia (OA), and on 13 testicular samples from patients with non-obstructive azoospermia (NOA). Five ejaculated sperm samples from normozoospermic fertile donors were evaluated as a control group. RESULTS: Both epididymal sperm samples showed normal FISH results for the parameters analysed when compared with those of the control group. FISH results were abnormal in 29% (two of seven) of testicular samples from OA patients and in 54% (seven of 13) of those from NOA patients, although this difference was not statistically significant. Testicular samples from OA patients showed a significant increase of disomy for sex chromosomes (P<0.01), whereas NOA patients displayed significantly higher rates of diploidy (P<0.0001) and disomy for chromosomes 13 (P<0.0001), 21 (P<0.001) and sex chromosomes (P<0.0001) than the control group. CONCLUSIONS: Testicular spermatozoa from azoospermic patients present increased rates of chromosomal abnormalities, mainly of the sex chromosomes, which are particularly high in NOA patients.     

Genetic evaluation of infertile men.

Recently, microdeletions in the azoospermic factor region of the Y chromosome, in addition to chromosomal anomalies, have been detected in men with azoospermia or severe oligozoospermia. In this study we evaluated the molecular and cytogenetic defects of infertile men. The frequency of Y microdeletions among 105 azoospermic, 28 oligozoospermic and 32 fertile men was tested on lymphocyte DNA using a series of 20 sequence-tagged sites. In addition, microdeletions were evaluated on testicular-derived DNA among 26 azoospermic patients who underwent testicular biopsy and in whom no sperm cells could be identified. Karyotype analysis was performed on 72 of the infertile patients. Deletions were detected in 6.7% azoospermic and 3.6% oligozoospermic men. No deletions were identified among the fertile men. Identical results were obtained with DNA derived either from lymphocytes or testicular tissue. The frequency of chromosomal aberrations in the 72 infertile patients tested (62 azoospermic, 10 oligozoospermic) was 16.6%, with a high percentage of gonosome anomalies. Additional andrological parameters (hormone values, cryptorchidism) failed to identify men at risk for having microdeletions before the test. Our findings support the recommendation to perform genetic defect screening among infertile men before their enrollment in an intracytoplasmic injection/in-vitro fertilization programme.     

Human male infertility and Y chromosome deletions: role of the AZF-candidate genes DAZ, RBM and DFFRY.

Microdeletions in Yq11 overlapping three distinct 'azoospermia factors' (AZFa-c) represent the aetiological factor of 10-15% of idiopathic azoospermia and severe oligozoospermia, with higher prevalence in more severe testiculopathies, such as Sertoli cell-only syndrome. Using a PCR-based screening, we analysed Yq microdeletions in 180 infertile patients affected by idiopathic Sertoli cell-only syndrome and different degrees of hypospermatogenesis, compared with 50 patients with known causes of testicular alteration, 30 with obstructive azoospermia, and 100 normal fertile men. In idiopathic severe testiculopathies (Sertoli cell-only syndrome and severe hypospermatogenesis), a high prevalence of microdeletions (34.5% and 24.7% respectively) was found, while milder forms were not associated with Yq alteration. No deletions were found in testiculopathies of known aetiology, obstructive azoospermia, normal fertile men and male relatives of patients with deletions. Deletions in the AZFc region involving the DAZ gene were the most frequent finding and they were more often observed in severe hypospermatogenesis than in Sertoli cell-only syndrome, suggesting that deletions of this region are not sufficient to cause complete loss of the spermatogenic line. Deletions in AZFb involving the RBM gene were less frequently detected and there was no correlation with testicular phenotype, with an apparent minor role for such gene in spermatogenesis. The DFFRY gene was absent in a fraction of patients, making it a candidate AZFa gene. Our data suggest that larger deletions involving more than one AZF-candidate gene are associated with a more severe testicular phenotype.     

Reproductive capacity of spermatozoa from men with testicular failure.

Controversial reports have been published about the influence of sperm source and of the underlying testicular pathology on success rates of intracytoplasmic sperm injection (ICSI). In this controlled study, ICSI treatment cycles with testicular spermatozoa from men with obstructive and non-obstructive azoospermia were compared with ICSI ejaculated sperm cycles with semen parameters < or = 5 x 10(6)/ml and < or = 10% progressive motility. The control cases were matched for female age, rank of trial, female basal follicle-stimulating hormone serum concentrations and close proximity to the study group's procedure. The fertilization, cleavage, pregnancy and abortion rates were similar in matched groups irrespective of the type of azoospermia. However, the implantation rate in the non-obstructive azoospermic patient group was significantly lower than that in the matched ejaculated sperm group (13.4% versus 26%, P = 0.05). On the other hand, no impairment of the implantation rate was observed in the obstructive azoospermic patient group. These data show that testicular pathology has a negative impact on reproductive performance of testicular spermatozoa, resulting in a decreased implantation potential without any apparent effect on fertilization and early preimplantation development.     

Insulin dependant diabetes mellitus: implications for male reproductive function

BACKGROUND: Diabetes mellitus (DM) is increasing in men of reproductive age. Despite this, the prevalence of diabetes in men attending fertility clinics is largely unknown. Furthermore, studies examining the effects of DM on sperm fertility potential have been limited to conventional semen analysis. METHODS: Conventional semen analysis (semen volume, sperm count, motility and morphology) was performed for 27 diabetic (mean age 34+/-2 years) and 29 non-diabetic subjects (control group, men undergoing routine infertility investigations, mean age 33+/-1 years). Nuclear DNA (nDNA) fragmentation was assessed using the alkaline Comet assay and mitochondrial DNA (mtDNA) deletions by Long-PCR. RESULTS: Other than a small, but significant, reduction in semen volume in diabetic men (2.6 versus 3.3 ml; P<0.05), conventional semen parameters did not differ significantly from control subjects. Diabetic subjects had significantly higher mean nDNA fragmentation (53 versus 32%; P<0.0001) and median number of mtDNA deletions (4 versus 3; P<0.05) compared with control subjects. CONCLUSIONS: Diabetes is associated with increased sperm nuclear and mtDNA damage that may impair the reproductive capability of these men.     

Efficient treatment of infertility due to sperm DNA damage by ICSI with testicular spermatozoa

BACKGROUND: Sperm DNA damage (fragmentation) is a recently discovered cause of male infertility for which no efficient treatment has yet been found. Previous findings have suggested that clinically relevant sperm DNA damage may occur at the post-testicular level. This study was undertaken to assess the clinical usefulness of ICSI with testicular spermatozoa in this indication. METHODS: The percentage of spermatozoa with fragmented DNA, assessed by terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling assay, and ICSI outcomes were compared in two sequential attempts performed, respectively, with ejaculated and testicular spermatozoa in 18 men with increased sperm DNA fragmentation. RESULTS: The incidence of DNA fragmentation was markedly lower in testicular spermatozoa as compared with ejaculated spermatozoa. No differences in fertilization and cleavage rates and in embryo morphological grade were found between the ICSI attempts performed with ejaculated and with testicular spermatozoa. However, eight ongoing clinical pregnancies (four singleton and four twin) were achieved by ICSI with testicular spermatozoa (44.4% pregnancy rate; 20.7% implantation rate), whereas ICSI with ejaculated spermatozoa led to only one pregnancy which was spontaneously aborted. CONCLUSIONS: These data show that ICSI with testicular spermatozoa provides the first efficient assisted reproduction treatment option for men with high levels of sperm DNA damage.     

Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa

The objectives of this cross-sectional observational study were: (i) to detect DNA damage and plasma membrane translocation of phosphatidylserine in purified sperm populations of high and low motility, and (ii) to analyse their relationship with the endogenous generation of reactive oxygen species. Ejaculates from infertile men were examined following gradient centrifugation. The main outcome measures were: sperm motion parameters (assessed with a computer analyser), generation of reactive oxygen species (measured by chemiluminescence), DNA damage (detected by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling and monoclonal antibody labelling of single-stranded DNA) and translocation of membrane phosphatidylserine (examined with annexin V staining). DNA fragmentation and membrane translocation of phosphatidyl-serine were observed in the fractions with low and high sperm motility in all patients. The fractions with low sperm motility had significantly higher proportion of cells with DNA damage and production of reactive oxygen species than the fractions with high sperm motility (P < 0.005). DNA fragmentation was significantly and positively correlated with the generation of reactive oxygen species (r = 0.42; P = 0.02). In conclusion: (i) spermatozoa from infertile men display translocation of membrane phosphatidylserine as diagnosed by annexin V positive staining; (ii) DNA damage (fragmentation and presence of single-stranded DNA) can be detected in ejaculated spermatozoa from infertile men in fractions with low and high sperm motility, and (iii) there is a relationship between DNA damage and oxidative stress.     

Screening for Y chromosome microdeletions in 226 Slovenian subfertile men.

BACKGROUND: The objective of this study was to estimate the frequency of Y chromosome microdeletions in the Slovenian population of infertile men and to analyse the consequences of mutation in respect to clinical severity and prognosis. METHODS: In a controlled clinical study at the university-based medical genetics service and infertility clinic, 226 infertile men undergoing ICSI were tested. The main outcome measures included polymerase chain reaction amplification of 16 genes and gene families and 42 sequence-tagged sites in the non-recombining region of the Y chromosome, semen, testicular volume and testicular histological analysis, serum FSH concentrations, fertilization and respective pregnancy rates. RESULTS: The incidence of deletions was 4.4%: 8.6% in men with azoospermia and 1.5% in men with oligoasthenoteratozoospermia. Isolated gene deletions were not identified. No statistically significant differences in clinical outcome measures were found in patients with mutations versus patients without mutations. High fertilization (49%) and pregnancy (43%) rates with sperm of patients with Y chromosome deletions were obtained. CONCLUSIONS: Testing for gene-specific microdeletions does not contribute significantly to the sensitivity of microdeletion test. Fertilization and pregnancy rates obtained using sperm of patients with Y chromosome deletions were comparable with those achieved in conventional IVF.     

Both protamine-1 to protamine-2 mRNA ratio and Bcl2 mRNA content in testicular spermatids and ejaculated spermatozoa discriminate between fertile and infertile men

BACKGROUND: Human sperm contain similar amounts of protamine-1 (P1) and protamine-2 (P2). Although aberrant protamine ratios have been observed in infertile men, functional evidence is provided by protamine knockout mice exhibiting male infertility. As sperm DNA integrity is known to be linked with DNA fragmentation and apoptosis, we investigated whether the protamine ratio or Bcl2 content represent a reliable biomarker to discriminate fertile and infertile men. METHODS: Real-time quantitative RT-PCR was used for P1, P2 and the apoptotic marker Bcl2 in testicular biopsies (TB; 74 infertile men versus 17 controls) and ejaculates (E; 95 infertile men versus 10 controls). RESULTS: The P1-P2 mRNA ratio differed significantly between groups, namely 1:4 versus 1:3.2 in TB (P = 0.0038) and 1:1.7 versus 1:1 in E (P = 0.0002), for infertile men and controls, respectively. Bcl2 mRNA content was correlated with protamine mRNA ratio (P = 0.0250 for TB; P = 0.0003 for E). Infertile men exhibit a more than 10-fold (P = 0.0155 for TB; P = 7.0 x 10(-6) for E) higher Bcl2 mRNA content versus controls. No correlation was found between absolute sperm density and the protamine mRNA ratio or Bcl2 mRNA content. No significant correlation was demonstrated with fertilization rate after ICSI and either protamine ratio or Bcl2 content. CONCLUSIONS: We found significantly aberrant protamine ratios and a higher Bcl2 content in TB and E of infertile men compared to controls, suggesting that these molecules may be useful biomarkers for predicting male infertility.     

Significance of human testicular mast cells and their subtypes in male infertility

The mast cell populations in the human testis were examined using immunohistochemical techniques in five fertile volunteers and 12 patients with obstructive azoospermia, seven patients with idiopathic azoospermia, and 30 patients with varicocele. The number of mast cells per seminiferous tubular section was significantly increased (P < 0.05) in the men with idiopathic azoospermia. In the normal testes, mast cells containing only tryptase were the predominant subtype. In the patient groups, the predominant subtype of mast cell was shifted to that containing both tryptase and chymase. The average number of mast cells containing both tryptase and chymase per seminiferous tubular section was significantly increased (P < 0.05) compared with the controls in patients with obstructive azoospermia, idiopathic azoospermia, and varicocele. The number of mast cells containing only tryptase was not increased in infertile men. The selective expansion of the mast cell population containing both tryptase and chymase may be related to spermatogenetic disorders and testicular fibrosis.     

Effects of vasectomy on spermatogenesis and fertility outcome after testicular sperm extraction combined with ICSI

BACKGROUND: Each year 40,000 men have a vasectomy in the UK whilst another 2400 request a reversal to begin a second family. Sperm can now be obtained by testicular biopsy and subsequently used in assisted conception with ICSI. The study aims were to compare sperm yields of men post-vasectomy or with obstructive azoospermia (OA) of unknown aetiology with yields of fertile men and to assess any alteration in the clinical pregnancy rates after ICSI. METHODS: Testicular tissue was obtained by Trucut needle from men who had undergone a vasectomy >5 years previously or had OA from other causes and from fertile men during vasectomy. Seminiferous tubules were milked to measure sperm yields. Numbers of Sertoli cells and spermatids and thickness of the seminiferous tubule walls were assessed using quantitative computerized analysis. RESULTS and CONCLUSIONS: Sperm yields/g testis were significantly decreased in men post-vasectomy and in men with OA, relative to fertile men. Significant reductions were also observed in early (40%) and mature (29%) spermatid numbers and an increase of 31% was seen in the seminiferous tubule wall (basal membrane and collagen thickness) of vasectomized men compared with fertile men. Clinical pregnancy rates in couples who had had a vasectomy were also significantly reduced.     

线粒体DNA与人精子活力间的相关性分析

目的 探讨线粒体ＤＮＡ与人精子活力间的关系。方法 用长链ＰＣＲ技术,对６０例精子活力正常和４０例精子活力异常不育患者的精子线粒体ＤＮＡ（ｍｔＤＮＡ）进行了多重缺失的分析。结果 两组不育患者中共有８例具有ｍｔＤＮＡ的多重缺失（其中精子活力正常不育患者６名,精子活力异常不育患者２名）,但缺失型ｍｔＤＮＡ（Ｓ５除外）在总ｍｔＤＮＡ中所占比例很小（０．１６％～１．８５％）,１例精子活力正常的不育患者（Ｓ５     

Andrology

In patients with obstructive azoospermia in whom standard microsurgicalprocedures fail or are unfeasible, the only source of spermatozoais the testicle. In addition, in some azoospermic patients withsevere spermatogenic failure, a few spermatozoa may be presentin testicular biopsyspecimens despite high serum follicle stimulatinghormone concentrations. In all these cases, intra cytoplasmicsperm injection (ICSI) with testicular biopsy-extracted spermatozoamayoffer the chance of pregnancy. To assess the efficacy of thisprocedure, we compared the results of twoseries of ICSI cyclesperformed during the same time period: 21 cycles using testicularbiopsy-extracted spermatozoa and 83 cycles using ejaculatedspermatozoa. Mean fertilization rates (59% with testicular and68% with ejaculated spermatozoa), mean cleavage rates (93% withtesticular and 90% with ejaculated spermatozoa), embryoquality(77% good quality embryos in the testicular sperm group and77% in the ejaculated sperm group) and clinical pregnancy rates(36.8% in the testicular sperm group and 28% in the ejaculatedsperm group) were not significantly different in both groups.We conclude that high fertilization, cleavage and pregnancyrates can be achieved with intra cytoplasmic testicular sperminjection, reaching levels comparable with those of ICSI usingejaculated spermatozoa.     

Assisted reproduction with in-vitro-cultured testicular spermatozoa in cases of severe germ cell apoptosis: a pilot study.

BACKGROUND: Apoptosis-related cell damage is known to compromise success rates of assisted reproduction with ejaculated spermatozoa. This study was undertaken to determine whether the frequency of apoptosis-related cell damage and reproductive performance of testicular spermatozoa from men with non-obstructive azoospermia can be improved by in-vitro culture. METHODS: Testicular tissue samples were cultured for 2 days in the presence of 50 IU/l FSH and 1 micromol/l testosterone. The frequency of spermatozoa showing DNA strand breakage and plasma membrane phosphatidylserine externalization was compared in before-culture and after-culture samples. The after-culture samples were used in assisted reproduction attempts. RESULTS: In a group of 11 azoospermic patients with at least two previous intracytoplasmic sperm injection (ICSI) failures, the incidence of DNA strand breakage was high in living testicular spermatozoa from before-culture samples, but significantly lower in after-culture samples (96 versus 30%, P < 0.001). The same applied to the incidence of phosphatidylserine externalization in the motile sperm subpopulation from the before-culture and after-culture samples (83 versus 6%, P < 0.001). Seven ongoing clinical pregnancies (six with fresh embryos and one with cryopreserved embryos) were established. CONCLUSIONS: Severe testicular sperm apoptosis may become a new indication for testicular tissue in-vitro culture before ICSI.     

Testicular needle biopsy, open biopsy, epididymal aspiration and intracytoplasmic sperm injection in obstructive azoospermia

Testicular or epididymal spermatozoa were obtained for in-vitrofertilization and intracytoplasmic sperm injection ICSI) in27 cycles out of 33 (in six men the azoospermia proved to havetesticular causes). Testicular needle biopsy carried out inaddition to surgical open biopsy proved to be an effective methodto obtain spermatozoa for ICSI from patients with obstructiveazoospermia. Thus it might be possible to replace scrotal operationsby simple needle biopsies. Embryos resulting from ICSI withtesticular spermatozoa were used in 19 transfers that resultedin six pregnancies. One pregnancy resulted from six embryo transfersfrom ICSI after microsurgical-epididymal sperm aspiration (MESA).The normal fertilization rates with testicular (37.3%) and MESAspermatozoa (53.7%) did not differ significantly from each other,but with testicular spermatozoa the rate was significantly lowerthan that obtained with ejaculated spermatozoa and ICSI (59.7%)in the matched couples. The abnormal fertilization of oocyteswith one pronucleus was significantly higher with testicularspermatozoa than with ejaculated spermatozoa in the controlcouples.     

Assessment of DNA integrity and morphology of ejaculated spermatozoa from fertile and infertile men before and after cryopreservation

Cryopreservation of human spermatozoa is extensively used in artificial insemination and IVF programmes. Despite various advances in cryopreservation methodology, the recovery rate of functional post-thaw spermatozoa remains mediocre, with sperm motility being significantly decreased after freezing. This aim of this study was to investigate the effects of cryopreservation on both DNA integrity and morphology of spermatozoa from fertile and infertile men. Semen samples were obtained from 17 fertile and 40 infertile men. All samples were prepared by discontinuous Percoll density centrifugation (95.0:47.5). Samples were divided into aliquots to allow direct comparison of fresh and frozen spermatozoa from the same ejaculate. Aliquots for cryopreservation were mixed with a commercial cryoprotectant and frozen by static phase vapour cooling before plunging into liquid nitrogen. Thawing was carried out slowly at room temperature. Sperm DNA integrity was determined using a modified alkaline single cell gel electrophoresis (comet) assay and sperm morphology analysed using the Tygerberg criteria. DNA of semen and prepared spermatozoa from fertile men was found to be unaffected by cryopreservation. In marked contrast, spermatozoa from infertile men were significantly damaged by freeze-thawing. Cryopreservation had a detrimental effect on morphology of semen and prepared samples from fertile and infertile men.