Predictive value of sperm chromatin condensation (aniline blue staining) in the assessment of male fertility

A case control study was carried out to determine the value of sperm chromatin condensation in the assessment of male fertility. A total of 165 semen samples from 90 patients (cases) and 75 healthy donors (control) were examined for chromatin condensation (aniline blue staining), as well as conventional sperm parameters, notably sperm morphology, sperm count, and progressive motility. Whereas only 55 +/- 12.0% of the samples from the infertile patients were unstained by aniline blue (chromatin condensed), 78 +/- 19.0% of the samples in the control group did not take up the stain (chromatin condensed). A significant difference (p < .001) was observed between the two groups. Similarly, the difference between the mean percentage of morphologically normal spermatozoa for the infertile patients (12.1 +/- 1.2%) and the control (23.9 +/- 1.9%) was very significant (p < .001). In addition, only 50 out of the 90 patients (55.5%) had a normal sperm count, whereas all the 75 (100%) were normal in the control group. By comparing between the two groups a significant difference (p < .001) was also observed. Furthermore, a significant difference (p < .001) was also found between the cases and the control with regard to the percentage of spermatozoa illustrating linear progressive motility (40 +/- 9.7% vs. 70 +/- 12.3%). However, no correlation was found between sperm chromatin condensation and morphology, count, and motility. This study suggests that chromatin condensation constitutes a valuable parameter in the assessment of male fertility, completely independent of conventional sperm parameters. Consequently, the inclusion of chromatin condensation to routine laboratory investigations of semen prior to assisted reproduction is strongly recommended.     

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.     

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     

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.     

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.     

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.     

Application of the hypo-osmotic swelling test to spermatozoa prepared by swim-up and discontinuous Percoll separation

The quality of spermatozoa prepared by washing and swim-up or by discontinuous Percoll centrifugation, was compared by applying the hypo-osmotic swelling (HOS) test to semen samples from 116 men of infertile couples. The HOS test performed on 95 normal semen samples showed that the percentage of swollen spermatozoa separated by both techniques was significantly higher than in the initial ejaculate ( p <0.001). The percentage of HOS-positive spermatozoa separated by the Percoll gradient technique was significantly higher than that separated by the swim-up technique ( p <0.001). On the contrary, in 21 abnormal semen samples, there was no significant difference in the percentage of spermatozoa which were positive in the HOS test between the Percoll gradient and the swim-up technique ( p =0.44). It is suggested that the Percoll gradient technique appears to be preferable to the swim-up technique when semen parameters are normal, but there is no significant difference between these two techniques in abnormal semen.     

Correlation between defective motility (asthenospermia) and ATP reactivation of demembranated human spermatozoa

Human spermatozoa treated with 0.05% Triton X-100 to remove the cell membranes became immotile but flagellar movement was reinitiated by addition of 0.06 to 1 mM adenosine triphosphate (ATP). The percentage of spermatozoa showing flagellar movement 2 minutes after addition of 1 mM ATP from men with idiopathic asthenospermia (mean +/- SD, 34 +/- 15%), oligozoospermia (17 +/- 21%), sperm autoimmunity (17 +/- 12%), vasoepididymostomy (20 +/- 22%), or idiopathic zero motility (0%) was significantly lower than with spermatozoa from normal men (54 +/- 12%). The percentage of reactivated spermatozoa was correlated with the proportion of live cells (Eosin Y stain, r = 0.602, P less than 0.001), percentage of motile cells (r = 0.761, P less than 0.001), and motility index (r = 0.759, P less than 0.001) in the same semen samples. When expressed relative to initial sperm motility, the proportion reactivated was similar for idiopathic asthenospermia (85%) and normospermia (82%). Thus, failure to produce ATP does not appear to be a frequent cause of low sperm motility in man.     

Assessment of human sperm morphology by strict criteria: comparison of wet preparation versus stained with the modified Diff-Quik method

Routine semen examination remains an important tool for the diagnosis and treatment in human subfertility. Of all semen parameters, sperm morphology seems to be one of the most powerful indicators of a man's fertilizing potential in vitro and in vivo. Lack of standardization of sperm morphology assessments remains the main reason for the usefulness of this parameter. The aim of this study was to analyze the agreement between the wet-stained preparations versus those stained with modified Diff-Quik for sperm morphology. A total of 100 unselected semen samples from infertile couples were analyzed. Sperm morphology was evaluated with unstained specimens and following modified Diff-Quik staining according to the strict (Kruger classification) criteria by two different examiners (intralaboratory blind assessment). Mean percentages of morphologically normal spermatozoa were identical on wet and stained preparation slides (4.79 vs. 4.61, p >.05). Wide divergence of results was found with respect to the percentage of sperm with head and midpiece defects with the two different preparations (p >.001). The percentage of sperm tail defects was similar in both methods (p >.05). Simple linear regression analysis between the two methods revealed very good correlation for the morphologically normal spermatozoa (r =.83), but poor correlation for the sperm head, midpiece, and tail defects (r =.25,.25, and.28, respectively). Wet preparation is suitable only for the morphologically normal spermatozoa, but to determine the percentage of the defective spermatozoa, staining the smear is recommended.     

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.     

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.     

Noninsulin-dependent diabetes mellitus: effects on sperm morphological and functional characteristics, nuclear DNA integrity and outcome of assisted reproductive technique

The aim of the study was to compare the semen characteristics and nuclear DNA fragmentation in spermatozoa of diabetic and nondiabetic men undergoing assisted reproduction and correlate them with pregnancy outcome. Semen characteristics and nuclear DNA fragmentation were analysed using computer-aided semen analysis system and sperm chromatin dispersion assay (SCD), respectively. Spermatozoa from diabetic patients showed significantly lower progressive (Type A) motility (14.64 ± 9.60 versus 17.99 ± 11.51, P < 0.02) and increased nuclear DNA fragmentation (37.05 ± 12.68 versus 21.03 ± 10.13, P < 0.001). Furthermore, a positive correlation was observed in diabetic patients in terms of blastocyst formation rate (38.13% versus 55.46%, P < 0.001), pregnancy rate (28.57% versus 46.34%, P < 0.001) and miscarriage rate (50.0% versus 24.56%, P < 0.001). The higher percentage of sperm DNA damage because of oxidative stress seen in diabetic patients may be responsible for the poor embryonic development and pregnancy outcome in these individuals.     

Correlation Between Defects in Chromatin Condensation of Human Spermatozoa Stained by Aniline Blue and Semen Characteristics

Human sperm heads which present disturbances of chromatin condensation are stained by acidic aniline, blue. To determine whether the proportion of unstained heads, i.e. with well condensed chromatin, can be considered as an index of sperm quality, a study was undertaken in 157 men during an infertility evaluation. In addition to the usual sperm characteristics, the percentages of unstained heads and of morphologically normal and abnormal forms were concomitantly evaluated. In a total of 15760 spermatozoa, the percentage of unstained heads was much higher in the population of morphologically normal forms than in that of abnormal forms (79.1% and 49.4% respectively, p less than 10(-9]. Among spermatozoa with structural abnormalities, it was much higher in cells with a single anomaly than in those with associated anomalies (53.9% and 40.6% respectively, p less than 10(-9]. When morphology was taken into account, only vitality was found to vary significantly with the percentage of unstained heads.     

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.     

Fast track recovery of elderly coronary bypass surgery patients.

BACKGROUND: To ascertain whether early extubation and fast-track treatment protocols are feasible in elderly patients, we analyzed 487 consecutive patients who had isolated coronary artery bypass grafting between January 1995 and June 1997, constituting the experience of a single surgeon. METHODS: Management consistently applied to all patients emphasized early extubation protocol, tepid cardioplegia and normothermic bypass to reduce pump times, early mobilization and chest tube removal, and protocol treatment of atrial fibrillation. Elderly patients at least 70 years old (n = 176, mean age 75 years) were compared with younger patients (n = 311, mean age 58 years). RESULTS: The hospital mortality rate was 0.8% (4 of 487 patients), and there was no difference in the operative mortality rate of the older cohort versus the younger cohort (0.6% versus 0.9%; p > 0.05). Older patients had a higher incidence of peripheral vascular disease, congestive heart failure, prior strokes, renal failure, and cerebrovascular disease (p < 0.05). Early extubation was achieved in 71% of younger patients versus 57% of older cohort (95% confidence interval, 14%+/-9%; p = 0.002). Older patients had significantly higher incidence of postoperative atrial fibrillation (27% versus 14%; 95% CI, 13%+/-7%; p < 0.001), a factor responsible for shorter length of stay among younger patients (5.6+/-2.8 days versus 7.2+/-3.7 days; 95% CI, 1.6+/-0.3 days; p < 0.001). Nonetheless discharge before the fifth postoperative day was achieved in 34% of the elderly patients. CONCLUSIONS: Although elderly patients have a higher acuity of illness, critical pathways for accelerated discharge are safe and feasible in most elderly patients.     

Association between sperm cell chromatin condensation, morphology based on strict criteria, and fertilization, cleavage and pregnancy rates in an IVF program

Summary. In this study, a total of 95 ejaculates from infertile patients were investigated morphologically according to Kruger's strict criteria and 78 of the 95 ejaculates were stained for chromatin condensation with acidic aniline blue. Patients were divided into two groups based on the percentage of morphologically normal spermatozoa as follows: Men with normal sperm morphology <14% (Group I), and men with normal morphology >14% (Group 2). The relationship between percentage of normal sperm morphology and fertilization, cleavage and pregnancy rate was analysed. The rates were 33.7%, 57.1% and 0.0% respectively, in the first group. The corresponding values for the second group were 76.1%, 68.2% and 32.1%. The fertilization and pregnancy rates correlate significantly with morphologically normal spermatozoa.
In regard to the percentage of morphologically normal spermatozoa stained with aniline blue, patients were divided into two groups: patients with 0–20% stained spermatozoa (Group I) and those with >20% (Group 2). Fertilization and pregnancy rates were higher in the first group than in the second group (79.9%, 52.8% vs. 58.8%, 29.5%).
The results demonstrate that chromatin condensation visualized by aniline blue staining is a good predictor for IVF outcome and should be considered besides morphology by sperm assessment for patients undergoing IVF treatment.—     

Extra-epididymal spermatozoa express nuclear abnormalities

Extra-epididymal spermatozoa account for approximately a third of all spermatozoa found in the normal human ejaculate. Whilst remaining outside of the testes at core body temperature, the functional competence of spermatozoa, including cell motility and fertilizing capacity, diminishes. By examining spermatozoa found in the seminal fluid of recently vasectomized men, this study has investigated the nuclear changes that occur in spermatozoa whilst persisting in sites distal to the epididymis. Spectral recordings of spermatozoa stained with the nucleic acid dye, toluidine blue and the sperm chromatin structure assay (SCSA) were performed. Toluidine blue staining of human sperm DNA is an effective predictor of abnormal protamine disulphide crosslinking and chromatin condensation. Using flow cytometry, the SCSA determines the sensitivity of sperm DNA to acid-induced denaturation, providing a measure of chromatin and DNA damage. Abnormal protamine disulphide crosslinking and chromatin condensation was significantly higher in spermatozoa from patients after vasectomy when compared to normozoospermic controls (p < 0.01). Additionally, spermatozoa from vasectomized donors were significantly more sensitive to acid-induced denaturation than were normozoospermic donors (p < 0.05). The results indicate that spermatozoa surviving in extra-epididymal sites are more likely to possess DNA and chromatin abnormalities than those present in the testes and epididymis. These changes may partly explain the depletion of cell viability and fertilizing capacity of extra-epididymal spermatozoa which has been reported previously.     

Immunomagnetic removal of cryo-damaged human spermatozoa

AIM: To estimate the dissipation of mitochondrial transmembrane potential (mTMP, DeltaPsim) and activation of sperm caspases (aCP) as signs of apoptosis in human spermatozoa during cryopreservation and to evaluate the efficiency of immunomagnetic cell separation (MACS) of these spermatozoa via annexin V-binding. METHODS: The mTMP and aCP in fresh and cryopreserved spermatozoa were detected by fluorescence microscopy and by Western blots. The sperm suspensions were divided into two sperm fractions (with intact and deteriorated membranes) by magnetic cell separation (MiniMACS, Miltenyi Biotec, Bergisch Gladbach, Germany) in dependence on their binding to superparamagnetic annexin V-microbeads (AN-MB). RESULTS: The cryopreservation decreased the portion of spermatozoa with intact mTMP from 80.1 % +/- 7.2 % to 53.5 % +/- 13.1 % and increased the spermatozoa with activated pan-caspases (aCP) from 21.8 % +/- 2.6 % to 47.7 % +/- 5.8 % (n=10; mean +/- SEM; P <0.01). The activation of caspases 1, 3, 8, and 9 in the cryopreserved spermatozoa was confirmed by Western blots (n=22). MACS reduced significantly the percentage of cryopreserved spermatozoa with dissipated mTMP to 8.1 +/- 3.9 (P <0.01) and also those with aCP to 9.3 % +/- 2.2 %. Western blot analyses confirmed the increase of the activated caspase3, 9, and 8 in the AN-MB-positive fraction (P <0.05) compared with the AN-MB-negative fraction. The MACS separation effect was confirmed by anti-annexin V-antibodies. There was no significant influence of the separation column and the magnetic field on the sperm functions. CONCLUSION: The cryopreservation impaired the mTMP and enhanced the activation status of caspases in human spermatozoa. The immunomagnetic sperm separation via binding of AN-MB could deplete low quality spermatozoa from cryopreserved semen samples.     

Influence of extended incubation time on Human sperm chromatin condensation,sperm DNA strand breaks and their effect on fertilisation rate

The purpose of this study was to determine influence of extended incubation time on sperm chromatin condensation and DNA strand breaks and their effect on fertilisation rate. Forty couples undergoing ICSI therapy were included. Semen was prepared by PureSperm gradient centrifugation and divided into two parts. The first part (G1) was used immediately for ICSI, whereas the second part (G2) was kept in the incubator at 37°C, 5% and 90% Humidity for 5 hr, and thereafter, the capacitated spermatozoa were used for ICSI. The TUNEL test and chromomycin CMA3 were used to evaluate the DNA strand breaks and chromatin condensation respectively. The percentage of condensed chromatin was 73.92 ± 12.70 in the group 1 and 81.13 ± 10.31% in group 2 (p = .001). However, the double‐strand breaks were 11.15 ± 8.67% in G.1 and 16.30 ± 11.12% in G.2. (p = .001). Fertilisation rate in the (Group 1) was 62.45% and 69.17% in (Group 2). There was a positive correlation between condensed chromatin and fertilisation rate (r = 0.846, p = .001) and a negative correlation with DNA double‐strand breaks (r = ?0.802; p = .001). In conclusion, the prolonged sperm incubation (5 hr) leads to a higher chromatin condensation and to a significantly increased number of DNA strands double breaks with no influence on fertilisation rates.