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Sperm DNA fragmentation and recurrent pregnancy loss: a systematic review and meta-analysis
Sperm DNA fragmentation and recurrent pregnancy loss: a systematic review and meta-analysis Dana B. McQueen, M.D., M.A.S., John Zhang, Ph.D., and Jared C. Robins, M.D. Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Northwestern University, Chicago, Illinois
Objective: To investigate the rate of sperm DNA fragmentation in male partners of women with recurrent pregnancy loss and fertile control women. Design: Systematic review and meta-analysis. Setting: Not applicable. Patient(s): A total of 579 male partners of women with recurrent pregnancy loss and 434 male partners fertile control women. Intervention(s): Prospective studies were identified through a Pubmed search. Recurrent pregnancy loss was defined as two or more previous pregnancy losses. Fertile control women had a history of a live birth or ongoing pregnancy. Main Outcome Measure(s): The primary outcome was the rate of sperm DNA fragmentation. The summary measures were reported as mean difference with 95% confidence interval (CI). Result(s): Fifteen prospective studies were included in a qualitative review. Pooled data from 13 studies with sufficient data for metaanalysis suggest that male partners of women with a history of recurrent pregnancy loss have a significantly higher rate of sperm DNA fragmentation compared to the partners of fertile control women: mean difference 11.91, 95% CI 4.97–18.86. Conclusion(s): These findings support an association between sperm DNA fragmentation and recurrent pregnancy loss. However, given the significant heterogeneity between studies and lack of prospective pregnancy outcome data, further large prospective studies are needed. (Fertil SterilÒ 2019;112:54–60. Ó2019 by American Society for Reproductive Medicine.) El resumen está disponible en Español al final del artículo. Key Words: Recurrent pregnancy loss, recurrent miscarriage, sperm, DNA fragmentation Discuss: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/users/16110-fertilityand-sterility/posts/43916-27219
R
ecurrent pregnancy loss (RPL) is a devastating reproductive issue facing couples trying to increase their family size. Unfortunately, despite extensive testing, more than 40% of patients are left without an answer (1). This is challenging for couples seeking an explanation for their losses and an effective treatment. Therefore, research to identify additional causes for RPL is critically important. Research has primarily focused on maternal causes for RPL, but there is growing evidence demonstrating the
impact of male factors. One focus area has been on the sperm DNA fragmentation index (DFI), a measure of the percentage of sperm with DNA damage present in an ejaculate. In an analysis of 2,969 couples, Robinson et al. demonstrated that sperm DNA fragmentation is associated with sporadic miscarriage, with a relative risk of 2.16, 95% confidence interval [CI] 1.54–3.03 (2). As a result, there is growing interest in whether there is a similar relationship between sperm DNA fragmentation and recurrent pregnancy loss.
Received October 29, 2018; revised February 15, 2019; accepted March 4, 2019; published online May 2, 2019. D.B.M. has nothing to disclose. J.Z. has nothing to disclose. J.C.R. has nothing to disclose. Reprint requests: Dana B. McQueen, M.D., M.A.S., 676 N. St. Clair, 18th Floor, Suite 1845, Chicago, IL 60611 (E-mail: [email protected]). Fertility and Sterility® Vol. 112, No. 1, July 2019 0015-0282/$36.00 Copyright ©2019 American Society for Reproductive Medicine, Published by Elsevier Inc. https://doi.org/10.1016/j.fertnstert.2019.03.003 54
Sperm DNA is highly compact to protect the genome from external damage. Although the majority of the genome is tightly packaged by protamines, portions of DNA are located peripherally, more loosely bound to histones and susceptible to oxidative damage. DNA fragmentation in these areas may result in abnormal embryonic development after the activation of the paternal genome (3). Protamine deficiency, reactive oxygen species, and failure to repair DNA damage have been associated with increased sperm DNA fragmentation, whereas dietary antioxidants and varicocele repair can improve sperm DNA fragmentation. One limitation to sperm DNA fragmentation testing is inconsistency in the type of assay used by research VOL. 112 NO. 1 / JULY 2019
Fertility and Sterility® protocols and consequently a wide variability in results. The major assays that measure sperm DNA fragmentation include the sperm chromatin structure assay (SCSA), the terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) assay, the sperm chromatin dispersion (SCD) test, and the comet assay. The SCSA and TUNEL assays use flow cytometry to measure DNA fragmentation, with the SCSA measuring the susceptibility of sperm to denaturation and the TUNEL assay assessing the quantity of DNA strand breaks. The SCD test images sperm directly by means of fluorescent microscopy. Sperm with low DNA fragmentation have a large halo appearance whereas sperm with DNA fragmentation have a small or absent halo. Finally, the comet assay is a method of single-cell electrophoresis and directly measures DNA fragmentation. An advantage of the comet assay is the ability to measure both single-stranded and doublestranded DNA breaks. Although sperm DNA fragmentation is associated with male infertility; specific cutoff points have not been established for each assay. Therefore, the American Society for Reproductive Medicine (ASRM) and the American Urological Society state that there is insufficient evidence to perform routine DNA fragmentation testing in the infertility population (4). The purpose of the present systematic review and meta-analysis was to determine whether there is a significant relationship between sperm DNA fragmentation and recurrent pregnancy loss.
MATERIALS AND METHODS Search and Selection Strategy The analysis was performed according to the Preferred Reporting Item for Systematic Reviews and Meta-analysis (PRISMA) Statement. The review was submitted for registration with the PROSPERO International Prospective Register of Systematic Reviews (ID CRD42018112929). A systematic review of Pubmed was conducted in October 2018. A single author (D.M.) performed independent extraction of articles with the use of predefined Pubmed Medical Subject Headings (MeSH) key words. The MeSH search string combined key words: (Sperm OR (DNA Fragmentation)) AND ((Recurrent pregnancy loss) OR (Recurrent miscarriage)). Articles in languages other than English were excluded. All abstracts were first reviewed for inclusion. The full text articles were then screened (n ¼ 17). Two articles were excluded because they lacked a fertile control group.
Study Selection We included clinical studies comparing the rate of sperm DNA fragmentation in an RPL cohort and a fertile control cohort. RPL was defined by the original publications and included two to three previous pregnancy losses. All control cohorts had proven fertility with one or more live birth or ongoing pregnancy. All assays to assess sperm DNA fragmentation were included. Although our search criteria did not limit results to idiopathic RPL or prospective studies, all studies identified by means of our search criteria identified themselves as prospective and included only couples with an RPL evaluation. VOL. 112 NO. 1 / JULY 2019
Data Extraction and Risk of Bias The Cochrane Handbook for Systematic Reviews was used to evaluate the risk of bias in each study. One author (D.M.) assessed the inclusion criteria, risk of bias at the study level and data extraction (Supplemental Figs. 1 and 2; Supplemental Figs. 1–4 are available online at www.fertstert.org). The primary outcome was the rate of sperm DNA fragmentation. Individual authors were contacted when necessary data was missing from the original publications.
Data Analysis The data analysis was performed by a single author (D.M) with the use of Revmed 5.3. Higgins I2 statistic was used to assess heterogeneity between studies. Because of high heterogeneity (I2 >70%), a random- effect model was used to obtain the pooled estimate. The results were reported as mean difference (MD) with 95% CI, with MD >1 indicating a higher risk of sperm DNA fragmentation in male partners of women with RPL compared with the partners of fertile control women. A funnel plot assessed publication bias (Supplemental Fig. 3). A P value of < .05 was considered to be statistically significant. A subgroup analysis was performed to differentiate the effect size when RPL was defined as two versus three or more pregnancy losses. A second subgroup analysis stratified by type of DNA fragmentation assay was performed.
RESULTS A flow diagram of the systematic review (PRISMA template) is shown in Supplemental Figure 4. A total of 235 trials were initially identified by the search criteria. Seventeen trials were reviewed and two were excluded because of the lack of a fertile control group. Fifteen trials were included in the qualitative analysis, all of which were prospective (3, 5–18). A total of 579 men with RPL were evaluated for sperm DNA fragmentation and compared with 434 control men. The included studies are presented in Table 1. Seven trials defined RPL as a history of two or more pregnancy losses and eight trials defined RPL as a history of three or more pregnancy losses. Five trials required pregnancy losses to be consecutive. All publications included only idiopathic RPL. One manuscript (7) did not describe the specific RPL workup performed. Of the 14 other studies, all required negative antiphospholipid antibodies and uterine cavity evaluation. Thirteen studies required normal thyroid function, ten required normal parental karyotypes, two screened for diabetes, and two required normal prolactin. Four of the 15 reports specifically excluded RPL subjects with concomitant infertility (9, 10, 12, 15), whereas 11 did not report the fertility status of the RPL group. Four publications came from India, two from the United States, two from Iran, two from Tunisia, two from China, one from France, one from the United Kingdom, and one from Spain. To measure sperm DNA fragmentation, six studies used TUNEL assay, three used SCSA, six used SCD test, one used comet assay, one used acridine orange, and one used aniline blue. Four studies used more than one method to detect DNA fragmentation. Eight studies used frozen sperm and seven 55
Qualitative analysis of studies identified in the systematic review. Author, year
Location
Recurrent pregnancy loss
Esquerre-Lamare, 2018
France
R3 losses at <12 wk (n ¼ 33)
R1 live birth (n ¼ 27)
Zidi-Jrah, 2016 Bareh, 2016 Coughlan, 2014
Tunisia USA UK
R2 losses (n ¼ 22) R2 losses at <20 wk (n ¼ 26) R3 consecutive losses (n ¼ 16)
R1 live birth (n ¼ 20) R1 live birth (n ¼ 31) R1 live birth (n ¼ 7)
Ruixue, 2013
China
Currently pregnant (n ¼ 63)
Khadem, 2012 Ribas, 2012
Iran Spain
Kumar, 2012 Zhang, 2012
India China
Absalan, 2012
Iran
R3 pregnancy losses during first trimester (n ¼ 68) R3 losses (n ¼ 30) R2 first or early second trimester losses (n ¼ 20) R3 losses at <20 wk (n ¼ 45) R2 consecutive losses in the first trimester (n ¼ 111) R3 losses at <20 wk (n ¼ 30)
Imam, 2011
India
Brahem, 2011
Tunisia
Venkatesh, 2011 Bhattacharya, 2008
India India
Carrell, 2003
USA
R3 consecutive losses at <20 wk (n ¼ 20) R2 consecutive losses in first or early second trimester (n ¼ 31) R2 losses <24 wk (n ¼ 32) %2 consecutive losses at <8 wk (n ¼ 74) R3 losses at <20 wk (n ¼ 21)
Control
Currently pregnant (n ¼ 30) R1 live birth (n ¼ 25) R1 live birth (n ¼ 20) R1 live birth (n ¼ 30)
Sperm preparation Frozen semen (with cryoprotectant) Washed with PBS, then frozen Fresh semen Density centrifugation gradient vs. fresh semen Fresh semen
Abstinence 3–6 d
SCSA and TUNELa
3–5 d 2–3 d 3–4 d
TUNEL TUNEL SCD and TUNELa
3–5 d
Aniline blue
Fresh semen Frozen semen (with cryoprotectant) Frozen semen Fresh semen
2–3 d >2 d
SCD Comet and SCDa
4d 2–3 d
SCSA SCD
‘‘Fertile’’ and partner with no history of pregnancy loss (n ¼ 30) R1 live birth (n ¼ 20)
Fresh semen
2–5 d
SCD
Frozen semen
3–4 d
SCSA
R1 live birth (n ¼ 20)
Frozen semen
3–4 d
TUNEL
R1 live birth (n ¼ 20) R1 live birth (n ¼ 65)
Frozen semen Fresh semen
4d 2–3 d
SCSA Acridine orange
R1 live birth (Donors) (n ¼ 26)
Frozen semen (with cryoprotectant)
2–5 d
SCD and TUNELa
Note: PBS ¼ phosphate-buffered saline solution; SCD ¼ sperm chromatin dispersion test; SCSA ¼ sperm chromatin structure assay; TUNEL ¼ terminal deoxynucleotide transferase–mediated dUTP nick-end labeling assay. a Assay used for pooled meta-analysis when more than one assay was used in the study. McQueen. Sperm DNA fragmentation and RPL. Fertil Steril 2019.
Assay
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TABLE 1
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FIGURE 1
Primary outcome in overall analysis. McQueen. Sperm DNA fragmentation and RPL. Fertil Steril 2019.
used fresh sperm. Of the studies using frozen sperm, three added cryoprotectant before freezing and four froze whole semen. One study performed density-gradient sperm sorting, whereas all of the other studies used semen without first selecting for motile sperm. All manuscripts performed a semen analysis in addition to the DNA fragmentation testing. Eight of 15 studies reported a significantly lower proportion of spermatozoa with normal morphology among men with RPL compared with control
men. Brahem et al. (12) reported a significant correlation between sperm DNA fragmentation and abnormal sperm morphology (correlation coefficient 0.553; P¼ .018). However, Bhattacharya et al. (16) found no correlation between sperm DNA integrity and sperm parameters. Venkatesh et al. (5) separated men with RPL into two groups: normal semen analysis and abnormal semen analysis. Both groups were found to have significantly higher sperm DNA fragmentation than control samples.
FIGURE 2 Recurrent pregnancy loss as 2 or more losses
Recurrent pregnancy loss as 3 or more losses
Subgroup analysis by definition of recurrent pregnancy loss (RPL). McQueen. Sperm DNA fragmentation and RPL. Fertil Steril 2019.
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FIGURE 3 TUNEL assay
SCD assay
SCSA assay
Comet assay
Subgroup analysis by sperm DNA fragmentation assay. SCD ¼ sperm chromatin dispersion; SCSA ¼ sperm chromatin structure assay; TUNEL ¼ terminal deoxynucleotide transferase–mediated dUTP nick-end labeling. McQueen. Sperm DNA fragmentation and RPL. Fertil Steril 2019.
Synthesis of Results Three studies did not report standard deviations of their outcomes in the original articles. One of the authors provided unpublished means and SDs and the other two studies were excluded from meta-analysis. A total of 901 men were included in the meta-analysis: 517 (57.4%) partners of women with RPL and 384 (42.6%) partners of fertile control women. The pooled analysis of the primary outcome is shown in Figure 1. Male partners of women with RPL had a significantly higher rate of sperm DNA fragmentation than those of fertile control women: MD 10.7, 95% CI 5.82–15.58. Statistical heterogeneity was considerable. A funnel plot is shown in Supplemental Figure 3. A subgroup analysis was performed to differentiate the effect size when RPL was defined as two versus three or more pregnancy losses. The pooled analysis remained statistically significant for both definitions with MDs of 58
12.51 (2.14–22.89) and 9.12 (3.16–15.08), respectively (Fig. 2). A second subgroup analysis was performed to evaluate the various assays used to measure sperm DNA fragmentation. The pooled MD was highest for the TUNEL assay at 14.25 (4.86–23.64) compared with MDs of 3.54 (3.30–10.38), 5.18 (0.31–10.05), and 10.10 (2.10–18.10) for SCD test, SCSA, and Comet assays, respectively (Fig. 3). The statistical heterogeneity remained high despite subgroup analysis.
DISCUSSION We report evidence that male partners of women experiencing RPL have a significantly higher rate of sperm DNA fragmentation compared with male partners of fertile control women. Sperm DNA is tightly bound to protamine, protecting the DNA from external damage. If DNA fragmentation does occur, subtle damage can be repaired by the oocyte after fertilization. VOL. 112 NO. 1 / JULY 2019
Fertility and Sterility® However, significant DNA fragmentation, beyond the threshold repairable by the oocyte, may contribute to poor blast development, implantation failure, and miscarriage (19). Although there was consistently elevated sperm DNA fragmentation in the RPL population, there was considerable heterogeneity among studies. Several factors likely contributed to the heterogeneity measured. First, there are multiple definitions of idiopathic RPL, with some studies including two or more losses and others restricting the cohort to three or more consecutive losses. In addition, the RPL evaluation was inconsistent among studies, with the majority deviating significantly from the recommended ASRM RPL work-up. Second, there are multiple assays used to measure sperm DNA fragmentation, each with different sensitivities. Third, some studies used cryopreserved semen, which can introduce DNA fragmentation, and others used fresh semen. Finally, each study had different criteria for periods of abstinence. Each of these factors may have significantly affected sperm DNA fragmentation in the individual studies as well as the meta-analysis heterogeneity. This heterogeneity persisted despite a subgroup analysis by definition of RPL and type of assay. The vast majority of current literature uses tests of sperm DNA fragmentation on whole semen. By including both motile and nonmotile sperm, nonviable sperm with a higher DNA fragmentation level are included in the overall result. Although this is unlikely to significantly bias results toward RPL or fertile control subjects, a measurement of DNA fragmentation in motile sperm may be more applicable to an RPL cohort that conceives spontaneously. Another potential limitation of the current literature is the inclusion of subjects with concomitant infertility. Four manuscripts described in this review specifically excluded subjects with infertility, while 11 manuscripts did not report the fertility status of the RPL cohort. Elevated DNA fragmentation is increased in the infertile population and therefore this is a potential important confounder. Among those studies limited to a fertile RPL population, two studies found a significant elevation in sperm DNA fragmentation in the RPL cohort (10, 12), whereas two studies showed no difference between RPL and control cohorts (9, 15). In addition, although all studies performed a semen analysis as part of their investigation, only five studies performed a statistical evaluation to adjust for semen parameters when analyzing DNA fragmentation results. In conclusion, this systematic review and meta-analysis suggests that sperm DNA fragmentation is associated with RPL. However, this association does not equate to cause. For sperm DNA fragmentation to become a routine part of the male RPL evaluation, it should be correlated with clinical outcomes. Therefore, future studies should prospectively assess the impact of elevated DNA fragmentation on miscarriage and live birth rates. In addition, to strengthen the evidence that sperm DNA fragmentation affects pregnancy outcomes, the effect of correcting elevated sperm DNA fragmentation with the use of oral antioxidants, microfluidic sperm sorting, and testicular sperm should be investigated in a randomized controlled fashion (20).
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Stephenson MD. Frequency of factors associated with habitual abortion in 197 couples. Fertil Steril 1996;66:24–9. Robinson L, Gallos I, Conner S, Rajkowa M, Miller D, Lewis S, et al. The effect of sperm DNA fragmentation on miscarriage rates: a systematic review and meta-analysis. Hum Reprod 2012;27:2908–17. Imam S, Shamsi M, Kumar K, Deka D, Dada R. Idiopathic recurrent pregnancy loss: role of paternal factors; a pilot study. J Reprod Infertil 2011; 12:267–76. ASRM Practice Committee. The clinical utility of sperm DNA integrity testing: a guideline. Fertil Steril 2013. Venkatesh S, Thilagavathi J, Kumar K, Deka D, Talwar P, Dada R. Cytogenetic, Y chromosome microdeletion, sperm chromatin and oxidative stress analysis in male partners of couples experiencing recurrent spontaneous abortions. Arch Gynecol Obstet 2011;284:1577–84. Khadem N, Poorhoseyni A, Jalali M, Akbary A, Heydari ST. Sperm DNA fragmentation in couplesl with unexplained recurrent spontaneous abortions. Andrologia 2014;46:126–30. Absalan F, Ghannadi A, Kazerooni M, Parifar R, Jamalzadeh F, Amiri S. Value of sperm chromatin dispersion test in couples with unexplained recurrent abortion. J Assist Reprod Genet 2012;29:11–4. Ruizue W, Hongli Z, Zhihong Z, Rulin D, Dongfeng G, Ruizhi L. The impact of semen quality, occupational exposure to environmental factors and lifestyle on recurrent pregnancy loss. J Assist Reprod Genet 2013;30:1513–8. Esquerre-Lamare C, Walschaerts M, Debordeaux L, Moreau J, Bretelle F, Isus F, et al. Sperm aneuploidy and DNA fragmentation in unexplained recurrent pregnancy loss: a multicenter case-control study. Basic Clin Androl 2018;28:4. Zidi-Jrah I, Hajlaoui A, Mougou-Zerelli S, Kammoun M, Meniaoui I, Sallem A, et al. Relationship between sperm aneuploidy, sperm DNA integrity, chromatin packaging, traditional semen parameters, and recurrent pregnancy loss. Fertil Steril 2016;105:58–64. Bareh G, Jacoby E, Binkley P, Chang T, Schenken R, Robinson R. Sperm Deoxyribonucleic Acid (DNA) Damage Assessment in Normozoospermic Male Partners of Couples with Unexplained Recurrent Pregnancy Loss: A Prospective Cohort Study. Fertil Steril 2016;105:329–36. Brahem S, Mehdi M, Landolsi H, Mougou S, Elghezal H, Saad A. Semen parameters and sperm DNA fragmentation as causes of recurrent pregnancy loss. Urology 2011;75:792–6. Coughlan C, Clarke H, Cutting R, Saxton J, Waite S, Ledger W, et al. Sperm DNA Fragmentation, recurrent implantation failure and recurrent miscarriage. Asian J Androl 2015;17:681–5. Carrell D, Liu L. Sperm DNA Fragmentation is increased in couples with unexplained recurrent pregnancy loss. Arch Androl 2003;49:49–55. Zhang L, Wang L, Zhang X, Xu G, Zhang W, Wang K, et al. Sperm chromatin integrity may predict future fertility for unexplained spontaneous abortion patients. Int J Androl 2012;35:752–7. Bhattacharya S. Association of various sperm parameters with unexplained repeated early pregnancy loss—which is most important? Int Urol Nephrol 2008;40:391–5. Ribas-Maynou J, Garcia-Piero A, Fernandez-Encinas A, Amengual M, Prada E, Cortes P, et al. Double stranded sperm DNA breakes, measured by comet assay, are associated with unexplained recurrent miscarriage in couples without a female factor. PLoS One 2012;7:1–9. Kumar K, Deka D, Singh A, Mitra D, Vanitha B, Dada R. Predictive value of DNA integrity analysis in idiopathic recurrent pregnancy loss following spontaneous conception. J Assist Reprod Genet 2012;29:861–7. Agarwal A, Allamaneni S. The effect of sperm DNA damage on assisted reproduction outcomes. A review. Minerva Ginecol 2004;56:235– 45. Cho CL, Agarwal A, Majzoub A, Esteves S. Clinical utility of sperm DNA fragmentation testing: concise practice recommendations. Transl Androl Urol 2017;6(Suppl 4):S366–73.
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ORIGINAL ARTICLE: ANDROLOGY Fragmentacion de ADN espermatico y perdida recurrente de embarazo: Una revisi on sistem atica y meta-an alisis Objetivo: Investigar la tasa de fragmentaci on del ADN espermatico en parejas masculinas de mujeres con perdida de embarazo recurrente y mujeres control fertiles. ~o: Revisi Disen on sistematica y meta-analisis. Entorno: No aplicable. Paciente(s): Un total de 579 parejas masculinas de mujeres con perdida recurrente de embarazo y 434 parejas masculinas de mujeres control fertiles. Intervencion(es): Los estudios prospectivos se identificaron a traves de una b usqueda en Pubmed. La perdida de embarazo recurrente se definio como dos o mas perdidas de embarazos anteriores. Las mujeres fertiles control tenían antecedentes de nacidos vivos o embarazos evolutivos. Principales medidas de resultado: El resultado primario fue la tasa de fragmentaci on de ADN espermatico. Las medidas resumidas se informaron como la diferencia de medias con intervalo de confianza (CI) del 95%. Resultados: En una revisi on cualitativa, se incluyeron quince estudios prospectivos. Datos agrupados de 13 estudios con suficientes datos para el meta-analisis sugieren que las parejas masculinas de mujeres con antecedentes de perdida recurrente de embarazo tienen una tasa significativamente mayor de fragmentacion de ADN espermatico en comparaci on con las parejas de mujeres fertiles control: diferencia de medias 11.91, 95% CI : 4.97 a 18.86. Conclusion: Estos hallazgos apoyan una asociaci on entre la fragmentaci on de ADN espermatico y la perdida recurrente de embarazo. Sin embargo, dada la gran heterogeneidad entre los estudios y la ausencia de datos prospectivos sobre el resultado de embarazo, son necesarios estudios prospectivos mas amplios.
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Assessment of risk of bias. McQueen. Sperm DNA fragmentation and RPL. Fertil Steril 2019.
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SUPPLEMENTAL FIGURE 2
Assessment of risk of bias. McQueen. Sperm DNA fragmentation and RPL. Fertil Steril 2019.
SUPPLEMENTAL FIGURE 3
Funnel plot for assessing publication bias. MD ¼ mean difference; SE(MD) ¼ standard error of the mean difference. McQueen. Sperm DNA fragmentation and RPL. Fertil Steril 2019.
60.e2
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Flow diagram of studies identified in the systematic review (PRISMA template). McQueen. Sperm DNA fragmentation and RPL. Fertil Steril 2019.
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Objective: To investigate the rate of sperm DNA fragmentation in male partners of women with recurrent pregnancy loss and fertile control women. Design: Systematic review and meta-analysis. Setting: Not applicable. Patient(s): A total of 579 male partners of women with recurrent pregnancy loss and 434 male partners fertile control women. Intervention(s): Prospective studies were identified through a Pubmed search. Recurrent pregnancy loss was defined as two or more previous pregnancy losses. Fertile control women had a history of a live birth or ongoing pregnancy. Main Outcome Measure(s): The primary outcome was the rate of sperm DNA fragmentation. The summary measures were reported as mean difference with 95% confidence interval (CI). Result(s): Fifteen prospective studies were included in a qualitative review. Pooled data from 13 studies with sufficient data for metaanalysis suggest that male partners of women with a history of recurrent pregnancy loss have a significantly higher rate of sperm DNA fragmentation compared to the partners of fertile control women: mean difference 11.91, 95% CI 4.97–18.86. Conclusion(s): These findings support an association between sperm DNA fragmentation and recurrent pregnancy loss. However, given the significant heterogeneity between studies and lack of prospective pregnancy outcome data, further large prospective studies are needed. (Fertil SterilÒ 2019;112:54–60. Ó2019 by American Society for Reproductive Medicine.) El resumen está disponible en Español al final del artículo. Key Words: Recurrent pregnancy loss, recurrent miscarriage, sperm, DNA fragmentation Discuss: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/users/16110-fertilityand-sterility/posts/43916-27219
R
ecurrent pregnancy loss (RPL) is a devastating reproductive issue facing couples trying to increase their family size. Unfortunately, despite extensive testing, more than 40% of patients are left without an answer (1). This is challenging for couples seeking an explanation for their losses and an effective treatment. Therefore, research to identify additional causes for RPL is critically important. Research has primarily focused on maternal causes for RPL, but there is growing evidence demonstrating the
impact of male factors. One focus area has been on the sperm DNA fragmentation index (DFI), a measure of the percentage of sperm with DNA damage present in an ejaculate. In an analysis of 2,969 couples, Robinson et al. demonstrated that sperm DNA fragmentation is associated with sporadic miscarriage, with a relative risk of 2.16, 95% confidence interval [CI] 1.54–3.03 (2). As a result, there is growing interest in whether there is a similar relationship between sperm DNA fragmentation and recurrent pregnancy loss.
Received October 29, 2018; revised February 15, 2019; accepted March 4, 2019; published online May 2, 2019. D.B.M. has nothing to disclose. J.Z. has nothing to disclose. J.C.R. has nothing to disclose. Reprint requests: Dana B. McQueen, M.D., M.A.S., 676 N. St. Clair, 18th Floor, Suite 1845, Chicago, IL 60611 (E-mail: [email protected]). Fertility and Sterility® Vol. 112, No. 1, July 2019 0015-0282/$36.00 Copyright ©2019 American Society for Reproductive Medicine, Published by Elsevier Inc. https://doi.org/10.1016/j.fertnstert.2019.03.003 54
Sperm DNA is highly compact to protect the genome from external damage. Although the majority of the genome is tightly packaged by protamines, portions of DNA are located peripherally, more loosely bound to histones and susceptible to oxidative damage. DNA fragmentation in these areas may result in abnormal embryonic development after the activation of the paternal genome (3). Protamine deficiency, reactive oxygen species, and failure to repair DNA damage have been associated with increased sperm DNA fragmentation, whereas dietary antioxidants and varicocele repair can improve sperm DNA fragmentation. One limitation to sperm DNA fragmentation testing is inconsistency in the type of assay used by research VOL. 112 NO. 1 / JULY 2019
Fertility and Sterility® protocols and consequently a wide variability in results. The major assays that measure sperm DNA fragmentation include the sperm chromatin structure assay (SCSA), the terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) assay, the sperm chromatin dispersion (SCD) test, and the comet assay. The SCSA and TUNEL assays use flow cytometry to measure DNA fragmentation, with the SCSA measuring the susceptibility of sperm to denaturation and the TUNEL assay assessing the quantity of DNA strand breaks. The SCD test images sperm directly by means of fluorescent microscopy. Sperm with low DNA fragmentation have a large halo appearance whereas sperm with DNA fragmentation have a small or absent halo. Finally, the comet assay is a method of single-cell electrophoresis and directly measures DNA fragmentation. An advantage of the comet assay is the ability to measure both single-stranded and doublestranded DNA breaks. Although sperm DNA fragmentation is associated with male infertility; specific cutoff points have not been established for each assay. Therefore, the American Society for Reproductive Medicine (ASRM) and the American Urological Society state that there is insufficient evidence to perform routine DNA fragmentation testing in the infertility population (4). The purpose of the present systematic review and meta-analysis was to determine whether there is a significant relationship between sperm DNA fragmentation and recurrent pregnancy loss.
MATERIALS AND METHODS Search and Selection Strategy The analysis was performed according to the Preferred Reporting Item for Systematic Reviews and Meta-analysis (PRISMA) Statement. The review was submitted for registration with the PROSPERO International Prospective Register of Systematic Reviews (ID CRD42018112929). A systematic review of Pubmed was conducted in October 2018. A single author (D.M.) performed independent extraction of articles with the use of predefined Pubmed Medical Subject Headings (MeSH) key words. The MeSH search string combined key words: (Sperm OR (DNA Fragmentation)) AND ((Recurrent pregnancy loss) OR (Recurrent miscarriage)). Articles in languages other than English were excluded. All abstracts were first reviewed for inclusion. The full text articles were then screened (n ¼ 17). Two articles were excluded because they lacked a fertile control group.
Study Selection We included clinical studies comparing the rate of sperm DNA fragmentation in an RPL cohort and a fertile control cohort. RPL was defined by the original publications and included two to three previous pregnancy losses. All control cohorts had proven fertility with one or more live birth or ongoing pregnancy. All assays to assess sperm DNA fragmentation were included. Although our search criteria did not limit results to idiopathic RPL or prospective studies, all studies identified by means of our search criteria identified themselves as prospective and included only couples with an RPL evaluation. VOL. 112 NO. 1 / JULY 2019
Data Extraction and Risk of Bias The Cochrane Handbook for Systematic Reviews was used to evaluate the risk of bias in each study. One author (D.M.) assessed the inclusion criteria, risk of bias at the study level and data extraction (Supplemental Figs. 1 and 2; Supplemental Figs. 1–4 are available online at www.fertstert.org). The primary outcome was the rate of sperm DNA fragmentation. Individual authors were contacted when necessary data was missing from the original publications.
Data Analysis The data analysis was performed by a single author (D.M) with the use of Revmed 5.3. Higgins I2 statistic was used to assess heterogeneity between studies. Because of high heterogeneity (I2 >70%), a random- effect model was used to obtain the pooled estimate. The results were reported as mean difference (MD) with 95% CI, with MD >1 indicating a higher risk of sperm DNA fragmentation in male partners of women with RPL compared with the partners of fertile control women. A funnel plot assessed publication bias (Supplemental Fig. 3). A P value of < .05 was considered to be statistically significant. A subgroup analysis was performed to differentiate the effect size when RPL was defined as two versus three or more pregnancy losses. A second subgroup analysis stratified by type of DNA fragmentation assay was performed.
RESULTS A flow diagram of the systematic review (PRISMA template) is shown in Supplemental Figure 4. A total of 235 trials were initially identified by the search criteria. Seventeen trials were reviewed and two were excluded because of the lack of a fertile control group. Fifteen trials were included in the qualitative analysis, all of which were prospective (3, 5–18). A total of 579 men with RPL were evaluated for sperm DNA fragmentation and compared with 434 control men. The included studies are presented in Table 1. Seven trials defined RPL as a history of two or more pregnancy losses and eight trials defined RPL as a history of three or more pregnancy losses. Five trials required pregnancy losses to be consecutive. All publications included only idiopathic RPL. One manuscript (7) did not describe the specific RPL workup performed. Of the 14 other studies, all required negative antiphospholipid antibodies and uterine cavity evaluation. Thirteen studies required normal thyroid function, ten required normal parental karyotypes, two screened for diabetes, and two required normal prolactin. Four of the 15 reports specifically excluded RPL subjects with concomitant infertility (9, 10, 12, 15), whereas 11 did not report the fertility status of the RPL group. Four publications came from India, two from the United States, two from Iran, two from Tunisia, two from China, one from France, one from the United Kingdom, and one from Spain. To measure sperm DNA fragmentation, six studies used TUNEL assay, three used SCSA, six used SCD test, one used comet assay, one used acridine orange, and one used aniline blue. Four studies used more than one method to detect DNA fragmentation. Eight studies used frozen sperm and seven 55
Qualitative analysis of studies identified in the systematic review. Author, year
Location
Recurrent pregnancy loss
Esquerre-Lamare, 2018
France
R3 losses at <12 wk (n ¼ 33)
R1 live birth (n ¼ 27)
Zidi-Jrah, 2016 Bareh, 2016 Coughlan, 2014
Tunisia USA UK
R2 losses (n ¼ 22) R2 losses at <20 wk (n ¼ 26) R3 consecutive losses (n ¼ 16)
R1 live birth (n ¼ 20) R1 live birth (n ¼ 31) R1 live birth (n ¼ 7)
Ruixue, 2013
China
Currently pregnant (n ¼ 63)
Khadem, 2012 Ribas, 2012
Iran Spain
Kumar, 2012 Zhang, 2012
India China
Absalan, 2012
Iran
R3 pregnancy losses during first trimester (n ¼ 68) R3 losses (n ¼ 30) R2 first or early second trimester losses (n ¼ 20) R3 losses at <20 wk (n ¼ 45) R2 consecutive losses in the first trimester (n ¼ 111) R3 losses at <20 wk (n ¼ 30)
Imam, 2011
India
Brahem, 2011
Tunisia
Venkatesh, 2011 Bhattacharya, 2008
India India
Carrell, 2003
USA
R3 consecutive losses at <20 wk (n ¼ 20) R2 consecutive losses in first or early second trimester (n ¼ 31) R2 losses <24 wk (n ¼ 32) %2 consecutive losses at <8 wk (n ¼ 74) R3 losses at <20 wk (n ¼ 21)
Control
Currently pregnant (n ¼ 30) R1 live birth (n ¼ 25) R1 live birth (n ¼ 20) R1 live birth (n ¼ 30)
Sperm preparation Frozen semen (with cryoprotectant) Washed with PBS, then frozen Fresh semen Density centrifugation gradient vs. fresh semen Fresh semen
Abstinence 3–6 d
SCSA and TUNELa
3–5 d 2–3 d 3–4 d
TUNEL TUNEL SCD and TUNELa
3–5 d
Aniline blue
Fresh semen Frozen semen (with cryoprotectant) Frozen semen Fresh semen
2–3 d >2 d
SCD Comet and SCDa
4d 2–3 d
SCSA SCD
‘‘Fertile’’ and partner with no history of pregnancy loss (n ¼ 30) R1 live birth (n ¼ 20)
Fresh semen
2–5 d
SCD
Frozen semen
3–4 d
SCSA
R1 live birth (n ¼ 20)
Frozen semen
3–4 d
TUNEL
R1 live birth (n ¼ 20) R1 live birth (n ¼ 65)
Frozen semen Fresh semen
4d 2–3 d
SCSA Acridine orange
R1 live birth (Donors) (n ¼ 26)
Frozen semen (with cryoprotectant)
2–5 d
SCD and TUNELa
Note: PBS ¼ phosphate-buffered saline solution; SCD ¼ sperm chromatin dispersion test; SCSA ¼ sperm chromatin structure assay; TUNEL ¼ terminal deoxynucleotide transferase–mediated dUTP nick-end labeling assay. a Assay used for pooled meta-analysis when more than one assay was used in the study. McQueen. Sperm DNA fragmentation and RPL. Fertil Steril 2019.
Assay
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FIGURE 1
Primary outcome in overall analysis. McQueen. Sperm DNA fragmentation and RPL. Fertil Steril 2019.
used fresh sperm. Of the studies using frozen sperm, three added cryoprotectant before freezing and four froze whole semen. One study performed density-gradient sperm sorting, whereas all of the other studies used semen without first selecting for motile sperm. All manuscripts performed a semen analysis in addition to the DNA fragmentation testing. Eight of 15 studies reported a significantly lower proportion of spermatozoa with normal morphology among men with RPL compared with control
men. Brahem et al. (12) reported a significant correlation between sperm DNA fragmentation and abnormal sperm morphology (correlation coefficient 0.553; P¼ .018). However, Bhattacharya et al. (16) found no correlation between sperm DNA integrity and sperm parameters. Venkatesh et al. (5) separated men with RPL into two groups: normal semen analysis and abnormal semen analysis. Both groups were found to have significantly higher sperm DNA fragmentation than control samples.
FIGURE 2 Recurrent pregnancy loss as 2 or more losses
Recurrent pregnancy loss as 3 or more losses
Subgroup analysis by definition of recurrent pregnancy loss (RPL). McQueen. Sperm DNA fragmentation and RPL. Fertil Steril 2019.
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FIGURE 3 TUNEL assay
SCD assay
SCSA assay
Comet assay
Subgroup analysis by sperm DNA fragmentation assay. SCD ¼ sperm chromatin dispersion; SCSA ¼ sperm chromatin structure assay; TUNEL ¼ terminal deoxynucleotide transferase–mediated dUTP nick-end labeling. McQueen. Sperm DNA fragmentation and RPL. Fertil Steril 2019.
Synthesis of Results Three studies did not report standard deviations of their outcomes in the original articles. One of the authors provided unpublished means and SDs and the other two studies were excluded from meta-analysis. A total of 901 men were included in the meta-analysis: 517 (57.4%) partners of women with RPL and 384 (42.6%) partners of fertile control women. The pooled analysis of the primary outcome is shown in Figure 1. Male partners of women with RPL had a significantly higher rate of sperm DNA fragmentation than those of fertile control women: MD 10.7, 95% CI 5.82–15.58. Statistical heterogeneity was considerable. A funnel plot is shown in Supplemental Figure 3. A subgroup analysis was performed to differentiate the effect size when RPL was defined as two versus three or more pregnancy losses. The pooled analysis remained statistically significant for both definitions with MDs of 58
12.51 (2.14–22.89) and 9.12 (3.16–15.08), respectively (Fig. 2). A second subgroup analysis was performed to evaluate the various assays used to measure sperm DNA fragmentation. The pooled MD was highest for the TUNEL assay at 14.25 (4.86–23.64) compared with MDs of 3.54 (3.30–10.38), 5.18 (0.31–10.05), and 10.10 (2.10–18.10) for SCD test, SCSA, and Comet assays, respectively (Fig. 3). The statistical heterogeneity remained high despite subgroup analysis.
DISCUSSION We report evidence that male partners of women experiencing RPL have a significantly higher rate of sperm DNA fragmentation compared with male partners of fertile control women. Sperm DNA is tightly bound to protamine, protecting the DNA from external damage. If DNA fragmentation does occur, subtle damage can be repaired by the oocyte after fertilization. VOL. 112 NO. 1 / JULY 2019
Fertility and Sterility® However, significant DNA fragmentation, beyond the threshold repairable by the oocyte, may contribute to poor blast development, implantation failure, and miscarriage (19). Although there was consistently elevated sperm DNA fragmentation in the RPL population, there was considerable heterogeneity among studies. Several factors likely contributed to the heterogeneity measured. First, there are multiple definitions of idiopathic RPL, with some studies including two or more losses and others restricting the cohort to three or more consecutive losses. In addition, the RPL evaluation was inconsistent among studies, with the majority deviating significantly from the recommended ASRM RPL work-up. Second, there are multiple assays used to measure sperm DNA fragmentation, each with different sensitivities. Third, some studies used cryopreserved semen, which can introduce DNA fragmentation, and others used fresh semen. Finally, each study had different criteria for periods of abstinence. Each of these factors may have significantly affected sperm DNA fragmentation in the individual studies as well as the meta-analysis heterogeneity. This heterogeneity persisted despite a subgroup analysis by definition of RPL and type of assay. The vast majority of current literature uses tests of sperm DNA fragmentation on whole semen. By including both motile and nonmotile sperm, nonviable sperm with a higher DNA fragmentation level are included in the overall result. Although this is unlikely to significantly bias results toward RPL or fertile control subjects, a measurement of DNA fragmentation in motile sperm may be more applicable to an RPL cohort that conceives spontaneously. Another potential limitation of the current literature is the inclusion of subjects with concomitant infertility. Four manuscripts described in this review specifically excluded subjects with infertility, while 11 manuscripts did not report the fertility status of the RPL cohort. Elevated DNA fragmentation is increased in the infertile population and therefore this is a potential important confounder. Among those studies limited to a fertile RPL population, two studies found a significant elevation in sperm DNA fragmentation in the RPL cohort (10, 12), whereas two studies showed no difference between RPL and control cohorts (9, 15). In addition, although all studies performed a semen analysis as part of their investigation, only five studies performed a statistical evaluation to adjust for semen parameters when analyzing DNA fragmentation results. In conclusion, this systematic review and meta-analysis suggests that sperm DNA fragmentation is associated with RPL. However, this association does not equate to cause. For sperm DNA fragmentation to become a routine part of the male RPL evaluation, it should be correlated with clinical outcomes. Therefore, future studies should prospectively assess the impact of elevated DNA fragmentation on miscarriage and live birth rates. In addition, to strengthen the evidence that sperm DNA fragmentation affects pregnancy outcomes, the effect of correcting elevated sperm DNA fragmentation with the use of oral antioxidants, microfluidic sperm sorting, and testicular sperm should be investigated in a randomized controlled fashion (20).
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Stephenson MD. Frequency of factors associated with habitual abortion in 197 couples. Fertil Steril 1996;66:24–9. Robinson L, Gallos I, Conner S, Rajkowa M, Miller D, Lewis S, et al. The effect of sperm DNA fragmentation on miscarriage rates: a systematic review and meta-analysis. Hum Reprod 2012;27:2908–17. Imam S, Shamsi M, Kumar K, Deka D, Dada R. Idiopathic recurrent pregnancy loss: role of paternal factors; a pilot study. J Reprod Infertil 2011; 12:267–76. ASRM Practice Committee. The clinical utility of sperm DNA integrity testing: a guideline. Fertil Steril 2013. Venkatesh S, Thilagavathi J, Kumar K, Deka D, Talwar P, Dada R. Cytogenetic, Y chromosome microdeletion, sperm chromatin and oxidative stress analysis in male partners of couples experiencing recurrent spontaneous abortions. Arch Gynecol Obstet 2011;284:1577–84. Khadem N, Poorhoseyni A, Jalali M, Akbary A, Heydari ST. Sperm DNA fragmentation in couplesl with unexplained recurrent spontaneous abortions. Andrologia 2014;46:126–30. Absalan F, Ghannadi A, Kazerooni M, Parifar R, Jamalzadeh F, Amiri S. Value of sperm chromatin dispersion test in couples with unexplained recurrent abortion. J Assist Reprod Genet 2012;29:11–4. Ruizue W, Hongli Z, Zhihong Z, Rulin D, Dongfeng G, Ruizhi L. The impact of semen quality, occupational exposure to environmental factors and lifestyle on recurrent pregnancy loss. J Assist Reprod Genet 2013;30:1513–8. Esquerre-Lamare C, Walschaerts M, Debordeaux L, Moreau J, Bretelle F, Isus F, et al. Sperm aneuploidy and DNA fragmentation in unexplained recurrent pregnancy loss: a multicenter case-control study. Basic Clin Androl 2018;28:4. Zidi-Jrah I, Hajlaoui A, Mougou-Zerelli S, Kammoun M, Meniaoui I, Sallem A, et al. Relationship between sperm aneuploidy, sperm DNA integrity, chromatin packaging, traditional semen parameters, and recurrent pregnancy loss. Fertil Steril 2016;105:58–64. Bareh G, Jacoby E, Binkley P, Chang T, Schenken R, Robinson R. Sperm Deoxyribonucleic Acid (DNA) Damage Assessment in Normozoospermic Male Partners of Couples with Unexplained Recurrent Pregnancy Loss: A Prospective Cohort Study. Fertil Steril 2016;105:329–36. Brahem S, Mehdi M, Landolsi H, Mougou S, Elghezal H, Saad A. Semen parameters and sperm DNA fragmentation as causes of recurrent pregnancy loss. Urology 2011;75:792–6. Coughlan C, Clarke H, Cutting R, Saxton J, Waite S, Ledger W, et al. Sperm DNA Fragmentation, recurrent implantation failure and recurrent miscarriage. Asian J Androl 2015;17:681–5. Carrell D, Liu L. Sperm DNA Fragmentation is increased in couples with unexplained recurrent pregnancy loss. Arch Androl 2003;49:49–55. Zhang L, Wang L, Zhang X, Xu G, Zhang W, Wang K, et al. Sperm chromatin integrity may predict future fertility for unexplained spontaneous abortion patients. Int J Androl 2012;35:752–7. Bhattacharya S. Association of various sperm parameters with unexplained repeated early pregnancy loss—which is most important? Int Urol Nephrol 2008;40:391–5. Ribas-Maynou J, Garcia-Piero A, Fernandez-Encinas A, Amengual M, Prada E, Cortes P, et al. Double stranded sperm DNA breakes, measured by comet assay, are associated with unexplained recurrent miscarriage in couples without a female factor. PLoS One 2012;7:1–9. Kumar K, Deka D, Singh A, Mitra D, Vanitha B, Dada R. Predictive value of DNA integrity analysis in idiopathic recurrent pregnancy loss following spontaneous conception. J Assist Reprod Genet 2012;29:861–7. Agarwal A, Allamaneni S. The effect of sperm DNA damage on assisted reproduction outcomes. A review. Minerva Ginecol 2004;56:235– 45. Cho CL, Agarwal A, Majzoub A, Esteves S. Clinical utility of sperm DNA fragmentation testing: concise practice recommendations. Transl Androl Urol 2017;6(Suppl 4):S366–73.
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ORIGINAL ARTICLE: ANDROLOGY Fragmentacion de ADN espermatico y perdida recurrente de embarazo: Una revisi on sistem atica y meta-an alisis Objetivo: Investigar la tasa de fragmentaci on del ADN espermatico en parejas masculinas de mujeres con perdida de embarazo recurrente y mujeres control fertiles. ~o: Revisi Disen on sistematica y meta-analisis. Entorno: No aplicable. Paciente(s): Un total de 579 parejas masculinas de mujeres con perdida recurrente de embarazo y 434 parejas masculinas de mujeres control fertiles. Intervencion(es): Los estudios prospectivos se identificaron a traves de una b usqueda en Pubmed. La perdida de embarazo recurrente se definio como dos o mas perdidas de embarazos anteriores. Las mujeres fertiles control tenían antecedentes de nacidos vivos o embarazos evolutivos. Principales medidas de resultado: El resultado primario fue la tasa de fragmentaci on de ADN espermatico. Las medidas resumidas se informaron como la diferencia de medias con intervalo de confianza (CI) del 95%. Resultados: En una revisi on cualitativa, se incluyeron quince estudios prospectivos. Datos agrupados de 13 estudios con suficientes datos para el meta-analisis sugieren que las parejas masculinas de mujeres con antecedentes de perdida recurrente de embarazo tienen una tasa significativamente mayor de fragmentacion de ADN espermatico en comparaci on con las parejas de mujeres fertiles control: diferencia de medias 11.91, 95% CI : 4.97 a 18.86. Conclusion: Estos hallazgos apoyan una asociaci on entre la fragmentaci on de ADN espermatico y la perdida recurrente de embarazo. Sin embargo, dada la gran heterogeneidad entre los estudios y la ausencia de datos prospectivos sobre el resultado de embarazo, son necesarios estudios prospectivos mas amplios.
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Assessment of risk of bias. McQueen. Sperm DNA fragmentation and RPL. Fertil Steril 2019.
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Assessment of risk of bias. McQueen. Sperm DNA fragmentation and RPL. Fertil Steril 2019.
SUPPLEMENTAL FIGURE 3
Funnel plot for assessing publication bias. MD ¼ mean difference; SE(MD) ¼ standard error of the mean difference. McQueen. Sperm DNA fragmentation and RPL. Fertil Steril 2019.
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Flow diagram of studies identified in the systematic review (PRISMA template). McQueen. Sperm DNA fragmentation and RPL. Fertil Steril 2019.
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