Effect of varicocelectomy on the abnormal retention of residual cytoplasm by human spermatozoa.

Abnormal retention of cytoplasmic residues by human spermatozoa is associated with the generation of reactive oxygen species (ROS) in semen and defective sperm function. We have examined the effect of varicocelectomy on the retention of residual cytoplasm by human spermatozoa. Clinical reports of 43 men who underwent microsurgical varicocelectomy at our institution during a 1 year period beginning July 1996 were reviewed. Standard semen parameters (concentration, motility and morphology) and residual cytoplasm retention (monitored by Papanicolaou stain) were assessed before and 6 months after varicocelectomy. The percentage of spermatozoa with residual cytoplasm decreased significantly following varicocelectomy compared to pre-operatively (25.8 versus 18.1% respectively). The percentages of motile spermatozoa and normal forms increased significantly (P = 0.0003, P = 0.005 respectively) following varicocelectomy (22.6 versus 32.9% and 46.4 versus 54.4% respectively). Our data suggest that varicocelectomy can improve the disposal of residual sperm cytoplasm by the testis and/or epididymis in infertile men with varicocele. These data also suggest that varicocelectomy reduces the potential for ROS generation by human spermatozoa in these men.     

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.     

Potential adverse effect of sperm DNA damage on embryo quality after ICSI

BACKGROUND: Sperm DNA damage is prevalent amongst infertile men and has been shown to strongly impact adversely natural reproduction, intrauterine insemination-assisted reproduction and to a lesser degree IVF/ICSI fertilization. The objective of this study was to examine further the relationship between sperm DNA denaturation (DD) and reproductive outcomes after ICSI. METHODS: We evaluated infertile couples (n = 60) undergoing IVF/ICSI at a single centre. Sperm DD was assessed by flow cytometry analysis of Acridine Orange-treated sperm and expressed as the percentage of sperm with DD. Couples were sub-grouped according to sperm DD results: group 1: 0-15%; group 2: >15-30%; group 3: >30%. RESULTS: There were no differences between the three groups with regard to maternal age, sperm parameters, oocyte maturation, fertilization or pregnancy rates. Group 3 had a significantly higher rate of multinucleation among the embryo cohorts compared to either groups 1 or 2 (20% versus 10% and 8% respectively, P = 0.04). There was a statistically insignificant trend toward an increased spontaneous pregnancy loss rate in group 3 (P =0.50). CONCLUSION: Although we did not observe significant relationships between sperm DNA damage and either fertilization or pregnancy rates, the potential adverse effect of sperm DNA damage on embryo quality and spontaneous pregnancy loss is concerning.     

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.     

Bilateral increased apoptosis and bilateral accumulation of cadmium in infertile men with left varicocele

BACKGROUND: Varicoceles are associated with venous flux that may cause increased heat and interstitial pressure within the testes, but these effects are variable. Some men with varicocele have infertility, but others do not. We question whether other factors contribute to the infertility, and whether these other factors could be identified by specific molecular/genetic markers. Can such markers predict the outcome of varicocele repair? Can these markers be demonstrated bilaterally in unilateral left varicocele? METHODS: Limited bilateral testes biopsies were obtained by ultrasonically guided percutaneous aspiration at the time of varicocelectomy. In each specimen, cadmium levels were determined by atomic absorption and the percentage apoptosis within the seminiferous tubules was quantified. RESULTS: The percentage of apoptotic nuclei and cadmium levels were high in some men with varicocele. There was a concordance of these values in both testes despite the presence of left-sided varicocele only. These values were inversely related to an increase in sperm concentration after varicocelectomy. CONCLUSIONS: Cadmium, a metal ion inducer of apoptosis, may contribute to this form of male infertility. Apoptosis may deplete the sperm concentration among men with varicocele and infertility. Pre-operative measurements of apoptosis and cadmium content may predict the outcome of varicocele repair.     

Azoospermia factor deletions in varicocele cases with severe oligozoospermia

BACKGROUND: Varicocele is the most common cause of male infertility. The etiology and pathophysiology of varicocele are multifactorial. When low sperm counts are associated with varicocele, varicocelectomy can partially restore spermatogenesis and fertility. Few recent studies have reported that in some varicocele cases, there may be an associated genetic etiology. Presence of a genetic factor like azoospermia factor microdeletions may lead to irreversible spermatogenic arrest in these cases, but very few reports support these findings. However, it is still not understood why some cases improve after varicocelectomy and why some cases show no improvement in semen parameters postoperatively. AIM: It is important to distinguish varicocele cases from Yq microdeletions as these cases have irreversible testicular damage and thus carry a poor prognosis after varicocelectomy. SETTINGS: Research and Referral tertiary care hospital. Design: Prospective study. MATERIALS AND METHODS: Seventy-two infertile men with varicocele were referred for Yq microdeletion analysis from the infertility clinic of AIIMS and Army Research and Referral Hospital. Genomic DNA was isolated from blood and polymerase chain reaction microdeletion screening was done in these cases to determine the presence or deletion of AZF loci. RESULTS: In this study 7 (9.7%) varicocele cases harbored Yq microdeletion. The sperm count in cases which harbored Yq microdeletion was significantly lower than in cases without Yq microdeletion. CONCLUSION: Varicocele cases with Yq microdeletion do not show improvement in semen parameters post-varicocelectomy. Detection of Yq microdeletion determines prognosis and future management in such cases.     

Late, but not early, paternal effect on human embryo development is related to sperm DNA fragmentation

BACKGROUND: It is known that repeated failure of assisted reproduction treatment (ART) can be due to a paternal effect. This study was undertaken to analyse the possible relationship between ART failure and sperm DNA fragmentation. METHODS: Zygote morphology and the percentage of spermatozoa with fragmented DNA (assessed by TUNEL) were compared in two groups using donor oocytes for ICSI attempts. The experimental group consisted of 18 infertile couples who had each undergone three previous failed ART attempts. The control group included 18 randomly selected infertile couples undergoing their first ICSI attempt. Both groups used sibling oocytes from the same donors. RESULTS: In 10 couples of the experimental group, the adverse paternal effect was evident as early as the zygote stage. This early paternal effect was not associated with sperm DNA fragmentation. In eight couples of the experimental group, the adverse paternal effect did not produce any perceptible deterioration of zygote morphology. However, this late paternal effect was associated with an increased percentage of spermatozoa with fragmented DNA. CONCLUSIONS: Early paternal effect can compromise ART outcomes in the absence of increased sperm DNA fragmentation. Evaluation of sperm DNA integrity is useful to detect late paternal effect, which is not associated with morphological abnormalities at the zygote and early cleavage stages.     

Sperm chromatin structure assay parameters as predictors of failed pregnancy following assisted reproductive techniques

The predictive value of sperm chromatin integrity for pregnancy outcome following in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) was studied in 24 men attending a university-based assisted reproductive techniques laboratory using the flow cytometric sperm chromatin structure assay (SCSA). The SCSA is a measure of the susceptibility of sperm DNA to low pH-induced denaturation in situ. The mean percentage of spermatozoa in the neat sample demonstrating DNA denaturation was significantly lower in the seven men that initiated a pregnancy (15.4 +/- 4.6, P = 0.01) than in the 14 men who did not initiate a pregnancy (31.1 +/- 3.2). No pregnancies resulted if > or =27% of the spermatozoa in the neat semen sample showed DNA denaturation. These data demonstrate that SCSA parameters are independent of conventional semen parameters. Furthermore, the SCSA may allow physicians to identify male patients for whom IVF and ICSI will be unlikely to result in pregnancy initiation.     

The varicocele dilemma

There is probably no subject that is more controversial in the area of male infertility than varicocele. The overwhelming majority of non-urologist infertility specialists in the world are extremely sceptical of the role of varicocele or varicocelectomy in the treatment of male infertility. Directors of most assisted reproductive technologies (ART) programmes view the enthusiasm with which urologists approach varicocelectomy as a potential impediment to the couple that is getting older and do not have much time left to become pregnant using ART. There are many credible, well-controlled studies which show no effect of varicocelectomy on fertility. There are also a few 'controlled' studies that favour varicocelectomy, but all can be criticised on the basis of patient selection bias. Thus the great weight of evidence from controlled studies is against varicocelectomy and the reports supporting varicocelectomy are extremely weak. Finally, the reports that semen parameters are improved by varicocelectomy is flawed by uncontrolled observations and the failure to take into account the variability of semen analysis in infertile men and its regression toward the mean. Many control studies have demonstrated that, because of this variability, men with an initially low sperm count tend later to have higher sperm counts in the absence of any treatment whatsoever.     

Chromatin status in human ejaculated spermatozoa from infertile patients and relationship to seminal parameters

The aim of this study was to evaluate the chromatin status in different groups of patients. Five groups of men were selected: pre-vasectomy; male factor infertility; varicocele; immunological male infertility; and idiopathic infertility. Chromatin status was evaluated using flow cytometry after staining the DNA with the fluorochrome propidium iodide. Differences were observed in the state of sperm chromatin between the male factor and varicocele groups with respect to the others. These two groups presented poorer quality chromatin, as evidenced fundamentally by a lower degree of condensation. These deficiencies in chromatin status were usually accompanied by alterations in the other standard parameters of semen analysis. Individuals who are infertile due to male factor and those presenting varicocele have spermatozoa with less condensed chromatin which might, in part, explain their sterility.     

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.     

Detection of Antibodies Toward Epididymal Sperm Antigens—An Obligatory Step in Evaluation of Human Immunologic Infertility?

PROBLEM : To test the relative impact of epididymal versus ejaculated sperm in immunologic infertility. METHOD : Human antibody binding to epididymal and ejaculated spermatozoa was compared by flow cytometry (FCM) since it allows quantitative analysis of viable sperm while ignoring nonsperm cells. To select sera for FCM, GAT, TAT, and ELISA were applied on 145 sera from fertile men, idiopathically infertile and varicocele patients. RESULTS : All GAT/TAT-positive infertile patients, a representative group of varicocele patients and the fertile control, were assessed by FCM. Higher reactivity toward epididymal sperm revealed 18/22 sera while only four out of them bound to ejaculated sperm stronger than the control. All varicocele sera were positive against epididymal while negative against ejaculated spermatozoa. CONCLUSIONS : Epididymal sperm antigens may play a predominant role in some cases of immunologic infertility. Such patients might not be adequately diagnosed and respectively treated due to the limitations of diagnostic procedures applying only ejaculated spermatozoa.     

Increased sperm DNA damage in patients with varicocele: relationship with seminal oxidative stress

BACKGROUND: The pathophysiology of the testicular damage in varicocele has not been completely understood. Oxidative stress and related sperm DNA damage have been identified as significant causes of male infertility. The current study was designed to determine the extent of sperm nuclear DNA damage in patients with varicocele and to examine its relationship with oxidative stress. METHODS: Semen samples from 55 patients with clinical varicocele and 25 normozoospermic donors were examined. Varicocele sperm samples were classified as normal or abnormal according to World Health Organization guidelines. Sperm DNA damage was evaluated by the sperm chromatin structure assay/flow cytometry and by the terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay. Levels of reactive oxygen species (ROS) and total antioxidant capacity were assessed by a chemiluminescence assay. RESULTS: DNA fragmentation index (DFI) (percentage of sperm with denatured DNA) values and the percentage of TUNEL-positive cells were significantly greater in patients with varicocele, either with normal (DFI, 20.7 +/- 4.0; TUNEL positive, 26.1 +/- 3.2) or with abnormal (DFI, 35.5 +/- 9.0; TUNEL positive, 32.2 +/- 4.1) semen profile, compared with controls (DFI, 7.1 +/- 0.9; TUNEL positive, 14.2 +/- 1.2). Similarly, ROS levels were significantly higher (P < 0.01) in both groups of patients with varicocele. CONCLUSIONS: The presence of a varicocele is associated with high levels of DNA-damage spermatozoa even in the presence of normal semen profile. The results also indicate that oxidative damage is associated with sperm DNA damage in these patients.     

Reactive oxygen species: potential cause for DNA fragmentation in human spermatozoa

The objective of this study was to evaluate the effect of the generation of reactive oxygen species (ROS) on the integrity of the DNA of human spermatozoa, and to determine if pretreatment with antioxidants can reduce DNA damage. Samples were obtained from 47 men undergoing infertility investigation. ROS were generated in the samples by the addition of xanthine/xanthine oxidase (X/XO) with or without antioxidants. After incubation at timed intervals (0-2 h) with X/XO, the percentage of spermatozoa with DNA fragmentation was determined using the method of TdT-mediated DNA end-labelling (TUNEL). Time intervals were selected to mimic the clinical situation in which spermatozoa are held for a period of time after swim-up while the oocytes are prepared for ICSI. A significant increase in sperm DNA damage was evident when samples were incubated in the presence of ROS for intervals of 1 and 2 h, but not when incubated with ROS for <1 h (P = 0.0001). The addition of antioxidants significantly decreased the amount of DNA damage induced by ROS generation (P < 0.04). ROS can cause an increase in DNA fragmentation and pretreatment with antioxidants can reduce DNA damage.      

ICSI in cases of sperm DNA damage: beneficial effect of oral antioxidant treatment

BACKGROUND: Most studies examining the use of ICSI for cases of elevated sperm DNA fragmentation report poor pregnancy and implantation rates. ICSI with testicular sperm samples has recently been suggested for these cases. Here we test a less invasive approach based on oral antioxidant treatment prior to ICSI with ejaculated spermatozoa. METHODS: Thirty-eight men with an elevated (> or =15%) percentage of DNA-fragmented spermatozoa in the ejaculate were treated with antioxidants (1 g vitamin C and 1 g vitamin E daily) for 2 months after one failed ICSI attempt. In 29 (76%) of these cases this treatment led to a decrease in the percentage of DNA-fragmented spermatozoa, and a second ICSI attempt was performed. Outcomes of the two attempts were compared. RESULTS: No differences in fertilization and cleavage rates or in embryo morphology were found between the ICSI attempts performed before and after the antioxidant treatment. However, a marked improvement of clinical pregnancy (48.2% versus 6.9%) and implantation (19.6% versus 2.2%) rates was observed after the antioxidant treatment as compared with the pretreatment ICSI outcomes. CONCLUSIONS: Oral antioxidant treatment appears to improve ICSI outcomes in those patiens with sperm DNA damage, in whom this treatment reduces the percentage of damaged spermatozoa.     

Elevation of serum testosterone and free testosterone after embolization of the internal spermatic vein for the treatment of varicocele in infertile men

BACKGROUND: To evaluate the effect of internal spermatic vein (ISV) embolization on levels of serum testosterone and free testosterone and on spermatogenesis. METHODS: The files of 83 infertile men treated for varicocele were reviewed for changes in serum testosterone, free testosterone and spermatogenesis after ISV embolization. RESULTS: Mean serum testosterone concentration rose after embolization by 43%, from 12.07 +/- 6.07 nmol/l to 17.22 +/- 8.43 nmol/l (P<0.001). Mean serum free testosterone concentration rose by 72%, from 5.93 +/- 2.44 nmol/l to 10.21 +/- 7.69 nmol/l (P<0.001). Mean sperm concentration increased from 7.49 +/- 1.73 x 10(6)/ml to 18.14 +/- 2.36 x 10(6)/ml (P<0.001); mean sperm motility increased from 21.74 +/- 2.47 to 34.47 +/- 2.27% (P<0.001); and mean sperm morphology increased from 6.63 +/- 1.07 to 13.08 +/- 1.44% (P<0.001). CONCLUSIONS: ISV embolization apparently induces an increase in both serum testosterone and free testosterone concentrations and in sperm parameters in infertile patient with varicocele, regardless of the size of the varicocele.     

Clinical adaptation of the sperm ubuquitin tag immunoassay (SUTI): relationship of sperm ubiquitylation with sperm quality in gradient-purified semen samples from 93 men from a general infertility clinic population

BACKGROUND: The proteolytic chaperone peptide ubiquitin accumulates in defective human spermatozoa. Immunodetection of ubiquitin in human sperm samples correlates with semen quality and male fertility. METHODS: Semen samples from 93 men from couples seeking infertility treatment were separated on a PureSperm density gradient and screened by immunofluorescence microscopy with anti-ubiquitin antibodies. The percentage of spermatozoa with head ubiquitylation was recorded and compared with clinical semen evaluation and embryo development data after IVF or ICSI. Subjects were divided into the following four groups based on the initial clinical diagnosis of the couples; group 1, male factor; group 2, idiopathic infertility; group 3, female infertility with neither partner having children previously; and group 4, female infertility with male partners having children from previous relationships. RESULTS: The percentage of sperm with ubiquitylated heads remaining after PureSperm separation in the respective groups was 4.0% (male factor), 2.5% (idiopathic infertility), 0.7% (female infertility and presumed fertile male) and 0.9% (female infertility with established fertile male). Negative correlations between sperm ubiquitin and several parameters reflective of embryo development after assisted fertilization were found within the male factor group. CONCLUSIONS: Use of this simplified ubiquitin-based sperm quality assay is feasible in a clinical environment. Since the gradient separation does not completely deplete the defective spermatozoa, the modified light microscopic sperm ubiquitin tag immunoassay could add a new level of stringency to the selection of human spermatozoa for ICSI.     

Differences in nuclear DNA fragmentation and mitochondrial integrity of semen and prepared human spermatozoa

Sperm DNA integrity is essential for accurate transmission of genetic material to offspring. Fragmentation of genomic DNA is an initial hallmark of apoptosis (programmed cell death). The aim of this study was to determine sperm nuclear DNA integrity and mitochondrial function, to quantify possible apoptosis and to investigate any relationship between these parameters. Semen samples (n = 25) were prepared by discontinuous Percoll density centrifugation (95.0:47.5). DNA integrity was determined using a modified alkaline single cell gel electrophoresis (Comet) assay. DNA fragmentation, possibly indicative of apoptosis, was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL). Mitochondrial transmembrane potential was determined using the mitochondrial probe 5,5',6,6'-tetrachloro-1,1', 3,3'-tetraethylbenzimidazolyl carbocyanine iodide (JC-1). The DNA integrity of prepared spermatozoa was significantly greater than that of semen (P < 0.005). Further, the percentage of spermatozoa with fragmented DNA and the degree of fragmentation within these cells in prepared spermatozoa is significantly less than in semen (P < 0.005). There is a significant correlation between DNA damage quantified using the Comet assay and DNA fragmentation determined using TUNEL (R = 0.562, P < 0.01). The percentage of spermatozoa with dysfunctional, possibly apoptotic, mitochondria was significantly lower in prepared spermatozoa than in neat semen samples (P < 0.001). There was a negative correlation between the percentage of spermatozoa with dysfunctional mitochondria and the percentage of progressively motile spermatozoa (R = -0.67, P < 0.01).     

Glutathione and hypotaurine in vitro: effects on human sperm motility, DNA integrity and production of reactive oxygen species

Sperm DNA integrity is of paramount importance for the accurate conveyance of genetic material. DNA damage may be a major contributory factor in male infertility as DNA from sperm of infertile men has been found to be more susceptible to induced DNA damage in vitro than DNA from fertile men. Reactive oxygen species (ROS) are a significant source of DNA damage and human sperm are extremely sensitive to ROS attack due to their high content of polyunsaturated fatty acids and lack of capacity for DNA repair. Seminal plasma, which contains a wealth of antioxidants, provides sperm with crucial protection against oxidative insult. However, during preparation for use in assisted conception techniques, sperm are separated from seminal plasma and deprived of that essential protection. The aim of this study was to determine the effects of supplementation with glutathione and hypotaurine during sperm preparation on subsequent sperm motility, DNA integrity, induced DNA damage and ROS generation. Semen samples (n = 45) were divided into aliquots and prepared by Percoll density centrifugation (95.0-47.5%) using medium which had been supplemented with these antioxidants to a number of different concentrations all within physiological levels. Control aliquots were included which had no glutathione or hypotaurine added. Sperm motility was determined using computer-assisted semen analysis. DNA damage was induced using H(2)O(2) and DNA integrity was determined using a modified alkaline single cell gel electrophoresis (Comet) assay, while ROS generation was measured using chemiluminescence. Addition of glutathione and hypotaurine, either singly or in combination, to sperm preparation medium had no significant effect on sperm progressive motility or baseline DNA integrity. Despite this, sperm were still afforded significant protection against H(2)O(2)-induced damage and ROS generation.     

Abnormalities for chromosomes 13 and 21 detected in spermatozoa from infertile men

Sperm samples from infertile men with oligozoospermia or teratozoospermiawere studied by multicolour fluorescence in-situ hybridization(FISH) using DNA probes for chromosomes 13 and 21. A total of90 809 sperm nuclei from nine infertile men and 182 799 spermnuclei from 18 control donors were analysed. There was a highlysignificant increase in the frequency of spermatozoa disomicfor chromosome 13 in infertile patients (0.28%) compared tocontrol donors (0.13%) (two-tailed Z statistic P <0.0001and for chromosome 21 (0.48% in infertile men versus 0.37% incontrols, P <0.0001). Also there was a significantly increasedfrequency of diploid spermatozoa in infertile men (0.85%) comparedto control donors (0.66%) (P <0.0001). Our previous studieson these same infertile patients demonstrated increased frequenciesof sperm disomy for chromosomes 1 and XY. This suggests thatinfertile men, who are prime candidates for intracytoplasmicsperm injection, may be at a very small increased risk of aneuploidoffspring.