The effect of sperm DNA fragmentation on intracytoplasmic sperm injection outcome

Our study objective was to assess the effect of various sperm DNA fragmentation levels on clinical intracytoplasmic sperm injection outcome. This retrospective study included 392 patients who underwent ICSI and performed sperm DNA fragmentation testing before the procedure. Based on sperm DNA fragmentation cut-off values, the patients were differentiated into 3 groups as <20%, 20%–30% and >30%. According to the female status, patients were differentiated into favourable group (n = 259) with female age <35 years and anti-Mullerian hormone level ≥7.1 pmol/L; and unfavourable group (n = 133) with female age ≥35 years and anti-Mullerian hormone level ≤7.1 pmol/L. The patient's medical records were reviewed, and patient's demographic, laboratory data including semen analysis, sperm DNA fragmentation determined by means of sperm chromatin dispersion, hormonal profile and data regarding intracytoplasmic sperm injection cycle were collected. This cohort reported that the clinical reproductive outcomes of intracytoplasmic sperm injection showed no statistical significance with increase sperm DNA fragmentation levels. In sperm DNA fragmentation above 30%, favourable females had significantly higher clinical pregnancy rate and live birth rate than unfavourable females, while fertilisation rate and miscarriage rate showed no significance between the subgroups. High sperm DNA fragmentation is linked to poor semen parameters.     

人精子染色质损伤及其检测的研究进展

人精子染色质损伤是常见的影响男性生殖能力的重要原因之一,受到遗传、环境和生活习惯等多因素的影响,且与男性不育、复发性流产等有着密切的联系。随着人们对精子染色质结构和功能认识的加深,对精子染色质结构分析技术的不断改进和推广,以及辅助生殖技术越来越广泛的应用,精子DNA损伤逐渐被认识到是一个新的评价精子质量的重要检测指标,对男性生殖力的评估和辅助生殖技术的选择具有重要的临床意义。     

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.     

Aneuploidy and DNA fragmentation in morphologically abnormal sperm

In light of the relative success of ICSI in the treatment of male infertility, much importance has been made to the selection of morphologically viable sperm. However, correlation between specific sperm morphology and chromosomal abnormalities is still relatively limited and less is known about the connection between sperm morphology and DNA integrity. Sperm obtained from isolated teratozoospermic men ( n  = 10) and control men ( n  = 9) were analysed using FISH (for chromosomes 13, 18, 21, X and Y) and TUNEL assays to determine the level of aneuploidy and DNA fragmentation. Sperm morphology was evaluated on its ability to identify the level of chromosomal abnormalities or fragmented DNA in sperm. Sperm from teratozoospermic men, compared with fertile men, had higher rates of total chromosomal abnormality ( p  < 0.05), total aneuploidy ( p  < 0.01) and chromosome 13 disomy ( p  < 0.01). Associations between particular types of sperm morphology and chromosomal abnormalities were observed in both control (tapered heads) and teratozoospermic (amorphous heads and tail abnormalities) samples. Levels of DNA fragmented sperm were higher in teratozoospermic men than in the control men (60.28 ± 21.40% vs. 32.40 ± 17.20%, p  < 0.05) and positively correlated to sperm with bent necks in control samples and round heads in teratozoospermic samples ( p  < 0.05). Sperm of isolated teratozoospermic men have higher rates of chromosomal abnormalities and DNA fragmentation than that of the fertile controls. Specific abnormal sperm morphology can be correlated to chromosomal abnormalities and level of DNA fragmentation in sperm.     

不同来源精子对ICSI治疗后妊娠结局的影响

目的分析不同精子来源和数量对单精子卵胞浆内注射术(ICSI)妊娠结局的影响。方法2000年1月～2005年1月在本中心进行ICSI治疗而妊娠的354例,据精子来源与数量分为精液正常组(47例)、少弱精子组(194例)与手术取精子组(113例)。比较3组的临床妊娠率、流产率、分娩率、妊娠并发症、分娩孕周、新生儿出生体重、畸形、围产期死亡率等组间差异。结果3组患者的年龄、不孕年限、临床妊娠率(41.3%、39.7%、40.4%)、流产率(14.9%、18.6%、15.9%)、分娩率(78.7%、75.8%、79.6%)、双胎率(34.4%、35.4%、37.8%)、妊高症发生率(10.8%、10.2%、11.1%)、前置胎盘发生率(5.4%、6.8%、6.7%)、早产率(27.0%、27.9%、27.8%)、分娩孕周(36.4±5.4)w、(37.6±2.3)w、(37.2±2.8)w、胎儿出生体重(2765.4±624.9)g、(2749.0±523.5)g、(2763.5±684.4)g、先天性畸形发生率(4.5%、5.0%、4.0%)、围产期死亡率(40‰、45‰、48‰),差异均无显著性(P>0.05)。结论不同精子来源与数量不影响ICSI治疗后的妊娠和围产儿结局。     

Relationship between sperm quality and chromatin condensation measured by sperm DNA fluorescence using flow cytometry

DNA flow cytometry of sperm from 100 randomly chosen men undergoing fertility investigation revealed a general association between reduced sperm quality, as judged by conventional parameters, and the appearance of sperm with lower degrees of chromatin condensation in the ejaculate as measured by DNA fluorescence intensity. Chromatin hypocondensation, as measured by increased fluorescence, was manifested to different degrees in different samples. In many cases of more extreme sperm pathology, such as oligoasthenoteratozoospermia (OAT), the whole population of spermatozoa appeared to be affected. Significant numbers of hypercondensed spermatozoa were present in both normozoospermic men and men with different degrees of disturbance in sperm quality. All of the different parameters of sperm quality could be correlated significantly with certain of the flow parameters, although not one in particular could be used to predict deviations from the normal flow profile. In several asthenoteratozoospermic men and a small proportion of men with OAT, the DNA profiles were normal, implying that in these cases the disturbance may not be so fundamental. The presence of leucocytes in the ejaculate was associated with a general increase in the preponderance of hypocondensed subpopulations of spermatozoa in men with OAT as well as in normozoospermic subjects, emphasizing the effect of inflammatory conditions in the reproductive tract on sperm quality.     

Sperm chromatin quality and DNA integrity in partial versus total globozoospermia

Globozoospermia is a severe form of teratozoospermia with low incidence in infertile patients, considered as one of the important causes of male infertility. The objective was to investigate the chromatin/DNA integrity as well as apoptosis in ejaculated spermatozoa of cases with partial or total globozoospermia. Fifty‐seven semen samples were divided into three groups of partial globozoospermia (n = 17), total globozoospermia (n = 10) and normozoospermia (control; n = 30). Sperm chromatin condensation, DNA integrity and apoptosis were assessed using cytochemical assays. The results showed significant differences in sperm parameters of count and motility between two case groups versus controls. The percentages of spermatozoa with abnormal chromatin packaging and protamine deficiency were significantly higher in total and partial globozoospermic men compared to normozoospermic samples. Also, the rates of TUNEL‐positive spermatozoa were significantly increased in both globozoospermic cases with respect to the control (18.3 ± 10.1 and 12.3 ± 9.2 versus 5.9 ± 3 respectively). However, no significant differences were noticed between two subgroups of patients with regard to sperm DNA denaturation, DNA fragmentation and apoptosis. Abnormal chromatin packaging, DNA damage and apoptosis were significantly higher in cases than controls. The sperm chromatin/DNA anomalies may be considered as one of the main aetiology of ART failure in globozoospermic patients.     

精子DNA完整性研究及其临床意义

精子DNA完整性检测反应了精子DNA的损伤程度,其发生机制可能与精子细胞发生过程中的凋亡发生异常、染色质组装异常或氧化应激反应有关。目前常用的精子DNA完整性检测方法有单细胞凝胶电泳法、原位标记法、精子染色质结构分析法、吖啶橙试验、精子染色质扩散试验和8-羟基脱氧鸟苷（8-OHdG）测定法等。精子DNA完整性检测对揭示男性不育的病因、预测辅助生殖结局以及指导临床治疗具有重要意义。     

Different outcomes after intracytoplasmic sperm injection without oocyte activation in two patients with different types of globozoospermia

Different outcomes after intracytoplasmic sperm injection (ICSI) without oocyte activation in two patients with different types of round‐headed spermatozoa (globozoospermia) are reported. After controlled ovarian hyperstimulation and oocyte pick‐up, retrieved oocytes were underwent ICSI without oocyte activation and a 33.33% (4/12) fertilisation rate was obtained in the first case, whereas an abnormal fertilisation was achieved in the second case. The transfer of two grade II embryos in the first couple resulted in clinical pregnancy with a healthy livebirth. It was concluded that the main problem of cases with globozoospermia was a low fertilisation rate or failure fertilisation, and even though ICSI and artificial oocyte activation have been employed to increase this rate, it is not necessarily needed to achieve a pregnancy.     

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

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

MII期卵母细胞质量对卵胞浆内单精子注射结局的影响

目的　探讨MII期卵母细胞质量对卵胞浆内单精子注射 (ICSI)结局的影响。　方法　对ICSI中去除颗粒细胞后的MII期卵母细胞从第一极体、卵周隙和胞浆形态三者综合评价。按形态表现分为 5级 ,比较各级卵母细胞的受精率、优质胚胎率及种植率、临床妊娠率。　结果　I级卵母细胞受精率、优质胚胎率 ,明显高于其他各级含异常形态的卵母细胞 (P <0 .0 1) ;种植率和临床妊娠率也明显高于其他组 (P <0 .0 5 )。　结论　根据MII期卵母细胞的第一极体、卵周隙及胞浆形态进行的卵母细胞分级可作为胚胎移植时选择胚胎的依据。     

Sperm chromatin structure assay （SCSA）： a tool in diagnosis and treatment of infertility

Diagnosis of male infertility has mainly been based on the World Health Organization (WHO) manual-based semen parameter's concentration, motility and morphology. It has, however, become apparent that none of these parameters are reliable markers for evaluation of the fertility potential of a couple. A search for better markers has led to an increased focus on sperm chromatin integrity testing in fertility work-up and assisted reproductive techniques. During the last couple of decades, numerous sperm DNA integrity tests have been developed. These are claimed to be characterized by a lower intraindividual variation, less intralaboratory and interlaboratory variation and thus less subjective than the conventional sperm analysis. However, not all the sperm chromatin integrity tests have yet been shown to be of clinical value. So far, the test that has been found to have the most stable clinical threshold values in relation to fertility is the sperm chromatin structure assay (SCSA), a flow cytometric test that measures the susceptibility of sperm DNA to acid-induced DNA denaturation in situ. Sperm DNA fragmentation as measured by SCSA has shown to be an independent predictor of successful pregnancy in first pregnancy planners as well as in couples undergoing intrauterine insemination, and can be used as a tool in investigation, counseling and treatment of involuntary childlessness. More conflicting data exist regarding the role of sperm DNA fragmentation in relation to fertilization, pre-embryo development and pregnancy outcome in in vitro fertilization and intracytoplasmic sperm injection (ICSI).     

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.     

Sperm DNA fragmentation in subfertile men: the effect on the outcome of intracytoplasmic sperm injection and correlation with sperm variables

OBJECTIVE

To present the first UK data on sperm DNA fragmentation levels in subfertile men and fertile controls, the correlation with semen variables, and to assess the effect on the outcome of intracytoplasmic sperm injection (ICSI).

PATIENTS, SUBJECTS AND METHODS

In all, 56 subfertile men undergoing ICSI (28 with positive and 28 with a negative outcome for paternity) and 10 control fertile semen donors were recruited. The sperm DNA fragmentation index (DFI) was assessed on raw pre‐preparation samples using the sperm chromatin structure assay. A mean of 5212 sperm were analysed per sample and DFI data are presented by fertility status, ICSI outcome and correlated with semen variables (assessed using World Health Organisation criteria).

RESULTS

Total DFI was significantly higher in subfertile men than in fertile controls (mean and median of 22.8% and 17.0% vs 8.4% and 5.0%; P < 0.001), as was the proportion of both moderate DFI (16.4% and 13.0% vs 6.4% and 4.0%; P = 0.001) and high DFI (6.2% and 6.1 vs 2.0% and 1.0%; P = 0.01). This difference remained significant when the control men were compared only with the subfertile men with successful paternity. There was no significant difference in DFI in the subfertile men when analysed by ICSI outcome (mean and median of 24.5% and 17.0% vs 22.3% and 21.0% for successful and unsuccessful cycles, respectively; P = 0.94). There was a positive statistically significant correlation (r = 0.37; P = 0.02) between the DFI and sperm morphology.

CONCLUSIONS

This study confirms a relationship between male subfertility and sperm DFI; we discuss the correct role for genetic testing of sperm in the evaluation of subfertile men. Although DNA fragmentation data might help to decide a suitable treatment, once it is decided to proceed with ICSI, DFI levels have no effect on the outcome.     

Higher frequency of Yq microdeletions in sperm DNA as compared to DNA isolated from blood

Aim： To determine if Yq microdeletion frequency and loci of deletion are similar in two tissues （blood and sperm） of different embryological origin. Methods： The present study included 52 infertile oligozoospermic cases. In each case, DNA was isolated from blood and sperms and polymerase chain reaction （PCR） microdeletion analysis was done from genomic DNA isolated from both the tissues. The PCR products were analyzed on a 1.8% agarose gel. PCR amplifications found to be negative were repeated at least three times to confirm the deletion of a given marker. Results： Only 1 case harbored microdeletion in blood DNA, whereas 4 cases harbored microdeletion in sperm DNA. Conclusion： The frequency of Yq microdeletions is higher in germ cells as compared to blood. As the majority of infertile couples opt for assisted reproduction procreation techniques （ART）, Yq microdeletion screening from germ cells is important to understand the genetic basis of infertility, to provide comprehensive counseling and most adapted therapeutics to the infertile couple.     

Etiologies of sperm DNA damage and its impact on male infertility

Male factor is responsible for up to 50% of infertility cases in the world. Semen analysis is considered the cornerstone of laboratory evaluation of male infertility, but it has its own drawbacks and fails to predict the male fertility potential with high sensitivity and specificity. Different etiologies have been linked with male infertility, of which sperm DNA damage has gained significant attention with extensive research on sperm function tests. The associations between sperm DNA damage and a variety of disorders such as varicocele, obesity, cancer, radiation and lifestyle factors are explored in this review. Furthermore, we discuss the mechanisms of DNA damage as well as its impact in different scenarios of male infertility, associated with spontaneous and assisted reproduction. Finally, we review the clinical applicability of sperm DNA fragmentation testing in the management of male infertility.     

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.     

The role of paternal chromosomes and sperm morphology on the outcome of intracytoplasmic sperm injection

The study investigated the possible relationship between the X/Y chromosomes bearing spermatozoa and the outcome of intracytoplasmic sperm injection (ICSI) therapy and morphological appearance/shape (Tygerberg criteria) of the sperm cell injected into the oocyte during ICSI therapy and fertilisation. Thirty-nine patients were recruited for the study from an assisted reproductive programme. Semen samples were prepared by using gradient centrifugation techniques. Prior to injection sperm images were captured using high-quality video graphic equipment. Sperm selection was based on the concept of 'best-looking' spermatozoa i.e. spermatozoa lacking gross and obvious malformations such as broken necks, cytoplasmic droplets, amorphous or elongated heads. Photomicrographs of each sperm cell were produced from video footage. The photographical material was used to determine the basic shape and the actual length-to-width ratio of the injected sperm heads. Embryo biopsies and fluorescent in situ hybridisation (FISH) was performed on 12 randomly selected couples from a set of 39. Embryos were evaluated on day 3 for development and embryo transfer. Embryo biopsies and FISH analyses were performed on those embryos that showed no developmental potential following injection. It was found that 70% of the embryos that showed no developmental potential were Y chromosome-bearing embryos. The sperm selection process for ICSI based on the approach of choosing the 'best-looking' spermatozoon in the ejaculate seems to provide cells that can be classified as normal based on the length-to-width ratio set by the World Health Organization for normal cells.     

微流控芯片精子分选法对精子常规参数及DNA完整性的影响

目的:研究微流控芯片精子优选技术对精子常规参数及DNA完整性的影响。方法:自行设计制作微流控芯片,利用芯片处理技术和上游法对40例精液标本进行精子优选,通过计算机辅助精液分析系统及染色质扩散试验从精子常规参数及DNA完整性两个方面评价精液体外处理对精子的影响。结果:精液经微流控芯片法和上游法处理后,精子活力、精子正常形态率以及精子尾部肿胀率均有显著提高(P<0.01),精子的DNA损伤率明显降低(P<0.01)。微流控芯片法与上游法相比,优选后前者精子DNA损伤率明显低于后者[(8.4±5.8)%vs(16.4±9.2)%,P<0.01],而其他参数差异无显著性。结论:微流控芯片技术在精子优选中能获得精子DNA损伤程度小的高质量精子。