Article
Sperm DNA fragmentation is a novel biomarker for early pregnancy loss

https://doi.org/10.1016/j.rbmo.2020.09.016Get rights and content

Highlights

  • Men with miscarriages have twice the sperm DNA damage than fertile donors

  • Alkaline Comet assay shows links between miscarriage and poor sperm DNA quality

  • Average, Low and High Comet Scores are all robust biomarkers for miscarriage

  • Sperm DNA testing may provide diagnosis and novel options for miscarriage couples

Abstract

Research question

Spontaneous pregnancy loss affects 10–15% of couples, with 1–2% suffering recurrent pregnancy loss and 50% of miscarriages remaining unexplained. Male genomic integrity is essential for healthy offspring, meaning sperm DNA quality may be important in maintaining a pregnancy. Does sperm DNA fragmentation measured by alkaline Comet assay act as a biomarker for early pregnancy loss?

Design

Sperm DNA fragmentation was measured by alkaline Comet test in 76 fertile donors and 217 men whose partners had recently experienced miscarriage. Couples were divided into five groups for analysis: one miscarriage after spontaneous conception; two or more miscarriages after spontaneous conception; one miscarriage after fertility treatment; two or more miscarriages after fertility treatment and biochemical pregnancy.

Results

Receiver operator characteristic curve analysis was used to determine ability of the average Comet score (ACS), low Comet score (LCS) and high Comet score (HCS) to diagnose miscarriage and develop clinical thresholds comparing men whose partners have miscarried with men with recently proven fertility. Male partners of women who had miscarried had higher sperm DNA damage (ACS 33.32 ± 0.57%) than fertile men (ACS 14.87 ± 0.66%; P < 0.001). Average Comet score, HCS and LCS all have promise as being highly predictive of sporadic and recurrent miscarriage using clinical thresholds from comparisons with fertile men's spermatozoa: receiver operating characteristic curve AUC for ACS ≥26%, 0.965; LCS ≤70%, 0.969; HCS ≥2%, 0.883; P <0.0001.

Conclusions

Sperm DNA damage measured by the alkaline Comet has promise as a robust biomarker for sporadic and recurrent miscarriage after spontaneous or assisted conception, and may provide novel diagnoses and guidance for future fertility pathways.

Introduction

Miscarriage is a devastating condition affecting a couple's current wellbeing and their relationship and life expectations. A sporadic miscarriage occurs in 15% of all clinical pregnancies, after detecting a fetal heartbeat. For women using home pregnancy testing kits and proving positive for a biochemical pregnancy, the incidence can reach 50% (Chard, 1991; Wilcox et al., 1998). Recurrent miscarriage is defined as ‘the loss of two or more pregnancies from the time of conception until 24 weeks of gestation’, as per the current European guideline, which has been reclassified from three consecutive pregnancy losses (Bender Atik et al., 2018). This is even more distressing to the couple and, despite a plethora of studies, few conclusive reasons exist for its occurrence (Aitken and De Iuliis, 2010); up to 30% remain unexplained and hence no robust treatments are available.

The spermatozoa and egg make equal contributions to the genome of the embryo; therefore, either could lead to fatal flaws at this stage of embryonic development, resulting in miscarriage. Yet, it is only recently that a link between sperm DNA and miscarriage has been explored. This has proved to be a useful route of enquiry as the integrity of sperm DNA is crucial for the accurate transmission of the father's genetic information to the embryo. Five systematic reviews and meta-analyses (Zini et al., 2008; Robinson et al., 2012; Osman et al., 2015; Zidi-Jrah et al., 2016; Tan et al., 2019) have reported that sperm DNA damage is significantly correlated to an increased risk of miscarriage after natural conceptions and after IVF and intracytoplasmic sperm injection (ICSI). At the time of publication of these reviews, few studies using the sensitive Comet assay had been undertaken (Kirkman-Brown and De Jonge, 2017).

The aims of the present study were to compare low, high and average damage levels between semen samples from fertile donors and men whose partners have had sporadic or recurrent miscarriage after natural conception and assisted reproductive technology (ART) conception to determine if the alkaline Comet assay can act as a biomarker for early pregnancy loss.

Section snippets

Materials and methods

All reagents were sourced from Sigma-Aldrich, USA.

The effect of age, smoking and alcohol consumption of men

Demographics of paternal age (Kruskal–Wallis, chi-squared 4 = 2.932, P = 0.569) and self-reported lifestyle hazards, such as smoking tobacco (Fishers exact test, P = 0.133 [two- tailed]) and drinking alcohol (Fishers exact test, P = 0.564 [two-tailed] showed no statistically significant differences between the five study groups (Table 1). Male age, female partner age and number of miscarriages experienced are presented in Table 2. Age of the female partner who had experienced miscarriage older

Discussion

The present study identifies distinct differences in average damage and proportions of both lowly and highly damaged spermatozoa between men with proven fertility and men whose partners have experienced miscarriages either sporadically or recurrently and by both natural conception and ART conception.

Men have been excluded from investigations into the causes of a couple's miscarriage until recently, partly because of the historical belief that if a man's spermatozoa achieved a pregnancy, any

Acknowledgements

Thanks to Cryos International for providing fertile donor samples and Examenlab Ltd for funding the study. This study was funded by Examenlab Ltd.

Lesley Haddock is the research and development manager at Examen Lab Ltd, a diagnostic company focusing on male infertility. Her research activities focus on the measurement of sperm DNA fragmentation.

Key message

Average Comet score, low Comet score and high Comet score measures of sperm DNA damage by the alkaline Comet may all be robust biomarkers for sporadic and recurrent miscarriage after either spontaneous or assisted conception and may provide pioneering diagnosis and

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  • Cited by (0)

    Lesley Haddock is the research and development manager at Examen Lab Ltd, a diagnostic company focusing on male infertility. Her research activities focus on the measurement of sperm DNA fragmentation.

    Key message

    Average Comet score, low Comet score and high Comet score measures of sperm DNA damage by the alkaline Comet may all be robust biomarkers for sporadic and recurrent miscarriage after either spontaneous or assisted conception and may provide pioneering diagnosis and guidance for future routes to fertility, if confirmed by other studies.

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