Full-term pregnancies achieved with ICSI despite high levels of sperm chromatin damage

BACKGROUND: Sperm DNA integrity is essential for the accurate transmission of genetic information. The clinical significance of this assessment lies in its association with not only natural conception rates, but also the success of assisted reproduction technology (ART). It has been reported that sperm chromatin structure assay (SCSA) identified thresholds for negative pregnancy outcome after ART when the DNA fragmentation index (DFI), previously known as COMPalphat, was >30%. METHODS: In a prospective clinical study, we examined 34 male infertile patients, the husbands of women undergoing conventional IVF or ICSI. SCSA and ART were carried out on semen aliquots taken from the same ejaculate. Fertilization rate, embryo quality and pregnancy rates were correlated to SCSA parameters, DFI and highly DNA stainable (HDS) cells. RESULTS: No differences were seen in SCSA parameter values between patients initiating pregnancies and not doing so in either ICSI or conventional IVF. Pregnancies and normal delivery were obtained even with high levels of DFI. CONCLUSIONS: There is still controversy over whether analytical techniques currently in use are able to identify the level of damage to spermatozoa. Large-scale studies should be conducted in different clinical settings to determine the effects of sperm DNA damage on the outcome of ART.     

Intra-individual variation in sperm chromatin structure assay parameters in men from infertile couples: clinical implications

BACKGROUND: Sperm DNA integrity is an important factor in the prognosis of male fertility. In this study, we investigated intra-individual variation of sperm chromatin structure assay (SCSA) parameters in infertility patients undergoing assisted reproductive techniques (ARTs). METHODS: Retrospective study of 282 consecutive patients referred for ART [intrauterine insemination (IUI), IVF or ICSI] with repeated (between 2 and 5) SCSA measurements. RESULTS: Mean coefficient of variation (CV) of DNA Fragmentation Index (DFI) for repeated SCSA measurements was 29%. A high proportion [37%; 95% confidence interval (CI): 27%, 49%] of patients with DFI >30% in the first test had DFI <30% in the second test. Also, a considerable proportion (27%; 95% CI : 16%, 40%) of patients with 21-30% DFI values in the first test had DFI >30% in the second test. CONCLUSIONS: Intra-individual variability in DFI is significant, therefore repeated SCSA measurements are recommended. The biological mechanisms behind these variations remain to be elucidated.     

The predictive value of sperm chromatin structure assay (SCSA) parameters for the outcome of intrauterine insemination, IVF and ICSI

INTRODUCTION: Sperm chromatin integrity assessment has been suggested as a fertility predictor. The aim of this study was to examine the relationship between the results of sperm chromatin structure assay (SCSA) and the outcome of IVF, ICSI and intrauterine insemination (IUI). METHODS: A total of 306 consecutive couples undergoing assisted reproduction were included. IUI was performed in 131, IVF in 109 and ICSI in 66. SCSA results were expressed as DNA fractionation index (DFI) and highly DNA stainable (HDS) cell fractions. Reproductive outcome parameters were biochemical pregnancy (BP), clinical pregnancy (CP) and delivery (D). RESULTS: For IUI, the chance of pregnancy/delivery was significantly higher in the group with DFI 27% or HDS >10%. The odds ratios (ORs) (95% confidence intervals) were 20 (2.3-117), 16 (1.9-137) and 14 (1.6-110) for BP, CP and D, respectively. No statistical difference between the outcomes of IVF versus ICSI was observed in the group with DFI 27% group, however, the results of ICSI were significantly better than those of IVF. Comparing ICSI with IVF, the OR (95% CI) for BP was 26 (1.9-350). CONCLUSIONS: SCSA is a useful method for prediction of the outcome of assisted reproduction.     

The sperm chromatin structure assay as a diagnostic tool in the human fertility clinic

BACKGROUND: Sperm DNA integrity has been shown to be necessary for achieving and sustaining embryo development. The objective was to evaluate the sperm chromatin structure assay (SCSA) as a diagnostic tool in clinical practice for intrauterine insemination (IUI), in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) treatments. METHODS: A total of 385 semen samples from 234 couples were frozen for SCSA, and smears were prepared for morphology: 48 IUI, 139 IVF and 47 ICSI. The main SCSA variables were DNA fragmentation index (DFI), standard deviation of DFI (SD-DFI) and high DNA stainability (HDS), and the reproductive outcomes were biochemical pregnancy (BP), clinical pregnancy (CP) and implantation ratio (IR). RESULTS: The results showed no significant difference in the fertility variables BP, CP and IR when <27% DFI was used between the IVF and ICSI groups. A low number of patients received IUI with low success rate, and statistical analysis was therefore not performed. Ongoing pregnancy was achieved for both IVF and ICSI couples with DFI levels >27%, and six couples in ICSI treatment achieved CP full-term. DFI >27% had a high prognostic power for predicting no CP for IVF patients, with a specificity of 97%. Couples diagnosed with male infertility had a significantly higher level of DFI compared to couples with idiopathic fertility. Sperm head morphology showed low but significant correlations with the SCSA variables. CONCLUSION: SCSA is a useful tool in andrological diagnosis and contributes with a prognosis for the fertility outcome of conventional IVF. Although full-term pregnancy can be achieved with assisted reproductive techniques with a DFI >27%, the probability of a successful pregnancy may be reduced.     

Sperm DNA damage in potentially fertile homozygous beta-thalassaemia patients with iron overload

BACKGROUND: To test the hypothesis that human sperm DNA could sustain iron-induced oxidative damage and reduce its fertilizing ability, we studied patients with homozygous beta-thalassaemia major (HbTh) as a model of iron overload. METHODS: Sperm from six thalassaemic patients and five age-matched controls were assessed by the sperm chromatin structure assay (SCSA) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay. Semen parameters, endocrine markers of testicular function, iron profiles and the presence of organ dysfunction were also determined. RESULTS: All patients with HbTh were iron overloaded (median ferritin: 2251 microg/l) and had evidence of spontaneous spermatogenesis. Thalassaemic patients had more sperm DNA damage than the controls (P < 0.01). The sperm DNA damage by SCSA and TUNEL were positively correlated (P < 0.05). Sperm motility and TUNEL results were negatively correlated (P < 0.05), while the age of onset of chelation and sperm DNA damage were positively associated with both SCSA (R(2) = 0.80, P = 0.016) and TUNEL data (R(2) = 0.67, P < 0.044). No other biochemical or clinical data were associated with sperm DNA damage. CONCLUSIONS: The increase in sperm DNA damage and the negative correlation between sperm motility and DNA damage suggest that iron overload in HbTh predisposes sperm to oxidative injury. This finding has important implications in assisted reproductive procedures such as ICSI where there is increased risk of transmitting defective DNA to the offspring.     

Characterization of sperm chromatin quality in testicular cancer and Hodgkin's lymphoma patients prior to chemotherapy

BACKGROUND: Although the incidences of testicular cancer and Hodgkin's lymphomahave increased in young men over the past decade, combinationchemotherapy has improved survival. As fertility is of importanceto these patients, characterization of sperm chromatin structureis needed. We assessed sperm chromatin in testicular cancerand Hodgkin's lymphoma patients prior to chemotherapy, in comparisonwith control community and idiopathic infertile volunteers. METHODS: DNA damage was assessed with the sperm chromatin structure assay(SCSA), terminal deoxynucleotidyl transferase-mediated dUTPnick end labeling (TUNEL) and comet assays; reactive thiols(SH) and DNA compaction were determined with the monobromobimane(mBBr) and chromomycin A3 (CMA3) assays, respectively. RESULTS: Both testicular cancer (37%) and Hodgkin's lymphoma (81%) patientshad normospermic samples with increased DNA damage, comparedwith controls. Cancer patients also had higher reactive thiolsand CMA3 staining, indicating low DNA compaction. CONCLUSIONS: Sperm DNA integrity and compaction were affected in testicularcancer and Hodgkin's lymphoma patients prior to chemotherapy.Although SCSA, TUNEL and comet assays all detected DNA damage,the latter was optimal for use in cancer patients. A combinationof the comet assay with tests that evaluate sperm DNA compaction,such as flow cytometry-based CMA3 and mBBr assays, is a reliablestrategy to characterize sperm chromatin quality in cancer patientsat the time of sperm banking.     

Sperm chromatin structure assay parameters measured after density gradient centrifugation are not predictive for the outcome of ART

BACKGROUND: The sperm chromatin structure assay (SCSA) parameter DNA fragmentation index (DFI) has been shown to predict in vivo and in vitro fertility. So far most SCSA studies have been based on SCSA analysis performed on neat semen. The aim of this study is to assess whether SCSA analysis of sperm prepared by density gradient centrifugation (DGC) could add more information in regard to the prediction of treatment outcome. METHODS: The study included 510 assisted reproductive technique (ART) cycles. SCSA was performed in neat semen and post DGC. SCSA results were expressed in terms of DFI and high DNA stainability (HDS) cell fractions. The outcome parameter was clinical pregnancy (CP). RESULTS: Scatter-plot diagrams demonstrated that for DGC samples, no DFI cut-off values could be set for in vivo or in vitro fertility. In intrauterine insemination, IVF and ICSI groups the mean difference (95% CI) in DFI post DGC between those who achieved CP and those who did not was 0.2% (-1.7 to 2.0%), 0.4% (-1.9 to 2.8%) and 1.3% (-3.1 to 5.9%), respectively, none of these being statistically significant. The corresponding differences for HDS were 0.1% (-1.3 to 1.5%), 0.1% (-0.7 to 0.9%) and 0.6% (-1.6 to 2.7%), respectively (all P-values >0.6). CONCLUSIONS: SCSA performed in semen prepared by DGC cannot predict the outcome of ART.     

Increased sperm DNA damage in patients with varicocele: relationship with seminal oxidative stress

BACKGROUND: The pathophysiology of the testicular damage in varicocele has not been completely understood. Oxidative stress and related sperm DNA damage have been identified as significant causes of male infertility. The current study was designed to determine the extent of sperm nuclear DNA damage in patients with varicocele and to examine its relationship with oxidative stress. METHODS: Semen samples from 55 patients with clinical varicocele and 25 normozoospermic donors were examined. Varicocele sperm samples were classified as normal or abnormal according to World Health Organization guidelines. Sperm DNA damage was evaluated by the sperm chromatin structure assay/flow cytometry and by the terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay. Levels of reactive oxygen species (ROS) and total antioxidant capacity were assessed by a chemiluminescence assay. RESULTS: DNA fragmentation index (DFI) (percentage of sperm with denatured DNA) values and the percentage of TUNEL-positive cells were significantly greater in patients with varicocele, either with normal (DFI, 20.7 +/- 4.0; TUNEL positive, 26.1 +/- 3.2) or with abnormal (DFI, 35.5 +/- 9.0; TUNEL positive, 32.2 +/- 4.1) semen profile, compared with controls (DFI, 7.1 +/- 0.9; TUNEL positive, 14.2 +/- 1.2). Similarly, ROS levels were significantly higher (P < 0.01) in both groups of patients with varicocele. CONCLUSIONS: The presence of a varicocele is associated with high levels of DNA-damage spermatozoa even in the presence of normal semen profile. The results also indicate that oxidative damage is associated with sperm DNA damage in these patients.     

Sperm DNA integrity assessment in prediction of assisted reproduction technology outcome

BACKGROUND: The sperm chromatin structure assay (SCSA) has beensuggested as a predictor of fertility in vivo as well as invitro. The available data however, have been based on limitednumbers of treatments. We aimed to define the clinical roleof SCSA in assisted reproduction. METHODS: A total of 998 cycles[387 intrauterine insemination (IUI), 388 IVF and 223 ICSI]from 637 couples were included. SCSA results were expressedas DNA fragmentation index (DFI) and high DNA stainable (HDS)cell fractions. Outcome parameters were biochemical pregnancy(BP), clinical pregnancy (CP) and delivery (D). RESULTS: ForIUI, the odds ratios (ORs) for BP, CP and D were significantlylower for couples with DFI >30% as compared with those withDFI 30%. No statistical difference between the outcomes of ICSIversus IVF in the group with DFI 30% was seen. In the DFI >30%group, the results of ICSI were significantly better than thoseof IVF. CONCLUSIONS: DFI can be used as an independent predictorof fertility in couples undergoing IUI. As a result, we proposethat all infertile men should be tested with SCSA as a supplementto the standard semen analysis. When DFI exceeds 30%, ICSI shouldbe the method of choice.     

Exposure to PCB and p, p'-DDE in European and Inuit populations: impact on human sperm chromatin integrity

BACKGROUND: Persistent organochlorine pollutants (POP), such as polychlorinated biphenyls (PCB) and dichlorodiphenyldichloroethylene (p, p'-DDE), are widely found in the environment and considered potential endocrine-disrupting compounds (EDC). Their impact on male fertility is still unknown. METHODS: To explore the hypothesis that POP is associated with altered sperm chromatin integrity, a cross-sectional study involving 707 adult males (193 Inuits from Greenland, 178 Swedish fishermen, 141 men from Warsaw, Poland, and 195 men from Kharkiv, Ukraine) was carried out. Serum levels of 2,2',4,4',5,5'-hexachlorobiphenyl (CB-153), as a proxy of the total PCB burden, and of p,p'-DDE were determined. Sperm chromatin structure assay (SCSA) was used to assess sperm DNA/chromatin integrity. RESULTS: We found a strong and monotonically increasing DNA fragmentation index with increasing serum levels of CB-153 among European but not Inuit men, reaching a 60% higher average level in the highest exposure group. No significant associations were found between SCSA-derived parameters and p, p'-DDE serum concentrations. CONCLUSION: These results suggest that human dietary PCB exposure might have a negative impact on the sperm chromatin integrity of adult males but additional issues, including differences in the genetic background and lifestyle habits, still need to be elucidated.     

Sleep duration is associated with sperm chromatin integrity among young men in Chongqing,China

This study explores whether sleep duration is associated with sperm chromatin integrity. To do so, we conducted a three‐phase panel study of 796 male volunteers from colleges in Chongqing (China) from 2013 to 2015. Sleep duration was measured using a modified Munich Chronotype Questionnaire. Sperm DNA integrity was examined via Sperm Chromatin Structure Assay and Comet assay. Setting 7–7.5 h day^?1 of sleep duration as a reference, either longer or shorter sleep duration was associated negatively with high DNA stainability (HDS ) (P  = 0.009), which reflected the immaturity of sperm chromatin. The volunteers with > 9.0 h day^?1 sleep and those with ≤ 6.5 h day^?1 sleep had 40.7 and 30.3% lower HDS than did volunteers with 7–7.5 h day^?1 sleep. No association was found between sleep duration and DNA fragmentation index or Comet assay parameters. This study suggests that sleep duration is associated with sperm chromatin integrity. Further studies are required to validate these findings and investigate the mechanism underlying this association.     

Value of the sperm chromatin dispersion test in predicting pregnancy outcome in intrauterine insemination: a blind prospective study

BACKGROUND: Sperm DNA integrity has been used as a new marker of sperm quality in the prediction of pregnancy. Nevertheless, no previous study has been performed by analysing the same samples that were employed in assisted reproduction. The main objective of this work was to correlate sperm chromatin dispersion (SCD), measured by the SCD test, with semen parameters and pregnancy outcome in intrauterine insemination (IUI). METHODS: A total of 100 semen samples obtained from males of couples undergoing IUI were analysed by the SCD test before and after swim-up, and the results were correlated with semen parameters and pregnancy outcome. RESULTS: SCD was negatively correlated with sperm motility in both ejaculated and processed semen. Sperm recovered by swim-up did not show a significant improvement in DNA integrity. No correlation was found between SCD and pregnancy outcome in IUI. CONCLUSIONS: DNA dispersion, as measured by the SCD test, is not correlated with pregnancy outcome in IUI.     

The relationship between human sperm apoptosis, morphology and the sperm deformity index

BACKGROUND: This study aimed to assess the relationship between apoptosis in human ejaculated spermatozoa, sperm morphology and the novel sperm deformity index (SDI). METHODS: Semen specimens from 50 healthy donors were prepared by density-gradient centrifugation followed by incubating the prepared sperm with paramagnetic annexin V-conjugated microbeads and subjecting this to magnetic cell sorting (MACS). The procedure delivers two sperm fractions: annexin-negative (non-apoptotic) and annexin-positive (apoptotic). Activated caspase-3 levels and the integrity of the sperm mitochondrial membrane potential (MMP) were assessed as markers of apoptosis in the annexin-negative and -positive aliquots following MACS. Sperm morphology and the SDI scores were assessed using the strict criteria. RESULTS: Compared with the apoptotic sperm subpopulations, the non-apoptotic sperm subpopulations had an improved sperm morphology profile as demonstrated by significantly higher proportions of sperm with normal morphology and significantly lower SDI scores and percentages of sperm with acrosomal defects, midpiece defects, cytoplasmic droplet and tail defects. There was a significant correlation between sperm morphology attributes studied and the expressed apoptotic markers - caspase-3 activation and MMP integrity. CONCLUSIONS: Non-apoptotic sperm fractions have morphologically superior quality sperm compared with apoptotic fractions as reflected by significantly lower SDI scores. The study results may support abortive apoptosis, where the apoptotic mechanism of sperm is already triggered prior to ejaculation.     

Sperm chromatin structure assay parameters as predictors of failed pregnancy following assisted reproductive techniques

The predictive value of sperm chromatin integrity for pregnancy outcome following in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) was studied in 24 men attending a university-based assisted reproductive techniques laboratory using the flow cytometric sperm chromatin structure assay (SCSA). The SCSA is a measure of the susceptibility of sperm DNA to low pH-induced denaturation in situ. The mean percentage of spermatozoa in the neat sample demonstrating DNA denaturation was significantly lower in the seven men that initiated a pregnancy (15.4 +/- 4.6, P = 0.01) than in the 14 men who did not initiate a pregnancy (31.1 +/- 3.2). No pregnancies resulted if > or =27% of the spermatozoa in the neat semen sample showed DNA denaturation. These data demonstrate that SCSA parameters are independent of conventional semen parameters. Furthermore, the SCSA may allow physicians to identify male patients for whom IVF and ICSI will be unlikely to result in pregnancy initiation.     

Dual use of Diff-Quik-like stains for the simultaneous evaluation of human sperm morphology and chromatin status

BACKGROUND: Sperm chromatin status and nuclear DNA damage can be detectedusing well-established assays. However, most techniques aretime-consuming and/or involve elaborate protocols and equipment.We have recently developed a simple and fast method to monitorsperm chromatin status in field conditions using the Diff-Quikassay which is employed in fertility clinics to assess spermmorphology with standard bright field microscopy. In the presentstudy, we demonstrate that any Diff-Quik-like stain can easily,reproducibly and routinely monitor human sperm chromatin statusas well. METHODS: Different Diff-Quik-like stains were used to assess sperm morphologyand the presence of abnormal dark nuclear staining in humansperm from four ART centres. The TUNEL assay was performed inthe same samples, and fertility outcomes were assessed. RESULTS: A significant correlation was found between TUNEL-positive spermand dark sperm nuclei. Moreover, associations were also foundbetween the percentage of dark sperm nuclei and seminal parameters,embryo development rate, embryo quality and clinical pregnancy,as well as with cryptorchidism, and there was a tendency towardsan association with age. A value of 32% abnormal staining issuggested as a predictive threshold for embryo development andpregnancy. CONCLUSIONS: Our results show that any Diff-Quik-like stain, already implementedin most laboratories to assess sperm morphology, can be adaptedas an indicator for chromatin status in human sperm.     

不明原因复发性流产与精子DNA完整性的关系

目的探讨不明原因复发性流产与男方精子DNA完整性的关系。方法精子DNA完整性检测采用精子染色质扩散试验（sperm chromatin dispersion,SCD）,以DNA断裂指数（DNA fragmentation index,DFI）表示。分别对不明原因复发性流产男方与无流产史男方精子进行精液常规分析和DFI分析。结果流产组精子密度、活动率、前向运动率和DFI与生育组比较差异有统计学意义（P〈0.05）。结论不明原因复发性流产可能与精子DNA损伤存在一定关系。     

厦门地区不同职业对男性不育患者精子DNA完整性和精子参数的影响

目的探讨厦门地区不同职业对精子DNA完整性和精子参数的影响。方法将就诊的1509例男性不育患者按照职业分为司机组、计算机工作者组、工人组。将600例正常生育组做为健康对照组。采用SQA—V全自动精子分析仪进行精液常规分析,精子DNA完整性检测采用精子染色质扩散试验（SCD）,以DNA断裂指数（DFI）表示。结果司机组、计算机工作者,工人组的精子密度、活动率、前向运动率和DFI与对照组比较差异有统计学意义（P〈0．05）;司机组和计算机工作者组的精子活动率、前向运动率、精子密度及和DFI与工人组比较差异有统计学意义（P〈0．05）;4组的精液量、精液pH值差异无统计学意义（P〉0．05）。结论司机、计算机工作者和工人可以影响精液质量,从而导致男性不育。