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Sperm DNA fragmentation is a novel biomarker for early pregnancy loss

Published:September 21, 2020DOI:https://doi.org/10.1016/j.rbmo.2020.09.016

      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.

      KEYWORDS

      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 T.
      Frequency of implantation and early pregnancy loss in natural cycles.
      ;
      • Wilcox A.J.
      • Weinberg C.R.
      • Baird D.D.
      Post-ovulatory ageing of the human oocyte and embryo failure.
      ). 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 R.
      • Christiansen O.B.
      • Elson J.
      • Kolte A.M.
      • Lewis S.
      • Middeldorp S.
      • Nelen W.
      • Peramo B.
      • Quenby S.
      • Vermeulen N.
      • Goddijn M.
      ESHRE guideline: recurrent pregnancy loss.
      ). This is even more distressing to the couple and, despite a plethora of studies, few conclusive reasons exist for its occurrence (
      • Aitken R.J.
      • De Iuliis G.N.
      On the possible origins of DNA damage in human spermatozoa.
      ); 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 A.
      • Boman J.
      • Belzile E.
      • Reproduction A.C.-H.
      U., 2008. Sperm DNA damage is associated with an increased risk of pregnancy loss after IVF and ICSI: systematic review and meta-analysis.
      ;
      • Robinson L.
      • Gallos I.D.
      • Conner S.J.
      • Rajkhowa M.
      • Miller D.
      • Lewis S.
      • Kirkman-Brown J.
      • Coomarasamy A.
      The effect of sperm DNA fragmentation on miscarriage rates: A systematic review and meta-analysis.
      ;
      • 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.
      ;
      • Zidi-Jrah I.
      • Hajlaoui A.
      • Mougou-Zerelli S.
      • Kammoun M.
      • Meniaoui I.
      • Sallem A.
      • Brahem S.
      • Fekih M.
      • Bibi M.
      • Saad A.
      • Ibala-Romdhane S.
      Relationship between sperm aneuploidy, sperm DNA integrity, chromatin packaging, traditional semen parameters, and recurrent pregnancy loss Presented at the 17th World Congress on in Vitro Fertilization, Tunis, Tunisia, on September 4-7, 2013.
      ;
      • Tan J.
      • Taskin O.
      • Albert A.
      Online, M.B.-R. biomedicine
      ) 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 J.C.
      • De Jonge C.
      Sperm DNA fragmentation in miscarriage – a promising diagnostic, or a test too far?.
      ).
      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.

      Materials and methods

      All reagents were sourced from Sigma-Aldrich, USA.

       Study populations

      Group A included 76 fertile donors. Inclusion criteria were time to pregnancy less than 12 months of unprotected intercourse; healthy; normal semen according to the world Health Organization (

      World Health Organization, 2010. Examination and processing of human semen. World Health Edition, F, 286.

      ); no hereditary diseases; seronegative for human immunodeficiency virus, syphilis, hepatitis B and C, herpes and cytomegalovirus; and no bacterial infections. All men were aged 18–45 years in accordance with Danish law.
      Group B included male partners of women who had experienced first-trimester miscarriages (n = 217). Two hundred and two men aged 25–45 years were recruited. Only 15 men were aged over 45 years and, of these, five men were aged over 50 years. These men were subdivided into groups depending on their partner's miscarriage history: first miscarriage after natural conception (n = 18); two or more miscarriages after natural conception (n = 147); first miscarriage after ART conception (n = 19); two or more miscarriages after ART conception (n = 7) or miscarriage after biochemical pregnancy (n = 67).
      The study was retrospective as couples were included after they had experienced at least one miscarriage. Semen samples were provided after the miscarriage at the review appointment. This was within 3 months of the miscarriage, so the spermatozoa tested were within the same spermatogenic cycle as the most recent miscarriage.
      Products of conception from these couples had not been available for laboratory investigation after their miscarriages; therefore, no history on cause of miscarriage was available.

       Sperm DNA damage assessment by Comet assay

      Semen samples were collected by masturbation after 2–5 days of abstinence. Fertile samples were cryopreserved using a cryoprotectant supplemented, fast freezing protocol. Samples from miscarriage partners were plunge frozen without cryoprotectant. In validation of our plunge freeze without cryoprotectant, data from a range of fertile and infertile men showed 30.1 ± 4.6% (mean ± SEM) DNA damage in fresh semen compared with 34.3 ± 5% DNA damage in plunge frozen semen without cryoprotectant (P = 0.13 [n = 157]) (unpublished data).
      Sperm DNA fragmentation was assessed using the single cell gel electrophoresis (alkaline Comet) assay, previously modified for human spermatozoa (
      • Hughes C.M.
      • Lewis S.E.M.
      • Mckelvey-Martin V.J.
      • Thompson W.
      Reproducibility of human sperm DNA measurements using the alkaline single cell gel electrophoresis assay.
      ;
      • Donnelly E.T.
      • O'Connell M.
      • McClure N.
      • Lewis S.E.M.
      Differences in nuclear DNA fragmentation and mitochondrial integrity of semen and prepared human spermatozoa.
      ), which quantifies the level of DNA damage in individual spermatozoa and, therefore, can be used to determine the degree of heterogeneity of DNA quality in the whole sperm population in semen. Briefly, aliquots of native semen were adjusted using phosphate buffered saline to a sperm concentration of 2 × 106 ml−1 and embedded in agarose gel. Embedded cells were then subjected to membrane lysis, protamine and histone removal, electrophoresis, SYBR Gold staining and Comet scoring (Komet 7.0, Andor Technologies, Belfast, UK), with analysis of 50 sperm cells per slide in duplicate. All steps were carried out in a temperature and humidity-controlled environment to prevent induction of DNA damage during processing. Our previous study has reported an intra-assay coefficient of variation of 6% for this assay (
      • Donnelly E.T.
      • O'Connell M.
      • McClure N.
      • Lewis S.E.M.
      Differences in nuclear DNA fragmentation and mitochondrial integrity of semen and prepared human spermatozoa.
      ;
      • Agbaje I.M.
      • Rogers D.A.
      • McVicar C.M.
      • McClure N.
      • Atkinson A.B.
      • Mallidis C.
      • Lewis S.E.M.
      Insulin dependant diabetes mellitus: Implications for male reproductive function.
      ).

       Statistical analysis

      XLSTAT-Biomed (Addinsoft, New York, NY, USA) was used for statistical analyses. Study variables included the conventional average Comet score (ACS) (mean per cent fragmentation of all Comets scored) and the novel parameters: the low Comet score (LCS) (per cent of Comets with a low level of DNA fragmentation) and the high Comet score (HCS) (per cent of Comets with a high level of DNA fragmentation) as detailed by
      • Nicopoullos J.
      • Vicens-Morton A.
      • Lewis S.
      • Lee K.
      • Larsen P.
      • Ramsey J.
      • Yap T.
      • Minhas S.
      Novel use of COMET parameters of sperm DNA damage may increase its utility to diagnose male infertility and predict live births following both IVF and ICSI.
      , sporadic or recurrent miscarriage, conception type and fertility. Comet profiles were compared for each group by calculating the mean and SE. Comparisons of the distribution of DNA damage across the semen samples by ACS, LCS and HCS in men whose partners had experienced miscarriages and fertile men were carried out by Mann–Whitney U test. P < 0.05 was considered to be significant.
      To evaluate the ability of each Comet parameter to diagnose miscarriage, threshold values for the three Comet parameters were determined by receiver operating characteristic curve analysis, using the maximum sum of sensitivity and specificity. A total of 76 fertile sperm donors (CRYOS, Aarhus, Denmark) were compared with 217 partners of women who had miscarried.

       Ethical approval

      The study received approval by an internal review board on 27 November 2019.

      Results

       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 or younger than 38 years showed no significant difference in relation to levels of sperm DNA damage (U = 41160.000, n = 553, P = 0.114) (Table 3), with a median of 32.59% DNA damage in the partners of women experiencing miscarriage who were younger than 38 years and a median of 33.96% DNA damage in the partners of women experiencing miscarriage who were older than 38 years (weighted analysis was used to give more equal representation of the two groups; <38 years original n = 134, weight = 2; >38 years original n = 57, weight = 5). When divided into the five miscarriage groups, comparison of Comet scores of men whose partners were either younger than 38 years or 38 years or older showed no significant difference except for the HCS in the biochemical pregnancy group (P = 0.017) (Table 3).
      TABLE 1LIFESTYLE FACTORS OF MEN WHOSE PARTNERS HAVE EXPERIENCED MISCARRIAGES
      GroupAverage male age (range)Smokers, %No alcohol, %Low alcohol, %Moderate alcohol, %High alcohol, %
      Total (n = 217)38 (25–71)11.9316.5943.7831.342.76
      Sporadic miscarriage after natural conception (n = 18)38 (32–46)11.1116.6750.0011.115.56
      Recurrent miscarriage after natural conception (n = 147)37 (25–55)12.2418.3744.2231.293.40
      Sporadic miscarriage after ART conception (n = 19: ICSI n = 3, IVF n = 16)40 (31–71)21.0515.7942.1131.585.26
      Recurrent miscarriage after ART conception (n = 7: ICSI n = 3, IVF n = 3, IVF/ICSI n = 1)37 (31–42)42.860.0057.1428.570.00
      Biochemical pregnancy (n = 67)38 (28–71)8.9613.4352.2429.850.00
      Significance testsKruskal–Wallis

      P = 0.569
      Fisher's exact

      P = 0.133
      Fisher's exact

      P = 0.564
      Mean age, self-reported smoking levels and self-reported alcohol consumption of 217 men whose partners had experienced miscarriages divided into those who have experienced sporadic miscarriage after natural conception; recurrent miscarriage after natural conception; sporadic miscarriage after assisted reproductive technology (ART) conception; recurrent miscarriage after ART conception; and those with biochemical pregnancy.
      Regarding alcohol consumption (n = 3, n = 4, n = 1, n = 1 and n = 3 men in each group, respectively, did not answer the question).
      ICSI, intracytoplasmic sperm injection.
      TABLE 2MALE AND FEMALE PARTNER AVERAGE AGES, AGE RANGES AND MISCARRIAGE HISTORY FOR EACH MISCARRIAGE GROUP
      GroupAverage male age (range)Average female age (range)Average number of miscarriages experienced (range)
      Total (n = 217 male; n = 191 female)38 (25–71)36 (25–46)3 (0–10)
      Sporadic miscarriage after natural conception (n = 18 male; n = 17 female)38 (32–46)38 (32–45)0
      Recurrent miscarriage after natural conception (n = 147 male; n = 130 female)37 (25–55)36 (25–45)3 (2–10)
      Sporadic miscarriage after ART conception (n = 19 male; n = 17 female)40 (31–71)39 (32–46)0
      Recurrent miscarriage after ART conception (n = 7 male; n = 5 female)37 (31–42)38 (34–40)3 (2–5)
      Biochemical pregnancy (n = 67 male, n = 55 female)38 (28–71)37 (27–46)1 (0–10)
      Groups: sporadic miscarriage after natural conception; recurrent miscarriage after natural conception; sporadic miscarriage after assisted reproductive technology (ART) conception; recurrent miscarriage after ART conception; and biochemical pregnancy.
      Those who had experienced recurrent miscarriage had an average of three miscarriages and as many as 10.
      TABLE 3EFFECT OF FEMALE PARTNER'S AGE ON THREE COMET ASSAY MEASURES FOR EACH MISCARRIAGE GROUP
      GroupAverage female age (range)Average Comet scoreLow Comet scoreHigh Comet score
      Median % (IQR)SignificanceMedian % (IQR)SignificanceMedian % (IQR)Significance
      <38≥38<38≥38<38≥38
      Total (n = 191: <38 years n = 134; ≥38 years n = 57)36 (25–46)32.59 (8.43)33.96 (11.00)U
      U = Mann–Whitney test.
      41160.00047.17 (35.50)46.25 (31.00)U
      Weighted analysis.
      42335.00013.64 (17.00)14.93 (14.00)U
      Weighted analysis.
      40860.000
      n
      df = degrees of freedom.
      553n
      df = degrees of freedom.
      553n
      df = degrees of freedom.
      553
      P
      n = sample size
      0.114P
      n = sample size
      0.642P
      n = sample size
      0.788
      Sporadic miscarriage after natural conception (n = 17: <38 years n = 9, ≥38 years n = 8)38 (32–45)33.00 (14.46)32.29 (4.33)t
      t = t-test.
      0.62746.00 (40.00)49.00 (9.25)t
      U = Mann–Whitney test.
      –0.41412.00 (28.00)6.50 (9.75)U
      Weighted analysis.
      1.730
      df
      df = degrees of freedom.
      15df
      t = t-test.
      15n
      n = sample size
      17
      P
      P = probability value
      0.540P
      n = sample size
      0.685P
      n = sample size
      0.421
      Recurrent miscarriage after natural conception (n = 130: <38 years n = 97; ≥38 years n = 33)36 (25–45)32.20 (9.70)36.46 (8.09)U
      Weighted analysis.
      1940.00048.00 (30.00)39.00 (30.00)t
      U = Mann–Whitney test.
      1.1878.00 (12.00)9.00 (14.00)U
      Weighted analysis.
      1778.500
      n
      df = degrees of freedom.
      130df
      t = t-test.
      128n
      df = degrees of freedom.
      130
      P
      n = sample size
      0.069P
      n = sample size
      0.237P
      n = sample size
      0.340
      Sporadic miscarriage after ART conception (n = 17: <38 years n = 8, ≥38 years n = 9)39 (32–46)30.81 (6.76)34.00 (14.94)t
      U = Mann–Whitney test.
      0.46455.00 (21.50)48.00 (38.00)t
      U = Mann–Whitney test.
      –0.49410.00 (14.50)7.00 (19.00)t
      U = Mann–Whitney test.
      0.461
      df
      t = t-test.
      15df
      t = t-test.
      15df
      t = t-test.
      15
      P
      n = sample size
      0.649P
      n = sample size
      0.628p
      n = sample size
      0.651
      Recurrent miscarriage after ART conception (n = 5: <38 years n = 3, ≥38 years n = 2)38 (34–40)29.58 (4.50)28.62 (13.02)t
      U = Mann–Whitney test.
      0.21760.00 (13.00)57.50 (31.50)t
      U = Mann–Whitney test.
      –0.11112.00 (4.50)14.50 (12.50)t
      U = Mann–Whitney test.
      –0.483
      df
      t = t-test.
      3df
      t = t-test.
      3df
      t = t-test.
      3
      P
      n = sample size
      0.842P
      n = sample size
      0.919P
      n = sample size
      0.662
      Biochemical pregnancy (n = 55: <38 years n = 35; ≥38 years n =20)37 (27–46)30.00 (9.63)36.62 (8.70)t
      U = Mann–Whitney test.
      –1.61855.00 (30.50)37.00 (25.75)t
      U = Mann–Whitney test.
      1.5487.00 (9.00)14.00 (15.50)U
      Weighted analysis.
      486.000
      df
      t = t-test.
      53df
      t = t-test.
      53n
      df = degrees of freedom.
      55
      P
      n = sample size
      0.112P
      n = sample size
      0.128P
      n = sample size
      0.017
      Groups: sporadic miscarriage after natural conception; recurrent miscarriage after natural conception; sporadic miscarriage after assisted reproductive technology (ART) conception; recurrent miscarriage after ART conception and biochemical pregnancy. Each of the five miscarriage groups were divided based on the female partner's age as either <38 years or ≥38 years and tested for any difference in sperm DNA fragmentation results between the groups using t-tests and Mann–Whitney tests. Only the high Comet score in the biochemical pregnancy group showed significant difference, being significantly lower in the men who had partners younger than 38 years. For analysis of the total group weighted analysis was used to account for difference in size of the two groups; <38 years original n = 134, weight = 2; >38 years original n = 57, weight = 5.
      a Weighted analysis.
      b U = Mann–Whitney test.
      c t = t-test.
      d df = degrees of freedom.
      e n = sample size
      f P = probability value
      As these men were not attending an ART clinic in which semen analysis could be carried out, their samples were not subjected to that analysis and, as such, results are not available for inclusion in this study.

       Comparison of novel Comet parameters in fertile men with those after miscarriage

      Sperm DNA quality in 76 recently proven fertile sperm donors were compared with 217 male partners of couples presenting with miscarriage (Figure 1). The fertile male distribution demonstrated a marked skew to the left indicating that most spermatozoa in this group had good-quality DNA with low damage levels, calculated as 90% of their spermatozoa. In contrast, the distribution of DNA damage was skewed to the right in the partners of couples with miscarriage group, with only 47% of spermatozoa having good-quality DNA and the rest having medium to high DNA damage. Statistically significant differences were found between the fertile group and the miscarriage group for all three Comet parameters (P < 0.001) (Table 4).
      Figure 1
      Figure 1Average Comet score comparison of 76 fertile men and 217 men whose partners have experienced miscarriages showing the distribution of per cent DNA damage in each population. Grey = fertile men; black = men whose partners have had miscarriages.
      TABLE 4DISTRIBUTION OF SPERM DNA DAMAGE ACROSS A SEMEN SAMPLE IN FERTILE MEN COMPARED WITH MEN WHOSE PARTNERS WHO HAD EXPERIENCED MISCARRIAGES
      Average Comet score (% ± SE)Low Comet score (% ± SE)High Comet score (% ± SE)
      Fertile men (n = 76)14.87 ± 0.6690.22 ± 1.051.97 ± 0.37
      Miscarriage (n = 217)33.32 ± 0.5747.01 ± 1.4814.52 ± 0.95
      Difference18.4543.2112.55
      Fertile versus miscarriage (Mann– Whitney U test)n
      Groups were weighted: fertile (3), miscarriage (1), owing to difference in numbers in the two groups, i.e. 76 compared with 217. IQR, interquartile range.
      445445445
      U47958.0001558.50043842.000
      P<0.001<0.001<0.001
      Miscarriage median (IQR)33.73 (10.77)47.00 (35.00)10.00 (17.00)
      Fertile median (IQR)14.51 (8.55)92.00 (14.00)1.00 (3.00)
      Mann–Whitney U tests were used to evaluate the significance of differences in average Comet score (ACS), low Comet score (LCS) and high Comet score (HCS) between 76 fertile men and 217 men whose partners have experienced miscarriages. All differences were significant.
      a Groups were weighted: fertile (3), miscarriage (1), owing to difference in numbers in the two groups, i.e. 76 compared with 217.IQR, interquartile range.
      The 217 men whose partners experienced a miscarriage were sub-divided into those with a single sporadic miscarriage after natural conception; several recurrent miscarriages after natural conception; single sporadic miscarriage after assisted conception; several recurrent miscarriages after assisted conception; and those with biochemical pregnancies. Initial investigation by Kruskal–Wallis testing found each of these sub-divided groups to be significantly different to the fertile group for ACS (chi-squared 5 = 140.801; P < 0.001), LCS (chi-squared 5 = 144.860; P < 0.001) and HCS (chi-squared 5 = 91.307; P < 0.001), with Bonferroni adjusted pairwise comparisons finding P ≤ 0.001 in all cases. For all three Comet parameters (ACS, LCS and HCS), both those with sporadic miscarriage after natural conception (Table 5) and those with recurrent miscarriage after natural conception (Table 6) differed significantly from the fertile group (P < 0.001). No significant differences were observed in sperm DNA damage with male age (data not shown).
      TABLE 5DISTRIBUTION OF SPERM DNA DAMAGE ACROSS A SEMEN SAMPLE IN FERTILE MEN COMPARED WITH MEN WHOSE PARTNERS EXPERIENCED SPORADIC MISCARRIAGES
      Average Comet score (% ± SE)Low Comet Score (% ± SE)High Comet Score (% ± SE)
      Fertile men (n = 76)14.87 ± 0.6690.22 ± 1.051.97 ± 0.37
      Sporadic miscarriage after natural conception (n = 18)35.37 ± 0.6345.89 ± 1.4819.28 ± 1.38
      Difference between fertile and sporadic miscarriage after natural conception20.544.3317.31
      Sporadic miscarriage after ART conception (n = 19: ICSI n = 3, IVF n = 16)32.16 ± 0.4551.16 ± 1.2611.89 ± 0.67
      Difference between fertile and sporadic miscarriage after ART Conception17.2939.069.92
      Fertile versus sporadic miscarriage after natural conception (Mann–Whitney U test)n
      Groups were weighted: fertile (1); miscarriage (4) owing to difference in numbers in the groups, i.e. 18 or 19 compared with 76. IQR, interquartile range.
      148148148
      U5408.00092.0004966.000
      P<0.001<0.001<0.001
      Miscarriage median (IQR)33.36 (10.04)48.50 (32.00)11.50 (20.00)
      Fertile median (IQR)14.51 (8.55)92.00 (14.00)1.00 (3.00)
      Fertile versus sporadic miscarriage after ART conception (Mann–Whitney U test)n
      Groups were weighted: fertile (1); miscarriage (4) owing to difference in numbers in the groups, i.e. 18 or 19 compared with 76. IQR, interquartile range.
      152152152
      U5615.000168.0004718.000
      P<0.001<0.001<0.001
      Miscarriage median (IQR)34.00 (11.00)48.00 (31.00)11.00 (17.00)
      Fertile median (IQR)14.51 (8.55)92.00 (14.00)1.00 (3.00)
      Average Comet score (ACS), low Comet score (LCS) and high Comet score (HCS) all differed significantly by Mann–Whitney U testing between the fertile group of 76 men and both men whose partners had experienced sporadic miscarriage after natural conception (n = 18) and after assisted reproductive technology (ART) conception (n = 19).
      a Groups were weighted: fertile (1); miscarriage (4) owing to difference in numbers in the groups, i.e. 18 or 19 compared with 76.IQR, interquartile range.
      TABLE 6DISTRIBUTION OF SPERM DNA DAMAGE ACROSS A SEMEN SAMPLE IN FERTILE MEN COMPARED WITH MEN WHOSE PARTNERS HAD EXPERIENCED RECURRENT MISCARRIAGES
      Average Comet score (% ± SE)Low Comet score (% ± SE)High Comet score (% ± SE)
      Fertile men (n = 76)14.87 ± 0.6690.22 ± 1.051.97 ± 0.37
      Recurrent miscarriage after natural conception (n = 146)32.66 ± 0.5547.50 ± 1.4412.66 ± 0.80
      Difference between fertile and recurrent miscarriage after natural conception17.7942.7210.69
      Recurrent miscarriage after ART conception (n = 7: ICSI n = 3, IVF n = 3, IVF/ICSI n = 1)31.90 ± 0.5951.86 ± 1.4214.29 ± 0.63
      Difference between fertile and recurrent miscarriage after ART conception17.0338.3612.32
      Fertile versus recurrent miscarriage after natural conception (Mann–Whitney U test)n
      Groups were weighted: fertile (2); recurrent miscarriage after natural conception (1); fertile (1); recurrent miscarriage after ART conception (12). Weights used owing to difference in numbers between the groups, i.e. 76 versus 146 and 76 versus 7. IQR, interquartile range.
      298298298
      U21498.000646.00019423.000
      P<0.001<0.001<0.001
      Miscarriage median (IQR)32.81 (11.15)47.50 (33.00)9.00 (14.00)
      Fertile median (IQR)14.51 (8.55)92.00 (14.00)1.00 (3.00)
      Fertile versus recurrent miscarriage after ART conception (Mann–Whitney U test)n
      Groups were weighted: fertile (2); recurrent miscarriage after natural conception (1); fertile (1); recurrent miscarriage after ART conception (12). Weights used owing to difference in numbers between the groups, i.e. 76 versus 146 and 76 versus 7. IQR, interquartile range.
      160160160
      U6000.000318.0005916.000
      P<0.001<0.001<0.001
      Miscarriage median (IQR)35.81 (8.10)44.00 (21.00)13.00 (9.00)
      Fertile median (IQR)14.51(8.55)92.00 (14.00)1.00 (3.00)
      Average Comet score (ACS), low Comet score (LCS) and high Comet score (HCS) all differed significantly by Mann–Whitney U testing between the group of 76 fertile men and both men whose partners had had recurrent miscarriage after natural conception (n = 146) and men whose partners had had recurrent miscarriage after assisted reproductive technology (ART) conception (n = 7).
      a Groups were weighted: fertile (2); recurrent miscarriage after natural conception (1); fertile (1); recurrent miscarriage after ART conception (12). Weights used owing to difference in numbers between the groups, i.e. 76 versus 146 and 76 versus 7.IQR, interquartile range.
      Those who had experienced miscarriage after ART also differed significantly from the fertile group (Table 5 and Table 6); (recurrent miscarriage after ART had a small group size of seven so may require caution in interpretation). The DNA damage in each ART group, whether spontaneous or recurrent, was similar in displaying more sperm DNA damage (ACS 31–32%) than the fertile group (ACS 14.87+/–0.66%) (Table 5 and Table 6). Those with biochemical pregnancy also differed significantly form the fertile group (Table 7).
      TABLE 7DISTRIBUTION OF SPERM DNA DAMAGE ACROSS A SEMEN SAMPLE IN FERTILE MEN COMPARED WITH MEN WHOSE PARTNERS HAVE HAD BIOCHEMICAL PREGNANCIES
      Average Comet score (% ± SE)Low Comet score (% ± SE)High Comet score (% ± SE)
      Fertile men (n = 76)14.87 ± 0.6690.22 ± 1.051.97 ± 0.37
      Biochemical pregnancy (n = 67)33.13 ± 0.5547.75 ± 1.5114.61 ± 0.92
      Difference between fertile and biochemical pregnancy18.2642.4712.64
      Fertile versus biochemical pregnancy Mann–Whitney U testn143143143
      U4944.00150.004542.500
      P<0.001<0.001<0.001
      Miscarriage median (IQR)33.00 (11.81)48.00 (33.00)11.00 (15.00)
      Fertile median (IQR)14.51 (8.55)92.00 (14.00)1.00 (3.00)
      Average Comet score (ACS), low Comet score (LCS) and high Comet score (HCS) all differed significantly by Mann–Whitney U testing between the 76 fertile men and the 67 men whose partners had had biochemical pregnancies.
      Receiver operator characteristic (ROC) analysis of the 76 fertile men and 217 men whose partners had experienced miscarriage showed all three Comet parameters were able to distinguish between live births and miscarriages (ACS area under the curve [AUC] = 0.965, LCS AUC = 0.969, HCS AUC = 0.883) (Table 8 and Figure 2).
      TABLE 8RECEIVER operating CHARACTERISTIC CURVE ANALYSIS OF SPERM DNA FRAGMENTATION IN INFERTILITY DIAGNOSIS
      Result parameterThreshold value, %AUC (95% CI)P-valueSensitivitySpecificityPPVNPV
      ACS≥25.620.965 (0.947 to 0.983)<0.00010.8351.0001.0000.679
      LCS≤70.000.969 (0.952 to 0.985)<0.00010.8490.9740.9890.692
      HCS≥2.000.883 (0.843 to 0.924)<0.00010.8670.7370.9040.659
      Seventy-six fertile men were compared with 217 men whose partners had had miscarriages. The high area under the curve (AUC) values and strong significance combined with high sensitivity, specificity and positive and negative predictive values indicate the strength of the associated thresholds as a tool in the prediction of male infertility. Threshold values were determined by maximizing the sum of sensitivity and specificity.
      ACS, average Comet score; HCS, high Comet score; LCS, low Comet score; NPV, negative predictive value; PPV, positive predictive value.
      Figure 2
      Figure 2Receiver operating characteristic (ROC) curves for A (average Comet score [ACS]); B (low Comet score [LCS]) and C (high Comet score [HCS]). Seventy-six fertile men were compared with 217 men whose partners had experienced miscarriages. AUC, area under the curve.

      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 abnormalities thereafter were linked exclusively to the female's oocyte quality or uterine environment. The few endeavours to investigate a male link with miscarriage used only a conventional semen analysis, and the results proved negative, so the entire line of enquiry was halted (

      Hill, J.A., Abbott, A.F., Joseph, B.S., Politch, A., 1994. Sperm morphology and recurrent abortion * t. https://doi.org/ 10.1016/S0015-0282(16)56661-8

      ;
      • Bhattacharya S.M.
      Association of various sperm parameters with unexplained repeated early pregnancy loss-which is most important?.
      ;
      • Brahem S.
      • Mehdi M.
      • Landolsi H.
      • Mougou S.
      • Elghezal H.
      • Saad A.
      Semen parameters and sperm DNA fragmentation as causes of recurrent pregnancy loss.
      ;
      • Zhang L.
      • Wang L.
      • Zhang X.
      • Xu G.
      • Zhang W.
      • Wang K.
      • Wang Q.
      • Qiu Y.
      • Li J.
      • Gai L.
      Sperm chromatin integrity may predict future fertility for unexplained recurrent spontaneous abortion patients.
      ;

      Eisenberg, M.L., Sapra, K.J., Kim, S.D., Chen, Z., Louis, G.M.B., 2017. Semen quality and pregnancy loss in a contemporary cohort of couples recruited before conception: data from the Longitudinal Investigation of Fertility and the Environment (LIFE) Study. https://doi.org/ 10.1016/j.fertnstert.2017.07.008

      ). The accuracy of such traditional biomarkers in diagnosing male infertility has been questioned (
      • Lamb D.
      Semen analysis in 21st century medicine: the need for sperm function testing.
      ;
      • Carrell D.T.
      • De Jonge C.J.
      The troubling state of the semen analysis.
      ). This standstill has led to a long-term disservice, in a lack of diagnosis for this all too common disorder for those wishing to have a family.
      In the pivotal meta-analyses of 2012, the more sensitive biomarker of sperm DNA damage was used, and close correlations between the paternal genome and miscarriage risk were found in 11 IVF and 14 ICSI studies (
      • Robinson L.
      • Gallos I.D.
      • Conner S.J.
      • Rajkhowa M.
      • Miller D.
      • Lewis S.
      • Kirkman-Brown J.
      • Coomarasamy A.
      The effect of sperm DNA fragmentation on miscarriage rates: A systematic review and meta-analysis.
      ). In fact, sperm DNA fragmentation doubled the risk of miscarriage (2.16; 95% CI 1.54 to 3.03, P < 0.0001). Since then, awareness and acceptance of the association between sperm quality and miscarriage has increased. This was further supported by the European guidelines in 2018, in which the body of evidence indicating this link of a male factor in the pathophysiology of miscarriage was sufficient to recommend men to be included for the first time (
      • Bender Atik R.
      • Christiansen O.B.
      • Elson J.
      • Kolte A.M.
      • Lewis S.
      • Middeldorp S.
      • Nelen W.
      • Peramo B.
      • Quenby S.
      • Vermeulen N.
      • Goddijn M.
      ESHRE guideline: recurrent pregnancy loss.
      ). It has been suggested that clinicians should inform the male partner of their possible contributions to sporadic and recurrent miscarriage and advise them on how to improve their sperm quality through lifestyle changes. The evidence, however, is not yet adequate for sperm DNA damage to be introduced routinely as a predictive factor for miscarriage (
      • Kirkman-Brown J.C.
      • De Jonge C.
      Sperm DNA fragmentation in miscarriage – a promising diagnostic, or a test too far?.
      ), although it is often used as an explanation after a miscarriage. More robust studies are needed. In the present study, the objective was to determine the relationship between the Comet test using its novel high and low score parameters with couples who had experienced miscarriages across the whole spectrum: first or second trimesters, sporadic or recurrent and after spontaneous or assisted conception.
      The Comet was used as the DNA test of choice as few data associating it with miscarriage are available, although its strong links with all other fertility checkpoints from fertilization to live birth are well recognized (
      • Lewis S.E.M.
      • Agbaje I.
      • Alvarez J.
      Sperm DNA tests as useful adjuncts to semen analysis.
      ;
      • Simon L.
      • Brunborg G.
      • Stevenson M.
      • Lutton D.
      • McManus J.
      • Lewis S.E.M.
      Clinical significance of sperm DNA damage in assisted reproduction outcome.
      ;
      • Simon L.
      • Castillo J.
      • Oliva R.
      • Lewis S.
      The relationship between human sperm protamines, DNA damage and assisted reproductive outcomes.
      ;
      • Simon L.
      • Lutton D.
      • McManus J.
      • Lewis S.
      Sperm DNA damage measured by the alkaline Comet assay as an independent predictor of male infertility and in vitro fertilization success.
      ;
      • Simon L.
      • Proutski I.
      • Stevenson M.
      • Jennings D.
      • McManus J.
      • Lutton D.
      • Lewis S.
      Sperm DNA damage has a negative association with live-birth rates after IVF.
      ;
      • Simon L.
      • Lewis S.E.M.
      Sperm DNA damage or progressive motility: Which one is the better predictor of fertilization in vitro?.
      ). In the systematic reviews and meta-analyses of both
      • Simon L.
      • Brunborg G.
      • Stevenson M.
      • Lutton D.
      • McManus J.
      • Lewis S.E.M.
      Clinical significance of sperm DNA damage in assisted reproduction outcome.
      ;
      • Simon L.
      • Castillo J.
      • Oliva R.
      • Lewis S.
      The relationship between human sperm protamines, DNA damage and assisted reproductive outcomes.
      ;
      • Simon L.
      • Lutton D.
      • McManus J.
      • Lewis S.
      Sperm DNA damage measured by the alkaline Comet assay as an independent predictor of male infertility and in vitro fertilization success.
      ;
      • Simon L.
      • Proutski I.
      • Stevenson M.
      • Jennings D.
      • McManus J.
      • Lutton D.
      • Lewis S.
      Sperm DNA damage has a negative association with live-birth rates after IVF.
      ) and
      • Cissen M.
      • Van Wely M.
      • Scholten I.
      • Mansell S.
      • De Bruin J.P.
      • Mol B.W.
      • Braat D.
      • Repping S.
      • Hamer G.
      Measuring sperm DNA fragmentation and clinical outcomes of medically assisted reproduction: A systematic review and meta analysis.
      , the TdT (terminal deoxynucleotidyl transferase) mediated dUDP nick-end labelling (TUNEL) and Comet tests are superior to other DNA tests (AUC 0.71 and 0.73, respectively) for clinical pregnancies compared with sperm chromatin structure assay or sperm chromatin dispersion tests (both AUC of 0.48), although TUNEL is not available commercially in the UK.
      Given the heterogeneity of a semen analysis even in fertile men (

      World Health Organization, 2010. Examination and processing of human semen. World Health Edition, F, 286.

      ), we have been interested to observe the contrasting homogeneity of sperm DNA quality in the population of fertile men (Table 4). Each of the three Comet parameters indicate that DNA fragmentation may be a robust diagnostic tool for miscarriage as well as for male fertility (
      • Nicopoullos J.
      • Vicens-Morton A.
      • Lewis S.
      • Lee K.
      • Larsen P.
      • Ramsey J.
      • Yap T.
      • Minhas S.
      Novel use of COMET parameters of sperm DNA damage may increase its utility to diagnose male infertility and predict live births following both IVF and ICSI.
      ). The only property of spermatozoa that matters after fertilization is its genetic integrity, so a wide range of motility and morphology values may not be critical, but DNA reliability is still clearly essential for a healthy live birth.
      The prevalence of sperm DNA damage in male partners within each of the five groups studied is remarkably uniform, with sperm DNA damage no higher in couples with recurrent rather than sporadic miscarriages. This is an encouraging factor as women who have experienced one miscarriage have a higher chance of subsequent miscarriages. From this study, the male does not appear to contribute to that increased risk. Therefore, in couples who have experienced recurrent miscarriage, additional male or female factors must be considered.
      The reduction in sperm quality observed here is not dependent on male age. In this group of men with wide ranging ages from 25–71 years, no correlation was found between age and DNA damage (r = 0.076, P = 0.265). This is contrary to other studies of fertile and infertile men where clear associations are observed with the usual ageing process (
      • Lewis S.E.M.
      • Kumar K.
      The paternal genome and the health of the assisted reproductive technology child, in: Asian Journal of Andrology.
      ). In this study, the small number of men aged over 45 years (n = 15), the age when the effects of advanced paternal age are believed to take effect, is likely the reason for no age effects being detected. The causes of diminished sperm DNA quality in the young men experiencing miscarriage are still unknown but it is likely to include oxidative stress as a major contributor (
      • Iommiello V.M.
      • Albani E.
      • Di Rosa A.
      • Marras A.
      • Menduni F.
      • Morreale G.
      • Levi S.L.
      • Pisano B.
      • Levi-Setti P.E.
      Ejaculate oxidative stress is related with sperm DNA fragmentation and round cells.
      ).
      Female age was only found to have a significant effect on the HCS in the biochemical pregnancy group. The high damage in this group may exceed the ability of older oocytes to repair sperm DNA, leading quickly to an inviable embryo and resulting in an early pregnancy loss. As the data are limited, it requires further investigation.
      Given the similarly strong relationship between sperm DNA and miscarriage after spontaneous conception, it is encouraging for couples to know that miscarriages are not exacerbated by assisted conception procedures. This supports the study of 1597 clinical IVF/ICSI pregnancies reporting miscarriages rates of 22% (
      • Tummers P.
      Risk of spontaneous abortion in singleton and twin pregnancies after IVF/ICSI.
      ); that is no higher than national miscarriage rates of 20–25% (; https://www.tommys.org/our-organisation/charity-research/pregnancy-statistics/miscarriage).
      The effect of sperm DNA fragmentation on pregnancy outcome is dependent on oocyte quality as elegantly demonstrated by
      • Meseguer M.
      • Santiso R.
      • Garrido N.
      Effect of sperm DNA fragmentation on pregnancy outcome depends on oocyte quality.
      . For every 10% increase in sperm damage, the chances of achieving a pregnancy decreased by 1.31. This effect was not evident when fertile donor oocytes were used with the same damaged spermatozoa. This group, however, has also reported that high sperm DNA fragmentation delays human embryo kinetics even when oocytes from young and healthy donors are microinjected (
      • Esbert M.
      • Pacheco A.
      • Soares S.R.
      • Amorós D.
      • Florensa M.
      • Ballesteros A.
      • Meseguer M.
      High sperm DNA fragmentation delays human embryo kinetics when oocytes from young and healthy donors are microinjected.
      ). The oocyte genome codes for maternal DNA repair machinery to be initiated to detect paternal DNA damage within minutes of the formation of the male pronucleus (
      • Derijck A.A.H.A.
      • van der Heijden G.W.
      • Giele M.
      • Philippens M.E.P.
      • van Bavel C.C.A.W.
      • de Boer P.
      γH2AX signalling during sperm chromatin remodelling in the mouse zygote.
      ;
      • Barton T.S.
      • Robaire B.
      • Hales B.F.
      DNA Damage Recognition in the Rat Zygote Following Chronic Paternal Cyclophosphamide Exposure.
      ). Recent studies have reported that oocytes from young women (<35 years) can repair up to 40% damage whereas older women's oocytes (>35 years) are less effective (
      • Cozzubbo T.
      • Neri Q.
      • Rosenwaks Z.
      • Palermo G.
      To what extent can oocytes repair sperm dna fragmentation?.
      ). Using an animal model, the importance of oocyte age for ability to repair sperm DNA damage has been confirmed (
      • Horta F.
      • Catt S.
      • Vollenhoven B.
      • Temple-Smith P.
      Oocyte DNA repair capacity of controlled sperm DNA damage is affected by female age.
      ). The mechanism may result in maternal mis-repair that can lead to de-novo mutations and fatal chromosomal anomalies (
      • Marchetti F.
      • Bishop J.
      • Gingerich J.
      • Wyrobek A.J.
      Meiotic interstrand DNA damage escapes paternal repair and causes chromosomal aberrations in the zygote by maternal misrepair.
      ). This provides clear evidence that oocytes can repair sperm DNA damage, so in the present study of miscarriage, the woman is also implicated; her oocytes seem to be unable to repair the damage in the fertilizing spermatozoa. The level of repair by oocytes is still unknown and, as the damage in these men's spermatozoa was so great, at 100% higher than in fertile men's spermatozoa, it may have overwhelmed even young oocyte repair mechanisms. Female age ranged from 25–46 years with 57 of the women aged 38 years or over and 134 aged younger than 38 years (interquartile range = 8, P = 0.114 using a weighted Mann–Whitney test to equalize group sizes) (Table 3).
      Damage (ACS) in each of the five study groups was more than double the fertile donor group (Table 5, Table 6 and Table 7). This suggests that DNA damage in the male genome is the common link to the non-viability of the embryo. DNA integrity of both gametes is crucial for the development of an embryo to full term. The clinical implications of sperm DNA damage for the embryo, however, may be delayed as the paternal genome exerts more influence on the embryo in later development. Couples can often achieve a pregnancy with damaged sperm DNA; however, the fetus cannot progress to term as the damage proves to be incongruous with viability when the paternal genome is fully activated (
      • Braude P.
      • Bolton V.
      • Moore S.
      Human gene expression first occurs between the four- and eight-cell stages of preimplantation development.
      ). In a previous study (
      • Simon L.
      • Proutski I.
      • Stevenson M.
      • Jennings D.
      • McManus J.
      • Lutton D.
      • Lewis S.
      Sperm DNA damage has a negative association with live-birth rates after IVF.
      ), we observed that, in men who did not achieve even a pregnancy, their mean DNA damage was over 45% compared with the mean damage here of 33%. In comparison, men who achieved live births after ART had means of less than 27% (
      • Nicopoullos J.
      • Vicens-Morton A.
      • Lewis S.
      • Lee K.
      • Larsen P.
      • Ramsey J.
      • Yap T.
      • Minhas S.
      Novel use of COMET parameters of sperm DNA damage may increase its utility to diagnose male infertility and predict live births following both IVF and ICSI.
      ).
      Our data support studies by Leach et al. (2015) using the sperm chromatin structure assay test,
      • Carlini T.
      • Paoli D.
      • Pelloni M.
      • Faja F.
      • Dal Lago A.
      • Lombardo F.
      • Lenzi A.
      • Gandini L.
      Sperm DNA fragmentation in Italian couples with recurrent pregnancy loss.
      using the TUNEL test and
      • Jayasena C.
      • Radia U.
      • Figueiredo M.
      • Revill L.
      • Dimakopoulou A.
      • Osagie M.
      • Vessey W.
      • Regan L.
      • Rai R.
      • Dhillo W.
      Reduced Testicular Steroidogenesis and Increased Semen Oxidative Stress in Male Partners as Novel Markers of Recurrent Miscarriage.
      using the Halo test. Only one small study of miscarriages after IVF (
      • Coughlan C.
      • Clarke H.
      • Cutting R.
      • Saxton J.
      • Waite S.
      • Ledger W.
      • Pacey A.
      Sperm DNA fragmentation, recurrent implantation failure and recurrent miscarriage.
      ) is in conflict, in which no associations were observed. These differences may be explained by the study size or by the female contributions to the couples before infertility. The novelty of this study over previous ones is the development of clinical thresholds for miscarriage by comparing the men with miscarriage with a large group of fertile men who have achieved live births for the first time.
      In Table 8, high levels of sensitivity and specificity in the development of predictive testing using the three clinical threshold values of 26% for ACS, 70% for LCS and 2% for HCS are reported; determined from ROC curve analysis by maximizing the sums of sensitivity and specificity.
      Once the couple has a diagnosis, they are finally able to take control of their situation and look for solutions, often starting with simple techniques to improve their sperm quality. Simple changes in lifestyle can improve DNA damage, such as reducing alcohol intake, cessation or reduction of smoking and avoidance of protein shakes at the gym as these often contain anabolic steroids detrimental to spermatozoa (
      • Ketheeswaran S.
      • Haahr T.
      • Povlsen B.
      Protein supplementation intake for bodybuilding and resistance training may impact sperm quality of subfertile men undergoing fertility treatment: a pilot study.
      ). Reductions in weight are also associated with improving sperm DNA damage (
      • Dupont C.
      • Faure C.
      • Sermondade N.
      Obesity leads to higher risk of sperm DNA damage in infertile patients.
      ). To combat DNA damage from oxidative stress, focusing on a healthy unprocessed diet, high in antioxidants and antioxidant supplementation may be protective; lifestyle enhancement can improve sperm quality within 3–6 months (
      • Aitken R.J.
      • De Iuliis G.N.
      On the possible origins of DNA damage in human spermatozoa.
      ).
      Men who have experienced miscarriage should visit a urologist, a specialist trained and focused on male reproductive problems. A clinical history and examination of the man and his spermatozoa can often lead to simple clinical interventions that improve sperm DNA. For example, many men unable to father a child have seminal infections that can be cured with antibiotics, and up to 48% of infertile men have a varicocele; varicoceles are a common cause of raised DNA damage and a simple outpatient varicocelectomy reduces both damage and miscarriage rates (
      • Mansour Ghanaie M.
      • Alaeddin Asgari S.
      • Dadrass N.
      • Allahkhah A.
      • Iran-Pour E.
      • Reza Safa- M.
      Effects of Varicocele Repair on Spontane-ous First Trimester Miscarriage A Randomized Clinical Trial.
      ;
      • Ibrahim Y.
      • Johnstone E.
      The male contribution to recurrent pregnancy loss.
      ).
      In conclusion, the present study provides novel evidence for the association between miscarriage and sperm DNA quality using the alkaline Comet assay. It gives couples a diagnosis and provides new opportunities for them to become parents.

      Acknowledgements

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

      References

        • Agbaje I.M.
        • Rogers D.A.
        • McVicar C.M.
        • McClure N.
        • Atkinson A.B.
        • Mallidis C.
        • Lewis S.E.M.
        Insulin dependant diabetes mellitus: Implications for male reproductive function.
        Hum. Reprod. 2007; 22 (https://doi.org/): 1871-1877https://doi.org/10.1093/humrep/dem077
        • Aitken R.J.
        • De Iuliis G.N.
        On the possible origins of DNA damage in human spermatozoa.
        Mol. Hum. Reprod. 2010; 16 (https://doi.org/): 3-13https://doi.org/10.1093/molehr/gap059
        • Barton T.S.
        • Robaire B.
        • Hales B.F.
        DNA Damage Recognition in the Rat Zygote Following Chronic Paternal Cyclophosphamide Exposure.
        Toxicol. Sci. 2007; 100 (https://doi.org/): 495-503https://doi.org/10.1093/toxsci/kfm242
        • Bender Atik R.
        • Christiansen O.B.
        • Elson J.
        • Kolte A.M.
        • Lewis S.
        • Middeldorp S.
        • Nelen W.
        • Peramo B.
        • Quenby S.
        • Vermeulen N.
        • Goddijn M.
        ESHRE guideline: recurrent pregnancy loss.
        Hum. Reprod. 2018; (Open 2018https://doi.org/)https://doi.org/10.1093/hropen/hoy004
        • Bhattacharya S.M.
        Association of various sperm parameters with unexplained repeated early pregnancy loss-which is most important?.
        Int. Urol. Nephrol. 2008; 40 (https://doi.org/): 391-395https://doi.org/10.1007/s11255-007-9282-y
        • 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; 78 (https://doi.org/): 792-796https://doi.org/10.1016/j.urology.2011.05.049
        • Braude P.
        • Bolton V.
        • Moore S.
        Human gene expression first occurs between the four- and eight-cell stages of preimplantation development.
        Nature. 1988; 332 (https://doi.org/): 459-461https://doi.org/10.1038/332459a0
        • Carlini T.
        • Paoli D.
        • Pelloni M.
        • Faja F.
        • Dal Lago A.
        • Lombardo F.
        • Lenzi A.
        • Gandini L.
        Sperm DNA fragmentation in Italian couples with recurrent pregnancy loss.
        Reprod. Biomed. Online. 2017; 34 (https://doi.org/): 58-65https://doi.org/10.1016/j.rbmo.2016.09.014
        • Carrell D.T.
        • De Jonge C.J.
        The troubling state of the semen analysis.
        Andrology. 2016; 4 (https://doi.org/): 761-762https://doi.org/10.1111/andr.12257
        • Chard T.
        Frequency of implantation and early pregnancy loss in natural cycles.
        Baillieres. Clin. Obstet. Gynaecol. 1991; 5 (https://doi.org/): 179-189https://doi.org/10.1016/S0950-3552(05)80077-X
        • Cissen M.
        • Van Wely M.
        • Scholten I.
        • Mansell S.
        • De Bruin J.P.
        • Mol B.W.
        • Braat D.
        • Repping S.
        • Hamer G.
        Measuring sperm DNA fragmentation and clinical outcomes of medically assisted reproduction: A systematic review and meta analysis.
        PLoS One. 2016; 11 (https://doi.org/)https://doi.org/10.1371/journal.pone.0165125
        • Coughlan C.
        • Clarke H.
        • Cutting R.
        • Saxton J.
        • Waite S.
        • Ledger W.
        • Pacey A.
        Sperm DNA fragmentation, recurrent implantation failure and recurrent miscarriage.
        Asian J. Androl. 2015; 17: 681-685
        • Cozzubbo T.
        • Neri Q.
        • Rosenwaks Z.
        • Palermo G.
        To what extent can oocytes repair sperm dna fragmentation?.
        Fertility and Sterility. 2014; (https://doi.org/)https://doi.org/10.1016/j.fertnstert.2014.07.208
        • Derijck A.A.H.A.
        • van der Heijden G.W.
        • Giele M.
        • Philippens M.E.P.
        • van Bavel C.C.A.W.
        • de Boer P.
        γH2AX signalling during sperm chromatin remodelling in the mouse zygote.
        DNA Repair (Amst). 2006; 5 (https://doi.org/): 959-971https://doi.org/10.1016/j.dnarep.2006.05.043
        • Donnelly E.T.
        • O'Connell M.
        • McClure N.
        • Lewis S.E.M.
        Differences in nuclear DNA fragmentation and mitochondrial integrity of semen and prepared human spermatozoa.
        Hum. Reprod. 2000; 15 (https://doi.org/): 1552-1561https://doi.org/10.1093/humrep/15.7.1552
        • Dupont C.
        • Faure C.
        • Sermondade N.
        Obesity leads to higher risk of sperm DNA damage in infertile patients.
        Asian J. Androl. 2013; 15: 622-625
      1. Eisenberg, M.L., Sapra, K.J., Kim, S.D., Chen, Z., Louis, G.M.B., 2017. Semen quality and pregnancy loss in a contemporary cohort of couples recruited before conception: data from the Longitudinal Investigation of Fertility and the Environment (LIFE) Study. https://doi.org/ 10.1016/j.fertnstert.2017.07.008

        • Esbert M.
        • Pacheco A.
        • Soares S.R.
        • Amorós D.
        • Florensa M.
        • Ballesteros A.
        • Meseguer M.
        High sperm DNA fragmentation delays human embryo kinetics when oocytes from young and healthy donors are microinjected.
        Andrology. 2018; 6 (https://doi.org/): 697-706https://doi.org/10.1111/andr.12551
      2. Hill, J.A., Abbott, A.F., Joseph, B.S., Politch, A., 1994. Sperm morphology and recurrent abortion * t. https://doi.org/ 10.1016/S0015-0282(16)56661-8

        • Horta F.
        • Catt S.
        • Vollenhoven B.
        • Temple-Smith P.
        Oocyte DNA repair capacity of controlled sperm DNA damage is affected by female age.
        Hum. Reprod. 2018; 33: 2
        • Hughes C.M.
        • Lewis S.E.M.
        • Mckelvey-Martin V.J.
        • Thompson W.
        Reproducibility of human sperm DNA measurements using the alkaline single cell gel electrophoresis assay.
        Mutation Research. 1997;
        • Ibrahim Y.
        • Johnstone E.
        The male contribution to recurrent pregnancy loss.
        Transl. Androl. Urol. 2018; 7: s317-s327
        • Iommiello V.M.
        • Albani E.
        • Di Rosa A.
        • Marras A.
        • Menduni F.
        • Morreale G.
        • Levi S.L.
        • Pisano B.
        • Levi-Setti P.E.
        Ejaculate oxidative stress is related with sperm DNA fragmentation and round cells.
        Int. J. Endocrinol. 2015; 2015 (https://doi.org/)https://doi.org/10.1155/2015/321901
        • Jayasena C.
        • Radia U.
        • Figueiredo M.
        • Revill L.
        • Dimakopoulou A.
        • Osagie M.
        • Vessey W.
        • Regan L.
        • Rai R.
        • Dhillo W.
        Reduced Testicular Steroidogenesis and Increased Semen Oxidative Stress in Male Partners as Novel Markers of Recurrent Miscarriage.
        Clin. Chem. 2019; 65: 161-169
        • Ketheeswaran S.
        • Haahr T.
        • Povlsen B.
        Protein supplementation intake for bodybuilding and resistance training may impact sperm quality of subfertile men undergoing fertility treatment: a pilot study.
        Asian J. Androl. 2019; 21: 208-211
        • Kirkman-Brown J.C.
        • De Jonge C.
        Sperm DNA fragmentation in miscarriage – a promising diagnostic, or a test too far?.
        Reprod. Biomed. Online. 2017; (https://doi.org/)https://doi.org/10.1016/j.rbmo.2016.12.002
        • Lamb D.
        Semen analysis in 21st century medicine: the need for sperm function testing.
        Asian J. Androl. 2010; 12: 64-70
        • Lewis S.E.M.
        • Agbaje I.
        • Alvarez J.
        Sperm DNA tests as useful adjuncts to semen analysis.
        Syst. Biol. Reprod. Med. 2008; (https://doi.org/)https://doi.org/10.1080/19396360801957739
        • Lewis S.E.M.
        • Kumar K.
        The paternal genome and the health of the assisted reproductive technology child, in: Asian Journal of Andrology.
        Medknow Publications. 2015; (https://doi.org/): 616-622https://doi.org/10.4103/1008-682X.153301
        • Mansour Ghanaie M.
        • Alaeddin Asgari S.
        • Dadrass N.
        • Allahkhah A.
        • Iran-Pour E.
        • Reza Safa- M.
        Effects of Varicocele Repair on Spontane-ous First Trimester Miscarriage A Randomized Clinical Trial.
        UROLOGY JOURNAL. 2012;
        • Marchetti F.
        • Bishop J.
        • Gingerich J.
        • Wyrobek A.J.
        Meiotic interstrand DNA damage escapes paternal repair and causes chromosomal aberrations in the zygote by maternal misrepair.
        Sci. Rep. 2015; 5 (https://doi.org/)https://doi.org/10.1038/srep07689
        • Meseguer M.
        • Santiso R.
        • Garrido N.
        Effect of sperm DNA fragmentation on pregnancy outcome depends on oocyte quality.
        Fertil. Steril. 2011; 95: 124-128
      3. Miscarriage statistics | Tommy's [WWW Document], n.d. URLhttps://www.tommys.org/our-organisation/charity-research/pregnancy-statistics/miscarriage (accessed 11.25.19).

        • Nicopoullos J.
        • Vicens-Morton A.
        • Lewis S.
        • Lee K.
        • Larsen P.
        • Ramsey J.
        • Yap T.
        • Minhas S.
        Novel use of COMET parameters of sperm DNA damage may increase its utility to diagnose male infertility and predict live births following both IVF and ICSI.
        Hum. Reprod. 2019; 151: 1-9
        • 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; (https://doi.org/)https://doi.org/10.1016/j.rbmo.2014.10.018
        • Robinson L.
        • Gallos I.D.
        • Conner S.J.
        • Rajkhowa M.
        • Miller D.
        • Lewis S.
        • Kirkman-Brown J.
        • Coomarasamy A.
        The effect of sperm DNA fragmentation on miscarriage rates: A systematic review and meta-analysis.
        Hum. Reprod. 2012; (https://doi.org/)https://doi.org/10.1093/humrep/des261
        • Simon L.
        • Brunborg G.
        • Stevenson M.
        • Lutton D.
        • McManus J.
        • Lewis S.E.M.
        Clinical significance of sperm DNA damage in assisted reproduction outcome.
        Hum. Reprod. 2010; 25 (https://doi.org/): 1594-1608https://doi.org/10.1093/humrep/deq103
        • Simon L.
        • Castillo J.
        • Oliva R.
        • Lewis S.
        The relationship between human sperm protamines, DNA damage and assisted reproductive outcomes.
        Fertil. Steril. 2011; 23: 724-734
        • Simon L.
        • Lewis S.E.M.
        Sperm DNA damage or progressive motility: Which one is the better predictor of fertilization in vitro?.
        Syst. Biol. Reprod. Med. 2011; 57 (https://doi.org/): 133-138https://doi.org/10.3109/19396368.2011.553984
        • Simon L.
        • Lutton D.
        • McManus J.
        • Lewis S.
        Sperm DNA damage measured by the alkaline Comet assay as an independent predictor of male infertility and in vitro fertilization success.
        Fertil. Steril. 2011; 95 (https://doi.org/): 652-657https://doi.org/10.1016/j.fertnstert.2010.08.019
        • Simon L.
        • Proutski I.
        • Stevenson M.
        • Jennings D.
        • McManus J.
        • Lutton D.
        • Lewis S.
        Sperm DNA damage has a negative association with live-birth rates after IVF.
        Reprod. Biomed. Online. 2013; 26 (https://doi.org/): 68-78https://doi.org/10.1016/j.rbmo.2012.09.019
        • Tan J.
        • Taskin O.
        • Albert A.
        • Online, M.B.-R. biomedicine
        Association between Sperm DNA Fragmentation and Idiopathic Recurrent Pregnancy Loss: A Systematic Review & Meta-Analysis. 38. Elsevier, 2019: 951-960
        • Tummers P.
        Risk of spontaneous abortion in singleton and twin pregnancies after IVF/ICSI.
        Hum. Reprod. 2003; 18 (https://doi.org/): 1720-1723https://doi.org/10.1093/humrep/deg308
        • Wilcox A.J.
        • Weinberg C.R.
        • Baird D.D.
        Post-ovulatory ageing of the human oocyte and embryo failure.
        Hum. Reprod. 1998; 13 (https://doi.org/): 394-397https://doi.org/10.1093/humrep/13.2.394
      4. World Health Organization, 2010. Examination and processing of human semen. World Health Edition, F, 286.

        • Zhang L.
        • Wang L.
        • Zhang X.
        • Xu G.
        • Zhang W.
        • Wang K.
        • Wang Q.
        • Qiu Y.
        • Li J.
        • Gai L.
        Sperm chromatin integrity may predict future fertility for unexplained recurrent spontaneous abortion patients.
        Int. J. Androl. 2012; 35 (https://doi.org/): 752-757https://doi.org/10.1111/j.1365-2605.2012.01276.x
        • Zidi-Jrah I.
        • Hajlaoui A.
        • Mougou-Zerelli S.
        • Kammoun M.
        • Meniaoui I.
        • Sallem A.
        • Brahem S.
        • Fekih M.
        • Bibi M.
        • Saad A.
        • Ibala-Romdhane S.
        Relationship between sperm aneuploidy, sperm DNA integrity, chromatin packaging, traditional semen parameters, and recurrent pregnancy loss Presented at the 17th World Congress on in Vitro Fertilization, Tunis, Tunisia, on September 4-7, 2013.
        Fertil. Steril. 2016; 105 (https://doi.org/): 58-64https://doi.org/10.1016/j.fertnstert.2015.09.041
        • Zini A.
        • Boman J.
        • Belzile E.
        • Reproduction A.C.-H.
        U., 2008. Sperm DNA damage is associated with an increased risk of pregnancy loss after IVF and ICSI: systematic review and meta-analysis.
        academic.oup.com. 2008; 23: 2663-2668

      Biography

      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.