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.     

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.     

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.     

Sperm chromatin structure assay results after swim-up are related only to embryo quality but not to fertilization and pregnancy rates following IVF

The aim of this study was to investigate whether the sperm chromatin structure assay (SCSA) results after swim-up are related to fertilization rates, embryo quality and pregnancy rates following in vitro fertilization (IVF). A total of 223 couples undergoing IVF in our hospital from October 2008 to September 2009 were included in this study. Data on the IVF process and sperm chromatin structure assay results were collected. Fertilization rate, embryo quality and IVF success rates of different DNA fragmentation index (DFI) subgroups and high DNA stainability (HDS) subgroups were compared. There were no significant differences in fertilization rate, clinical pregnancy or delivery rates between the DFI and HDS subgroups. However, the group with abnormal DFI had a lower good embryo rate. So, we concluded that the SCSA variables, either DFI or HDS after swim-up preparation, were not valuable in predicting fertilization failure or pregnancy rate, but an abnormal DFI meant a lower good embryo rate following IVF.     

Effect of different culture techniques used to induce capacitation on the chromatin stability of human sperm

To validate an in-vitro bioassay for assessing chromatin stability of human sperm, 38 semen samples from infertile men were studied using sodium dodecyl sulphate, an anionic detergent which disorganizes only the cytoplasmic membrane. Assay sensitivity was 50 sperm, whilst the within- and between-assay variation, and the between-observer variation were found to be within the accepted range for this type of bioassay. The influence of different in-vitro treatments currently used in some clinical assisted fertilization programmes was evaluated: a destabilizing effect occurred in Grade I (stable) and Grade III (swollen) sperm. In the former, all treatments reduced stable sperm; in the latter, a significant (P less than 0.001) increase in swollen sperm was shown with two methods that used Ham's F-10 as culture medium. Different chromatin patterns found in the treated sperm suggest the possibility that the recovered samples could be modified compared to their status at the time of isolation.     

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.     

Defective sperm decondensation: a cause for fertilization failure

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

精子染色质完整性对IVF/ICSI临床结局的预测价值

目的:通过分析精子染色质完整性与体外受精胚胎移植(IVF-ET)、卵细胞胞质内单精子注射(ICSI)结局之间的关系,探讨精子染色质完整性检测在辅助生殖技术(ART)中的预测价值。方法:采用精子染色质结构分析试验(SCSA)方法检测187个ART周期中精子的染色质完整性,以精子DNA损伤指数(DFI)为参数,分为高DFI组(DFI≥30%)和低DFI组(DFI<30%),两组中根据采用不同的体外受精方式分为IVF亚组和ICSI亚组。分别比较高DFI组、低DFI组中IVF亚组和ICSI亚组间受精率、卵裂率、D2天胚胎质量、D3天胚胎质量、临床妊娠率差异性。结果:在高DFI组中,行ICSI治疗的夫妇临床妊娠率显著高于行IVF治疗的夫妇,具有统计学意义;在行IVF治疗的夫妇中,比较高DFI组与低DFI组的各临床结局,未见统计学差异;在行ICSI夫妇中,比较高DFI组与低DFI组的各临床结局,未见统计学差异。结论:精子染色质完整性影响辅助生育技术的结局,在选择辅助生殖技术方案时,可作为常规精液检查的一个补充。     

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.     

Prognostic value of an automated sperm analysis in IVF or insemination therapy

Summary. During the course of sterility treatment semenograms of 271 IVF and 316 insemination patients were carried out by two automated semen analysers. The parameters of these analyses were correlated to pregnancies resulting from the treatment. Semen samples were analysed in the ejaculate and after swim-up preparation. In addition, the swim-up suspension of IVF patients was measured again 18 h after sperm preparation. Patients were divided into three groups: (1) couples who achieved a pregnancy, (2) couples who did not achieve a pregnancy, and (3) IVF patients with no fertilization of the oocytes. Because of large standard deviations in the quality of ejaculates, the results in the IVF group show no significant differences in the semen parameters of husbands of pregnant and non-pregnant women. In contrast husbands of women with no fertilization have a significantly lower sperm motility. After swim-up preparation these differences no longer occur. A further measurement, taken 18 h later, reveals that there are no differences in the sperm parameters between the pregnant and non-pregnant group. However, the semen quality in the group with no fertilization is significantly reduced. The results of the insemination patients are similar to those of the IVF group. Thus, the results from automated sperm analysers cannot replace either the microscopic or biochemical analysis of an ejaculate and, moreover, cannot be used as prognosis for the fertilization capacity of sperms or a following pregnancy.     

Clinical significance of the Acridine Orange test performed as a routine examination: comparison with the CASA estimates and strict criteria

The aim of the present study is to clarify the clinical significance of the Acridine Orange (AO) test (a sperm function test) as a routine examination by investigating the relationships between chromatin decondensation assessed by the AO test and routine semen analysis. Totally 543 semen samples were obtained from 286 infertile men. The AO test was performed, and spermatozoa displaying green fluorescence were considered as mature. The threshold of green AO fluorescence as 50% was adopted and the values <50% were considered as positive in the test. Computer-aided sperm analysis (CASA) and strict criteria were used for routine semen analysis. Twenty-two (4.05%) of 543 semen samples were positive in the AO test. In 20 semen samples obtained from men with severe male infertility treated by intracytoplasmic sperm injections (ICSI), the positive rate was 30.0%, which was significantly higher than that (2.63%) treated by conventional in vitro fertilization (IVF) (p=0.01). As for the basic sperm parameters, there were significant correlations between the AO test and sperm motility (p<0.001), and between the AO test and normal sperm morphology (p=0.02). However, there was no relationship between the AO test and sperm concentration (p=0.585). Sperm motion parameters assessed by CASA, including amplitude of lateral head displacement (ALH), curvilinear velocity (VCL) and rapid sperm movement (Rapid), were significantly correlated with the AO test. The information obtained using the AO test was indicated to be useful in planning treatment strategy for infertile couples.     

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.     

Sperm count and motility influence the results of human fertilization in vitro

In order to select sperm characteristics that can predict the outcome of in-vitro fertilization-embryo transfer (IVF-ET), semen samples delivered in conjunction with this treatment were studied carefully. We have analysed these data retrospectively in relation to the outcome of treatment. Ninety-one couples were treated for tubal infertility by IVF-ET. Fifteen women became pregnant. Sperm were isolated from semen using a swim-up technique and in most cases 40-80 x 10(3) (range 20-120 x 10(3)) motile sperm per ovum were used for insemination. The couples were divided into three groups: group A who achieved pregnancies, group B who achieved cleaved ova but no pregnancies, and group C who achieved no ova that were cleaved 48 h after oocyte recovery. Comparisons between these groups showed that some characteristics of the native semen samples and the swim-up preparations were significantly different: the sperm concentration (P = 0.001) and total sperm count (P = 0.003) in the native sample, the number of sperm recovered during 30 min of swim-up (P = 0.001), and the specific progressive motility of sperm in the swim-up preparation, both at the time of insemination and on each day, up to 5 days thereafter (P = 0.002-0.028). No pregnancy was achieved with a sperm concentration below 26 x 10(6) ml-1 in the native sample. Some of the sperm characteristics studied in this paper may be of value in the pretreatment evaluation for IVF treatment.     

优选前后精子顶体酶活性与IVF受精率相关性的研究

目的探讨优选处理前和处理后精子顶体酶活性的变化,及与体外受精(IVF)受精率的相关性。方法采用分光光度比色法,对接受IVF治疗的53例不育夫妇男方精液,分别测定优选处理前和处理后精子顶体酶活性,分析其与IVF受精率的相关性。结果优选处理后精子顶体酶活性与优选前比较有显著性差异(P<0.05);达到常规IVF标准并选择常规IVF治疗者,优选后的精子顶体酶活性与IVF受精率有相关性,精子顶体酶活性降低与IVF受精率降低有关。结论精子顶体酶活性与IVF受精率有相关性,并且通过精子顶体酶活性可以预测IVF受精率。     

Does computerized image analysis of sperm movement enhance the predictive value of semen analysis for in-vitro fertilization results?

The influence of semen quality on fertilization rates in an in-vitro fertilization (IVF) programme was studied by analysing both conventional semen parameters and computerized movement characteristics. The study was based on 407 inseminated oocytes which were obtained from 50 patients in 113 laparoscopies. Sperm concentration did not correlate strongly with the fertilization rate. Sperm motility and morphology were the most meaningful parameters in predicting fertilization success. A drop in fertilization rate was found when sperm motility or normal morphology were below 40%. Sperm velocity measured in semen was the only sperm movement parameter which correlated with the fertilization rate, albeit weakly. The latter was reduced when average sperm velocity in semen was less than 50 microns/sec. Conventional semen parameters seem to be more predictive of the fertilizing potential of an ejaculate than movement characteristics obtained by computerized image analysis.     

Separation of sperm cells by sedimentation technique is not suitable for in vitro fertilization purposes

Summary. Two methods of sperm preparation for in vitro fertilization were compared: the swim-up technique vs. the migration-sedimentation technique. The study comprised fresh semen samples obtained from 25 couples treated in the In Vitro Fertilization Unit. Oocytes aspirated in a single cycle were divided into two groups, each inseminated by sperm prepared by one of these techniques. Motility, degree of motility, and normal morphology were improved by both methods. The improvement was greater when the migration-sedimentation technique was applied. However, fertilization rate was significantly higher after the swim-up technique. In order to clarify this contradiction, an additional group of 26 semen samples was divided and then prepared by the swim-up or migration-sedimentation techniques. Sperm quality was examined up to 72 h after separation. Compared with the swim-up technique, sperm characteristics were better after separation by the migration-sedimentation technique. However, this difference abated after 24 h. The better results of the swim-up technique in the 'survival experiment' may explain its improved performance in in vitro fertilization, despite lower separation capacity. Thus, the migration-sedimentation technique is not recommended for sperm preparation in in vitro fertilization.
Migration-sedimentation technique (MST)—     

Effect of freeze–thawing procedure on chromatin stability, morphological alteration and membrane integrity of human spermatozoa in fertile and subfertile men

Cryopreservation is known to impair sperm motility and decrease the fertilization rate by detrimental effects on acrosomal structure and acrosin activity. However, the consequences of cryopreservation on the integrity of the sperm nucleus, chromatin stability and centrosome are less clear. The present study was designed to determine the effect of the freeze-thawing procedure on chromatin condensation (aniline blue staining) and the morphology (strict criteria) and membrane integrity of human spermatozoa. The structural and functional characteristics of the sperm plasma membrane were measured by the eosin-test and hypo-osmotic swelling test which were done separately. Sperm cryopreservation was performed on semen samples from two groups of men classified as fertile (n = 20) and subfertile (n = 72), based on their reproductive history and semen analysis according to WHO guidelines. The mean percentage of condensed chromatin, morphologically normal spermatozoa and membrane integrity in all semen samples investigated (n = 92) decreased significantly (p = 0.0001) after freeze-thawing, in comparison to the value observed prior to freezing. By comparing the semen samples between fertile and subfertile patients, significantly (p = 0.0009) greater damage was demonstrated in the subfertile than in the fertile group. Furthermore, no significant difference was observed between the two groups with regard to the morphological alteration and structural as well as functional damage of the sperm membrane. In conclusion, the freeze-thawing procedure significantly affects chromatin structure and sperm morphology, especially in the head and the tail regions, and this may explain the lower fertilization rate and IVF/ICSI outcome when frozen-thawed spermatozoa are used. In addition, this study demonstrates that chromatin condensation is a sensitive parameter for the evaluation of cryodamage of semen samples from fertile and subfertile patients, though subfertile patients with very poor semen characteristics have yet to be studied. It is therefore recommended that chromatin condensation be used as an additional parameter for the assessment of sperm quality after freeze-thawing.     

精子冻存技术及上游法精子处理技术对精子DNA完整性的影响

目的通过对冷冻前后及精子处理前后精子DNA完整性的比较,探讨冷冻技术、冷冻时间及上游法精子处理技术对精子DNA完整性的影响。方法（1）精液常规检测正常的患者30例,手淫法取精,精液液化混匀后分4份,分别用于精子染色质扩散（SCD）实验检测精子DNA完整性、上游法处理精液、以及两组冻存实验（不加保护剂直接冻存的为冻存1组;添加蛋黄葡萄糖保护剂冻存的为冻存2组）;（2）上游法处理后的精液一部分用于检测精子动力和形态,一部分用于精子DNA完整性检测;（3）冻存1组分别于冻存第7天和第90天解冻,SCD实验检测精子DNA完整性;（4）冻存2组分别于第7天和第90天解冻,0．1ml用于精子DNA完整性检测,剩余精液应用上游法处理,检测上游处理前后精子DNA的完整性。结果（1）冻存1组中,冻存90d后解冻的精子DNA损伤率[（25．6土7．3）％]显著高于冻存7d者[（22．4±7．4）％]（P〈0．05）,且均显著高于新鲜精液的精子DNA损伤率[（20．6±7．3）％]（P〈O．05）;冻存2组中,冻存90d后精子DNA损伤率显著高于冻存7d者[（25．9±7．2）％VS．（23．6土7．8）％]（P〈O．05）,且均显著高于新鲜精液（P〈0．05）;而冻存7d和90d后,两个冻存组间比较,精子DNA损伤率均无显著差异（P〉0．05）。（2）新鲜精液经上游法处理后,精子DNA损伤率由处理前的（20．6±7．3）％降为（6．4±2．5）％（P〈O．05）;冻存2组中精液冻存7d和90d后复苏上游法处理后,精子DNA损伤率较未经上游法处理者均显著降低[分别为（9．38±2．8）％VS．（23．6±7．8）％和（9．7±2．6）％VS．（25．9±7．2）％]（P％0．05）。结论冻存对精子DNA有损伤,冻存时间对于精子DNA完整性有影响。不添加保护剂直接冻存和添加保护剂对精子DNA完整性的影响无显著差异。不论是新鲜精液还是冻存复苏精液,上游法处理并不会增加精子DNA的损伤,且有利于筛选出具有更好DNA完整性的精子。     

A simple method to detect sperm chromatin abnormalities: cytochemical mechanism and possible value in predicting semen quality in assisted reproductive procedures

Protamine crosslinking by disulphide (-SS-) bonds is the main factor responsible for the stability of chromatin structure in mammalian spermatozoa. Sperm chromatin containing arginine/cysteine-rich protamines shows a deeply modified cytochemical reactivity (e.g. basophilia) when compared with somatic chromatin. After methanol or ethanol-acetic acid fixation and toluidine blue (TB) staining, most sperm heads in semen smears from human fertile donors exhibited a pale blue (orthochromatic) colour, while a few sperm heads exhibited violet-blue or violet (metachromatic) staining. Smears from infertile patients showed an increased amount of metachromatic sperm nuclei. After reduction of -SS- bonds by dithiothreitol, sperm heads from all smears were metachromatic, suggesting that DNA phosphates then become available for TB stacking. Micro- and macro-spectrophotometric studies confirmed the microscopic colour reaction of sperm nuclei. The ortho-/metachromatic ratio seems a useful parameter for evaluation of altered chromatin structure in spermatozoa cells. Taking into account the current interest in complementary staining tests for evaluation of semen quality, the metachromatic TB reaction represents a simple cytochemical approach for detecting sperm chromatin abnormalities based on differences in -SS- crosslinking.     

Comparative study on density gradients and swim-up preparation techniques utilizing neat and cryopreserved spermatozoa

AIM: To 1) compare post-wash and post-thaw parameters of sperm processed with PureSperm density gradient technique and swim-up method; and 2) test the efficacy of two commonly available density gradient media PureSperm and ISolate. METHODS: This prospective study used semen specimens from 22 patients. Specimens from nine patients were processed by both PureSperm density gradient and swim-up method. These specimens were then cryopreserved. Thirteen specimens were processed by both PureSperm (40 % and 80 %) and Isolate (50 % and 90 %) double density gradient techniques. The two fractions processed by both PureSperm and swim-up were analyzed for post-wash sperm characteristics. Post-thaw analysis was done after 24 hours. Sperm fractions obtained after processing with PureSperm and ISolate were compared for post-wash sperm characteristics and ROS levels. Results: Specimens prepared with PureSperm had significantly higher median total motile sperm counts (TMSC) (32.2 x 10(6) vs. 17.6 x 10(6)), recovery rates (69.2 % vs. 50.0 %), and longevity at 4 hours (83.0 % vs. 55.0 %) compared to specimen prepared by swim-up. Post-thaw specimens also had a higher recovery and longevity at 4 hours with PureSperm as compared to the swim-up. Semen specimens processed by PureSperm had significantly higher total sperm count, TMSC, and percentage recovery rates (30.0 % vs. 19.7 %) than ISolate. CONCLUSION: Semen quality is better preserved in fresh and cryopreserved semen prepared with PureSperm density gradient compared to swim-up. A significant enrichment of sperm is observed with PureSperm compared to ISolate. Higher recovery rates of mature motile sperm obtained after PureSperm sperm preparation may be beneficial for successful ART.