|Year : 2020 | Volume
| Issue : 3 | Page : 296-301
Sperm DNA fragmentation in Chinese couples with unexplained recurrent pregnancy loss
Xiao-Bin Zhu, Qian Chen, Wei-Min Fan, Zhi-Hong Niu, Bu-Fang Xu, Ai-Jun Zhang
Reproductive Medical Center of Ruijin Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai 200025, China
|Date of Submission||22-Sep-2018|
|Date of Acceptance||02-Apr-2019|
|Date of Web Publication||23-Jul-2019|
Reproductive Medical Center of Ruijin Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai 200025
Source of Support: None, Conflict of Interest: None
We aimed to study the association between sperm DNA fragmentation and recurrent pregnancy loss (RPL) in the Chinese population via a retrospective observational study of Chinese couples who had experienced RPL between May 2013 and August 2018. The study population included 461 men from couples with RPL and 411 men from a control group (couples with clinical pregnancy via in vitro fertilization owing to female causes). Routine semen analysis, sperm chromatin analysis, and microscopic (high-power) morphological analysis were performed using semen samples. Semen samples were assessed for volume, sperm count, and motility. The sperm DNA fragmentation index (DFI) was calculated, and the median DFI was obtained. Men were categorized as having normal (37.8%; DFI ≤ 15.0%), moderate (33.6%; 15.0% < DFI < 30.0%), or severe (28.6%; DFI ≥ 30.0%) DNA fragmentation levels. The percentage of men with severe DNA fragmentation was significantly higher in the RPL (42.3%) group than that in the control group (13.1%), whereas the percentage of men with normal levels of DNA fragmentation was significantly lower in the RPL group (22.8%) than that in the control group (54.7%). Subsequent analysis also demonstrated that the sperm DNA fragmentation rate had a moderate reverse correlation with the sperm progressive motility rate (r = −0.47, P < 0.001) and the total motile sperm count (r = −0.31, P < 0.001). We found a positive correlation between RPL and sperm DNA fragmentation. The results suggest that increased sperm DNA damage is associated with RPL.
Keywords: DNA fragmentation index; recurrent pregnancy loss; sperm chromatin structure assay
|How to cite this article:|
Zhu XB, Chen Q, Fan WM, Niu ZH, Xu BF, Zhang AJ. Sperm DNA fragmentation in Chinese couples with unexplained recurrent pregnancy loss. Asian J Androl 2020;22:296-301
|How to cite this URL:|
Zhu XB, Chen Q, Fan WM, Niu ZH, Xu BF, Zhang AJ. Sperm DNA fragmentation in Chinese couples with unexplained recurrent pregnancy loss. Asian J Androl [serial online] 2020 [cited 2020 May 30];22:296-301. Available from: http://www.ajandrology.com/text.asp?2020/22/3/296/263268 - DOI: 10.4103/aja.aja_60_19
Xiao-Bin Zhu, Qian Chen
These authors contributed equally to this work.
| Introduction|| |
Recurrent pregnancy loss (RPL) is defined as two or more consecutive miscarriages according to the American Society for Reproductive Medicine. The known etiological causes of RPL include genetic factors, uterine anatomy, hormonal factors, immune system dysfunction, and thrombosis. However, the etiology remains unidentified in about 50% of the RPL couples. In the past, among couples with RPL, it was always the women who primarily underwent numerous medical examinations, with the male contribution being commonly associated with karyotype abnormalities alone. However, evidence suggests that patients with karyotype 46, XY may present with a high percentage of spermatozoa with aneuploidies. Male gametes provide 50% of the genetic material, and there is a chance that these sperm with genetic abnormalities or epigenetic changes may fertilize the oocyte. However, this would lead to chromosomal damage, thereby seriously affecting early embryonic development.
Following extensive research, it has been confirmed that sperm DNA damage is associated with infertility, lower rate of pregnancy following artificial insemination, and reduced rates of high quality embryos and blastocysts after both in vitro fertilization (IVF) and intracytoplasmic sperm injection.,,,,, The testing of sperm DNA fragments has opened a new era in the comprehensive evaluation of infertility. It may also potentially contribute to the increasing success of assisted reproductive technology (ART) in the future. There have also been sporadic reports on the correlation between sperm DNA damage and RPL.,,,, These studies present conflicting results, which may be related to the detection methods employed or the sample size assessed. However, there are no large sample data to support this correlation.
There is increasing evidence that sperm DNA fragmentation (SDF) abnormalities not only severely affect fertility, but also have a close relationship with RPL. Therefore, the aim of this study was to examine a possible relationship between SDF and RPL due to no specific reason in Chinese women and as well as to evaluate the routine sperm parameters and SDF in their husbands' semen using the sperm chromatin structure assay (SCSA) method.
| Patients and Methods|| |
This retrospective observational study analyzed the data of couples with RPL who visited the Center of Reproductive Medicine of Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine in Shanghai, China, as outpatients between May 2013 and August 2018. Among the RPL cases, 339 couples had miscarried twice, 102 couples had miscarried 3 times, and 20 couples had miscarried 4 or more times. The average number of miscarriages was 2.32. We identified 461 couples who had normal karyotypes and tested negative for the presence of endocrine disorders, antiphospholipid and lupus antibodies, and coagulation defects. Women with uterine structural abnormalities or age above 40 years were excluded. Likewise, the men presenting for fertility evaluation were reviewed, and those included in this study had no history of orchitis, toxic exposure, chronic illness, or radiation exposure in the last 3 months. The control group comprised 411 IVF patients who visited our center between May 2013 and August 2018, and their data were retrospectively analyzed. Only women with tubal factors for infertility were recruited and the exclusion criteria were the same as those for the women in the RPL group. Furthermore, the analysis was restricted to women who had given birth to live babies or carried a pregnancy for more than 3 months following IVF treatments. To better compare the differences in SDF and high DNA stainability (HDS) between the two groups, as described in several other studies conducted in recent years,, we divided our study population into three groups based on the SDF levels. DNA fragmentation equal to or lower than 15% was regarded as normal, DNA fragmentation more than 15% and lower than 30% was considered as moderate, and DNA fragmentation equal to or more than 30% was considered as severe. Furthermore, we also divided the study population into two groups based on sperm HDS levels, with the normal cutoff value being <15.0%. Informed consent was obtained from all participants. This study has been approved by the Research and Ethics Committee of Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine.
Semen samples were collected by masturbation after 3–7 days of abstinence. After liquefaction, the semen volume was measured, and sperm concentration, total motility, and progressive motility were analyzed using a Computer-assisted Sperm Analysis (CASA) system (Sperm Class Analyzer; Microptic, Barcelona, Spain), and the total motile sperm count (TMSC) (semen volume × sperm concentration × progressive motility) was calculated. Sperm morphology was determined according to the 5th World Health Organization guidelines. Morphological assessment was based on high-power microscopic evaluation of the sperm for intactness of membranes of the acrosome, head, neck, midpiece, and tail. The semen smears were fixed on slides, stained using Diff-Quik (Biomart, Shenzhen, China), and then observed through oil immersion light microscopy (BX41, Olympus, Center Valley, PA, USA) with a magnification of ×100.
Samples were run using a BD FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA), recording 5000 events. The SCSA protocol has been described elsewhere. Briefly, thawed samples (100 μl) were combined with 200 μl of acid detergent (pH 1.2) for 30 s. The sample was then stained with 600 μl of acridine orange (AO) staining solution (CellPro Biotech Co., Ltd., Ningbo, China) (600 μl AO 1.0 mg ml−1 to 100 ml staining buffer pH 6.0) and allowed to rest for a total of 3 min. Under AO stain, double-stranded DNA fluoresces green and single-stranded DNA fluoresces red. The extent of DNA damage was expressed as the DNA fragmentation index (DFI), which was calculated by assessing the ratio of red to total fluorescent cells using the flow cytometer software (DFIView 2010 Alpha11.15, CellPro Biotech Co., Ltd., Ningbo, China). HDS represents immature spermatozoa with incomplete chromatin condensation stained with the most intense green color.
Statistical analysis was performed using the Statistical Package for the Social Sciences software, version 15 (SPSS Inc., Chicago, IL, USA). Continuous variables were presented as mean value ± standard deviation (s.d.), and comparisons between the RPL and control groups were determined using the Student's t-test; categorical variables were presented as count (percentage), and comparisons between the RPL and control groups were determined using the chi-squared test or Mann–Whitney U test. The Spearman's correlation test was used to determine correlations between the DFI and the semen parameters. Receiver operating characteristic (ROC) curve analysis was applied to obtain the cutoff value of DFI to differentiate patients from controls, and the sensitivity and specificity for the best cutoff point were then assessed. Moreover, multivariate logistic regression analysis was used to determine the factors predicting RPL. All tests were two-sided. P < 0.05 was considered statistically significant.
| Results|| |
Sperm DFI analysis and routine semen analysis for semen parameters such as volume and pH of the sample as well as progressive motility, morphology, and HDS of the sperm were performed in 872 men. The relationship between the two groups was analyzed as shown in [Table 1]. Regarding semen parameters, there was a statistically significant difference between the RPL and control groups only for DFI (P < 0.001) and sperm progressive motility (P = 0.047). Those enrolled in the study included 461 women in the age range of 21–39 (mean: 30.88 ± 3.62) years who had experienced RPL and 411 women in the age range of 22–39 (mean: 30.40 ± 3.58) years as controls. There were no statistically significant differences between the ages of the women (P = 0.056).
To study the distribution of DFI in the RPL population, the RPL and control couples were subdivided into three groups on the basis of the cutoff of DFI, that is, 15.0% and 30.0%. Of the 461 RPL and 411 control samples analyzed, 105 (22.8%) and 225 (54.7%) had a sperm DFI of 0.0%–15.0%, 161 (34.9%) and 132 (32.1%) had a sperm DFI of 15.0%–30.0%, and 195 (42.3%) and 54 (13.1%) had a sperm DFI of 30.0%–100.0%, respectively. The RPL and control couples were also subdivided into two groups on the basis of the cutoff HDS of 15.0%. Of the 461 RPL samples analyzed, 424 (92.0%) had a sperm HDS of 0.0%–15.0%, and 37 (8.0%) had a sperm HDS of more than 15.0%. In the control group, 386 (93.9%) samples had sperm HDS of 0.0%–15.0% and 25 (6.1%) samples had a sperm HDS of more than 15.0%. The different DFI and HDS ranges in the two groups were compared, as shown in [Table 2]. There was a statistically significant difference in the percentage of DFI between the normal subgroup and the severe subgroup. With an increase in the DFI, the percentage of RPL also increased [Figure 1]a. There was no significant difference observed on comparison of the HDS ranges of the male partners in the RPL and control groups [Figure 1]b.
|Table 2: The comparison of different DNA fragmentation index and high DNA stainability ranges in recurrent pregnancy loss male partners and controls|
Click here to view
|Figure 1: Tendency regarding different DFI and HDS ranges for the RPL and control groups. (a) Sperm of men from couples with a history of RPL tended to have a higher DFI than do sperm of men from the control group. (b) There was no tendency for different HDS ranges for the RPL and control couples. with a history of RPL and the controls. RPL: recurrent pregnancy loss; DFI: DNA fragmentation index; HDS: high DNA stainability.|
Click here to view
The relationship between semen parameters and DNA fragmentation in 461 RPL couples is shown [Figure 2]. Using Spearman's rank correlation coefficient, a moderate inverse relationship was seen between sperm progressive motility and DNA fragmentation (r = −0.47, P < 0.001). SDF also showed a moderate inverse correlation with TMSC (r = −0.31, P < 0.01). A mild inverse correlation was seen between sperm concentration and DNA fragmentation (r = −0.16, P < 0.01), whereas a mild correlation was observed between sperm DNA fragmentation and male age (r = 0.15, P < 0.01). SDF showed no correlation with semen volume, sperm morphology, HDS, and number of RPL.
|Figure 2: Correlation between sperm DFI and semen parameters, age, and the number of RPL in recurrent pregnancy loss patients. The relationship between male age, semen variables, and sperm DFI was analyzed by Spearman's correlation test. (a) A mild correlation was seen in male age, (b) no correlation in semen volume, (c) a moderate inverse relationship in sperm progressive motility, (d) a mild inverse relationship in mean sperm concentration, (e) a moderate inverse relationship in TMSC, (f) no correlation in mean sperm normal forms, (g) sperm HDS, and (h) number of RPL with sperm DFI. RPL: recurrent pregnancy loss; DFI: DNA fragmentation index; HDS: high DNA stainability; TMSC: total motile sperm count.|
Click here to view
The area under the curve was 0.713 (P < 0.001; 95% CI: 0.679–0.747), with 81.8% sensitivity and 54.2% specificity [Figure 3]. According to the ROC curve analysis, a sperm DFI of approximately 24.6% was used as the threshold to distinguish between the RPL group and the control group. [Table 3] shows the results of regression models of the association between variables predicting RPL. A higher risk of RPL was observed with increased SDF (OR = 1.096, 95% CI: 1.078–1.115, P < 0.001), older age of the female partner (OR = 1.120, 95% CI: 1.062–1.182, P < 0.001), and lower progressive motility (OR = 0.905, 95% CI: 0.866–0.945, P = 0.003).
|Figure 3: Receiver operating curve analysis of DFI in PRL and control group. Using receiver operating characteristic curve analysis, a threshold value of 24.6% was obtained to discriminate from the control group. The area under the curve was 0.713 (P < 0.001; 95% CI: 0.679–0.747), with 81.8 % sensitivity and 54.2 % specificity. AUC: area under the curve; RPL: recurrent pregnanc loss; DFI: DNA fragmentation index; CI: confidence interval.|
Click here to view
|Table 3: Multivariate logistic regression analyses of factors affecting recurrent pregnancy loss|
Click here to view
| Discussion|| |
In this study, we investigated the relationship between the rate of RPL in women and the sperm DFI in their respective partners in a relatively large sample in China. The main finding from this study was that increased SDF may be a risk factor in couples experiencing RPL. Two different statistical analyses showed that sperm from men in the RPL group have a higher percentage of DNA damage than do sperm from men in the control group, as shown by two different statistical analyses (P < 0.001). These data were in accordance with the results of other studies,,,,, and suggest an association between increased SDF and a history of RPL. As for the other semen parameters, there was no significant difference between the two groups except for sperm progressive motility, with lower progressive motility being associated with an increased risk of RPL; older age of the female partner was also found to be associated with increased RPL. An inverse relationship was found between sperm progressive motility and DNA fragmentation, which may explain the reason for the a statistically significant difference between the RPL group and the controls in sperm progressive motility.
Although it is well known that sperm DNA integrity plays a vital role in the development of the embryo and in fetal wellbeing, and while there has also been growing interest in the use of DFI as a marker of evaluating RPL, the correlation between DFI and RPL has remained highly controversial. This diversity in opinion may be due to the limited caseload as well as the different methods used in the evaluation having different sensitivities and specificities. The most commonly used methods are SCSA, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and sperm chromatin dispersion (SCD).,,, The TUNEL assay detects both single- and double-stranded DNA breaks by labeling the free 3'-OH terminus with the large terminal deoxynucleotidyl transferase (TdTA) enzyme. The SCSA test determines the percentage of sperm stained with AO in a semen sample that fluoresces red (broken DNA) or green (intact DNA) following an acid denaturation step. The SCD test is based on the principle that DNA fragments of sperm cannot produce a “halo” of dispersed DNA rings after acidic denaturation and nucleoprotein removal. The number of sperm without DNA fragments was assessed by microscopy.
A meta-analysis of seven eligible papers on the research carried out by Zini et al. concluded that spontaneous pregnancy loss is associated with sperm DFI. The results were based on SCSA and TUNEL array, and the sample size ranged from 50 to 388. Leach et al. indicated that 108 couples with a history of RPL showed sperm with high levels of DNA fragmentation on evaluation using SCSA. In addition, Kumar et al. and Kamkar et al., using the same method, observed that the DFI was higher in the RPL group of 42 patients compared to that in a control group of 45 patients. Carlini et al. investigated 112 men from RPL couples, 114 infertile men with 1 or more impaired semen parameters, and 114 fertile men with high-quality semen by analyzing the SDF using TUNEL, and they found that the DFI was higher in the RPL group than that in the fertile controls (18.8% ± 7.0% vs 12.8% ± 5.3%, P < 0.001), and similar to that in infertile patients. Most of these studies used either the SCSA or the TUNEL method, and the study population was relatively large. Our results are in accordance with the above-mentioned research. However, there are some studies that have observed that the DFI was higher in the RPL group of patients than that in the control group using the SCD method.,
Others, in contrast, reported that there was no significant correlation between DNA fragmentation and RPL and concluded that DFI was not an important cause and predictive factor for RPL. Gil-Villa et al. evaluated the DFI in a control group (18.5% ± 4.2%) and RPL group (16.3% ± 4.0%) using the SCSA test and found no significant difference between 23 couples with history of RPL and 11 men with recent fertility. Bellver et al. found that there was no statistically significant difference in the DFI (using the SCD test method) between a group of 30 patients with RPL and the 30 controls, and Coughlan et al. came to the same conclusion using the SCD test in 16 RPL patients.
To our knowledge, the present study is the first investigation of the male factor in RPL following natural conception in a large cohort of Chinese patients. We also analyzed the correlation between semen parameters and DFI. Progressive motility of sperm, TMSC, sperm concentration, and male age showed correlation to the DFI, with no significant differences for other semen parameters. Previous studies conducted to demonstrate the correlation between SDF and semen parameters showed mixed results in RPL patients. Zhang et al. showed that there were no significant differences in ejaculate volume, sperm concentration, or percentage of abnormal forms between 111 RPL men and 30 healthy fertile controls. Brahem et al. found that sperm motility was higher in the control group (P < 0.001), and they did not find any statistically significant difference in other semen parameters between the RPL and control groups. Bhattacharya et al. studied 74 RPL men and 65 fertile men and found no significant difference in age and semen parameters. Further, Khadem et al. also did not find any statistically significant difference in semen parameters except for a negative correlation between SDF and progressive motility (r = −0.613; P < 0.001) and percentage of abnormal forms (r = −0.764; P < 0.001). Carlini et al. found that SDF had a positive correlation with sperm morphology, although this was not statistically significant. However, there was a moderate inverse correlation between SDF and progressive motility (r = −0.41, P < 0.001). Besides, they found no correlation between SDF and the total sperm count. The inconsistency in the results of different studies is due to the differences in the techniques used, sample size, and cutoff values of DFI.
In our findings, SDF showed moderate inverse correlations with progressive motility (r = −0.47, P < 0.001) and TMSC (r = −0.31, P < 0.001) and mild inverse correlations with sperm concentration (r = −0.16, P < 0.001), indicating that sperm DNA damage might be a key factor leading to the decrease in semen quality. It has been described by some authors that men with a history of RPL had a higher incidence of sperm with poor motility than that of men from a control group., Furthermore, Xue et al. found that sperm DFI, as an independent factor, could predict male fertility even better than routine semen parameters.
Some studies have shown that the sperm DFI tends to increase with increasing paternal age.,, Cohen-Bacrie et al. reported a significant correlation between SDF and paternal age in a prospective study of 1633 patients using TUNEL. Our data also suggest that increasing male age is correlated with decreasing sperm DNA integrity (r = 0.15, P < 0.01), which is consistent with the findings of these studies.
Despite a number of studies showing that RPL and sperm DFI have a strong correlation, the potential mechanism of the effects of high sperm fragmentation on RPL remains unclear. Sperm DNA integrity is an important requisite for the correct transmission of genetic material to the offspring, and its impairment increases the risk of abortion. The production of DNA fragments in sperm is usually caused by external factors, such as reactive oxygen species (ROS), rather than programmed cell death., This is supported by the fact that the use of antioxidant therapy in men reduced oxidative DNA damage. In their study, Menezo et al. reported that the use of oral antioxidant therapy could reduce the sperm DFI, especially in the setting of oxidative DNA damage, and significantly improve sperm DNA quality. DNA strand breaks usually occur during meiosis, and oxidative stress induces DNA degeneration, which results in single- and double-stranded breaks. The spermatozoa carrying damaged DNA can fertilize and bind to oocytes, but with the paternal genome activated, it may interfere with the development of the embryo, leading to regulation failure of paternal genes in early embryos.,,,,, Another factor that has a possible role in RPL with high sperm fragmentation is the repair mechanism of the oocyte on sperm DNA damage. Hamatani et al. reported that sperm DNA may be repaired by oocytes up to a threshold of female age ≤35 years. Our data also suggest that female age could be a risk factor for RPL (OR = 1.120, 95% CI = 1.062–1.182, P < 0.001).
ROC curves and the area under the ROC curve (AUC) were used to assess the feasibility of DFI in distinguishing RPL and control cases. The value (25%) of DFI from RPL cases by using ROC curve analysis was lower than the 30% value previously reported threshold for male infertility. A minor limitation of this study is that the two groups of women underwent different treatments, with the women from the control group having received ovarian stimulation. However, the control group completed the IVF cycle treatment within 1 month after the DFI test, so the quality of sperm was less likely to be affected by environmental and lifestyle factors.
On the basis of the results above, our study demonstrated that SDF is an important cause of RPL, and couples with a history of RPL showed a higher incidence of SDF and poor progressive motility of the sperm. These findings indicate that testing for DNA fragmentation has a certain predictive value in the assessment of the prospective risk of RPL; moreover, the higher the level of sperm DFI, the higher the risk of RPL. It is necessary to perform an SDF test in couples experiencing RPL.
| Author Contributions|| |
XBZ and QC designed the project, recruited the patients, reviewed and analyzed the data, and wrote the paper. WMF conducted semen tests and SCSA tests, and ZHN conducted the statistical analysis. BFX also recruited the patients and analyzed the data. AJZ conceived this study, performed data analysis, and prepared the manuscript. All authors have read and approved the final manuscript.
| Competing Interests|| |
The authors declared no competing interests.
| Acknowledgments|| |
This work was supported by grants from the National Natural Science Foundation of China (No 81100469, 81671517) and the Scientific Research Foundation of Shanghai Municipal Commission of Health and Planning (201840060).
| References|| |
Medicine PCotASfR. Evaluation and treatment of recurrent pregnancy loss: a committee opinion. Fertil Steril
2012; 98: 1103–11.
Stephenson M, Kutteh W. Evaluation and management of recurrent early pregnancy loss. Clin Obstet Gynecol
2007; 50: 132–45.
Gil-Villa AM, Cardona-Maya W, Agarwal A, Sharma R, Cadavid A. Assessment of sperm factors possibly involved in early recurrent pregnancy loss. Fertil Steril
2010; 94: 1465–72.
Burrello N, Vicari E, Shin P, Agarwal A, Palma AD, et al.
Lower sperm aneuploidy frequency is associated with high pregnancy rates in ICSI programmes. Hum Reprod
2003; 18: 1371.
Agarwal A, Majzoub A, Esteves SC, Ko E, Ramasamy R, et al.
Clinical utility of sperm DNA fragmentation testing: practice recommendations based on clinical scenarios. Transl Androl Urol
2016; 5: 935–50.
Benchaib M, Braun V, Lornage J, Hadj S, Salle B, et al.
Sperm DNA fragentation decreases the pregnancy rate in an assisted reproductive technique. Hum Reprod
2003; 18: 1023–8.
Bungum M, Humaidan P, Spano M, Jepson K, Bungum L, et al.
The predictive value of sperm chromatin structure assay (SCSA) parameters for the outcome of intrauterine insemination, IVF and ICSI. Hum Reprod
2004; 19: 1401–8.
Cissen M, Wely MV, Scholten I, Mansell S, Bruin JP, et al.
Measuring sperm dna fragmentation and clinical outcomes of medically assisted reproduction: a systematic review and meta-analysis. PLoS One
2016; 11: e0165125.
Osman A, Alsomait H, Seshadri S, El-Toukhy T, Khalaf Y. The effect of sperm DNA fragmentation on live birth rate after IVF or ICSI: a systematic review and meta-analysis. Reprod Biomed Online
2015; 30: 120–7.
Sadeghi MR, Lakpour N, Heidari-Vala H, Hodjat M, Amirjannati N, et al.
Relationship between sperm chromatin status and ICSI outcome in men with obstructive azoospermia and unexplained infertile normozoospermia. Rom J Morphol Embryol
2011; 52: 645–51.
Evenson D, Jost L. Sperm chromatin structure assay is useful for fertility assessment. Methods Cell Sci
2000; 22: 169–89.
Carlini T, Paoli D, Pelloni M, Faja F, Dal Lago A, et al.
Sperm DNA fragmentation in Italian couples with recurrent pregnancy loss. Reprod Biomed Online
2017; 34: 58–65.
Caseiro AL, Regalo A, Pereira E, Esteves T, Fernandes F, et al.
Implication of sperm chromosomal abnormalities in recurrent abortion and multiple implantation failure. Reprod Biomed Online
2015; 31: 481–5.
Khadem N, Poorhoseyni A, Jalali M, Akbary A, Heydari ST. Sperm DNA fragmentation in couples with unexplained recurrent spontaneous abortions. Andrologia
2014; 46: 126–30.
Kumar K, Deka D, Singh A, Mitra DK, Vanitha BR, et al.
Predictive value of DNA integrity analysis in idiopathic recurrent pregnancy loss following spontaneous conception. J Assist Reprod Genet
2012; 29: 861–7.
Leach M, Aitken RJ, Sacks G. Sperm DNA fragmentation abnormalities in men from couples with a history of recurrent miscarriage. Aust N Z J Obstet Gynaecol
2015; 55: 379–83.
Evenson DP, Larson KL, Jost LK. Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques. J Androl
2002; 23: 25–43.
Virro MR, Larson-Cook KL, Evenson DP. Sperm chromatin structure assay (SCSA) parameters are related to fertilization, blastocyst development, and ongoing pregnancy in in vitro
fertilization and intracytoplasmic sperm injection cycles. Fertil Steril
2004; 81: 1289–95.
Hamilton JA, Cissen M, Brandes M, Smeenk JM, de Bruin JP, et al.
Total motile sperm count: a better indicator for the severity of male factor infertility than the WHO sperm classification system. Hum Reprod
2015; 30: 1110–21.
World Health Organization. WHO Laboratory Manual for the Examination and Processing of Human Semen. 5th
ed. Geneva: World Health Organization; 2010.
Coughlan C, Clarke H, Cutting R, Saxton J, Waite S, et al.
Sperm DNA fragmentation, recurrent implantation failure and recurrent miscarriage. Asian J Androl
2015; 17: 681–5.
Bungum M. Sperm DNA integrity assessment: a new tool in diagnosis and treatment of fertility. Obstet Gynecol Int
2012; 2012: 531042.
Evenson DP, Darzynkiewicz Z, Melamed MR. Relation of mammalian sperm chromatin heterogeneity to fertility. Science
1980; 210: 1131–3.
Morris ID, Ilott S, Dixon L, Brison DR. The spectrum of DNA damage in human sperm assessed by single cell gel electrophoresis (Comet assay) and its relationship to fertilization and embryo development. Hum Reprod
2002; 17: 990–8.
Sharma R, Masaki J, Agarwal A. Sperm DNA fragmentation analysis using the TUNEL assay. Methods Mol Biol
2013; 927: 121–36.
Fernandez JL, Muriel L, Rivero MT, Goyanes V, Vazquez R, et al.
The sperm chromatin dispersion test: a simple method for the determination of sperm DNA fragmentation. J Androl
2003; 24: 59–66.
Zini A, Boman JM, Belzile E, Ciampi A. Sperm DNA damage is associated with an increased risk of pregnancy loss after IVF and ICSI: systematic review and meta-analysis. Hum Reprod
2008; 23: 2663–8.
Kamkar N, Ramezanali F, Sabbaghian M. The relationship between sperm DNA fragmentation, free radicals and antioxidant capacity with idiopathic repeated pregnancy loss. Reprod Biol
2018; 18: 330–5.
Absalan F, Ghannadi A, Kazerooni M, Parifar R, Jamalzadeh F, et al.
Value of sperm chromatin dispersion test in couples with unexplained recurrent abortion. J Assist Reprod Genet
2012; 29: 11–4.
Bellver J, Meseguer M, Muriel L, Garcia-Herrero S, Barreto MA, et al.
Y chromosome microdeletions, sperm DNA fragmentation and sperm oxidative stress as causes of recurrent spontaneous abortion of unknown etiology. Hum Reprod
2010; 25: 1713–21.
Zhang L, Wang L, Zhang X, Xu G, Zhang W, et al.
Sperm chromatin integrity may predict future fertility for unexplained recurrent spontaneous abortion patients. Int J Androl
2012; 35: 752–7.
Brahem S, Mehdi M, Landolsi H, Mougou S, Elghezal H, et al.
Semen parameters and sperm DNA fragmentation as causes of recurrent pregnancy loss. Urology
2011; 78: 792–6.
Bhattacharya SM. Association of various sperm parameters with unexplained repeated early pregnancy loss–which is most important? Int Urol Nephrol
2008; 40: 391–5.
Lu JC, Jing J, Chen L, Ge YF, Feng RX, et al.
Analysis of human sperm DNA fragmentation index (DFI) related factors: a report of 1010 subfertile men in China. Reprod Biol Endocrinol
2018; 16: 23.
Xue X, Wang WS, Shi JZ, Zhang SL, Zhao WQ, et al.
Efficacy of swim-up versus density gradient centrifugation in improving sperm deformity rate and DNA fragmentation index in semen samples from teratozoospermic patients. J Assist Reprod Genet
2014; 31: 1161–6.
Bojar I, Witczak M, Wdowiak A. Biological and environmental conditionings for a sperm DNA fragmentation. Ann Agric Environ Med
2013; 20: 865–8.
Cohen-Bacrie P, Belloc S, Menezo YJ, Clement P, Hamidi J, et al.
Correlation between DNA damage and sperm parameters: a prospective study of 1,633 patients. Fertil Steril
2009; 91: 1801–5.
Das M, Al-Hathal N, San-Gabriel M, Phillips S, Kadoch IJ, et al.
High prevalence of isolated sperm DNA damage in infertile men with advanced paternal age. J Assist Reprod Genet
2013; 30: 843–8.
Henkel R, Hajimohammad M, Stalf T, Hoogendijk C, Mehnert C, et al.
Influence of deoxyribonucleic acid damage on fertilization and pregnancy. Fertil Steril
2004; 81: 965–72.
Wright C, Milne S, Leeson H. Sperm DNA damage caused by oxidative stress: modifiable clinical, lifestyle and nutritional factors in male infertility. Reprod Biomed Online
2014; 28: 684–703.
Menezo YJ, Hazout A, Panteix G, Robert F, Rollet J, et al.
Antioxidants to reduce sperm DNA fragmentation: an unexpected adverse effect. Reprod Biomed Online
2007; 14: 418–21.
Badouard C, Menezo Y, Panteix G, Ravanat JL, Douki T, et al.
Determination of new types of DNA lesions in human sperm. Zygote
2008; 16: 9–13.
Brinkworth MH. Paternal transmission of genetic damage: findings in animals and humans. Int J Androl
2000; 23: 123–35.
Nagy ZP, Verheyen G, Tournaye H, Van Steirteghem AC. Special applications of intracytoplasmic sperm injection: the influence of sperm count, motility, morphology, source and sperm antibody on the outcome of ICSI. Hum Reprod
1998; 13 Suppl 1: 143–54.
Robinson WP, Beever C, Brown CJ, Stephenson MD. Skewed X inactivation and recurrent spontaneous abortion. Semin Reprod Med
2001; 19: 175–81.
Rubio C, Gil-Salom M, Simon C, Vidal F, Rodrigo L, et al.
Incidence of sperm chromosomal abnormalities in a risk population: relationship with sperm quality and ICSI outcome. Hum Reprod
2001; 16: 2084–92.
Hamatani T, Falco G, Carter MG, Akutsu H, Stagg CA, et al.
Age-associated alteration of gene expression patterns in mouse oocytes. Hum Mol Genet
2004; 13: 2263–78.
Evenson DP, Wixon R. Clinical aspects of sperm DNA fragmentation detection and male infertility. Theriogenology
2006; 65: 979–91.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]