The role of sperm DNA fragmentation testing in predicting intra-uterine insemination outcome: A systematic review and meta-analysis

European Journal of Obstetrics & Gynecology and Reproductive Biology 244 (2020) 8–15

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European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb

Review article

The role of sperm DNA fragmentation testing in predicting intra-uterine insemination outcome: A systematic review and meta-analysis Alessa Sugiharaa,* , Freya Van Avermaetea , Ella Roelantb , Usha Punjabia , Diane De Neubourga a b

Centre for Reproductive Medicine – Antwerp University Hospital, University of Antwerp, Wilrijkstraat 10, 2650 Edegem, Belgium Clinical Trial Centre – Antwerp University Hospital, University of Antwerp, Wilrijkstraat 10, 2650 Edegem, Belgium

A R T I C L E I N F O

A B S T R A C T

Article history: Received 14 May 2019 Received in revised form 23 September 2019 Accepted 8 October 2019 Available online xxx

There is a growing body of literature that recognizes the importance of sperm DNA fragmentation as a candidate test for the assessment of sperm function and thus male reproductive potential. Research on the subject has mostly been focused on couples undergoing IVF/ICSI treatment whilst much uncertainty still exists about the relationship between sperm DNA fragmentation and IUI. This study systematically reviews the literature, aiming to define the value of sperm DNA fragmentation measurement in predicting clinical pregnancy outcome in couples undergoing intra-uterine insemination From inception until March 2018, the relevant databases were searched for studies investigating the relationship between sperm DNA fragmentation as measured by SCSA, TUNEL, SCD or Comet assay and pregnancy outcome after IUI. The Quality in Prognosis Studies (QUIPS) tool was utilized for quality assessment. This review is reported according to the 2009 PRISMA statement. The literature search resulted in 433 studies of which we finally retained nine studies for the qualitative analysis and four studies for the meta-analysis, accounting for 940 IUI cycles. In summary, the observed effect of low sperm DNA fragmentation on clinical pregnancy after IUI as analyzed with the random effects model reveals a relative risk of 3.15 (95% CI: 1.46–6.79; I2 = 13.1%) and pooled sensitivity and specificity of respectively 94% (95% CI: 0.88; 0.97) and 19% (95% CI: 0.14; 0.26). Taken together, the included studies show a limited capacity of sperm DNA fragmentation in discriminating between couples who will benefit from the test, namely in either predicting IUI outcome or in advising for or against IUI as first choice of treatment instead of advancing to more invasive medically assisted reproduction. This review has thrown up many questions in need of further investigation. As such, future studies might explore issues such as determining relevant cut-off values for prediction of spontaneous pregnancy and pregnancy after IUI as well as the assessment of the stability of the test over time and before and after density gradient centrifugation. © 2019 Elsevier B.V. All rights reserved.

Keywords: Male infertility Artificial insemination Intra-uterine insemination Sperm DNA fragmentation Sperm damage

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . Materials and methods . . . . . . . . . . . . . Eligibility criteria . . . . . . . . . . . . . . . Search strategy and study selection Electronic searches . . . . . . . . . . Searching other resources . . . . . . . .

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* Corresponding author. E-mail addresses: [email protected] (A. Sugihara), [email protected] (F. Van Avermaete), [email protected] (E. Roelant), [email protected] (U. Punjabi), [email protected] (D. De Neubourg). https://doi.org/10.1016/j.ejogrb.2019.10.005 0301-2115/© 2019 Elsevier B.V. All rights reserved.

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A. Sugihara et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 244 (2020) 8–15

Study appraisal and synthesis methods Data analysis . . . . . . . . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Study selection and characteristics . . . Risk of bias . . . . . . . . . . . . . . . . . . . . . . Synthesis of results . . . . . . . . . . . . . . . . Descriptive review . . . . . . . . . . . . . Meta-analysis . . . . . . . . . . . . . . . . . Discussion/Conclusion . . . . . . . . . . . . . . . . . Main findings . . . . . . . . . . . . . . . . . . . . . Strengths and limitations . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . Implications . . . . . . . . . . . . . . . . . . . . . . Statements . . . . . . . . . . . . . . . . . . . . . . . . . . Disclosure statement . . . . . . . . . . . . . . . Funding sources . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Introduction Measuring sperm DNA fragmentation, sperm DNA damage in the male germ line in the form of single- or double-strand breaks occurring at testicular, epididymal or post-ejaculatory levels [1], has gained a considerable amount of interest over the past 15 years and has been proposed as a candidate test for the assessment of sperm function and thus male reproductive potential [2,3]. The current diagnosis of male factor infertility, contributing to 30–50% of clinical cases of infertility, is based on the classic semen parameters as defined by the WHO 2010 guidelines (WHO 2010). Although these guidelines suggest accuracy through a methodology harmonized across laboratories [4], the relevance for the choice of treatment and the predictive value for an infertile couple embarking on medically assisted reproduction (MAR) is questionable [3]. Moreover, a significant number of subfertile men have abnormal sperm DNA integrity despite normal semen parameters [5–7]. The integrity of the genetic information contained within the sperm cells is continuously challenged by both intrinsic factors (protamine deficiency, excess reactive oxygen species (ROS) levels, apoptosis) and extrinsic factors (e.g. testicular hyperthermia, environmental toxins) [8] resulting in different degrees of DNA damage. Various tests have been proposed to assess sperm DNA fragmentation (SDF). The most commonly used indirect methods, measuring the susceptibility to DNA denaturation, are the Sperm Chromatin Structure Assay (SCSA) and the sperm chromatin dispersion test (SCD) [9,10]. The TUNEL and Comet assay represent the most studied direct methods of sperm DNA fragmentation measurements [11,12]. Importantly, since there is a lack of standardization of the different assays, most laboratories perform the DNA fragmentation test according to the reference paper for that specific assay, e.g. Evensson and Jost for the SCSA [9]. Several lines of evidence suggest that sperm DNA fragmentation is associated with a low potential for natural conception and a prolonged time to pregnancy [6,9]. Numerous studies also support the existence of a significant association between sperm DNA fragmentation and pregnancy outcomes after in vitro fertilization (IVF) [13]. However, the current evidence on the measurement of sperm DNA fragmentation and its consequences for MAR outcome remain controversial [14,15]. To date, a large proportion of the available research concerning this topic has focused its attention to couples undergoing IVF/ICSI treatment, leaving the relationship between sperm DNA fragmentation and intra-uterine insemination (IUI) outcome on the background. This notwithstanding, IUI is considered a simple, noninvasive, less stressful and less expensive first line treatment

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with clinical pregnancy (CP) rates varying between 5–13% per IUI cycle [16–22]. Given the strong association between SDF and natural fertility and the fact that IUI remains one of the most easily accessible and most frequently used fertility treatments [23], exploring the relationship between IUI outcome and SDF as thoroughly as possible is warranted. We have therefore conducted a systematic review including available studies investigating the relationship between sperm DNA fragmentation as measured by SCSA, TUNEL, SCD, SCGE or Comet assay and pregnancy outcome after IUI. This systematic review aims to address the following research question: what is the independent value of sperm DNA fragmentation measurement in predicting clinical pregnancy outcome in couples undergoing intra-uterine insemination? Materials and methods This review is reported according to the 2009 PRISMA statement18 and MOOSE guidelines19 [21,22]. Details of the protocol for this systematic review were registered on PROSPERO with registration number CRD42018096637 and can be accessed at www.crd.york.ac.uk/ PROSPERO/display_record.asp?ID=CRD42018096637. Eligibility criteria Types of studies: We selected studies pertaining to couples undergoing intra-uterine insemination. The intervention is the evaluation of sperm DNA fragmentation in the diagnostic or IUI semen sample. As we did not expect to encounter randomized controlled trials, both prospective and retrospective cohort studies were included. There was no language restriction. Types of participants: Trials including couples undergoing autologous IUI in either natural or stimulated cycles were included. The search was restricted to human studies. Types of interventions: Trials were included if they included sperm DNA fragmentation measurement by SCSA, Comet or SCGE assay, SCD, TUNEL on a diagnostic or IUI semen sample. Types of outcome measures: Primary outcome: Clinical pregnancy rate (defined as the presence of a gestational sac, confirmed by ultrasound) per cycle Search strategy and study selection Electronic searches We searched the following databases (from inception to March 2018) using the Ovid and PubMed platforms: Cochrane Central Register of Controlled Trials (CENTRAL), Medline (126), Embase

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(313) and Cumulative Index to Nursing and Allied Health Literature (CINAHL). The following combinations of terms, both MeSH and text word when available, were used with use of the Boolean operators AND, OR to generate two subsets of citations relevant to our research question. The Pubmed search results included studies of (1 OR 2) AND 3 AND 4 1) "SDF", “DNA fragmentation”, “DNA quality”, "Chromatin", "DNA integrity", "deoxyribonucleic acid quality, "deoxyribonucleic acid integrity, "deoxyribonucleic acid damage", "DNA damage, “chromatin quality”, “chromatin integrity”, “chromatin damage” 2) "In Situ Nick-End Labeling", "TUNEL”, “Halosperm”, "sperm chromatin dispersion”, "SCSA", "sperm chromatin structure assay", "single cell gel electrophoresis", "SCGE” 3) "Spermatozoa", "Sperm Maturation", “sperm”, “semen" 4) “Insemination”, "AI”, "IUI We hand searched the reference lists of primary and review articles to identify additional articles not captured by the electronic search [7]. Searching other resources Appropriate journals were hand searched for trial conference abstracts. Research trial registers were searched for ongoing and recently completed trials.

Two researchers (A.S. and F.V.A.) independently identified and screened relevant titles and abstracts. If deemed relevant the full text was read by both researchers. Differences were discussed until consensus could be reached. In case the study was reported in a language other than English, data was extracted when possible from the English abstract, figures and tables. In case additional information was necessary, a native speaker was consulted. Abstracts and unpublished studies were screened for completeness and relevance, in case the provided information was too scarce to assess for quality assessment, the study was omitted from the systematic review. Study appraisal and synthesis methods One author (A.S.) extracted data from the included articles, this included: first author, year of publication, study design, type of semen sample for sperm DNA fragmentation analysis, sperm DNA fragmentation test, IUI treatment specifications, number of participants and relevant information such as infertility diagnosis/ age/ BMI/ type of outcome. When pertinent raw data was not available, we extracted it from the graphs and/or contacted the corresponding authors. The Quality in Prognosis Studies (QUIPS) tool was utilized for quality assessment by two authors (A.S. and F.V.A) This tool allowed us to estimate the risk of bias due study participation and attrition, prognostic factor and outcome measurement, study confounding and statistical analysis reporting.

Fig. 1. PRISMA 2009 Flow Diagram of the search and selection of literature on sperm DNA fragmentation measurement and IUI pregnancy outcome.

A. Sugihara et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 244 (2020) 8–15

Data analysis In each of the studies included in the SCSA meta-analysis we calculated the relative risk on pregnancy in the low DNA fragmentation group compared to the high DNA fragmentation group as determined by the cut-off values provided by the respective authors. A meta-analysis was applied to the relative risks (RRs) of the individual two-by-two tables. In case of an empty cell 0.5 was added to all values of the two-by-two table [23]. Fixed and random effects models for meta-analysis were used to calculate an overall RR and corresponding 95% CI. Sensitivity defined as the number of pregnancies with low DNA fragmentation in proportion to the total number of pregnant couples (other measures are defined accordingly), specificity, positive predictive value (PPV) and negative predictive value (NPV) were pooled using a fixed and a random effects model. The meta-analysis was done using R 3.3.2 (R core team, 2016). Results Study selection and characteristics The literature search resulted in 126 reports from Medline, 313 from Embase and 11 from CENTRAL. Of these 24 duplicates were removed. After a careful review of the body of literature and based on our inclusion criteria, 8 peer reviewed studies, of which the study by Yang et al. [24] was the only non-English publication, were found to evaluate the relationship between sperm DNA fragmentation and pregnancy after IUI and thus included in our systematic review (Fig. 1). Risk of bias Study participation risk of bias was moderate in two studies, either due to inadequate description of the source population or patient inclusion being limited by the availability of laboratory facilities. We judged all studies with a moderate risk of bias due to study attrition owing to a general absence of mention of: proportion of eligible patients versus participants and whether there were drop-outs or missing data. Prognostic factor and outcome measurement was overall judged to be at low risk. We estimated a moderate risk for study confounding in four studies due to missing information regarding possible confounders such as diagnosis or treatment protocol. One study was judged to have a high risk in this domain due to lack of disclosure of the participants’ age, which is potentially an important confounder in MAR. We judged two studies with a high risk of bias in statistical analysis due to the low number of participants. Worth mentioning is that none of the included studies reported an a priori sample size calculation (Table 2). Synthesis of results Descriptive review All studies of the systematic review are prospective cohort studies with clinical pregnancy as an outcome measure. The SCSA has been applied in five out of nine studies while the SCD and TUNEL were both performed in two studies. There were no results for relevant studies with SCGE. All studies have performed the sperm DNA fragmentation analysis on the IUI sample although there is considerable heterogeneity with respect to when the sperm aliquot has been drawn for DNA fragmentation analysis, e.g.: before or after sperm preparation or both. This specification would be highly interesting as different studies have shown a difference in sperm DNA fragmentation after either density

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gradient centrifugation or swim-up technique, as compared to the basal rates [25–27]. Furthermore, all studies, but one, have reported their results per IUI cycle meaning a subfertile couple might have contributed to the final results with a minimum of one cycle but no clearly defined maximum for most. Four studies report a statistically significant association between lower levels of sperm DNA fragmentation and higher pregnancy rates, albeit with different methodological design, whereas three studies report no significant association. Muriel et al. [28] did not find a significant association between sperm DNA fragmentation and IUI outcome. Regrettably a clear definition of this outcome, whether pertaining to a biochemical or clinical pregnancy or even delivery, is nowhere to be found. Moreover, the inclusion criteria only relate to male sperm characteristics (non-severe male infertility) whereas the female partners have various diagnoses ranging from unexplained to advanced age. Consequently, there is no transparency as to how many cycles were performed per couple in the heterogeneous female population which possibly might contribute to the lack of association between sperm DNA fragmentation and IUI. Thomson et al. [29] also did not find a significant association. Interestingly, in comparison to reported threshold values ranging from 4 to 20% by several authors using TUNEL [30–32], the mean sperm DNA fragmentation in this study was relatively low in both the pregnant (4.1%) and non-pregnant (7.3%) group after IUI. Boe-Hansen et al. [33] do not report a statistical analysis due to both low number of couples with high sperm DNA fragmentation and low treatment success rate. Saleh et al. [34] did not perform a statistical analysis but the mean sperm DNA fragmentation values for couples with a positive clinical pregnancy were lower compared to those of couples with a negative outcome, perhaps suggesting a trend of more clinical pregnancies after IUI in couples with lower sperm DNA fragmentation. Duran et al. [30] reported the degree of DNA fragmentation after preparation was significantly lower in cycles resulting in pregnancy than in those that did not (7.3  3.5 versus 13.9  10.8 respectively; p = 0.044). Vandekerckhove et al. [35] set out to determine a ROC curve for sperm DNA fragmentation and clinical pregnancy as measured by SCD both before and after sperm treatment, unfortunately either the AUC was too small or the association was insignificant. Important to mention is the restriction of inclusion of eligible patients based on laboratory facilities leading to analysis of only 21 couples. Bungum et al. [36] studied the predictive value of sperm DNA fragmentation by SCSA for both in vivo and vitro ART outcomes. They concluded the odds ratio for obtaining a positive CP was significantly lower with a DNA fragmentation index (DFI) >30% as compared with 30% based on 387 IUI cycles. Yang et al. [24] have included 428 IUI cycles and report sperm DNA fragmentation as a robust indicator for IUI outcome with a cut-off DNA fragmentation index of 25%. Inclusion was restricted to men aged between 21 and 40 years with a total progressive sperm count of more than 5 million before washing. Women aged between 21 and 40 years with at least one patent fallopian tube and proven ovulatory cycles were included. The absence of information regarding the diagnosis of endometriosis is worth mentioning. Different correlations between DNA fragmentation and WHO semen parameters were reported in the included studies. However, we did not encounter predictive models pertaining to the interaction of these variables as predictors of pregnancy outcome. In order to distill the available data in answering the question concerning the predictive value of sperm DNA fragmentation tests and IUI outcome, we proceeded to perform a meta-analysis. While four studies have investigated the association between sperm DNA

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fragmentation and IUI outcome, these studies report sperm DNA fragmentation as a continuous variable and do not mention cut-off values [28–30,34]. We were thus able to construct a two-by-two table based on low and high sperm DNA fragmentation, as determined by the cutoff value chosen by the author, and clinical pregnancy for 4 studies. Given that each test needs solitary consideration as they are quite different in the aspects of what they study, we felt it necessary to limit our meta-analysis to the studies performing SCSA. As such we performed a meta-analysis based on the two-by-two table constructed from the three SCSA studies, which refer to the SCSA procedure as described by Evenson et al [37]. Meta-analysis The relationship between sperm DNA fragmentation and clinical pregnancy was analyzed in 917 IUI cycles. The hypothesis of the meta-analysis being there is a difference in clinical pregnancy outcome after IUI in couples with low versus high sperm DNA fragmentation. Regarding the association between low sperm DNA fragmentation and clinical pregnancy as analyzed with the random effects model, we found a relative risk (RR) of 3.30 (95% CI: 1.16; 9.39). The pooled sensitivity and specificity were respectively 94% (95% CI: 0.88; 0.97) and 19% (95% CI: 0.14; 0.26). We calculated a positive predictive value of 17% (95% CI: 0.11; 0.26) and negative predictive value of 95% (95% CI: 0.90; 0.98). We notice significant heterogeneity for specificity and positive predictive value. We also acknowledge the negative correlation between sensitivity and specificity but due to low number of studies it was not possible to fit the bivariate model proposed by Reitsma et al. (2005) (Fig. 2). Discussion/Conclusion

DNA fragmentation index of <30% [38]. However, these results must be interpreted with caution since the meta-analysis was based on two studies from the same group, and arguably, from the same patient population with both papers stating an identical study setting and inclusion criteria. The systematic review of 3 studies by Zini in 2011, also concluded that sperm DNA damage is associated with and might even have the greatest effect on IUI pregnancy rates although further prospective IUI studies are necessary before implementing testing in the routine patient management [39]. The current literature fails to bring forward a clear recommendation for clinical practice due to conflicting results (Table 1). We have noticed a wide variety in data reporting among the included studies. Some authors performed the statistical analysis on more than one cycle per couple [36], others on the first cycle per couple [35] and others have reported their results per included couple [33]. Furthermore, none of the included studies reported an a priori sample size calculation. The number of included cycles or couples appears to be fairly random or restricted by the running time of the study rather than by the statistical power necessary for the hypothesis testing. Interestingly, there is no report of longitudinal data concerning sperm DNA fragmentation and IUI outcome even though couples often go through more than one IUI cycle before obtaining a positive result. Given that sperm DNA fragmentation can be influenced by a myriad of intrinsic and extrinsic factors it seems plausible that this parameter might change between IUI cycles or more importantly between time of diagnosis and IUI. Ensuing, we share the concern expressed by Zini and Sigman [40] that the included studies only report sperm DNA fragmentation in the sample used for IUI, meaning the proposed test can’t be applied to change the patient’s treatment course as the couple is already in the IUI treatment arm.

Main findings Strengths and limitations To our knowledge, this is the first review providing a comprehensive overview and systematic quality assessment of publications on the subject of SDF and IUI outcome. We also performed a meta-analysis and expressed the association between sperm DNA fragmentation and IUI pregnancy in relative risk. According to the calculated RR of 3.3, couples with low levels of sperm DNA fragmentation are three times more likely to conceive after IUI. However, caution is warranted due to the poor test performance with the test failing to diagnose failure to conceive in 81%. Alternatively, the negative predictive value of high sperm DNA fragmentation resulting in a clinical pregnancy is high, namely 95%. These findings are in line with those obtained by Evenson and Wixon in 2006 who reported an odds ratio of 7.3 suggesting the odds of achieving a clinical pregnancy were 7 times greater with a

The SCSA meta-analysis was conducted with data from the well-known large prospective study by Bungum et al. (387 cycles) and from Boe-Hansen et al. though pertaining low number of cycles (48 cycles). Additionally, we were able to retrieve data from the study from Yang et al. [24] including 482 IUI cycles which contributed considerably to the meta-analysis. Due to the limited number of studies, the results from the metaanalysis should be interpreted with reservation. Also, we weren’t able to perform sub-analyses comparing the diagnostic/prognostic value of the different types of sperm DNA fragmentation tests nor the intra-assay and inter-observer variability. Moreover, there was insufficient data available for correction of possible confounders such as female and male age, semen parameters, uni- or multifollicular growth.

Fig. 2. Forest plot of relative risk (with 95% CI) on clinical pregnancy comparing low to high fragmentation group.

Table 1 Characteristics of studies included in the systematic review. Test type

Cohort

Infertility

Stimulation

Age (years)

Sample Semen

Reported cut-off value

Cycles (n)

Which cycle(s)

Outcome

Results

1

Muriel [28]

SCD (Halosperm)

Prospective

Start day 3: recFSH 75U to 150U

Not specified

IUI

Neat, after swimup

No cut-off value provided

100

Limited to one, not specified if first

CP, D

No association between SCD and IUI outcome (p > 0.05)

2

Vandekerckhove [35]

SCD (Halosperm)

Prospective

Only males with following semencount: Motility >25% Concentration >10  10^6/ml Total insemination count > 3  10^6 Idiopathic

CC 50 mg day 3–7

IUI

Neat, after DGC

DF >20,25,30%

21

First

CP, OP

No significant correlation (p > 0.05 or AUC < 0.70 cm2 )

3

Boe-Hansen [33]

SCSA

Prospective

Not specified

Not specified

Mean (SD) female age: 33.8 (4.1) Mean (SD) male age: 35.8 (8.2) Women: <34 years Men: not specified

IUI

Neat

DFI > 27%

48

Not specified

BP, CP

4

Bungum [36]

SCSA

Prospective

Idiopathic

Day 3–7: CC; day 7–9: recFSH 75U

IUI

Neat

DFI > 30%

387

Not specified

CP, D

No statistical analysis due to low success rate of treatment and only 2 couples with DFI > 27% A significantly lower odds of obtaining a CP was seen in the group with a DFI > 30% (OR = 0.1; 95% CI (0.02–0.42)

5

Saleh [34]

SCSA

Prospective

Idiopathic/ Male factor infertility

IUI

Not specified

Excellent: DFI < 15%; Good: 15% < DFI < 24%;

19

Not specified

CP

No statistical analysis report concerning the correlation between DFI and IUI outcome

Significant lower pregnancy rate after IUI in couples with DFI > 25% compared with DF  25% (OR: 0.38; 95% CI (0.16–0.97)) The percentage of DNA fragmentation after preparation was significantly lower in cycles resulting in pregnancy than in those that did not (p < 0.05)

6

Yang [24]

SCSA

Prospective

7

Duran [30]

TUNEL

Prospective

Idiopathic/ Non-severe male infertility (TMSP > 5 million) Not specified

8

Thomson [29]

TUNEL

Prospective

Mixed

Median (range) female age in the group DFI  30%: 29.9 (21.2–40.6); group DFI > 30% 32.1 (23.7–38.9) Median (range) male age in the group DFI  30%:31.1 (23.3–56.7); group DFI > 30%: 33.1(26.2–46.2) Not specified Mean (range) female age of cohort with idiopathic infertility: 30 (29–36); male factor infertility: 33 (30–35) Mean (range) male age of the cohort with idiopathic infertility: 33 (30–36); male factor infertility: 33 (31–37) Different Female age in the group protocols DFI  25%: 29.46 (3.19); group DFI > 25%: 29.65(3.82) Male age in the group DFI  25%: 31.16(3.84); group DFI > 25%: 31.73(4.74) CC 100 mg Mean (range) female age: day 3–7 or 34.6 (22–45) Mean (range) male age: CC 100 mg day 3–7 + start day 9: 36.5 (21–55) Gonadotrophins 150U or Gonadotrophines only: start day 3 75–150U recFSH Mean (SD) female age: 33.3 (3.8) Mean (SD) male age: 35.4 (6.1)

Fair: 25% < DFI < 30%; Poor: DFI > 30%

IUI

After DGC

DFI > 25%

482

Not specified

BP, CP

IUI

After DGC

No cut-off value provided

154

Mixed

CP

IUI

After DGC

No appropriate 53 sperm DNA fragmentation threshold value could be determined for predicting success

First 3 cycles

CP

A. Sugihara et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 244 (2020) 8–15

Study

Trend towards significant correlation between DF and IUI outcome; ROC analysis for IUI and CP show AUC = 0.675 cm2 (p = 0.08; 95% CI (0.5–0.85))

13

14

A. Sugihara et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 244 (2020) 8–15

Table 2 Study quality assessment according to the QUIPS (Quality In Prognosis Studies) tool Risk of bias.

Boe-Hansen [33] Bungum [36] Duran [30] Muriel [28] Saleh [34] Thomson [29] Vandekerckhove [35] Yang [24]

Study participation

Study Attrition

Prognostic Factor Measurement

Outcome measurement

Study Confounding

Statistical Analysis and Reporting

Moderate Low Low Low Low Low Moderate Low

Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate

Low Low Low Low Low Low Low Low

Low Low Low Low Low Low Low Low

Moderate Low Low High Moderate Low Low Moderate

High Low Low Moderate Low Low High Low

Conclusion

References

Our systematic review concludes that the current literature does not offer enough evidence to use sperm DNA fragmentation tests in the routine work-up of couples undergoing intra-uterine insemination. So far, the available studies show a limited capacity of sperm DNA fragmentation in discriminating between couples who will benefit from the test, namely in predicting IUI outcome or in advising for or against IUI as first choice of treatment or in advancing to more invasive medically assisted reproduction. This is in line with the conclusion formulated by the ASRM Opinion Committee [41] which recognizes the value of the research on DNA fragmentation but does not recommend routine testing in the clinical setting as the results, so far, do not independently predict pregnancy outcome after natural conception, IUI or IVF. However, we should take into account that this lack of evidence might be related to poor quality evidence, whether related to flawed study design, limited study size or potential confounders, rather than the true absence of an association.

[1] Lewis SE, Aitken RJ. DNA damage to spermatozoa has impacts on fertilization and pregnancy. Cell Tissue Res 2005;322(1):33–41. [2] Shamsi MB, Imam SN, Dada R. Sperm DNA integrity assays: diagnostic and prognostic challenges and implications in management of infertility. J Assist Reprod Genet 2011;28(11):1073–85. [3] Hamilton JA, Cissen M, Brandes M, Smeenk JM, de Bruin JP, Kremer JA, et al. Total motile sperm count: a better indicator for the severity of male factor infertility than the WHO sperm classification system. Hum Reprod 2015;30 (5):1110–21. [4] Wang C, Swerdloff RS. Limitations of semen analysis as a test of male fertility and anticipated needs from newer tests. Fertil Steril 2014;102(6):1502–7. [5] Kodama H, Yamaguchi R, Fukuda J, Kasai H, Tanaka T. Increased oxidative deoxyribonucleic acid damage in the spermatozoa of infertile male patients. Fertil Steril 1997;68(3):519–24. [6] Spano M, Bonde JP, Hjollund HI, Kolstad HA, Cordelli E, Leter G. Sperm chromatin damage impairs human fertility. The danish first pregnancy planner study team. Fertil Steril 2000;73(1):43–50. [7] Zini A, Bielecki R, Phang D, Zenzes MT. Correlations between two markers of sperm DNA integrity, DNA denaturation and DNA fragmentation, in fertile and infertile men. Fertil Steril 2001;75(4):674–7. [8] Zini A. Antioxidants and sperm DNA damage. Syst Biol Reprod Med 2009;55 (1):29–31. [9] Evenson DP, Jost LK, Marshall D, Zinaman MJ, Clegg E, Purvis K, et al. Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic. Hum Reprod 1999;14(4):1039–49. [10] Balasuriya A, Speyer B, Serhal P, Doshi A, Harper JC. Sperm chromatin dispersion test in the assessment of DNA fragmentation and aneuploidy in human spermatozoa. Reprod Biomed Online 2011;22(5):428–36. [11] Mitchell LA, De Iuliis GN, Aitken RJ. The TUNEL assay consistently underestimates DNA damage in human spermatozoa and is influenced by DNA compaction and cell vitality: development of an improved methodology. Int J Androl 2011;34(1):2–13. [12] Simon L, Lutton D, McManus J, Lewis SEM. 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(2):652–7. [13] Collins JA, Barnhart KT, Schlegel PN. Do sperm DNA integrity tests predict pregnancy with in vitro fertilization? Fertil Steril 2008;89(4):823–31. [14] Cissen M, Wely MV, Scholten I, Mansell S, Bruin JP, Mol BW, et al. Measuring sperm DNA fragmentation and clinical outcomes of medically assisted reproduction: a systematic review and meta-analysis. PLoS One 2016;11(11)e0165125. [15] Simon L, Zini A, Dyachenko A, Ciampi A, Carrell DT. A systematic review and meta-analysis to determine the effect of sperm DNA damage on in vitro fertilization and intracytoplasmic sperm injection outcome. Asian J Androl 2017;19(1):80–90. [16] Bensdorp AJ, Cohlen BJ, Heineman MJ, Vandekerckhove P. Intra-uterine insemination for male subfertility. Cochrane Database Syst Rev 2007(4) Cd000360. [17] Goverde AJ, McDonnell J, Vermeiden JP, Schats R, Rutten FF, Schoemaker J. Intrauterine insemination or in-vitro fertilisation in idiopathic subfertility and male subfertility: a randomised trial and cost-effectiveness analysis. Lancet (London, England) 2000;355(9197):13–8. [18] Guzick DS, Carson SA, Coutifaris C, Overstreet JW, Factor-Litvak P, Steinkampf MP, et al. Efficacy of superovulation and intrauterine insemination in the treatment of infertility. National Cooperative Reproductive Medicine Network. N Engl J Med 1999;340(3):177–83. [19] Steures P, van der Steeg JW, Mol BW, Eijkemans MJ, van der Veen F, Habbema JD, et al. Prediction of an ongoing pregnancy after intrauterine insemination. Fertil Steril 2004;82(1):45–51. [20] Group ECW. Intrauterine insemination. Hum Reprod Update 2009;15 (3):265–77. [21] Tummon IS, Asher LJ, Martin JS, Tulandi T. Randomized controlled trial of superovulation and insemination for infertility associated with minimal or mild endometriosis. Fertil Steril 1997;68(1):8–12. [22] Verhulst SM, Cohlen BJ, Hughes E, Te Velde E, Heineman MJ. Intra-uterine insemination for unexplained subfertility. Cochrane Database Syst Rev 2006 (4) Cd001838.

Implications More studies are needed concerning this highly interesting topic. In order to fully delineate the clinical value of sperm DNA fragmentation testing and to aid the clinical decision making these studies should ideally also address the following issues: To assess the chances of a spontaneous pregnancy and live birth in comparison to IUI for a certain sperm DNA fragmentation cut-off value; to determine the role of sperm DNA fragmentation measurement at diagnosis in predicting the IUI outcome, both independently or in combination with other clinical and semen parameters; to define the appropriate cut-off value for a specific assay/protocol in regards to predicting IUI outcome. Statements Disclosure statement The authors have no conflicts of interest to declare. Funding sources The study was conducted by employees of the Antwerp University Hospital, Belgium and funded by a governmental grant Fonds voor Wetenschappelijk onderzoek-TBMT007016N.

Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.ejogrb.2019. 10.005.

A. Sugihara et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 244 (2020) 8–15 [23] Veltman-Verhulst SM, Hughes E, Ayeleke RO, Cohlen BJ. Intra-uterine insemination for unexplained subfertility. Cochrane Database Syst Rev 2016;2: Cd001838. [24] Yang XY, Zhang Y, Sun XP, Cui YG, Qian XQ, Mao YD, et al. Sperm chromatin structure assay predicts the outcome of intrauterine insemination. Zhonghua Nan Ke Xue 2011;17(11):977–83. [25] Oguz Y, Guler I, Erdem A, Mutlu MF, Gumuslu S, Oktem M, et al. The effect of swim-up and gradient sperm preparation techniques on deoxyribonucleic acid (DNA) fragmentation in subfertile patients. J Assist Reprod Genet 2018;35 (6):1083–9. [26] Punjabi U, Van Mulders H, Goovaerts I, Peeters K, Clasen K, Janssens P, et al. Sperm DNA fragmentation in the total and vital fractions before and after density gradient centrifugation: significance in male fertility diagnosis. Clin Biochem 2018;62:47–54. [27] Donnelly ET, Kristine Steele E, McClure N, Lewis SEM. Assessment of DNA integrity and morphology of ejaculated spermatozoa from fertile and infertile men before and after cryopreservation. Hum Reprod 2001;16(6):1191–9. [28] Muriel L, Meseguer M, Fernandez JL, Alvarez J, Remohi J, Pellicer A, et al. Value of the sperm chromatin dispersion test in predicting pregnancy outcome in intrauterine insemination: a blind prospective study. Hum Reprod 2006;21 (3):738–44. [29] Thomson LK, Zieschang JA, Clark AM. Oxidative deoxyribonucleic acid damage in sperm has a negative impact on clinical pregnancy rate in intrauterine insemination but not intracytoplasmic sperm injection cycles. Fertil Steril 2011;96(4):843–7. [30] Duran EH, Morshedi M, Taylor S, Oehninger S. Sperm DNA quality predicts intrauterine insemination outcome: a prospective cohort study. Hum Reprod 2002;17(12):3122–8. [31] Borini A, Tarozzi N, Bizzaro D, Bonu MA, Fava L, Flamigni C, et al. Sperm DNA fragmentation: paternal effect on early post-implantation embryo development in ART. Hum Reprod 2006;21(11):2876–81.

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[32] Huang CC, Lin DP, Tsao HM, Cheng TC, Liu CH, Lee MS. Sperm DNA fragmentation negatively correlates with velocity and fertilization rates but might not affect pregnancy rates. Fertil Steril 2005;84(1):130–40. [33] Boe-Hansen GB, Fedder J, Ersboll AK, Christensen P. The sperm chromatin structure assay as a diagnostic tool in the human fertility clinic. Hum Reprod 2006;21(6):1576–82. [34] Saleh RA, Agarwal A, Nada EA, El-Tonsy MH, Sharma RK, Meyer A, et al. Negative effects of increased sperm DNA damage in relation to seminal oxidative stress in men with idiopathic and male factor infertility. Fertil Steril 2003;79(Suppl 3):1597–605. [35] Vandekerckhove FW, De Croo I, Gerris J, Vanden Abbeel E, De Sutter P. Sperm chromatin dispersion test before sperm preparation is predictive of clinical pregnancy in cases of unexplained infertility treated with intrauterine insemination and induction with clomiphene citrate. Front Med (Lausanne) 2016;3:63. [36] Bungum M, Humaidan P, Axmon A, Spano M, Bungum L, Erenpreiss J, et al. Sperm DNA integrity assessment in prediction of assisted reproduction technology outcome. Hum Reprod 2007;22(1):174–9. [37] Evenson DP, Larson KL, Jost LK. Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques. J Androl 2002;23(1):25–43. [38] Bungum M, Humaidan P, Spano M, Jepson K, Bungum L, Giwercman A. The predictive value of sperm chromatin structure assay (SCSA) parameters for the outcome of intrauterine insemination, IVF and ICSI. Hum Reprod 2004;19 (6):1401–8. [39] Zini A. Are sperm chromatin and DNA defects relevant in the clinic? Syst Biol Reprod Med 2011;57(1-2):78–85. [40] Zini A, Sigman M. Are tests of sperm DNA damage clinically useful? Pros and cons. J Androl 2009;30(3):219–29. [41] ASRM. The clinical utility of sperm DNA integrity testing: a guideline. Fertil Steril 2013;99(3):673–7.