Evaluation of gram stain as an alternative in the assessment of human spermatozoa quality

INTRODUCTION: During spermiogenesis, protaminosis and sperm chromatin condensation are important prerequisites for the preservation of DNA integrity in spermatozoa. The aim of this study is to assess Gram stain as an alternative technique for the evaluation of human sperm chromatin condensation status. Patients and METHODS: Aniline blue and Gram staining were applied to semen samples from 34 donors in order to determine the relationship between sperm chromatin condensation and infertility. In addition, the possible correlation between morphology and vitality (eosin-Y staining) of spermatozoa compared with their nuclear status (aniline blue and Gram staining) was studied. RESULTS: Chromatin condensation and sperm vitality were significantly higher in fertile men compared to the subfertile. A significant correlation was found between chromatin condensation and (a) sperm vitality (p < 0.01), and (b) nuclear protein status (p < 0.01). CONCLUSIONS: Gram staining may be used as a routine method in assisted reproduction laboratories and could assist in the evaluation of sperm quality as well as in the selection of the appropriate fertilization technique.     

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.     

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.     

The effect of chilled storage and cryopreservation on the sperm DNA fragmentation dynamics of a captive population of koalas

This experiment documented the incidence and variability of sperm characteristics found in freshly collected and ex vivo-manipulated semen samples from a population of disease-free captive koalas with a special emphasis on the dynamic aspects of DNA fragmentation. These changes were analyzed in light of the putative negative effect of iatrogenic damage after chilled storage and cryopreservation with respect to different semen extender compositions to maximize sperm longevity. Sperm DNA fragmentation (SDF) dynamics (SDF assessment after a varying period of time) was investigated with the sperm chromatin dispersion assay after either dilution in tris-citrate media and chilled preservation at 4°C for upward of 16 days or cryopreservation in either glycerol or dimethylacetamide (DMA) tris-citrate-based cryoprotectant media; corresponding data on progressive sperm motility, plasma membrane integrity, and the proportion of koala sperm with relaxed chromatin were also recorded. SDF analysis of the captive koala population revealed a low mean (±SEM) basal level of only 6.7% ± 0.9%. The percentage of progressive sperm motility, percentage of intact sperm plasma membranes, and the percentage of relaxed chromatin did not correlate significantly with that of basal SDF. Moreover, despite the absence of cysteine residues in marsupial protamines, koala spermatozoa showed remarkable stability in terms of their DNA integrity after the incubation of either fresh, chilled, or frozen-thawed semen samples; observations of progressive motility (P < .05) and plasma membrane integrity (P < .05) revealed that chilled koala spermatozoa declined after 4 days, whereas the incidence of relaxed chromatin increased after 8 days. Although koala SDF increased significantly (P < .05) with the period of chilled storage, these values remained less than 16% after 16 days storage and subsequent incubation at 35°C for a further 48 hours. Survivorship of prefreeze sperm DNA damage was not different when compared with sperm frozen in DMA or between sperm frozen in DMA or glycerol; however, spermatozoa frozen in glycerol showed a higher (P = .042) rate of DNA fragmentation than prefreeze spermatozoa. This result differed from that of observations of progressive motility, plasma membrane integrity, and relaxed chromatin, which were all adversely affected (P < .05) after cryopreservation in either glycerol or DMA; however, the postthaw characteristics of sperm cryopreserved in either glycerol or DMA were not different. After thawing, koala sperm chromatin tended to decondense; however, the incidence of sperm DNA fragmentation was not correlated with the incidence of sperm chromatin relaxation after glycerol (R = .2) or DMA (R = -.04) cryopreservation.     

Cytochemical evaluation of sperm chromatin and DNA integrity in couples with unexplained recurrent spontaneous abortions

The aim of this study was to examine the possible relationship between sperm DNA integrity and chromatin packaging evaluated by cytochemical assays, traditional sperm parameters and recurrent spontaneous abortion (RSA) of unknown origin. In this cohort study, 40 couples with a history of RSA and 40 couples with proven fertility were considered as case and control groups respectively. The semen samples of all husbands were analysed for sperm parameters and also sperm chromatin and DNA integrity assessed using cytochemical tests including aniline blue (AB), chromomycin A3 (CMA3), toluidine blue (TB), acridine orange (AOT) and nuclear chromatin stability assay. Among different sperm parameters, only slow motility was significantly different between the two groups. In sperm chromatin evaluations, there were significant differences between the two groups in all of the tests. In addition, the majority of semen samples in RSA patients exhibited upper percentages of abnormal spermatozoa than the cut-off values regarding different cytochemical assays. Our study showed that in the cases of RSA, slow motility had a significant reduction in comparison with controls and also spermatozoa of men from RSA group had less chromatin condensation and poorer DNA integrity than spermatozoa that obtained from fertile men with no history of RSA.     

Comparison between human sperm preservation medium and TEST-yolk buffer on protecting chromatin and morphology integrity of human spermatozoa in fertile and subfertile men after freeze-thawing procedure.

The aim of this study was to identify the detrimental effect of the freeze-thaw process on chromatin integrity and morphology of human spermatozoa, and to determine whether human sperm preservation medium (HSPM) or TEST-yolk buffer (TYB) offers a better protection to spermatozoa from cryodamage after the freeze-thaw procedure. Thirty-five semen samples obtained from couples childless because of male factor infertility (subfertile men, group 1) and 25 semen samples from healthy, normal volunteers of proven fertility (group 2) were included in the study. Each semen sample was divided into 2 parts, the first part was mixed with HSPM and the other with TYB (1:1), and frozen with a controlled slow-stage freezer, before plunging into liquid nitrogen. Twelve smears from each semen sample were made before (n = 4) and after (n = 8) the freeze-thaw process. Chromatin structure was evaluated after staining using the acridine orange (AO) test, whereas morphology was analyzed according to strict criteria. The mean percentage of spermatozoa that exhibited normal morphology and intact chromatin structure was decreased after freeze-thaw in all samples treated with HSPM or TYB in comparison with the value observed in the native semen samples of both groups. However, TYB preserved chromatin and morphology significantly better than HSPM did (9.3% +/- 5.6% and 88.7% +/- 11.2% vs. 7.8% +/- 4.2% and 85.5% +/- 12.5%, respectively). Therefore, TYB could be recommended as a first choice cryoprotectant for semen preservation in order to avoid extra chromatin structure damage and morphology alterations of spermatozoa not only for patients pursuing assisted reproduction, but also for donor samples.     

Effect of storage in short--and long-term commercial semen extenders on the motility, plasma membrane and chromatin integrity of boar spermatozoa

For artificial insemination (AI) in pigs, preservation of liquid boar semen at 16-20 degrees C is still common practice as sperm cryopreservation remains suboptimal in this species. To meet the different needs of the swine industry, several extenders have been developed to preserve semen in liquid form for short--and long-term storage. In the present study, three different commercial extenders devised for short-term (BTS+) or long-term preservation (MR-A and X-Cell), were used to test whether storage of semen from four mature, fertile boars at 17 degrees C for 96 h would affect sperm characteristics relevant for fertility, such as motility, membrane integrity and chromatin stability. Computer-assisted sperm analysis, and stainings with the acylated membrane dye SYBR-14/propidium iodide, and acridine orange in connection with flow cytometry were used to evaluate these variables. Percentages of total motile spermatozoa decreased slightly, but significantly, after 72-96 h. While membrane integrity values varied during the period of study, no significant changes in either membrane integrity or chromatin stability were, however, registered. This suggests a customary 96-day storage at 17 degrees C in these extenders was too short an interval to cause losses of integrity in nuclear DNA in the boar population studied.     

Association between freezing agent and acrosome damage of human spermatozoa from subnormal and normal semen

This experimental study compares the effects of human sperm preservation medium (HSPM) with TEST-yolk buffer (TYB) as cryoprotectants of human spermatozoa with respect to the integrity of the acrosome after the freeze-thawing procedure. Fifty-six semen samples were included in this study; 18 were subnormal (G1) and 38 were normal (G2) based on World Health Organization criteria, except for morphology, which was evaluated according to strict criteria. Each semen sample was divided into two parts: the first part was prepared for cryopreservation by the addition of HSPM (1:1) and the second by addition of TYB (1:1). Freezing was performed in liquid nitrogen vapour. Smears were made before freezing and after the thawing process for evaluation of acrosome integrity using fluorescent-lectin labelling. The mean percentage of spermatozoa with intact acrosomes in the subnormal group was 77.0 +/- 7.2% before freezing and decreased significantly (P < 0.001) after thawing: to 63.7 +/- 8.2% with the use of HSPM and 66.8 +/- 8.7% with the use of TYB. The corresponding values in the normal semen samples were 83.4 +/- 9.2%, 76.0 +/- 8.8% and 77.9 +/- 9.2%, respectively. It is obvious that the decrease in the mean percentage of spermatozoa with intact acrosome was significantly higher when using HSPM in comparison with TYB, not only for G1 (-14.9 +/- 1.9% versus -11.8 +/- 1.4%) but also for G2 samples (-13.8 +/- 1.5% versus -11.9 +/- 1.3%). In conclusion, TYB should be recommended for freeze-thawing of human spermatozoa as the first-choice cryoprotectant, for normal as well as subnormal semen samples, in order to protect the sperm acrosome from the deleterious effects of the freeze-thawing procedure.     

Assessment of sperm quality, DNA integrity and cryopreservation protocols in men diagnosed with testicular and systemic malignancies

Men diagnosed with malignancy are often referred for semen banking to preserve their fertility prior to cancer treatment. The chances of cancer patients for achieving future fecundity will be determined by the sperm quality including the integrity of the genomic material in the frozen samples. The objectives of this study were to compare the sperm quality and DNA integrity in men diagnosed with testicular and systemic malignancies before receiving treatment and to identify the optimum cryopreservation protocol for their samples including a remote semen collection option. In comparison with fertile donors, patients with testicular malignancies had significantly lower sperm concentration, while both testicular and systemic malignancy patients had significantly lower sperm motility and cryosurvival rates. In addition, the SCSA defined DNA fragmentation index was significantly higher in patients with testicular and systemic malignancies compared with fertile donors. It was noted that the extent of deterioration in sperm quality and DNA integrity seen in cancer patients did not reach the previously defined statistical threshold for impaired fertility. Freezing spermatozoa with the seminal plasma offers the highest protection against cryo-injury. Nevertheless, remote semen collection can still be used as it yields adequate results.     

Fertilization of hamster ova by human spermatozoa in relation to other semen parameters

Different indices of the zona-free hamster ovum test system were interrelated. High correlations were found between the fertilization percentage and the average number of penetrated spermatozoa/ovum (r = 0.99) and between the fertilization percentage and the number of spermatozoa attached to the ova surfaces (r = 0.72). Fertilization percentages from 63 subfertile patients were correlated with different semen factors assessed by multiple exposure photography (MEP). Low correlations were found between sperm concentration and fertilization percentage (r = 0.29) and between fertilization percentage and motile sperm count (r = 0.33). Spermatozoa progressing with high average velocity had low fertilization rates compared to specimen with moderate average velocity. Effects of sperm washing on the in vitro fertilizing capacity were studied in a group of 40 subfertile men. The fertilization percentages of 8 specimens -with visual seminal plasma abnormalities- increased significantly when immediate dilution of semen was applied.     

An evaluation of chromatin condensation and DNA integrity in the spermatozoa of men with cancer before and after therapy

Cancer has been known for a long time to have a depressive effect on sperm number and quality. Cytotoxic agents and radiotherapy have also been shown to impair spermatogenesis. The aim of this study was to assess DNA integrity and chromatin condensation in the spermatozoa of men with cancer before and after treatment. Chromatin condensation was evaluated using flowcytometric assessment with propidium iodide, DNA integrity was determined using the comet assay. Thirty-three men with cancer (testicular cancer, lymphoma and leukaemia) and 14 men with proven fertility took part in the study. The study found that in men with cancer, the percentage of spermatozoa with highly condensed DNA was less than that of controls. DNA integrity when assessed using the comet assay was also reduced by cancer. Percentage head DNA intact and percentage of condensed chromatin in the spermatozoa of men with cancer after treatment were less than those in fertile men. This study, although small, does demonstrate a detrimental effect on chromatin condensation and DNA integrity of cancer and its treatment. These findings are important because of the potential effects impaired chromatin and DNA integrity could have on fertilization, blastocyst and embryo development.     

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     

Effect of varicocele on chromatin condensation and DNA integrity of ejaculated spermatozoa using cytochemical tests

Varicocele occurs in approximately 15% to 20% of the general male population and it is the most common cause of poor semen production and decreased semen quality. It has been demonstrated that patients with varicocele have a significantly higher DNA fragmentation index (DFI) and spermatozoa with nuclear anomalies than healthy fertile men. Therefore, the aim of this study was to evaluate sperm chromatin integrity in these patients. Sixty men referring to the andrology laboratory were categorised into three different groups: 20 infertile men with varicocele, 20 infertile men with abnormal semen parameters and 20 fertile men who had normal spermatogram were considered as control group. Semen analysis was performed according to WHO criteria. To evaluate sperm chromatin quality and DNA integrity, after fixation of sperm smears, aniline blue, toluidine blue, chromomycin A₃ and acridine orange staining were applied in three groups. The slides were analysed by light and fluorescent microscopy and to determine the percentage of mature or immature spermatozoa, 200 spermatozoa were counted in each slide. The results showed that the rates of aniline blue-reacted spermatozoa were significantly higher in infertile and varicocele patients than in the normal group ( P  < 0.001). In addition, with regard to chromomycin A₃, acridine orange and toluidine blue staining, there was a significant difference between the three groups ( P  < 0.001). The results showed that the varicocele samples contain a higher proportion of spermatozoa with abnormal DNA and immature chromatin than those from fertile men as well as infertile men without varicocele. Therefore, varicocele results in the production of spermatozoa with less condensed chromatin and this is one of the possible causes of infertility due to varicocele.     

Expression of lectin receptors on the membrane surface of sperm of fertile and subfertile boars by flow cytometry

Studies suggest that carbohydrates are important in different stages of fertilization. Plasma membrane changes accompanying in vitro capacitation and acrosome reaction (AR), such as removal or appearance of specific glycoproteins, have been studied using lectins that bind specifically to carbohydrate residues. In specialized artificial insemination farms and semen production centers, identification of boars with decreased fertilization ability (subfertility) is a newborn necessity. This investigation is a sequential study to determine the kinetics of surface carbohydrates turnover during in vitro capacitation and AR in fertile and subfertile boar sperm. Flow cytometry determinations of the binding of three FITC-labeled lectins were assessed. WGA binding was significantly lower in fresh, capacitated, and acrosome-reacted sperm of subfertile boars than in fertile boars. Con-A binding was not significantly different in fresh sperm of fertile and subfertile boars. However. Con-A labeling in capacitated, and acrosome-reacted sperm differed significantly in both groups. UEA binding increased only in capacitated sperm of subfertile boars. These findings could be used as indicators of capacitation and AR and may also be a good indicator of sperm fertilizing ability in boars.     

Accurate sperm morphology assessment predicts sperm function

Sperm morphology has been associated with in vitro as well as in vivo fertilisation. The study aimed to evaluate the possible relation between the percentage of spermatozoa with normal morphology and the following sperm functional assays: (i) zona-induced acrosome reaction (ZIAR); (ii) DNA integrity; (iii) chromatin condensation; (iv) sperm apoptosis; and (v) fertilisation rates. Regression analysis was employed to calculate the association between morphology and different functional tests. Normal sperm morphology correlated significantly with the percentages of live acrosome-reacted spermatozoa in the ZIAR (r = 0.518; P < 0.0001; n = 92), DNA integrity (r = -0.515; P = 0.0018; n = 34), CMA(3) -positive spermatozoa (r = -0.745; P < 0.0001; n = 92), sperm apoptosis (r = -0.395; P = 0.0206; n = 34) and necrosis (r = -0.545; P = 0.0009; n = 34). Negative correlations existed between for the acrosome reaction, and DNA integrity, while negative associations were recorded with the percentages of CMA(3) -positive spermatozoa, apoptotic and necrotic spermatozoa. Sperm morphology is related to sperm dysfunction such as poor chromatin condensation, acrosome reaction and DNA integrity. Negative and significant correlations existed between normal sperm morphology and chromatin condensation, the percentage of spermatozoa with abnormal DNA and spermatozoa with apoptotic activity. The authors do not regard sperm morphology as the only test for the diagnosis of male fertility, but sperm morphology can serve as a valuable indicator of underlying dysfunction.     

Evaluation of the effect of cooling and of the addition of collagenase on llama sperm DNA using toluidine blue

The effect cryopreservation has on sperm chromatin condensation has been studied in many species but not in South American camelids. The objectives of this study were to evaluate with toluidine blue (TB) the effects of cooling and of adding collagenase on llama sperm DNA condensation. The optimum incubation time (30 s, 1.5 and 3 min) with a reducing agent (dithiothreitol) was also determined. When comparing cooled samples with the raw ejaculate, a significant increase in sperm showing a high degree of decondensation (TB positive) was observed (P = 0.005). A positive correlation was observed, both in raw and cooled semen, between sperm head morphological abnormalities observed in TB-stained cells and TB-positive sperm (highly decondensed DNA), but not with TB-intermediate spermatozoa (moderately decondensed DNA). No significant differences (P > 0.05) were observed in samples incubated with or without 0.1% collagenase. In cooled semen, but not in raw, a significant increase (P = 0.000) in reacted sperm (TB positive) was observed using 3-min incubation with 1% dithiothreitol (DTT). To conclude, cooling would seem to produce an increase in llama sperm chromatin decondensation. Also, 0.1% collagenase in H-TALP-BSA could be added to raw semen to aid its manipulation as it would not seem to increase DNA decondensation.     

Comparative analysis of tests used to assess sperm chromatin integrity and DNA fragmentation

Male infertility has a complex etiology, and many times, the cause is unknown. While routine semen analysis provides an overview of basic semen parameters, such as sperm concentration, motility, viability and morphology, a significant overlap of these parameters has been reported in fertile and infertile men. Moreover, conventional semen parameters do not reveal the cellular or molecular mechanisms of sperm dysfunctions leading to infertility. Therefore, sperm functional parameters, including sperm chromatin integrity, are evaluated to provide information on subtle sperm defects that are not routinely identified. Incomplete or defective sperm chromatin condensation increases the susceptibility of the sperm DNA to oxidative damage or other factors. To evaluate sperm chromatin integrity, different methods with varying degrees of diagnostic and prognostic capabilities are available. Among these assays, SCSA, TUNEL and SCD assays are most commonly used. While these assays rather evaluate the DNA directly for damages, the aniline blue and chromomycin A3 stains test for the quality of chromatin condensation. Thus, this review discusses and compares different methods used to evaluate sperm chromatin integrity and condensation, and their inclusion in the routine evaluation of the male infertility.     

Relationship between chromatin condensation, DNA integrity and quality of ejaculated spermatozoa from infertile men

Normal chromatin condensation is important for sperm fertilising ability. However, routine semen analysis does not identify defects in sperm chromatin structure. This study aimed to investigate the condensation of chromatin and DNA integrity in spermatozoa of infertile men and deduce the relationship with sperm quality, as measured by conventional semen parameters. Semen analysis was carried out to assess sperm quality according to World Health Organization criteria. The remaining aliquot of each sample was processed for transmission electron microscopy, chromomycin A3 (CMA3) and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assays. The ultrastructural analysis of spermatozoa from infertile men showed heterogeneity in sperm nuclear morphology. Some spermatozoa displayed a round nucleus with incomplete chromatin condensation. Immunoreactivity with antitransitional protein and antiprotamine antibodies indicated nuclear maturation defects in the spermatozoa of infertile men. Spearman's correlation analysis indicated a positive correlation between the percentages of CMA3- and TUNEL-positive spermatozoa. In addition, these two parameters were negatively correlated with sperm concentration, motility and normal morphology. This study demonstrated that men with morphologically normal spermatozoa of <30% have greater degree of protamine deficiency and DNA damage than men with morphologically normal spermatozoa of >30%. Evaluation of chromatin integrity appears to be a useful tool for assessing male fertility.     

Kruger strict morphology and post-thaw progressive motility in cryopreserved human spermatozoa

The purpose of this prospective study was to evaluate Kruger strict morphology and conventional semen analysis in predicting cryosurvival and the progressive motility recovery rate of frozen spermatozoa. Our study included 56 semen samples with >10 million spermatozoa per ejaculate. The main outcome measures were conventional semen analysis, strict morphology analysis by the Kruger method, cryosurvival rate and post-thaw sperm motility. A significant reduction in sperm motility after cryopreservation was demonstrated. The freeze-thawing process caused a 66% reduction in rapid progressive motile spermatozoa, a 45% reduction in slow progressive motile spermatozoa and a 2% reduction in nonprogressive motile spermatozoa. The cryosurvival and progressive motility recovery rates were not correlated with parameters of conventional semen analysis, such as sperm concentration, motility, WHO morphology and total motile count, but the progressive motility recovery rate was significantly correlated with the percentage of spermatozoa exhibiting Kruger normal morphology (P = 0.028). The recovery rate of rapidly progressive motility was profoundly decreased compared with slow progressive motility following the frozen-thaw procedure of semen. Kruger strict morphology assessment was a better predictor of the progressive motility recovery rate following the freezing-thaw procedure than parameters of conventional semen analysis.     

Improvement in semen quality and sperm fertilizing ability after filtration through the L4 membrane: comparison of results with swim up technique.

The L4 filtration membrane was used at our laboratory on 30 semen specimens (19 from suspected subfertile patients and 11 from fertile donors) to assess changes in semen quality after routine sperm washing and swim up versus filtration through the L4 membrane. Of these 30 samples 17 (11 suspected subfertile patients and 6 fertile donors) were chosen to evaluate differences in sperm fertilizing ability between the 2 sperm preparation methods. Liquified specimens were analyzed on a Cell Soft semen analyzer. Semen specimens were divided equally, and 1 aliquot was processed by sperm washing and the swim up technique and the other by filtration. In all subjects the results of semen analysis and the hamster-egg penetration test showed significant improvement in sperm quality and fertilizing ability after filtration through the L4 membrane. Sperm motility was 58% after the swim up technique compared to 79% after filtration (p less than 0.0001) and velocity was 47 mu. per second after the swim up method compared to 52 mu. per second after filtration (p less than 0.048). Penetration rate and penetration index also showed a significant increase after filtration over values for the swim up method. Our results demonstrate that sperm filtration through the L4 membrane provides results superior to those of the traditional swim up technique. Due to significant savings in time associated with its use the L4 membrane can be used during sperm processing for intrauterine insemination and in vitro fertilization procedures.