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.     

Sperm decondensation and semen parameters: utilization of a simple staining technique for the evaluation of human sperm decondensation

Summary. The ability of spermatozoa to fertilize an oocyte depends on a sequence of events ending ultimately in the decondensation of the sperm chromatin on penetration of the oocyte. Knowledge of what percentage of sperm decondenses is useful, especially in patients where other functional tests and sperm quality fail to explain the reported poor in vitro fertilization (IVF) rates. The objective of this study was (1) to compare sperm decondensation induced by either SDS/EDTA or heparin with semen parameters (volume, concentration, motility and morphology), and (2) to evaluate the use of a simplified staining technique (Diff Quik^R [DQ]) in comparison with the standard phase contrast method (Rose Bengal-[RB]). Randomly selected semen samples from 31 men attending an assisted reproductive programme were analysed for basic semen parameters and decondensation with SDS/EDTA and heparin. Two staining methods for the evaluation of decondensation were compared (phase contrast microscopy after Rose Bengal [RB] staining and light microscopy after Diff Quik^R (DQ) staining). Moderate and grossly swollen sperm heads were recorded. Semen samples included both fertile and unfertile semen parameters. Sperm decondensation results showed poor to moderate correlations with semen parameters. The SDS/EDTA (DQ) (moderate forms) showed a significant negative correlation (r = -0.46) with seminal volume and and a significant positive correlation (r = 0.41) with normal sperm morphology. The heparin (DQ) (moderate forms) decondensation showed a significant positive correlation with motility (r = 0.61) and sperm concentration (r = 0.43). The DQ method was preferred over the RB method due to its optical and storage advantage. Sperm decondensation by SDS/EDTA and heparin have limited use in the IVF laboratory as they correlate poorly with semen parameters. Future studies should investigate the use of an ooplasmic factor similar to nucleoplasmin in Xenopus laevis egg.     

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.     

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.     

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.     

Comparison between computerized slow-stage and static liquid nitrogen vapour freezing methods with respect to the deleterious effect on chromatin and morphology of spermatozoa from fertile and subfertile men

The purpose of this study was to determine the negative effects (cryodamage) on human spermatozoa after freeze-thawing and to determine whether freeze-thawing of spermatozoa with a programmed slow freezer is better than freezing with liquid nitrogen vapour (rapid freezing) with regard to alterations in sperm chromatin and morphology in semen from fertile (donor) and subfertile, IVF/ICSI, patients. Ninety-five semen samples were obtained either from patients attending our IVF unit for treatment (n=34) or from donors (n=25) with proven fertility and normal sperm quality according to WHO guidelines. Each semen sample was divided into two parts after liquefaction and addition of the cryoprotectant. The first part was frozen using a programmed biological freezer and the second part was frozen by means of liquid nitrogen vapour. Smears were made before the freezing and after the thawing procedure to assess morphology (strict criteria) and chromatin condensation (Acridine Orange test). The mean percentage of chromatin condensed spermatozoa in the samples from donors (control group) was 92.4 +/- 8.4% before freezing and this decreased significantly (p < 0.0001) to 88.7 +/- 11.2% after freeze-thawing with the computerized slow-stage freezer and to 87.2 +/- 12.3% after using static liquid nitrogen vapour (p < 0.001). The corresponding values for semen obtained from patients was 78.9 +/- 10.3% before freezing which decreased to 70.7 +/- 10.8 and 68.5 +/- 14.8%, respectively (p < 0.001). On the other hand, the mean percentage of normal sperm morphology in the control group decreased from 26.3 +/- 7.5% before freezing to 22.1 +/- 6.4% (p < 0.0001) after thawing with the computerized slow-stage freezer and to 22.2 +/- 6.6% (p < 0.0001) after the use of static liquid nitrogen vapour. In the patient group, the mean percentage of normal morphology decreased from 11.7 +/- 6.1% after freezing with the biological freezer to 9.3 +/- 5.6% and to 8.0 +/- 4.9% after freezing with static liquid nitrogen vapour. This study demonstrates that chromatin packaging and morphology of human spermatozoa decrease significantly after the freeze-thawing procedure, not only after the use of static liquid nitrogen vapour but also after the use of a computerized slow-stage freezer. However, the chromatin of semen samples with normal semen parameters (donor sperm) withstand the freeze-thaw injury better than those with low quality semen samples. Therefore, the computerized slow stage freezer could be recommended for freezing of human spermatozoa, especially for subnormal semen samples, for example, ICSI and ICSI/TESE candidates and from patients with testicular tumours or Hodgkin's disease, in order to avoid further damage to the sperm chromatin structure.     

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.     

Sperm head ultramorphology and chromatin stability of males with unexplained infertility who fail to fertilize normal human ova in vitro

Summary. An in vitro nuclear chromatin decondensation test, and quantitative nuclear ultramorphology analysis, were performed on 18 males judged to be infertile, by two failures in in vitro fertilization, and 16 fertile males. These two clinical groups only differed significantly in (1) the direction of their chromatin stability change, which took place 30–120 min post-ejaculation while stored in the seminal plasma, and (2) in the incidence of the hypoelongated sperm-head. Generally, the fertile male group exhibited positive chromatin stability change after prolonged storage, and low incidence of hypoelongated sperm heads, and vice versa in the unexplained infertile group. When the nuclear chromatin decondensation test and quantitative ultramorphology analysis were performed in step-wise fashion, it was possible to correctly classify 94% of the fertile cases with 6% of false-negative, and 89% of the unexplained infertile cases with 11% of false-positive. Therefore, it appears that these tests might be of benefit clinically for identifying functional properties of sperm-cells in unexplained infertile males, which cannot be detected by routine semen analysis.     

Altered protamine 2 expression is uncommon in donors of known fertility, but common among men with poor fertilizing capacity, and may reflect other abnormalities of spermiogenesis.

During the spermatid elongation stage of spermiogenesis approximately 85% of sperm nuclear histones are replaced by protamines. Protamines increase the packing ratio of sperm chromatin, presumably facilitating sperm motility and function. In this study we evaluated the incidence of abnormal protamine expression in 75 patients undergoing in vitro fertilization (IVF) and 50 donors of known fertility by isolation of sperm nuclear proteins, quantitative gel electrophoresis, and Western blot analysis. In addition, we evaluated the relationship between abnormal protamine expression and semen quality, sperm penetration ability, chromatin stability, and IVF outcome. Seventeen percent (13/75) of IVF patients had no measurable protamine 2 (P2) versus 0% (0/50) of donors of known fertility (P < .005). Sperm penetration rates were decreased in 12 of 13 patients without P2, and mean penetration rates (4.6 +/- 1.2 vs 32.8 +/- 2.9, P < .005), normal morphology (22.4 +/- 3.6 vs 48.7 +/- 4.2, P < .05), and progressive motility (22.3 +/- 2.5 vs 35.4 +/- 2.1, P < .05) were all significantly decreased compared with patients with measurable P2. The mean sperm concentration was not significantly different. The presence of protamine precursor bands was also associated with a diminished penetration capacity (18.4 +/- 2.8 vs 36.7 +/- 3.0, P < .05). Sperm chromatin decondensation following exposure to heparin sulfate was significantly increased in patients without a measurable P2 band. Twelve patients with no measurable P2 underwent intracytoplasmic sperm injection (ICSI), with 6 patients (6/12, 50%) becoming pregnant. ICSI fertilization and subsequent embryo cleavage were not different in patients without P2 compared with other patients undergoing ICSI. These data indicate that abnormal sperm protamine levels are a common defect in infertility patients, but not in donors of known fertility. It appears that abnormal protamine levels may reflect defects of late spermiogenesis, including sperm penetration capacity.     

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.     

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.     

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.     

Nucleons II: cryopreservation and metabolic activity.

The establishment of intracytoplasmatic sperm injection (ICSI) as a routine procedure in assisted fertilization has been used in the treatment of male infertility. The major technical problem that has arisen with the use of immotile sperm for ICSI has been differentiating between live and dead cells. Nucleons from human, pig, hamster, mouse, rat, and bull have been able to induce their chromatin decondensation by the action of heparin/GSH. Cryopreservation is deleterious to sperm function, killing more than 50% of the spermatozoa during the process. Nucleon cryostorage was performed at 5 and -5 degrees C and analyzed for total area (mu2), perimeter (mu), width (mu), and length (mu), using Metamorph Imaging System software. On the other hand, fluorescein diacetate (FDA) is hydrolyzed by intracellular estereases to produce fluorescein, which exhibits green fluorescence when excited by blue light. This fact is a striking result since the presence of this metabolic activity opens the possibility to select the nucleons for ICSI. In the present study, the authors decided to search for a suitable metabolic test, which might reflect the metabolism and viability of these chromatin structures. This is a simple cryostorage technique that after months of cryopreservation, allow the use of nucleons for ICSI with suitable fertilization and pregnancies rates.     

Comparison of sperm preparation methods: effect on chromatin and morphology recovery rates and their consequences on the clinical outcome after in vitro fertilization embryo transfer

The aim of this study was to evaluate the efficacy of swim-up, PureSperm gradient centrifugation and glass-wool filtration methods for semen preparation and to assess the possible enhancement of the quality of the subpopulation of spermatozoa in terms of sperm concentration, morphology and chromatin condensation. Moreover, to determine the effect of this semen processing technique on the clinical outcome after in vitro fertilization embryo transfer (IVF-ET). A total of 180 semen samples of patients' husbands who were undergoing IVF therapy were prepared by swim-up (G1, n = 60), PureSperm gradient centrifugation (G2, n=60) or glass-wool (G3, n=60) methods. Chromatin condensation was assessed by Chromomycin (CMA3), whereas sperm morphology was evaluated according to strict criteria. In all three semen processing methods, the percentage of chromatin condensed and morphologically normal spermatozoa was higher after semen processing in comparison with native semen samples. The proportion of normal chromatin condensed spermatozoa prepared in glass-wool filtration was significantly higher than that in swim-up (G.I, p=0.02) or PureSperm (G.II, p=0.001). In addition semen processing with PureSperm yields significantly a higher percentage of morphologically normal spermatozoa than swim-up (p < 0.001) or glass-wool method (p < 0.002). However, the fertilization, implantation and pregnancy rates, in turn were similar in all semen preparation methods. In conclusion, PureSperm gradient centrifugation yields a higher percentage of morphologically normal spermatozoa than shown in traditional swim-up or glass-wool filtration. However, the percentage of chromatin condensed spermatozoa was significantly higher after semen processing via glass-wool in comparison with the other two methods. Nevertheless, there were no significant difference in the fertilization, implantation and pregnancy rates of sperm prepared by means of swim-up, PureSperm or glass-wool filtration. Therefore, glass-wool filtration should be recommended as the first choice for semen preparation for Intracytoplasmic sperm injection (ICSI) technique as the natural selection is bypassed. Whereas, swim-up and PureSperm should be used for semen processing in IVF programme.     

Spermatozoa DNA damage measured by sperm chromatin structure assay (SCSA) and birth characteristics in children conceived by IVF and ICSI

High levels of spermatozoa DNA damage hinder fertility in vivo but not in vitro. It is a source of worry that following in vitro fertilization (IVF) spermatozoa DNA damage, if not repaired by the oocyte, might have a negative impact on the offspring. The aim of this study was to assess if a high spermatozoa DNA Fragmentation Index (DFI) is associated with alterations in birthweight (BW) and/or gestational length in IVF children. One hundred and thirty-one singleton pregnancies established by standard IVF or intracytoplasmic sperm injection (ICSI) were included in the study. DFI was measured by sperm chromatin structure assay (SCSA) in semen samples used for fertilization. DFI was categorized as low and high, using 20, 30, 40 and 50% as cut-off levels. Birthweight, gestational age, as well as gestational age adjusted BW score were used in a linear regression model as end points For none of the tested birth characteristics, statistically significant differences between the groups with low and high DFI were seen regardless of whether 20, 30, 40 or 50% were used as cut-off levels, both when the IVF and ICSI data were merged or analysed separately. Spermatozoa DNA damage as assessed by SCSA is not associated with BW or gestational length in IVF and ICSI children.     

Computer-assisted assessment of sperm morphology: comparison with conventional techniques

The aim of the present study was to compare conventional and computer-assisted morphology assessment of spermatozoa. Sixty-two semen samples from patients undergoing in vitro fertilization (IVF) and 40 samples from patients undergoing an intracytoplasmic sperm injection (ICSI) were studied using both techniques. The percentage of normal spermatozoa found was closely correlated between the techniques (r=0.788, p < 0.0001). The intra-operator variation was low for both techniques but the inter-operator variation was much higher with the conventional than with the computer-assisted method (coefficient of variation = 0.43 vs. 0.08, respectively, for conventional and computer-assisted assessments). The percentage of spermatozoa with normal morphology, as well as sperm motility, was significantly enhanced after PureSperm preparation, whatever the method used for assessment. In the IVF study, fertilization rate was poorly correlated with sperm morphology using both methods. However, combined with motility, morphology assessed with the computer allowed discrimination of two groups of patients with significantly different fertilization rates (30.5 +/- 5.4% vs. 63.1 +/- 5.4%, p < 0.0001). In contrast, the fertilization rate in ICSI was influenced neither by sperm morphology nor by motility. In conclusion, computer-assisted assessment of sperm morphology has a slightly better predictive value for ART than conventional assessment, but above all is much more reproducible, allowing standardization.     

Comparison between the quality and function of sperm after semen processing with two different methods

Aim: To compare the recovery rate of morphologically normal and chromatin condensed spermatozoa from native se-men samples using the SpermPrep~(TM) filtration columns and Percoll gradient centrifugation and to determine the influenceof the two processing techniques on fertilization and pregnancy rates in an IVF-ET program. Methods: Sixteen se-men samples obtained from patient's husband were included in this study. Each was divided into two aliquots. The firstaliquot was processed with SpermPrep~(TM) filtration columns and the second, Percoll gradient centrifugation. Smears weremade before and after semen processing with both methods for the evaluation of chromatin condensation (chromomycineCMA3) as well as morphology (strict criteria) of spermatozoa. One hundred and seventy oocytes were retrieved fromthe patients and the oocytes from each patient were subdivided into two sets : one set was inseminated using spermatozoaprocessed with SpermPrep~(TM) and the other inseminated after semen processing with Pe     

Human spermatozoa ultrastructure assessment in the infertility treatment by assisted reproduction technique

The aim of this study was to prospectively investigate the spermatozoa ultrastructure in relation to the results of in vitro fertilization-embryo transer (IVF-ET). Forty-nine consecutive couples admitted for IVF-ET were prospectively evaluated for electron microscopic spermatozoa morphology and the outcome of IVF-ET. Thirty-four couples revealed successful fertilization, defined as presence of two pronuclei 14-16 hours after spermatozoa administration, while the remaining 15 formed the failure group. Spermatozoa fixed with 2.5% glutaraldehyd and embedded in Spurr's resin were analyzed with JAM 100 S transmission electron microscope (TEM) for the following ultrastructure abnormalities: head deformity, cytoplasmic residues, chromatin condensation failures, acrosomal alterations, neck defects, mid-piece defects, principal piece and end-piece defects and immature forms. Successful IVF-ET couples revealed a significantly higher percentage of normal spermatozoa utrastructure (32.0 +/- 13.1% versus 17.1 +/- 13.4%, p < 0.001). Failed IVF-ET couples represented a significantly higher percentage of chromatin condensation failures (9.8 +/- 5.1% versus 5.7 +/- 5.3%, p < 0.05) and tail defects (16.7 +/- 11.5% versus 7.2 +/- 7.2%, p < 0.001). A positive correlation between normal ultrastructure spermatozoa percentage and fertilized oocytes percentage was found (r = 0.35, p < 0.05). Our data suggest that spermatozoa TEM findings correlate with IVF-ET results. Ultrastructural estimation of spermatozoa can improve the diagnosis of male fertility and may explain some reasons of failure in assisted reproduction methods. We consider systematic TEM spermatozoa examination in cases with failed IVF-ET prior to intracytoplasmic sperm injection (ICSI).     

Influence of Seminal Plasma on the Chromatin Stability of Ejaculated Human Spermatozoa

Nuclear chromatin decondensation (NCD) of human ejaculated spermatozoa exposed to sodium dodecyl sulphate (SDS) has been studied. A high proportion of NCD reacting spermatozoa was only found in semen samples with a relatively low activity of some prostatic factor(s) (i.e. zinc/fructose ratio below 0.18) in the seminal plasma. Exposure to SDS for one h was found sufficient to reveal the main proportion of spermatozoa undergoing NCD in such a solution.
Addition of seminal plasma with an apparently normal composition to a sperm population with a high NCD reactivity restored the sperm SDS resistance to normal, i.e. blocked the NCD-response. Other results indicated that NCD reactivity was decreased or abolished upon prolonged storage of the spermatozoa in the seminal plasma.
The various results indicated that some factor(s) in the seminal plasma can preserve the nuclear chromatin stability of human spermatozoa and that this factor most likely is of prostatic origin.,     

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.