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Add-ons in the laboratory: hopeful, but not always helpful

2019; Elsevier BV; Volume: 112; Issue: 6 Linguagem: Inglês

10.1016/j.fertnstert.2019.10.031

1556-5653

Sarah Armstrong, M. Atkinson, Jeanette MacKenzie, Allan Pacey, Cindy Farquhar,

Ovarian function and disorders

All the steps in an in vitro fertilization cycle are important but none more so than those that occur in the laboratory. To improve the chance of success, adjuncts, commonly referred to as ‘add-ons’, are offered. Yet as with other new interventions, add-ons in the laboratory require justification by well-designed studies prior to being offered as routine practice. Add-ons aim to improve the chance of a take-home baby, but, their safety and efficacy is less than clear. In addition, the financial burden from the use of add-ons is often borne by the couple. This review of the most commonly used laboratory add-ons did not find any high-quality evidence to support their use in routine practice. All the steps in an in vitro fertilization cycle are important but none more so than those that occur in the laboratory. To improve the chance of success, adjuncts, commonly referred to as ‘add-ons’, are offered. Yet as with other new interventions, add-ons in the laboratory require justification by well-designed studies prior to being offered as routine practice. Add-ons aim to improve the chance of a take-home baby, but, their safety and efficacy is less than clear. In addition, the financial burden from the use of add-ons is often borne by the couple. This review of the most commonly used laboratory add-ons did not find any high-quality evidence to support their use in routine practice. Discuss: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/users/16110-fertility-and-sterility/posts/55093-28992 Discuss: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/users/16110-fertility-and-sterility/posts/55093-28992 The interventions that occur in the fertility laboratory are of critical importance to the outcomes of an in vitro fertilization (IVF) cycle. It is not surprising that many scientists and clinicians seek improvements in outcomes by proposing adjuncts (or add-ons) to the traditional laboratory procedures involved in IVF. In the early days of IVF, few innovations were comprehensively evaluated. Some readers may remember gamete intrafallopian transfer, zygote intrafallopian tube transfer, and preimplantation genetic screening on a subset of chromosomes using fluorescence in situ hybridization, all now relegated to history. The IVF laboratory now faces many rapid changes and innovations with some advances being driven by industry. Most laboratory add-ons fall outside the pharmaceutical regulations and are therefore often not required to have rigorous evaluations before being marketed. As a result, many are unsupported by well-designed research (1Heneghan C. Spencer E.A. Bobrovitz N. Collins D.R.J. Nunan D. Plüddemann A. et al.Lack of evidence for interventions offered in UK fertility centres.BMJ. 2016; 355: 392-394Google Scholar, 2Harper J. Jackson E. Sermon K. Aitken R.J. Harbottle S. Mocanu E. et al.Adjuncts in the IVF laboratory: where is the evidence for 'add-on' interventions?.Hum Reprod. 2017; 32: 485-491Crossref PubMed Scopus (95) Google Scholar). Add-ons may rapidly become established as part of normal working practice well before high quality studies such as randomized controlled trials (RCTs) have been completed. Finally, the cost of add-ons is often borne by patients in the belief that if it is offered, then it must have been properly evaluated. Clearly, it is important that add-ons are properly evaluated so that benefits and harms can be reported. The UK’s Human Fertilisation and Embryology Authority (HFEA) defines IVF add-ons for patients as, “… optional extras you may be offered on top of your normal fertility treatment, often at an additional cost. They’re sometimes emerging techniques that may have shown some promising results in initial studies, or they may have been around for a number of years, but haven’t necessarily been proven to improve pregnancy or birth rates…” (3Human Fertilization and Embryology AuthorityTreatment add-ons 2018.https://www.hfea.gov.uk/treatments/explore-all-treatments/treatment-add-ons/Google Scholar). In this article we discuss the some of the most commonly offered laboratory add-ons, outlining the best available clinical evidence on their use (Fig 1). We have specifically not discussed preimplantation genetic testing for aneuploidy (PGT-A) as this has been debated extensively already. Embryos develop in an incubator, moving through the fertilization stage to cleavage stage and on to blastocyst stage in some cases. Embryologists check the developing embryos in order to select those most likely to implant and develop into a baby (4Hong K.H. Werner M.D. Franasiak J.M. Forman E.J. Prodoehl A. Upham K. et al.Embryologist interpretation of time-lapse imaging parameters at the blastocyst stage do not alter selection among transferred euploid blastocysts.Fertil Steril. 2014; 102: e305Abstract Full Text Full Text PDF Google Scholar, 5Scott L. The biological basis of non-invasive strategies for selection of human oocytes and embryos.Hum Reprod Update. 2003; 9: 237-249Crossref PubMed Scopus (93) Google Scholar, 6Scott L. Pronuclear scoring as a predictor of embryo development.Reprod Biomed Online. 2003; 6: 201-214Abstract Full Text PDF PubMed Scopus (130) Google Scholar, 7Shoukir Y. Campana A. Farley T. Sakkas D. Early cleavage of in-vitro fertilized human embryos to the 2-cell stage: a novel indicator of embryo quality and viability.Hum Reprod. 1997; 12: 1531-1536Crossref PubMed Scopus (246) Google Scholar, 8Alpha-ScientistsThe Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting.Hum Reprod. 2011; 26: 1270-1283Crossref PubMed Scopus (1033) Google Scholar, 9Cutting R. Morroll D. Roberts S.A. Pickering S. Rutherford A. Elective single embryo transfer: guidelines for practice British Fertility Society and Association of Clinical Embryologists.Hum Fertil. 2008; 11: 131-146Crossref PubMed Scopus (156) Google Scholar). Traditionally, embryos are removed from the controlled environment of the incubator and briefly placed under a microscope to be examined by an embryologist. In contrast, time-lapse systems (TLS) allow the embryologist to monitor the developing embryo without removing it from the incubator, and to select the best embryo for transfer based on morphology, and on the timing and synchronicity of early mitotic divisions and abnormal cleavage patterns that generate morphokinetic parameters (10Bhide P. Maheshwari A. Cutting R. Seenan S. Patel A. Khan K. et al.Time lapse imaging: is it time to incorporate this technology into routine clinical practice?.Hum Fertil. 2017; 20: 74-79Crossref PubMed Scopus (12) Google Scholar). There has been widespread uptake of TLS in IVF clinics worldwide (11VitrolifeGeneral 2015.http://www.vitrolife.com/en/Products/EmbryoScope-Time-Lapse-System/Support-Material/FAQ/General/Google Scholar). Potential advantages of TLS include: the availability of detailed digital images of developing embryos, which can be compiled to create a time-lapse sequence of their development; achievement of an undisturbed culture environment for embryos, which avoids exposing embryos to mechanical disturbance or changes in temperature, pH, humidity and gas composition (12Meseguer M. Rubio I. Cruz M. Basile N. Marcos J. Requena A. Embryo incubation and selection in a time-lapse monitoring system improves pregnancy outcome compared with a standard incubator: a retrospective cohort study.Fertil Steril. 2012; 98: 1481-1489.e10Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar); and the availability of embryo selection software, with complex algorithms based on a combination of morphokinetic parameters and selection and de-selection criteria which help the embryologist to select the optimal embryo for transfer (12Meseguer M. Rubio I. Cruz M. Basile N. Marcos J. Requena A. Embryo incubation and selection in a time-lapse monitoring system improves pregnancy outcome compared with a standard incubator: a retrospective cohort study.Fertil Steril. 2012; 98: 1481-1489.e10Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 13Armstrong S. Vail A. Mastenbroek S. Jordan V. Farquhar C. Time-lapse in the IVF-lab: how should we assess potential benefit?.Hum Reprod. 2015; 30: 3-8Crossref PubMed Scopus (53) Google Scholar, 14Conaghan J. Chen A.A. Willman S.P. Ivani K. Chenette P.E. Boostanfar R. et al.Improving embryo selection using a computer-automated time-lapse image analysis test plus day 3 morphology: results from a prospective multicenter trial.Fertil Steril. 2013; 100: 412-419.e5Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Potential disadvantages include the increased osmolarity in drops as some TLS cannot be humified, and the cost, which is approximately $1000 per cycle although not all clinics charge patients (1Heneghan C. Spencer E.A. Bobrovitz N. Collins D.R.J. Nunan D. Plüddemann A. et al.Lack of evidence for interventions offered in UK fertility centres.BMJ. 2016; 355: 392-394Google Scholar, 15Swain J.E. Schoolcraft W.B. Bossert N. Batcheller A.E. Media osmolality changes over 7 days following culture in a non-humidified benchtop incubator.Fertil Steril. 2016; 106: e362Abstract Full Text Full Text PDF Google Scholar). A 2019 Cochrane systematic review (16Armstrong S. Bhide P. Jordan V. Pacey A. Marjoribanks J. Farquhar C. Time-lapse systems for embryo incubation and assessment in assisted reproduction.Cochrane Database Syst Rev. 2019; 5: CD011320PubMed Google Scholar) included nine RCTs (2955 women). Compared with conventional incubation it was unclear if there was a difference in live birth rate with the use of the time-lapse images with an algorithm (odds ratio [OR] 1.12, 95% confidence interval [CI] 0.92–1.36; 3 RCTs). Overall the review concluded that there was insufficient good quality evidence of differences in live birth or ongoing pregnancy, miscarriage, stillbirth or clinical pregnancy to choose between TLS, with or without embryo selection software, and conventional incubation. The evidence was low or very low quality overall. The HFEA consider that the evidence is conflicting for TLS and that there is certainly not enough evidence to show that time-lapse imaging improves birth rates (3Human Fertilization and Embryology AuthorityTreatment add-ons 2018.https://www.hfea.gov.uk/treatments/explore-all-treatments/treatment-add-ons/Google Scholar). The zona pellucida is an acellular glycoprotein coat involved in different processes during fertilization and embryo development. Once the blastocyst reaches the uterus, the embryo needs to exit the zona pellucida so that it can interact with the endometrium and implant. It has been suggested that zona pellucida hardening may occur as a result of in vitro culture (17Cohen J. Assisted hatching of human embryos.J In Vitro Fert Embryo Transf. 1991; 8: 179-190Crossref PubMed Scopus (161) Google Scholar). The escape of the embryo may be inhibited by a thickened or hardened zona pellucida (18Cohen J. Alikani M. Trowbridge J. Rosenwaks Z. Implantation enhancement by selective assisted hatching using zona drilling of human embryos with poor prognosis.Hum Reprod. 1992; 7: 685-691Crossref PubMed Scopus (377) Google Scholar) and it is suggested that failure of the zona pellucida to rupture following blastocyst expansion may be a contributing factor in failure of embryo implantation. Assisted hatching can be performed with acid, a laser or mechanically. Acid tyrodes digests the zona pellucida leaving a breach. Partial zona dissection uses a micropipette to mechanically slice through the zona pellucida. Laser-assisted hatching uses photoablation to make a very precise and accurately controlled opening in the zona pellucida. Possible advantages of assisted hatching are that it may assist the embryo to hatch by allowing the embryo to come away from the zona pellucida more freely. However, possible disadvantages are that it may cause damage to the embryo and may increase the risk of a multiple pregnancy (19Carney S.K. Das S. Blake D. Farquhar C. Seif M.M. Nelson L. Assisted hatching on assisted conception (in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI).Cochrane Database Syst Rev. 2012; 12: CD001894PubMed Google Scholar). The reason for the increase in multiple pregnancies is difficult to explain as most of the studies transfer more than one embryo but it is possible that monozygotic twinning occurs because of embryo splitting occurring during artificial zona hatching (20Li D. Yang D.L. An J. Jiao J. Zhou Y.M. Wu Q.J. et al.Effect of assisted hatching on pregnancy outcomes: a systematic review and meta-analysis of randomized controlled trials.Sci Rep. 2016; 6: 31228Crossref PubMed Scopus (38) Google Scholar). A Cochrane review of 31 RCTs in 2012 demonstrated that although assisted hatching does appear to offer a significantly increased chance of achieving a clinical pregnancy (OR 1.13, 95% CI 1.01–1.27; moderate quality evidence) there was no increase in the live birth rate (9 RCTs; OR 1.03, 95% CI 0.85–1.26; moderate quality evidence) (19Carney S.K. Das S. Blake D. Farquhar C. Seif M.M. Nelson L. Assisted hatching on assisted conception (in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI).Cochrane Database Syst Rev. 2012; 12: CD001894PubMed Google Scholar). When restricting analysis of clinical pregnancy rate to those trials that went on to report live birth, which overall were better quality trials with a lower risk of bias, the clinical pregnancy result showed insufficient evidence of a difference between the assisted hatching and the control group. A different systematic review in 2016 had similar results with no difference in live birth rate (OR 1.09, 95% CI 0.92–1.30) but a small increase in clinical pregnancies OR 1.16, 95% CI 1.00–1.36) and multiple pregnancy rates (OR 1.50, 95% CI 1.11–2.01) with assisted hatching (20Li D. Yang D.L. An J. Jiao J. Zhou Y.M. Wu Q.J. et al.Effect of assisted hatching on pregnancy outcomes: a systematic review and meta-analysis of randomized controlled trials.Sci Rep. 2016; 6: 31228Crossref PubMed Scopus (38) Google Scholar). Overall, there was some evidence of increased multiple pregnancy in the assisted hatching group in both systematic reviews but as more than one embryo was usually transferred this is difficult to interpret (19Carney S.K. Das S. Blake D. Farquhar C. Seif M.M. Nelson L. Assisted hatching on assisted conception (in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI).Cochrane Database Syst Rev. 2012; 12: CD001894PubMed Google Scholar, 20Li D. Yang D.L. An J. Jiao J. Zhou Y.M. Wu Q.J. et al.Effect of assisted hatching on pregnancy outcomes: a systematic review and meta-analysis of randomized controlled trials.Sci Rep. 2016; 6: 31228Crossref PubMed Scopus (38) Google Scholar). Only the Cochrane review reported on monozygotic twinning but only 6 of the 31 trials reported this outcome and the data is inconclusive (19Carney S.K. Das S. Blake D. Farquhar C. Seif M.M. Nelson L. Assisted hatching on assisted conception (in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI).Cochrane Database Syst Rev. 2012; 12: CD001894PubMed Google Scholar). The prevalence of multiple pregnancy with monozygotic splitting after elective single embryo transfer was 1.36% and was associated with embryo manipulations including assisted hatching (20Li D. Yang D.L. An J. Jiao J. Zhou Y.M. Wu Q.J. et al.Effect of assisted hatching on pregnancy outcomes: a systematic review and meta-analysis of randomized controlled trials.Sci Rep. 2016; 6: 31228Crossref PubMed Scopus (38) Google Scholar). Another cohort study suggested that blastocyst transfer is more likely to lead to monozygotic twinning (21Mateizel I. Santos-Ribeiro S. Done E. Van Landuyt L. Van de Velde H. Tournaye H. et al.Do ARTs affect the incidence of monozygotic twinning?.Hum Reprod. 2016; 31: 2435-2441Crossref PubMed Scopus (40) Google Scholar). The quality of the evidence is overall poor to moderate secondary to selective reporting in several studies, and significant statistical heterogeneity between trials. The National Institute for Health and Care Excellence (NICE, UK) and the HFEA recommends against the use of assisted hatching, due to lack of evidence of benefit and safety (3Human Fertilization and Embryology AuthorityTreatment add-ons 2018.https://www.hfea.gov.uk/treatments/explore-all-treatments/treatment-add-ons/Google Scholar, 22NICEFertility problems: assessment and treatment (CG156) 2017.https://www.nice.org.uk/guidance/CG156Google Scholar). In 2014 the American Society for Reproductive Medicine (ASRM) published guidelines on assisted hatching, they stated there is good evidence that assisted hatching slightly improves clinical pregnancy rates in poor prognosis patients but insufficient evidence to conclude that assisted hatching improves live birth rates. The ASRM recommends that assisted hatching should not be recommended routinely for all patients undergoing IVF (23American Society for Reproductive, Society for Assisted Reproductive TechnologyRole of assisted hatching in in vitro fertilization: a guideline.Fertil Steril. 2014; 102: 348-351Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The rate of human implantation is low (10%-30%), and failed implantation is a common cause of IVF failure even in younger women with euploid embryos (24Schoolcraft W.B. Katz-Jaffe M.G. Comprehensive chromosome screening of trophectoderm with vitrification facilitates elective single-embryo transfer for infertile women with advanced maternal age.Fertil Steril. 2013; 100: 615-619Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Any improvement in the implantation rate may maximize the chance of achieving a pregnancy and livebirth. Improved implantation rates may also lead to a reduction in multiple embryo transfer in IVF and multiple pregnancy (25Bontekoe S. Heineman M.J. Johnson N. Blake D. Adherence compounds in embryo transfer media for assisted reproductive technologies.Cochrane Database Syst Rev. 2014; : CD007421PubMed Google Scholar). Hyaluronic acid (HA) is used as a supplement to conventional embryo transfer medium, with the aim of improving implantation rates via the following proposed mechanisms (26Fancsovits P. Lehner A. Murber A. Kaszas Z. Rigo J. Urbancsek J. Effect of hyaluronan-enriched embryo transfer medium on IVF outcome: a prospective randomized clinical trial.Arch Gynecol Obstet. 2015; 291: 1173-1179Crossref PubMed Scopus (29) Google Scholar): indirectly promote angiogenesis and improve cell-to-cell and cell-to matrix adhesion, thus assisting in embryo apposition and attachment to the endometrium; enhance embryo transfer and prevent expulsion of embryos from the uterine cavity after transfer because of HA’s high viscosity; and act as a receptor mediator, as the primary HA receptor is CD44, which is also expressed in the pre-implantation embryo and in the endometrium. A 2015 Cochrane review included 16 RCTs (n=3,687) using transfer medium supplemented with HA versus transfer medium not supplemented with HA and reported moderate quality evidence of improved live birth rates with the intervention (OR 1.41, 95% CI 1.17–1.69; 6 RCTs, n = 1,950, moderate-quality evidence). However multiple pregnancy rates were also increased, possibly due to combination of adherence compound as most studies transferred multiple embryos (25Bontekoe S. Heineman M.J. Johnson N. Blake D. Adherence compounds in embryo transfer media for assisted reproductive technologies.Cochrane Database Syst Rev. 2014; : CD007421PubMed Google Scholar). A subsequent RCT compared HA versus conventional transfer medium in 581 cycles and found no evidence of an improvement in live birth rates (26Fancsovits P. Lehner A. Murber A. Kaszas Z. Rigo J. Urbancsek J. Effect of hyaluronan-enriched embryo transfer medium on IVF outcome: a prospective randomized clinical trial.Arch Gynecol Obstet. 2015; 291: 1173-1179Crossref PubMed Scopus (29) Google Scholar). A raised multiple pregnancy rate is the expected natural consequence of increased implantation and pregnancy rates. This suggests that clinics using HA supplemented embryo transfer medium should adopt an elective single embryo transfer policy, closely monitor their multiple pregnancy rate and ensure that patients are aware of the increased chance of multiple pregnancy if multiple embryos are transferred (2Harper J. Jackson E. Sermon K. Aitken R.J. Harbottle S. Mocanu E. et al.Adjuncts in the IVF laboratory: where is the evidence for 'add-on' interventions?.Hum Reprod. 2017; 32: 485-491Crossref PubMed Scopus (95) Google Scholar). The HFEA considers the evidence to be conflicting and suggests that further high-quality studies are needed before patients, clinicians and embryologists can be confident of the benefits of hyaluronic acid. The quality of a sperm sample is not completely described by reporting the number and motility of spermatozoa (27Leuchtenberger C. Schrader F. Weir D.R. Gentile D.P. The desoxyribosenucleic acid (DNA) content in spermatozoa of fertile and infertile human males.Chromosoma. 1953; 6: 61-78Crossref PubMed Scopus (17) Google Scholar, 28De Jonge C. Semen analysis: looking for an upgrade in class.Fertil Steril. 2012; 97: 260-266Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Tests of sperm DNA fragmentation attempt to provide further information about sperm quality with the aim of improving outcomes such as live birth. A number of sperm DNA fragmentation tests have been developed and these include the: sperm chromatin structure assay; sperm chromatin dispersiontest; terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labelling; and single cell gel electrophoresis assay (29Cissen M. Wely M.V. Scholten I. Mansell S. Bruin J.P. Mol B.W. et al.Measuring Sperm DNA Fragmentation and Clinical Outcomes of Medically Assisted Reproduction: A Systematic Review and Meta-Analysis.PLoS One. 2016; 11: e0165125Crossref PubMed Scopus (173) Google Scholar, 30Collins J.A. Barnhart K.T. Schlegel P.N. Do sperm DNA integrity tests predict pregnancy with in vitro fertilization?.Fertil Steril. 2008; 89: 823-831Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 31Deng C. Li T. Xie Y. Guo Y. Yang Q.Y. Liang X. et al.Sperm DNA fragmentation index influences assisted reproductive technology outcome: A systematic review and meta-analysis combined with a retrospective cohort study.Andrologia. 2019; 51: e13263Crossref PubMed Scopus (66) Google Scholar, 32Evenson D. Wixon R. Meta-analysis of sperm DNA fragmentation using the sperm chromatin structure assay.Reprod Biomed Online. 2006; 12: 466-472Abstract Full Text PDF PubMed Scopus (218) Google Scholar, 33Li Z. Wang L. Cai J. Huang H. Correlation of sperm DNA damage with IVF and ICSI outcomes: a systematic review and meta-analysis.J Assist Reprod Genet. 2006; 23: 367-376Crossref PubMed Scopus (127) Google Scholar, 34Osman A. Alsomait H. Seshadri S. El-Toukhy T. Khalaf Y. The effect of sperm DNA fragmentation on live birth rate after IVF or ICSI: a systematic review and meta-analysis.Reprod Biomed Online. 2015; 30: 120-127Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 35Simon L. Zini A. Dyachenko A. Ciampi A. Carrell D.T. 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: 80-90PubMed Google Scholar, 36Zhao J. Zhang Q. Wang Y. Li Y. Whether sperm deoxyribonucleic acid fragmentation has an effect on pregnancy and miscarriage after in vitro fertilization/intracytoplasmic sperm injection: a systematic review and meta-analysis.Fertil Steril. 2014; 102: 998-1005.e8Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). Several underlying causes of sperm DNA fragmentation have been proposed. These include defective apoptosis, excessive reactive oxygen species production; and decreased seminal antioxidants. Exogenous factors have also been implicated including toxic effects of drugs, cigarette smoking, pollution, high testicular temperature associated with systemic fever or varicoceles, and advanced age (29Cissen M. Wely M.V. Scholten I. Mansell S. Bruin J.P. Mol B.W. et al.Measuring Sperm DNA Fragmentation and Clinical Outcomes of Medically Assisted Reproduction: A Systematic Review and Meta-Analysis.PLoS One. 2016; 11: e0165125Crossref PubMed Scopus (173) Google Scholar). A potential benefit of identifying high sperm DNA fragmentation could be that it motivates the couple to avoid such exposures. Ultimately, we must consider if measurement of sperm DNA fragmentation will bring benefit to a couple by influencing clinical management. This must be weighed against the potential disadvantages of the additional cost incurred by the patient for running the sperm DNA fragmentation test, and then the additional cost of any subsequent management strategies proposed. Multiple systematic reviews and meta-analyses have been performed regarding the clinical utility of sperm DNA fragmentation tests (29Cissen M. Wely M.V. Scholten I. Mansell S. Bruin J.P. Mol B.W. et al.Measuring Sperm DNA Fragmentation and Clinical Outcomes of Medically Assisted Reproduction: A Systematic Review and Meta-Analysis.PLoS One. 2016; 11: e0165125Crossref PubMed Scopus (173) Google Scholar, 30Collins J.A. Barnhart K.T. Schlegel P.N. Do sperm DNA integrity tests predict pregnancy with in vitro fertilization?.Fertil Steril. 2008; 89: 823-831Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 31Deng C. Li T. Xie Y. Guo Y. Yang Q.Y. Liang X. et al.Sperm DNA fragmentation index influences assisted reproductive technology outcome: A systematic review and meta-analysis combined with a retrospective cohort study.Andrologia. 2019; 51: e13263Crossref PubMed Scopus (66) Google Scholar, 32Evenson D. Wixon R. Meta-analysis of sperm DNA fragmentation using the sperm chromatin structure assay.Reprod Biomed Online. 2006; 12: 466-472Abstract Full Text PDF PubMed Scopus (218) Google Scholar, 33Li Z. Wang L. Cai J. Huang H. Correlation of sperm DNA damage with IVF and ICSI outcomes: a systematic review and meta-analysis.J Assist Reprod Genet. 2006; 23: 367-376Crossref PubMed Scopus (127) Google Scholar, 34Osman A. Alsomait H. Seshadri S. El-Toukhy T. Khalaf Y. The effect of sperm DNA fragmentation on live birth rate after IVF or ICSI: a systematic review and meta-analysis.Reprod Biomed Online. 2015; 30: 120-127Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 35Simon L. Zini A. Dyachenko A. Ciampi A. Carrell D.T. 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: 80-90PubMed Google Scholar, 36Zhao J. Zhang Q. Wang Y. Li Y. Whether sperm deoxyribonucleic acid fragmentation has an effect on pregnancy and miscarriage after in vitro fertilization/intracytoplasmic sperm injection: a systematic review and meta-analysis.Fertil Steril. 2014; 102: 998-1005.e8Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). Although there is some low quality evidence that antioxidant supplementation might improve live birth rates in subfertile men (37Smits R.M. Mackenzie-Proctor R. Yazdani A. Stankiewicz M.T. Jordan V. Showell M.G. Antioxidants for male subfertility.Cochrane Database Syst Rev. 2019; 3: CD007411PubMed Google Scholar), it should be noted as well that these studies have been performed in couples attending fertility clinics, rather than only including men with high DNA fragmentation levels. The question of whether DNA fragmentation tests should guide the decision for treatment is also unclear as although the chance of spontaneous conception is low with a DNA fragmentation index >20%, and approaches zero for DNA fragmentation index >30% to 40% (38Evenson D.P. Jost L.K. Marshall D. Zinaman M.J. 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: 1039-1049Crossref PubMed Scopus (921) Google Scholar, 39Spano M. Bonde J.P. Hjollund H.I. Kolstad H.A. Cordelli E. Leter G. Sperm chromatin damage impairs human fertility. The Danish First Pregnancy Planner Study Team.Fertil Steril. 2000; 73: 43-50Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar), there are no trials comparing the likelihood of spontaneous pregnancy to pregnancy following medically assisted reproduction with different DNA fragmentation thresholds (29Cissen M. Wely M.V. Scholten I. Mansell S. Bruin J.P. Mol B.W. et al.Measuring Sperm DNA Fragmentation and Clinical Outcomes of Medically Assisted Reproduction: A Systematic Review and Meta-Analysis.PLoS One. 2016; 11: e0165125Crossref PubMed Scopus (173) Google Scholar). The ASRM (40Practice Committee of the American Society for Reproductive M. The clinical utility of sperm DNA integrity testing: a guideline.Fertil Steril. 2013; 99: 673-677Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar) and the British Fertility Society (41Tomlinson M. Lewis S. Morroll D. British Fertility S. Sperm quality and its relationship to natural and assisted conception: British Fertility Society guidelines for practice.Hum Fertil. 2013; 16: 175-193Crossref PubMed Scopus (48) Google Scholar) have both concluded that current methods for assessing sperm DNA integrity do not reliably predict treatment outcomes and hence cannot be recommended routinely for clinical use. The limitation of sperm DNA fragmentation measurements in predicting pregnancy may be secondary to the fact that multiple other factors also influence pregnancy. In particular the quality and age of the ooc