Investigation of the association between the outcomes of sperm chromatin condensation and decondensation tests,and assisted reproduction techniques

The main purpose of this prospective study is to examine possible influences of abnormalities of sperm nuclear condensation and chromatin decondensation with sodium dodecyl sulphate (SDS)‐EDTA on outcomes of intrauterine insemination (IUI) or intracytoplasmic sperm injection (ICSI) cycles. Semen samples from 122 IUI and 236 ICSI cycles were evaluated. Before semen preparation for IUI or ICSI, basic semen analysis was performed and a small portion from each sample was spared for fixation. The condensation of sperm nuclear chromatin was evaluated with acidic aniline blue, followed by sperm chromatin decondensation by SDS‐EDTA and evaluation under light microscope. Ongoing pregnancy rate was 24% and 26.2% in the IUI and ICSI groups respectively. The chromatin condensation rate was significantly higher in the ongoing pregnancy‐positive group compared to the negative group, both in IUI (P = 0.042) and ICSI groups (P = 0.027), and it was positively correlated with ongoing pregnancy rate in both IUI and ICSI groups (P = 0.015, r = 0.214 and P = 0.014, r = 0.312 respectively). Chromatin decondensation rates were not significantly different in neither of the groups. These results indicate that IUI and ICSI outcome is influenced by the rate of spermatozoa with abnormal chromatin condensation. Sperm chromatin condensation with aniline blue is useful for selecting assisted reproduction techniques (ART) patients.     

Predictive value of chromatin decondensation in vitro on fertilization rate after intracytoplasmic sperm injection (ICSI)

This study was undertaken to identify the relationship between sperm chromatin decondensation after incubation with sodium dodecyl sulphate (SDS) and ethylene diamine tetra acetic acid (EDTA), or heparin at various points of time. Likewise, this study will determine chromatin stability within definite time intervals, chromatin decondensation after intracytoplasmic sperm injection (ICSI), and whether chromatin decondensation in vitro could be used as a predictive test for fertilization capability after ICSI. Sixty-five infertile couples undergoing ICSI therapy were included in this prospective study. Male factor infertility was the main indication for inclusion. One millilitre from each semen sample after washing was mixed with SDS-EDTA (group 1) or SDS/heparin (group 2) and incubated for 120 min. Many smears were made within 10 min of mixing the spermatozoa with detergent and the reducing agents and at the following points of time 30, 60 and 120 min and after 24 h. Chromatin decondensation was evaluated after staining with acridine orange (AO). The mean percentage of uncondensed chromatin of spermatozoa in the semen sample in the first group before addition of SDS/EDTA was 26.1 +/- 19.0 and 22.3 +/- 18.9% in the second one. After incubation of spermatozoa for 30, 60 and 120 min and 24 h, the chromatin decondensation increased in the first group to 64.0 +/- 28.6, 83.0 +/- 21.1, 87.9 +/- 14.6, 92.1 +/- 16.2 and 98.0 +/- 6.75%, respectively. The corresponding values in the second group were 69.5 +/- 29.9, 78.6 +/- 22.4, 86.9 +/- 17.1, 95.13 +/- 6.5 and 98.3 +/- 5.6%. On the other hand, no correlations were found between the chromatin decondensation or chromatin decondensation rate in vitro and the fertilization rates in all investigated groups. In conclusion, neither the chromatin decondensation ability in vitro nor the rate of chromatin decondensation between various points of time after using SDS/EDTA, SDS/heparin could predict the chromatin decondensation of spermatozoa (fertilization capability) after ICSI.     

Freeze‐dried stallion spermatozoa: evaluation of two chelating agents and comparative analysis of three sperm DNA damage assays

During the freeze‐drying procedure, sperm DNA might become damaged by both freezing and drying stresses. Sperm DNA status can be detected using well‐established assays; however, most techniques are expensive and involve elaborate protocols and equipment. Indirect assessments can provide alternative strategies. The objective of this study was to compare a simple test of DNA status using Diff‐Quik (DQ) with two established procedures: acridine orange test (AOT) and sperm chromatin dispersion (SCD) on freeze‐dried (FD) stallion spermatozoa. Ejaculated spermatozoa from three stallions were freeze‐dried in basic medium supplemented with two different chelating agents: EGTA or EDTA. After rehydration, the spermatozoa were subjected to DNA damage detection using a SCDt, AOT and DQ stain simultaneously. The results showed that the DNA damage levels in the EGTA group were significantly lower than those in the EDTA group. AOT detected a significantly higher proportion of spermatozoa with fragmented DNA than DQ and SCD. The results of the SCD test and DQ stain exhibited a significant positive correlation for DNA fragmentation (r = 0.528), whereas a negative correlation was observed between SCD, DQ and AOT (r = ?0.134 and r = ?0.332 respectively). The present study shows that both the SCD test and DQ assay are effective methods for detecting FD stallion sperm DNA fragmentation, whereas using of AOT is questionable.     

Nuclear chromatin decondensation of human sperm assessed by DNA measurement

A sperm nuclear decondensation ability test using 1% SDS + 6 mM EDTA was used to determine an objective method of quantification of the decondensation process by comparing two different methods of quantification: the usual histological method, after staining by the method of Shoor and classification of the sperm heads into normal heads, doubtful heads, swollen heads, and very swollen heads, and the measurement of the removal of DNA from the extremely swollen spermatozoa. After 5 min of exposure to SDS/EDTA, the percentage of removed DNA was correlated (negatively) to the percentage of normal heads, which assessed the two methods and confirmed that all decondensation results obtained after long periods of treatment with SDS/EDTA can be discarded since spermatozoa can be extremely swollen and disappear.     

Nuclear chromatin decondensation of spermatozoa in vitro: a method for evaluating the fertilizing ability of ovine semen

Spermatozoa obtained from testes, epididimydes and complete ejaculates of healthy rams during the breeding and non-breeding seasons were induced to show nuclear chromatin decondensation by controlled exposure to dithiotreitol (DTT) and sodium dodecyl sulphate (SDS) in vitro. A gradual resistance to decondensation was shown by sperm during epididymal transit, confirming a progressive increase in the prevalence of chromatinic disulphide bonds during sperm maturation in this species. A high % of stable (non-decondensed) sperm nuclei after treatment (79%) was found in semen from rams with normal fertility (64% non-return rate at first oestrus). Opposite changes were found in the semen from rams having low fertility rates (37%), as these showed only 31% of stable sperm nuclei. There were no differences in the spermiograms of these two groups. When semen from the same rams was tested during the non-breeding season, a similar relationship was found, although in both groups there was a higher % of sperm with stable nuclei than during the breeding season. The possible role of seminal plasma and of some of its constituents (e.g., zinc) on the decondensation of ram sperm nuclear chromatin was also studied. The presence of seminal plasma and the addition of zinc largely or completely inhibited the decondensation of ram sperm nuclear chromatin whilst the reverse situation was seen following the addition of chelating agents (e.g. EDTA) to the semen samples. The present results suggest that the induction of sperm nuclear decondensation by exposure to DTT and SDS under controlled conditions may provide a simple but reliable method for predicting in vitro the fertilizing ability of a ram semen sample.     

Metallothionein binding zinc inhibits nuclear chromatin decondensation of human spermatozoa

Summary.  Nuclear chromatin decondensation (NCD) of the human spermatozoa was induced by 1% sodium dodecyl sulphate (SDS). NCD of the spermatozoa induced in healthy and fertile men was significantly stronger and at higher rates than that in infertile men. In 1% SDS with 6 mM zinc chelating EDTA, metallothionein (MT) significantly enhanced NCD in a healthy man. In contrast MT alone significantly inhibited sperm NCD. Sperm NCD rate induced by 1% SDS in 10 infertile men was significantly inhibited by adding 75 or 750 μg ml^-1 of MT. By adding 1.5 mM zinc, MT at concentrations of 0.75, 7.5, 75, or 750 μg ml^-1, enhanced the inhibitory effect of 1.5 mM zinc. This suggested that thiols in the MT could, when liberated from zinc by zinc-chelating EDTA, induce sperm decondensation by cleaving stabilizing S-S bridges and that zinc bound to MT could exert a chromatin stabilizing effect mediated by the zinc dependent type of chromatin stability. The present study suggested that zinc bound to MT, which is secreted mainly from the prostate gland, is one factor that contributes to the chromatin stabilizing effect of human prostatic fluid.     

Effect of human sperm chromatin anomalies on fertilization outcome post-ICSI

The aim of this study was to evaluate the effect of sperm chromatin anomalies on fertilization outcome post-intracytoplasmic sperm injection (ICSI). Therefore, along with semen parameters, Chromomycin A3 (CMA3) staining for protamine deficiency, aniline blue staining for excessive histones, SDS for sperm chromatin stability and SDS + EDTA for the ability of sperm to undergo decondensation were carried out on 55 semen samples from patients referred to the Isfahan Fertility and Infertility Center for ICSI. The results showed that among the aforementioned tests and semen parameters only CMA3 showed a significant correlation with fertilization outcome post-ICSI. Patients were also grouped according to CMA3 level of <30% or >30% or fertilization rate of <50% or >50%. The results show that the mean percentage fertilization and mean percentage of CMA3 positivity is different in both groups, respectively. The area under receiver operator characteristics curve shows that CMA3 is a highly sensitive and specific test for prediction of fertilization outcome post-ICSI. In conclusion, that sperm protamine deficiency has profound effect on fertilization failure in ICSI.     

Variability of the chromatin decondensation ability test on human sperm

A sperm nuclear decondensation ability test using 1% SDS + 6 mM EDTA was used to evaluate: a system of classification and nomenclature for the decondensation of nuclear chromatin; the progress of decondensation as a function of the duration of exposure to SDS/EDTA; the residual variance, or "scoring error;" the within-subject variance (N = 5); and the between-subject variance (N = 10). The process of chromatin decondensation was found to be a continuous phenomenon, but a scheme of nomenclature using four categories along with a system of data analysis using class weightings were developed. A 5-min exposure to SDS/EDTA resulted in a minimum scoring error (8.34%). The within- and between-subject variances were not significantly different from each other, but both were individually different (p less than 0.001) from the residual variance.     

Cystic fibrosis transmembrane conductance regulator is correlated closely with sperm progressive motility and normal morphology in healthy and fertile men with normal sperm parameters

Cystic fibrosis transmembrane conductance regulator (CFTR) has been demonstrated to be expressed in mature spermatozoa and correlated with sperm quality. Sperm CFTR expression in fertile men is higher than that in infertile men suffering from teratospermia, asthenoteratospermia, asthenospermia and oligospermia, but it is unknown whether CFTR is correlated with sperm parameters when sperm parameters are normal. In this study, 282 healthy and fertile men with normal semen parameters were classified into three age groups, group (I): age group of 20–29 years (98 cases, 27.1 ± 6.2), group (II): age group of 30–39 years (142 cases, 33.7 ± 2.6) and group (III): age group of more than or equal to 40 years (42 cases, 44.1 ± 4.6). Sperm concentration, total count and progressive motility were analysed by computer‐assisted sperm analysis. Sperm morphology was analysed by modified Papanicolaou staining. Sperm CFTR expression was conducted by indirect immunofluorescence staining. There was a significant positive correlation (P < 0.001) between CFTR expression and sperm progressive motility (r = 0.221) and normal morphology (r = 0.202), but there were no correlations between sperm CFTR expression and semen volume, sperm concentration, sperm total count as well as male age (P > 0.05). Our findings show that CFTR expression is associated with sperm progressive motility and normal morphology in healthy and fertile men with normal sperm parameters, but not associated with the number of spermatozoa and male age.     

Heparin and glutathione II: correlation between decondensation of bull sperm cells and its nucleons.

The correlation between the kinetics of bull sperm nuclear and nucleon decondensation induced by the action of physiological concentrations of heparin/GSH was studied. Sperm and nucleon suspensions were incubated at 37 degrees C in salt medium, at a constant concentration of either heparin or GSH and increasing concentrations of the other reagent. Even though nucleons are pretreated with DTT/CTAB, when they are incubated alone with GSH for 96 h, they remain intact, no matter which concentration is employed, and it was impossible to observe the slightest sign of nuclei decondensation. Therefore, rupture of disulfide bridges is not the main mechanism to induce nuclei decondensation and perhaps the GSH role resides in potentate the heparin effect by increasing its negative charge. Nevertheless, nucleons reach 95% of chromatin decondensation in the presence of heparin plus GSH or heparin alone. The fact that the correlation between heparin and GSH concentrations needed to induce sperm nuclei decondensation was 3- to 4-fold greater that in nucleons might be due to the complete lack of nucleon membranes. Heparin/GSH seem to induce nuclei decondensation by an ionic chromatin charge neutralization mechanism.     

In vitro decondensation of nuclear chromatin of human spermatozoa: assessing fertilizing potential.

Spermatozoan nuclear chromatin is in a highly condensed state prior to fertilization. In vivo decondensation occurs in the ooplasm and is essential for successful fertilization and the formation of male pronucleus and the zygote to occur. The chromatin of spermatozoa and nucleus can undergo in vitro decondensation with sodium dodecyl sulfate (SDS) and 6 mM ethylene diamine tetraacetic acid (EDTA). The ability of sperm to decondense in vitro was compared with their ability to fertilize human oocytes in vitro. Spermatozoa from normal samples were studied for their decondensation ability as regards their fertilizing performance in an in vitro fertilization (IVF) program. Fertilization occurred when the decondensation percentage of sperm nuclear chromatin was more than 70%. The effective sperm count was significantly (p less than 0.05) lower in the unfertilized group. This is a new diagnostic technique to assess sperm-fertilizing potential at the initial evaluation of the male.     

Defective sperm decondensation: a cause for fertilization failure

The study aimed to evaluate the role of chromatin packaging (CMA3 staining), sperm morphology during sperm-zona binding, sperm decondensation and the presence of polar bodies in oocytes that failed in vitro fertilization (IVF). The percentage CMA3 staining categorized the data into three groups, < 44%, n = 10; > or = 44-59%, n = 10; and > or = 60%, n = 29. Morphology groups were < or = 4% (n = 11); > 4-14% (n = 19); and > 4% (n = 19). One hundred and seventy-two oocytes that failed IVF were evaluated for sperm-zona binding, ooplasma penetration and sperm decondensation. Odds ratio analyses indicated that being in the > or = 60% CMA3 staining group resulted in a 15.6 fold increase in the risk of decondensation failure, relative to CMA3, staining of < 44%. For morphology, there was a 2.17 fold decrease in the risk of fertilization failure in the morphology group with > 4-14% normal cells, while it increased 2.45 fold for the morphology group with < or = 4% normal cells. Using CMA3 fluorescence to discriminate, 51% of the oocytes in the group with elevated CMA3 fluorescence had no sperm in the ooplasma compared to 32% and 16% penetration failure in the CMA3 staining groups > or = 44-59% and < 44%, respectively. Sperm chromatin packaging quality and sperm morphology assessments are useful clinical indicators of human fertilization failure. Immunofluorescence techniques could be used to provide a clear diagnosis of failed fertilization.     

Effect of cryopreservation on the nuclear chromatin decondensation ability of human spermatozoa

The possible effect of cryopreservation on human sperm chromatin decondensation ability has been investigated. Comparisons of the actions of the decondensation-inducing agent 1% (w/v) sodium dodecyl sulphate + 6 mM EDTA were made on 30 ejaculates between spermatozoa in seminal plasma, spermatozoa in semen diluted with cryoprotective medium (CPM) and spermatozoa frozen and thawed in the semen-CPM mixture. The results, analyzed as paired series, showed no significant differences between the spermatozoa under the three treatment conditions. Thus, spermatozoa cryopreserved by a method routinely used for semen storage for subsequent artificial insemination showed a nuclear stability equivalent to that of fresh semen. The CPM by itself had no effect upon chromatin instability. No correlation was found between the percentage recovery of post-thaw motility (an usual index for judging semen cryopreservation) and the tests of chromatin decondensation.     

The micronutrient supplements,zinc sulphate and folic acid,did not ameliorate sperm functional parameters in oligoasthenoteratozoospermic men

We investigated the effects of folic acid and zinc sulphate supplementation on the improvement of sperm function in subfertile oligoasthenoteratozoospermic (OAT) men. Eighty‐three OAT men participated in a 16‐week intervention randomised, double‐blind clinical trial with daily treatment of folic acid (5 mg day^?1) and zinc sulphate (220 mg day^?1), or placebo. Before and after treatment, semen and blood samples were obtained for determining sperm concentration, motility, and morphology, sperm viability, sperm mitochondrial function, sperm chromatin status using toluidine blue, aniline blue, acridine orange and chromomycin A₃ staining; and semen and blood folate, zinc, B₁₂, total antioxidant capacity ( TAC) and malondialdehyde (MDA) concentrations. Sperm concentration (×10⁶ ml^?1) increased in subfertile men receiving the combined treatment of folic acid and zinc sulphate and also in the group receiving only folic acid treatment; however, it was not statistically significant (P = 0.056 and P = 0.05, respectively). Sperm chromatin integrity (%) increased significantly in subfertile men receiving only zinc sulphate treatment (P = 0.048). However, this improvement in sperm quality was not significant after adjusting placebo effect. This study showed that zinc sulphate and folic acid supplementation did not ameliorate sperm quality in infertile men with severely compromised sperm parameters, OAT. Male infertility is a multifactorial disorder, and also nutritional factors play an important role in results of administration of supplementation on sperm parameters. However, these results should be confirmed by multiple studies in larger populations of OAT men.     

Changes in human sperm chromatin stability during preparation for in-vitro fertilization

This study was designed to define the effects of sperm preparation on sperm chromatin stability in relation to in-vitro fertilization (IVF). Semen samples used for IVF-embryo transfer (ET) in the treatment of infertility due to tubal factors were studied. Cases with semen variables below reference limits in previous samples were excluded. Sperm were prepared by a swim-up technique employing either of two different tissue culture media, Ham's F-10 or Earle's balanced salt solution. Sperm chromatin stability was tested by exposure both to sodium dodecyl sulphate (SDS) only and SDS together with a zinc-chelating agent, disodium ethylene diamine tetraacetate (SDS-EDTA). Sperm head swell scores were defined under different experimental conditions and the relationship to sperm motility, morphology, fertilization rate and pregnancy occurrence was tested. No differences were seen between the chromatin stability of sperm from the original sample and that after swim-up preparation, neither immediately after completion of the swim-up procedure, nor at the time of insemination of ova. With time, the chromatin became more stable, which occurred to a similar extent both in the original sample and in swim-up preparations using Ham's F-10. Otherwise, sperm chromatin stability was unaffected by either of the two media used for swim-up. At higher incubation temperatures, decondensation in SDS was enhanced. Altogether, no correlation was found between sperm chromatin stability or enhancement of decondensation by temperature and the success of IVF treatment expressed in fertilization rates or pregnancies. The results are reassuring in that only small changes in sperm chromatin stability occurred during the preparation for IVF. As long as semen of presumably good quality is used, these changes in chromatin stability do not seem to be of clinical importance.     

Apoptotic sperm biomarkers and the correlation between conventional sperm parameters and clinical characteristics

The principal aim of this retrospective study was to examine the relationship between sperm apoptotic biomarkers and the patient's biclinical characteristics, the conventional sperm parameters and the results of assisted reproductive technology. Sperm analysis, activated caspases, annexin V staining for phosphatidylserine (PS) externalisation and labelling assay for DNA fragmentation were assessed in 122 males of infertile couples. Fifty‐seven couples were allocated to the natural conception group, and 65 couples underwent IVF or ICSI. Semen of IVF/ICSI patients showed a higher proportion of apoptotic spermatozoa in their spermatozoa when compared with a natural conception group (p < .05). Sperm apoptotic biomarkers correlated with age, FSH, and conventional sperm parameters. DNA fragmentation correlated positively with the percentage of semen having externalised PS (r = .78, p = 0) and activated caspases (r = .71, p = 0). Patients without clinical pregnancy had higher frequency of DNA fragmentation, externalised PS and activated caspases compared to patients with clinical pregnancy (p < .001). The best specificity and greater sensitivity were obtained with the test of the DNA fragmentation compared to the other biomarkers. Among the apoptotic biomarkers, only DNA fragmentation was found to predict natural or assisted pregnancy better than conventional sperm parameters.     

Chromatin decondensation of human sperm in vitro and its relation to fertilization rate after ICSI

The inability of sperm chromatin to decondense has been implicated in the failure of fertilization, This study was undertaken to identify the relationship between sperm chromatin decondensation in vitro after incubation with follicular fluid at various points in time and fertilization or pregnancy rates after intracytoplasmic sperm injection. Moreover, an attempt was made to determine whether this test could be used as a predictive test for the outcome of ICSI. Thirty-two infertile couples undergoing ICSI therapy were included in this prospective study. One milliter of semen from each sample was mixed with 1 mL of follicular fluid obtained from ICSI patients at the time of oocyte retrieval and incubated for 24 h. Many smears were made directly after semen liquefaction at the following time intervals: 30, 60, and 120 min and 24 h. Chromatin decondensation was evaluated with acridine orange staining. The mean percentage of uncondensed chromatin of spermatozoa in the native semen samples was 25 +/- 18.3%, which increased within 24 h to 91 +/- 9.5%. On the other hand, the fertilization and ongoing pregnancy rates were 64 +/- 21.7% and 20%, respectively. However, no correlations were found between chromatin decondensation at various point of time (30, 60, and 120 min and 24 h) and fertilization rate. No correlation was shown between the chromatin decondensation and sperm counts in the ejaculate. morphology, or the percentage of condensed chromatin. In light of this study, chromatin decondensation in vitro cannot be recommended for predicting the fertilization potential of spermatozoa and pregnancy rates in the ICSI program. Further research is necessary, especially in cases where ICSI is being considered as a therapeutic option.     

Correlation of biochemical constituents of seminal plasma with semen quality in Teddy goat (Capra hircus) bucks

This study was planned to determine the relationship between semen quality parameters and the levels of biochemical constituents of seminal plasma of Teddy (Capra hircus) buck semen. For this purpose, semen ejaculates were collected from five mature healthy Teddy bucks. All the experimental bucks were kept under natural environmental conditions. Semen was collected twice in a week for the duration of 6 weeks by Artificial Vagina (AV) in the breeding season (February‐April). Two successive ejaculates of single buck were pooled at time of collection, and a total of 60 semen samples were processed for semen analysis. Sperm per cent motility, sperm concentration, dead sperm percentage, morphological abnormal spermatozoa, plasma membrane integrity were correlated with biochemical constituents of seminal plasma. The mean per cent motility (89.18% ± 0.37%), sperm concentration (1.86 ± 0.04 × 10⁹/ml), dead sperm percentage (8.08% ± 0.29%), morphological abnormal spermatozoa (6.05% ± 0.29%) and plasma membrane integrity (88.22% ± 0.34%) were recorded. The seminal plasma contained Na⁺ (144.12 ± 1.59 mEq/L), K⁺ (27.38 ± 0.49 mEq/L), Cl^? (65.73 ± 0.45 mEq/L), Ca⁺⁺ (9.34 ± 0.22 mg/dl), P (19.32 ± 0.97 mg/dl), aspartate aminotransferase (AST; 26.48 ± 1.30 IU/L), alanine aminotransferase (ALT; 168.47 ± 5.18 IU/L), lactate dehydrogenase (LDH; 215.98 ± 6.06 IU/L), albumin (1.90 ± 0.10 g/dl), globulins (2.08 ± 0.11 g/dl) and total protein (3.98 ± 0.20 g/dl). The collected data were analysed by applying Pearson's correlation coefficients. Dead sperm percentage had negative correlation with sodium (r = ?.278, p < .05), albumin (r = ?.294, p < .05), globulin (r = ?.266, p < .05) and total protein (r = ?.295, p < .05). Phosphorus was negatively associated with sperm concentration (r = ?.262, p < .05). AST was negatively correlated with plasma membrane integrity (r = ?.292, p < .05). It was concluded that most of the semen quality parameters of Teddy bucks were positively correlated with biochemical constituents, but opposite trends were found in case of dead sperm percentage. The seminal biochemical constituents dynamically interact with each other.     

Stenotrophomonas maltophilia isolated from prostatic fluid as an infertility factor in a male dog

The aim of this case was to describe very rare infection of canine prostate gland with Stenotrophomonas maltophilia which had influence on male fertility. The bacterium was cultured from third fraction of the ejaculate collected by manual manipulation. The sperm concentration and motility parameters were evaluated by Hamilton‐Thorne Sperm Analyser, version IVOS 12.3, sperm morphology by Diff‐Quick staining and live/dead spermatozoa by eosin/nigrosin staining. After 3 weeks of treatment with targeted antibiotic, ciprofloxacin, there was no bacterial growth in prostate fluid. Semen parameters were improved after 60 days from the end of treatment, and females were successfully mated.     

Association of human sperm nuclear decondensation and in vitro penetration ability

Sperm nuclear decondensation is an integral step in fertilization which leads to the formation of the male pronucleus. The association between the in vitro spontaneous nuclear decondensation of human sperm and its fertilizing ability was studied in infertile male patients. The ability of sperm to fertilize an egg using the discontinuous two-layer Percoll method was significantly correlated to the percentage of decondensed swollen head (r = 0.43; P less than 0.005). The fertilizing ability of sperm processed with Test-Yolk buffer was correlated with the percentage of sperm at the fully decondensed stalk stage (r = 0.51; P less than 0.05). There were insignificant correlations for the whole-wash centrifugation, cryopreserved-thawed and swim-up methods. Samples of sperm that were positive (greater than 0% fertilization) in the sperm penetration assay had a higher percentage of decondensed sperm heads (66.7% vs. 20.6%) after Percoll wash or whole-wash centrifugation (60.5% vs. 44.3%) treatments compared with samples with no fertilization. Treatments that included Test-Yolk resulted in high percentages of decondensed swollen heads. The results suggest a positive association between sperm nuclear decondensation and the fertilizing ability of sperm, and affirm the importance of nuclear decondensation to the study of fertilization events.