Comparison of chromatin assays for DNA fragmentation evaluation in human sperm

Sperm chromatin integrity is vital for successful pregnancy and transmission of genetic material to the offspring. We evaluated chromatin integrity in sperm from 60 infertile men and 7 fertile donors comparing the sperm chromatin structure assay (SCSA), TdT-mediated-dUTP nick end labeling (TUNEL), the sperm chromatin dispersion (SCD) test, and acridine orange staining technique (AOT). The TUNEL and SCD assays showed a strong relationship with the SCSA (r > .866; P < .001) for sperm DNA fragmentation, both in infertile men and donors of known fertility. AOT did not show any relationship with SCSA. The breakdown of the DNA fragmentation index (DFI) into 3 categories (< or =15%, >15%-<30%, and > or =30%) showed that the SCSA, TUNEL, and SCD test predict the same levels of DNA fragmentation. AOT consistently showed higher levels of DNA fragmentation for each DFI category. DNA fragmentation in sperm between infertile men and donor sperm was significantly different (P < .05) under SCSA (22.0 +/- 1.6 vs 11.8 +/- 1.4), TUNEL (19.5 +/- 1.3 vs 11.1 +/- 0.9) and SCD (20.4 +/- 1.3 vs 10.8 +/- 1.1), respectively. DNA fragmentation in sperm evaluated by AOT did not differ (P > .05) between infertile men (31.3 +/- 2.4) and donors (32.7 +/- 4.8). AOT showed extreme variations for sperm DNA fragmentation in semen from both infertile men and donors. The problems of indistinct colors, rapid fading, and the heterogeneous staining were also faced. In conclusion, SCSA, TUNEL, and SCD show similar predictive values for DNA fragmentation, and AOT shows variable and increased levels of DNA fragmentation, which makes it of questionable value in clinical practice.     

Flow cytometric measurement of sperm nuclear DNA fragmentation in infertile men with normal standard sperm parameters

BackgroundMale-factor infertility plays a role in approximately 50% of infertile couples. In at least 30% of cases, repeated standard semen analyses of the male partner of an infertile couple reveal normal results. When diagnostic work-up of the female partner is also normal, they are classified as idiopathic. The objective of this study was to evaluate the levels of sperm nuclear DNA fragmentation in a population of infertile men with normal standard semen parameters and to compare their results with those from men who had abnormal semen parameters, as well as with a control group of fertile men.MethodsSemen samples were obtained from 202 infertile men and 30 fertile donors. Standard semen analysis was performed according to the World Health Organization guidelines. Flow cytometry has been extensively used to study sperm DNA fragmentation and the results are expressed as the percentage of sperm DNA fragmentation index (DFI).ResultsOf the 202 patients, 48 (23.8%) had normal standard sperm parameters, while 154 (76.2%) had an abnormality in one or more of these parameters. DFI in infertile men with normal sperm parameters was significantly higher than in fertile donors (p = 0.03), but not significantly different from infertile men with abnormal sperm parameters (p = 0.10). There were statistically significant negative correlations between DFI and the percentage of motile sperm from infertile men with abnormal and normal semen parameters, but not in fertile donors (r = ?0.26, p = 0.001 and r = ?0.48, p = 0.0001, respectively).ConclusionSperm from infertile men with normal standard sperm parameters may have significant levels of DNA fragmentation that are comparable to levels in infertile men with abnormal sperm parameters. Sperm DNA fragmentation analysis is an independent test of sperm quality and has an important diagnostic value in the evaluation of male infertility.     

Relationship between seminal ascorbic acid and sperm DNA integrity in infertile men

Ascorbic acid has recently been reported to protect sperm DNA from the damage induced by exogenous oxidative stress in vitro. But, there is no report on seminal ascorbic acid and sperm DNA fragmentation in infertile men. In this study, we asked whether sperm DNA damage correlates with seminal ascorbic acid levels. Sperm DNA fragmentation index (DFI) was analysed in 75 men by flow cytometry after acridine orange staining. We also measured the levels of seminal plasma ascorbic acid and total antioxidant capacity. Abnormal sperm DNA integrity (DFI >or= 30%) was observed in 12% of the patients with normal semen parameters and in 52% of the patients with abnormal semen parameters. There were significant correlations between the level of DFI and conventional semen parameters including sperm count, motility and morphology (r = -0.29, -0.55 and -0.53 respectively; p < 0.05). Seminal ascorbic acid level was significantly lower in the patients with leucospermia than the patient with normal semen parameters. Interestingly, a significantly greater percentage of men with abnormal DFI were observed in the patients with low levels of seminal ascorbic acid compared with those with normal or high levels of ascorbic acid (59% vs. 33%, p < 0.05). Men with insufficient seminal ascorbic acid frequently have sperm DNA damage.     

精子DNA碎片指数和精子畸形率对ICSI临床结局的影响

目的:通过评估ICSI治疗前的精子畸形率(SMR)和精子DNA碎片指数(DFI),探讨精子DFI和SMR对卵胞浆内单精子注射(ICSI)助孕结局的影响。方法:共入组79对因少弱精子症实施第一周期ICSI治疗的不孕夫妇,在进入治疗周期前3⁶个月,评价精子浓度、前向运动精子百分率、SMR及DFI。主要观察SMR和DFI与ICSI结局参数的关系。结果:79例少弱精子症患者DFI正常51例,异常28例,异常组的DFI值明显升高(14.18%vs 41.47%);巧合的是,SMR正常组同样为51例,异常组28例,异常组的SMR值亦明显升高(87.88%vs98.46%)。按DFI正常(DFI≤25%)与异常(DFI>25%)分组,或按SMR正常(≤96%)与异常(>96%)分组,组间的双方年龄、女方BMI、获卵数、移植胚胎数等基本情况差异无统计学意义。DFI正常和异常组间,SMR正常和异常组间的受精卵子数、可移植胚胎数、早期流产率无显著差异;异常组生化妊娠率(43.5%vs 61.5%)和临床妊娠率(39.1%vs 56.4%)降低,但差异无统计学意义(P=0.19及0.10)。精子DFI与SMR呈显著正相关(r=0.231,P<0.05)。结论:精子DFI增高(>25%),与按严格标准检测的SMR增高(>96%)男性行ICSI治疗,生化妊娠率和临床妊娠率降低,但与正常者比较未发现有统计学差异,可能与样本量小有关,有必要深入研究。     

Analysis of the outcome of intracytoplasmic sperm injection using fresh or frozen sperm

Study Type – Diagnostic (retrospective cohort)
Level of Evidence 2b What’s known on the subject? and What does the study add? The results of ICSI using fresh or frozen sperm on the site of sperm retrieval remains controversial with respect to outcome. The results of this study showed no difference in outcome using ICSI either with respect to the site of retrieval or whether the sperm used was fresh or frozen. It also showed that the outcome of ICSI is not related to the underlying cause of the azoospermia.

OBJECTIVES

? To compare the outcome of first‐attempt intracytoplasmic sperm injection (ICSI) ICSI–embryo transfer (ET) cycles using frozen‐thawed testicular sperm (FTTS), fresh testicular sperm (FTS), frozen‐thawed epididymal sperm (FTES) and fresh epididymal sperm (FES) so as to determine which of these has the most successful ICSI outcome with respect to fertilization rate (FR), pregnancy rate (PR) and birth rate. ? To assess the outcomes according to the underlying aetiology of azoospermia.

PATIENTS AND METHODS

? The records of 493 patients undergoing first‐attempt ICSI between 1993 and 2008 were reviewed retrospectively. FTS was used in 112 cycles, FTTS in 43 cycles, FES in 279 cycles, and FTES in 59 cycles. ? Within each group, the aetiology of the azoospermia was recorded according to history, clinical examination and histological analysis (n= 316). ? The FR, clinical PR and delivery rate were calculated for each group with respect to the type of sperm retrieval used.

RESULTS

? Analysis of the data showed no significant differences between any of the four groups in the FR, PR or delivery rate (P > 0.05). ? There were no significant differences seen between fresh sperm (FTS and FES) and frozen sperm (FTTS and FTES) or between epididymal sperm (FES and FTES) and testicular sperm (FTS and FTTS) in any of the outcomes measured (P > 0.05). However, sub‐set analysis showed a statistically higher FR and PR for FTTS over fresh sperm. ? When comparing aetiologies, there was no significant difference in the FR, clinical PR and delivery rate between obstructive azoospermia (OA) and non‐obstructive azoospermia (NOA) groups. However, sub‐set analysis showed a higher PR and birth rate for FTTS over fresh sperm in both OA and NOA groups.

CONCLUSIONS

? The results of the present study suggest that using frozen sperm in ICSI cycles is a reliable and favourable method with the same outcome as fresh sperm. ? Testicular and epididymal sperm have similar ICSI outcomes for both fresh and frozen samples. However, results suggest a tendency for higher PRs and birth rates for frozen than for fresh testicular sperm in both OA and NOA aetiologies. ? The aetiology of azoospermia does not significantly affect the outcome of first‐attempt ICSI. The higher rates in the frozen groups suggest that these patients have had better quality semen when they were initially harvested and frozen.     

Higher sperm DNA damage in semen from men with spinal cord injuries compared with controls

Semen from men with spinal cord injuries (SCI) and control subjects was investigated for sperm DNA damage using the sperm chromatin structure assay. Three experiments were performed. In experiment 1, the DNA fragmentation index (DFI) was compared in semen from SCI subjects and control subjects. In experiment 2, the % DFI was determined in repeated ejaculations to examine the effect of anejaculation on DFI. In experiment 3, the DFI was determined in neat vs processed semen to examine the effect of necrospermia or leukocytospermia on DFI. The results of experiment 1 showed a significantly higher mean (+/- SEM) DFI in the semen of SCI subjects (65.2% +/- 6.6%; range, 42.3%-90.8%) compared with control subjects (15.4% +/- 2.9%; range, 5.4%-33.5%; P < .001). In experiment 2, there was a high correlation between the DFIs obtained in the first semen specimens and the DFIs obtained 3 days later in semen of the same SCI subjects (r(s) = .94; P < .02). In experiment 3, the results showed no significant difference between mean DFI in aliquots of neat semen (79.3% +/- 9.9%) vs matched aliquots of semen processed to remove dead sperm and leukocytes in SCI subjects (75.2% +/- 16.1%). The DFI is higher in semen from men with SCI vs controls. The cause of this condition is unknown but does not seem to be due to prolonged anejaculation or to the proximate conditions of necrospermia or leukocytospermia. The relevance of these findings to fertility outcomes with SCI male partners remains to be determined.     

Pyriform head: a frequent but little-studied morphological abnormality of sperm

This study was conducted to investigate the frequency of sperm with a pyriform head in semen samples, to determine the percentage of the occurrence of this abnormal sperm form, and to assess its possible correlation with other semen parameters. The study was designed as a retrospective data analysis in the setting of an andrology laboratory at a tertiary-care academic hospital. Semen quality data were analyzed from 114 subfertile men and 60 fertile men. The Student's t test, the Mann-Whitney nonparametric test, and the Pearson correlation coefficient were used for statistical analysis. Sperm with a pyriform head were present in the semen samples of 98% of the subfertile men and 100% of the fertile men; the percentage of this abnormal sperm form was 22 +/- 14.9% in subfertile and 13% +/- 7.8 in fertile men (p <.001); 16% of the subfertile men presented a higher percentage of these abnormal sperm than the normal upper limit. In some subfertile men with a high percentage of sperm with a pyriform head, their subfertility could be attributed to the cause that produces this morphological abnormality. Moreover, morphological abnormalities in the neck and the tail, as also a cytoplasmic droplet, are significantly more frequent in sperm with a pyriform head than in sperm with a normal head.     

Testicular spermatozoon is superior to ejaculated spermatozoon for intracytoplasmic sperm injection to achieve pregnancy in infertile males with high sperm DNA damage

The purpose of this study was to compare the clinical outcome of testicular spermatozoon versus ejaculated spermatozoon in the treatment of infertile males with high sperm DNA damage, referred as sperm DNA fragmentation index (DFI), that attending intracytoplasmic sperm injection (ICSI) programme in terms of clinical pregnancy, births delivered as the primary and pregnancy loss and embryo fertilisation as the secondary outcome. A total of 102 males fulfilling the inclusion criteria were enrolled in the present study. Of the 102 males, 61 infertile males underwent testicular spermatozoon combined with ICSI while the remaining 41 males applied ejaculated spermatozoa in their first ICSI cycles, and the data of them were collected and analysed. In a 18‐month follow‐up, testicular spermatozoon achieved higher pregnancy rate and deliver rate than those in ejaculated sperm group (pregnancy rate, 36% vs. 14.6%, p = 0.017; deliver rate, 38.5% vs. 9.8%, p = 0.001). Nevertheless, there were no significant differences in the number of oocytes aspirated and number of embryos transferred between the two groups. Additionally, the fertilisation rate in the testicular sperm study cohort (70.4%) was also similar to that in the ejaculated sperm group (75.0%). Based on the current data, we conclude that testicular spermatozoon is the prior option in the treatment of infertile males with high sperm DFI in ICSI programme. More high‐quality studies with larger samples size are needed in the future due to the relative small size and the nonrandomized design of the present study.     

Predictors of pregnancy outcome for infertile couples attending IVF and ICSI programmes

The purpose of this study was to evaluate the predictors of pregnancy outcome for infertile couples attending in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) programmes. Infertile couples attending IVF or ICSI procedures were included in this study. Related data including semen parameters and male and female age and body mass index were collected and analysed. The main outcome was clinical pregnancy, defined as an ultrasound detection of foetal heartbeat 6 weeks after embryo transfer. A total of 1316 couples who underwent IVF and 266 who underwent ICSI were recruited for this study. A multivariate logistic regression with likelihood ratio test revealed the following predictors of pregnancy outcome: female age and sperm DNA fragmentation index (DFI) and acrosomal activity in IVF procedures (chi‐square of likelihood ratio = 26.42, d.f. = 3, P < 0.005) and female age and DFI in ICSI procedures (chi‐square of likelihood ratio = 18.88, d.f. = 2, P < 0.005). In conclusion, our study indicated that sperm DFI, female age and acrosomal levels have a significant effect on ART pregnancy outcome.     

Effect of varicocele on sperm function and semen oxidative stress

Study Type – Aetiology (case control) Level of Evidence 3b What’s known on the subject? and What does the study add? Varicocele leads to alterations in sperm DNA integrity even when alterations in semen quality are not yet observed in adolescents. In adults, alterations to sperm DNA are associated to altered sperm morphology, indicating that altered spermatogenesis may be an important cause for the increased sperm DNA fragmentation observed in these men. One other important cause of increased DNA fragmentation is oxidative stress, and we wished to verify if this was the case. The study adds the information that, in the adult varicocele, it is most likely that an altered testicular environment is leading to increased DNA fragmentation and decreased mitochondrial activity and acrosome integrity, because no increase in oxidative stress was observed.

OBJECTIVE

? To assess the effect of varicocele on sperm DNA integrity, mitochondrial activity, lipid peroxidation and acrosome integrity.

PATIENTS AND METHODS

? In all, 30 patients with a clinically diagnosed varicocele of grade II or III and 32 men without a varicocele were evaluated for sperm DNA fragmentation (comet assay), mitochondrial activity (3,3′‐diaminobenzidine assay), lipid peroxidation (malondialdehyde) and acrosome integrity (fluorescent probe labelled peanut agglutinin).

RESULTS

? The varicocele group showed fewer spermatozoa with intact DNA (grade II, P= 0.040), more cells with inactive mitochondria (class III, P= 0.001), fewer cells with active mitochondria (class I, P= 0.005) and fewer spermatozoa with intact acrosomes (P < 0.001). Finally, no significant differences were observed in lipid peroxidation levels.

CONCLUSION

? Men with varicocele showed an increase in sperm DNA fragmentation and a reduction in mitochondrial activity and acrosome integrity. However, lipid peroxidation levels remained unchanged.     

复方玄驹胶囊联合维生素E对不育患者精子染色质损伤的影响

目的:初步探讨复方玄驹胶囊联合维生素E治疗方案对轻度少精子症和/或弱精子症患者精子染色质损伤的临床疗效。方法:50例精液异常的男性不育患者随机分为实验组(n=24)和对照组(n=26),分别予以复方玄驹胶囊+维生素E和单纯维生素E治疗3个月,运用计算机辅助精液分析系统(CASA)及精子染色质结构分析(SCSA)方法分析两组患者治疗前后精液常规参数和精子DNA损伤指数(DFI),比较治疗前后精液常规参数及精子DFI的变化。结果:实验组治疗后前向运动精子率为(21.55±8.68),对照组为(21.47±11.53),两组相比差异没有统计学意义(P>0.05)。在实验组中治疗前DFI为34.09±10.32,治疗后DFI为29.57±12.19,与治疗前相比显著下降(P<0.05)。结论:复方玄驹胶囊联合维生素E治疗可有效改善不育患者精液质量,对精子染色质损伤有一定的改善作用。     

Correlation between semen parameters and the Hamster Egg Penetration Test (HEPT) among fertile and subfertile men in Singapore

The objective of this retrospective study was to distinguish between fertile and subfertile men based on their semen parameters and hamster egg penetration test (HEPT) outcome. This study involved 110 subfertile men recruited from an infertility clinic and 48 fertile men attending an antenatal clinic in Singapore. The men were required to donate a semen specimen for semen analysis and HEPT assay. The results indicated that the subfertile group had significantly lower normal sperm morphology according to the Tygerberg strict criteria, and lower progressive motility (P < .05). Semen volume, density, HEPT decondensation rate, and sperm penetration index were not significantly different between the 2 groups. Receiver operating characteristic curve analysis indicated that sperm morphology had the highest predictive power of 65.7% with a threshold value of 7%, and progressive motility had a predictive power of 61.8% with a threshold value of 50%. Using the tenth percentile of the fertile population as the cutoff, lower adjusted thresholds of 3% for sperm morphology and 28% for progressive motility were obtained, giving higher positive predictive values of 81.8% and 84.4%, respectively. This study shows that these new cutoff values can be used to screen the general population to identify subfertile men. In contrast, the HEPT proved to be an insensitive and unreliable assay in identifying subfertile males. To our knowledge the comparison of HEPT and semen parameters between subfertile and fertile men has not been previously reported in an Asian population.     

Clearance after vasectomy with a single semen sample containing < than 100 000 immotile sperm/mL: analysis of 1073 patients

Study Type – Diagnostic (non‐consecutive)
Level of Evidence 3b

OBJECTIVE

To evaluate the safety and efficacy of a new semen analysis protocol after vasectomy, where clearance is given to patients who provide a single semen sample with <100 000 immotile sperm/mL at ≥3 months after vasectomy.

PATIENTS AND METHODS

Between 1 July 2005 and 31 March 2008, 1073 men provided a first semen sample at ≥3 months after vasectomy. Semen was first evaluated on a wet‐slide preparation. Those samples with no (‘azoospermia’) or sporadic immotile spermatozoa could be cleared without further analysis. Samples with motile sperm were immediately labelled as potentially fertile, while those with a significant number of immotile sperm were re‐analysed using a Neubauer haemocytometer. All samples with <100 000 immotile sperm/mL were cleared.

RESULTS

Of men providing semen at 3 months after vasectomy, 96% could be cleared. No sperm were seen (‘azoospermia’) in 51.3% of samples, and 44.7% of samples contained <100 000 immotile sperm. No paternity has been reported in the cleared group after a follow‐up of at least 1 year.

CONCLUSIONS

A protocol stipulating that patients can be cleared after a single semen sample containing <100 000 immotile sperm/mL at ≥3 months after vasectomy is safe and dramatically reduces the number of men who cannot be cleared at 3 months after vasectomy.     

Normal sperm morphology and chromatin packaging: comparison between aniline blue and chromomycin A3 staining

The successful implementation of ICSI has provided a unique means of allowing couples suffering from severe male infertility to achieve their reproductive goals. However, despite the great therapeutic advantages of the technique, ICSI often provides solutions to clinicians in the absence of an aetiological or pathophysiological diagnosis. The development of a sequential diagnostic schedule for patients consulting for fertility disturbances would be an ideal method of approach. Since sperm morphology recorded by strict criteria has often been correlated with fertilization failure, the present study aimed to evaluate the relationship between normal morphology and chromatin staining among fertile and subfertile men. Both chromomycin A3 (CMA3) and acidic aniline blue (AAB) were employed to record chromatin packaging quality among 58 men visiting the andrology laboratory. Intra- and interassay variations were initially recorded for fertile sperm donors. The coefficients of variation (CV) for all intra- and inter-assay assessments were < 12%. Chromatin packaging was significantly and negatively correlated with normal sperm morphology, namely r = 0.40 (P = 0.001) and r = 0.33 (P = 0.001) for CMA3 and AAB, respectively. Receiver operator characteristics illustrated sensitivity and specificity values of 75% and 82% for CMA3 and 60% and 91% for AAB, respectively. Significantly different CMA3 and AAB staining was recorded among men with severe teratozoospermia (< 4% normal forms) when compared with normozoospermic men (> 14% normal forms), namely 49% vs. 29% for CMA3 and 51% vs. 26% for AAB staining, respectively. Chromatin packaging assessments should be a valuable addition to the sequential diagnostic programme in an assisted reproduction arena.     

Diagnostics of DNA fragmentation in human spermatozoa: Are sperm chromatin structure analysis and sperm chromatin dispersion tests (SCD‐HaloSpermG2®) comparable?

Men affected with idiopathic infertility often display basic spermiogramme values similar to fertile individuals, questioning the diagnostic impact of the World Health Organization (WHO) thresholds used. This study explored sperm DNA fragmentation in single ejaculates from 14 fertile donors and 42 patients with idiopathic infertility providing semen for assisted reproductive techniques in a university fertility clinic. Each ejaculate was simultaneously studied for sperm DNA fragmentation by the flow cytometer‐based sperm chromatin structure analysis (SCSA) and the new light‐microscopy‐based sperm chromatin dispersion assay (SCD‐HaloSpermG2^®), before and after sperm selection for in vitro fertilisation with a colloid discontinuous gradient. The WHO semen variables did not differ between groups, but DNA fragmentation after SCSA (DFI) or SCD (SDF) was significantly (p < 0.05) higher in patients (DFI: 40.2% ± 3.0 vs. SDF: 40.3% ± 1.4) than in fertile donors (DFI: 17.1% ± 2.1 vs. SDF: 20.9% ± 2.5). Sperm selection led to lower proportions of DNA‐fragmented spermatozoa (DFI: 11.9 ± 1.7 vs. SCD: 10.0 ± 0.9, p < 0.05). The techniques output correlated highly and significantly (r² = 0.82). DNA fragmentation is confirmed as a relevant variable for scrutinising patients with idiopathic infertility, beyond the evidently insufficient WHO semen analyses. Since both techniques yielded similar results, the reduced necessity of complex equipment when running SCD ought to be considered for a clinical setting.     

Evaluation of sperm nuclear morphology in specimens with abnormal sperm chromatin structural assays

Two recent tests have claimed to identify the subfertile male even when other semen parameters were normal: the sperm chromatin structure assay (SCSA) and abnormal sperm nuclear morphology using much higher magnification. The present study attempted to determine if having a high (> 30%) DNA fragmentation index (DFI), thus resulting in an abnormal SCSA test, is associated with a greater likelihood of sperm with abnormal nuclei. Four males with high DFI scores (57.6%, 65.4%, 31.0%, and 35.3%) had their nuclei evaluated by a complex microscope set-up that magnifies the sperm at least 6000x. The corresponding % of normal nuclei was 0%, 20.0%, 23.7% and 40.0%. The mean and median % of normal nuclei was 20.9+/-16.43 and 21.8, respectively. More studies of similarly matched refractory in vitro fertilization cases, where males have normal DFI scores, are needed to determine if having a high DFI index is associated with a lower percentage of normal nuclei.     

Association between obesity and alteration of sperm DNA integrity and mitochondrial activity

Study Type – Prognosis (cohort) Level of Evidence 3a What's known on the subject? and What does the study add? The relationship between high levels of BMI and changes in altered standard semen analysis parameters are described in the literature. However, the functional characteristics of the sperm are essential to complete the evaluation of male infertility. Thus, this study provides important information about the functionality of the sperm of men with different levels of BMI.

OBJECTIVE

• ? To assess the effect of obesity on semen analysis, sperm mitochondrial activity and DNA fragmentation.

MATERIALS AND METHODS

• ? A transversal study of 305 male patients, presenting for clinical evaluation, was carried out. The patients were divided into three groups according to body mass index (BMI) as follows: eutrophic (BMI < 25 kg/m², n= 82), overweight (BMI ≥ 25 kg/m² and <30, n= 187) and obese (BMI ≥ 30 kg/m², n= 36).
• ? The variables analysed were semen analysis, rate of sperm DNA fragmentation and sperm mitochondrial activity.
• ? Groups were compared using one‐way analysis of variance followed by a least significant difference post‐hoc test. A P‐value of <0.05 was considered to indicate statistical significance.

RESULTS

• ? No differences were observed in age, ejaculatory abstinence, ejaculate volume, sperm vitality, morphology or round cell and neutrophil count among the groups.
• ? The eutrophic group had a higher percentage of sperm with progressive motility (P= 0.001). Mitochondrial activity was lower in the obese group (P= 0.037) when compared to the eutrophic, and the percentage of sperm with DNA damage was higher in the obese group (P= 0.004) than the other two groups.

CONCLUSION

• ? Increased BMI values are associated with decreased mitochondrial activity and progressive motility and increased DNA fragmentation.

     

精子DNA完整性与体外受精-胚胎移植临床结局的相关性研究

目的 探讨精子DNA完整性与精液参数及体外受精-胚胎移植（IVF-ET）/卵胞浆内单精子注射（ICSI）临床结局的关系.方法 选择2008年6月～2009年6月在解放军105医院生殖医学中心接受IVF/ICSI治疗的179对不育夫妇作为研究对象,采用吖啶橙试验（AOT）对116例实施IVF和63例实施ICSI治疗的男性患者进行精子DNA完整性分析,根据精子DNA碎片指数（DFI）将患者分为DFI≤30%组和DFI＞30%组,比较两组间精液参数、受精率、卵裂率、优胚率、胚胎冷冻率、着床率和临床妊娠率.结果 DFI ＞30%组精子畸形率显著高于≤30%组（P＜0.01）,但两组间精子密度、活动率、前向运动精子（a＋b）均无显著性差异（P＞0.05）;DFI＞30%组IVF和ICSI的优胚率、ICSI的胚胎着床率和临床妊娠率均显著低于DFI≤30%组（P＜0.01,P＜0.05）.结论 精子DNA完整性与精子形态密切相关,精子DNA损伤在IVF/ICSI过程中对胚胎质量有负面影响,并显著影响ICSI的胚胎着床率和妊娠率,建议行ICSI前应对精子DNA完整性进行评估.     

Relationship of spermatozoal DNA fragmentation with semen quality in varicocele‐positive men

The aim of the study was to assess the semen quality and levels of spermatozoal nuclear DNA fragmentation in subfertile subjects clinically diagnosed with varicocele, subfertile subjects without varicocele and healthy fertile controls. Semen samples were obtained from 302 subjects. Of them, 115 were healthy fertile controls having normal semen characteristics, 121 subfertile men diagnosed with varicocele, both, clinically and on ultrasonography, while 66 subjects were subfertile with no varicocele. Spermatozoal concentration, percentage motility, morphology and DNA fragmentation were measured. In the study population, deterioration in semen quality‐decreased spermatozoal concentration, percentage motility and normal morphology was seen in subfertile subjects, especially with varicocele. Highest spermatozoal DNA fragmentation was observed in varicocele‐positive subjects as compared with varicocele‐negative subjects and healthy fertile controls. Significant negative correlation was seen between spermatozoal DNA fragmentation and concentration (r = ?0.310), motility (r = ?0.328) normal morphology, WHO method (r = ?0.221) and Tygerberg strict criteria (r = ?0.180) in the varicocele‐positive subfertile subjects. In conclusion, this study suggests existence of a negative relationship between spermatozoal DNA fragmentation and semen quality in varicocele‐positive subfertile subjects.     

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

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