Male factor testing in recurrent pregnancy loss cases: A narrative review
Recurrent pregnancy loss is a distinct disorder defined as the loss of at least 2 pregnancies before the 20th wk of gestation. With half of the genome of the embryo belonging to the father, the integrity of the sperm genome is crucial for a successful pregnancy. Semen analysis is recommended for men in such cases to evaluate sperm concentration, morphology, vitality and motility. However, other important sperm parameters such as sperm epigenetics, aneuploidy, Y chromosome microdeletion and chromatin integrity also correlate with successful pregnancy and delivery rate. This article examines the use of different sperm tests and their importance in male partners of women suffering from recurrent pregnancy loss.
Key words: DNA fragmentation, Sperm, Y chromosome, Recurrent pregnancy loss.
 Talebi AR, Fesahat F, Mangoli E, Ghasemzadeh J, Nayeri M, Sadeghian-Nodoshan F. Relationship between sperm protamine deficiency and apoptosis in couples with unexplained repeated spontaneous abortions. Int J Reprod Biomed 2016; 14: 199–204.
 Nematollahi Z, Hadinedoushan H, Aflatoonian A, Eslami G, Ghasemi N. The association between single nucleotide polymorphism in interleukin-27 gene and recurrent pregnancy loss in Iranian women. Iran J Reprod Med 2015; 13: 209–214.
 Talebi AR, Vahidi S, Aflatoonian A, Ghasemi N, Ghasemzadeh J, Dehghani Firoozabadi R, et al. Cytochemical evaluation of sperm chromatin and DNA integrity in couples with unexplained recurrent spontaneous abortions. Andrologia 2012; 44 (Suppl.): 462–470.
 Pourmasumi S, Ghasemi N, Talebi AR, Mehrabani M, Sabeti P. The effect of vitamin E and selenium on sperm chromatin quality in couples with recurrent miscarriage. Int J Med Lab 2018; 5: 1–10.
 Dehghani Firoozabadi R, Klantar M, Seyed-Hasani M, Ghasemi N, Asgharnia M, Sheikhha MH. Cytogenetic analysis in couples with recurrent spontaneous abortion. Iran J Reprod Med 2006; 4: 13–17.
 Sabeti P, Pourmasumi S, Rahiminia T, Akyash F, Talebi AR. Etiologies of sperm oxidative stress. Int J Reprod Biomed 2016; 14: 231–240.
 Sharma R, Agarwal A, Rohra VK, Assidi M, Abu-Elmagd M, Turki RF. Effects of increased paternal age on sperm quality, reproductive outcome and associated epigenetic risks to offspring. Reprod Biol Endocrinol 2015; 13: 35.
 Ramasamy R, Chiba K, Butler P, Lamb DJ. Male biological clock: A critical analysis of advanced paternal age. Fertil Steril 2015; 103: 1402–1406.
 Jenkins TG, Aston KI, Meyer T, Carrell DT. The sperm epigenome, male aging, and potential effects on the embryo. Adv Exp Med Biol 2015; 868: 81–93.
 Maconochie N, Doyle P, Prior S, Simmons R. Risk factors for first trimester miscarriage-results from a UK: Population based case-control study. BJOG 2007; 114: 170–186.
 Kleinhaus K, Perrin M, Friedlander Y, Paltiel O, Malaspina D, Harlap S. Paternal age and spontaneous abortion. Obstet Gynecol 2006; 108: 369–377.
 De La Rochebrochard E, Thonneau P. Paternal age: Are the risks of infecundity and miscarriage higher when the man is aged 40 years or over? Rev Epidemiol Sante Publique 2005; 53: 2S47–2S55.
 de La Rochebrochard E, Thonneau P. Paternal age ≥ 40 years: An important risk factor for infertility. Am J Obstet Gynecol 2003; 189: 901–905.
 Ghuman NK, Mair E, Pearce K, Choudhary M. Does age of the sperm donor influence live birth outcome in assisted reproduction? Hum Reprod 2016; 31: 582–590.
 Leridon H. Can assisted reproduction technology compensate for the natural decline in fertility with age? A model assessment. Hum Reprod 2004; 19: 1548–1553.
 Glazer CH, Bonde JP, Eisenberg ML, Giwercman A, Haervig KK, Rimborg S, et al. Male infertility and risk of nonmalignant chronic diseases: A systematic review of the epidemiological evidence. Semin Reprod Med 2017; 35: 282–290.
 Mima M, Greenwald D, Ohlander S. Environmental toxins and male fertility. Curr Urol Rep 2018; 19: 50.
 Skurla M, Rybar R. Obesity and reduced fertility of men. Ceska Gynekol 2018; 83: 212–217.
 Bendayan M, Alter L, Swierkowski-Blanchard N, Caceres- Sanchez L, Selva J, Robin G, et al. [Environment and lifestyle: Impacts on male fertility?]. Gynecol Obstet Fertil Senol 2018; 46: 47–56. (in French)
 Pourmasumi S, Sabeti P, Rahiminia T, Mangoli E, Tabibnejad N, Talebi AR. The etiologies of DNA abnormalities in male infertility: An assessment and review. Int J Reprod Biomed 2017; 15: 331–344.
 Robinson L, Gallos ID, Conner SJ, Rajkhowa M, Miller D, Lewis S, et al. The effect of sperm DNA fragmentation on miscarriage rates: A systematic review and metaanalysis. Hum Reprod 2012; 27: 2908–2917.
 Pourmasumi S, Khoradmehr A, Rahiminia T, Sabeti P, Talebi A, Ghasemzadeh J. Evaluation of sperm chromatin integrity using aniline blue and toluidine blue staining in infertile and normozoospermic men. J Reprod Infertil 2019; 20: 95–101.
 Saxena P, Misro MM, Chaki SP, Chopra K, Roy S, Nandan D. Is abnormal sperm function an indicator among couples with recurrent pregnancy loss? Fertil Steril 2008; 90: 1854–1858.
 Zolghadri J, Tavana Z, Alborzi S, Kazerooni T. Pregnancy outcome with intrauterine insemination in patients with unexplained recurrent abortion whose partners have abnormal hypo-osmotic swelling test. Iran J Med Sci 2009; 34: 172–176.
 Buckett WM, Luckas MJ, Aird IA, Farquharson RG, Kingsland CR, Lewis-Jones DI. The hypo-osmotic swelling test in recurrent miscarriage. Fertil Steril 1997; 68: 506–509.
 Talaei-Khozani T, Borzoei Z, Bahmanpour S, Zolghadr J, Dehbashi S, Zareh HR. Effects of sera taken from women with recurrent spontaneous abortion on sperm motility and apoptosis. Iran J Reprod Med 2011; 9: 125–130.
 Gupta S, Agarwal A, Banerjee J, Alvarez JG. The role of oxidative stress in spontaneous abortion and recurrent pregnancy loss: A systematic review. Obstet Gynecol Surv 2007; 62: 335–347.
 El Hachem H, Crepaux V, May-Panloup P, Descamps Ph, Legendre G, Bouet PE. Recurrent pregnancy loss: Current perspectives. Int J Womens Health 2017; 9: 331– 345.
 Hill JA, Abbott AF, Politch JA. Sperm morphology and recurrent abortion. Fertil Steril 1994; 61: 776–778.
 Sbracia S, Cozza G, Grasso JA, Mastrone M, Scarpellini F. Semen parameters and sperm morphology in men in unexplained recurrent spontaneous abortion, before and during a 3 year follow-up period. Hum Reprod 1996; 11: 117–120.
 Kavitha P, Malini SS. Positive association of sperm dysfunction in the pathogenesis of recurrent pregnancy loss. J Clin Diagn Res 2014; 8: OC07–OC10.
 Evgeni E, Charalabopoulos K, Asimakopoulos B. Human sperm DNA fragmentation and its correlation with conventional semen parameters. J Reprod Infertil 2014; 15: 2–14.
 Kovac JR, Addai J, Smith RP, Coward RM, Lamb DJ, Lipshultz LI. The effects of advanced paternal age on fertility. Asian J Androl 2013; 15: 723–728.
 Pourmasumi S, Nazari A, Fagheirelahee N, Sabeti P. Cytochemical tests to investigate sperm DNA damage: Assessment and review. J Family Med Prim Care 2019; 8: 1533–1539.
 Bellver J, Meseguer M, Muriel L, Garcia-Herrero S, Barreto MA, Garda AL, et al. Y chromosome microdeletions, sperm DNA fragmentation and sperm oxidative stress as causes of recurrent spontaneous abortion of unknown etiology. Hum Reprod 2010; 25: 1713–1721.
 Gorczyca W, Traganos F, Jesionowska H, Darzynkiewicz Z. Presence of DNA strand breaks and increased sensitivity of DNA in situ to denaturation in abnormal human sperm cells: Analogy to apoptosis of somatic cells. Exp Cell Res 1993; 207: 202–205.
 Loo DT. In situ detection of apoptosis by the TUNEL assay: An overview of techniques. Methods Mol Biol 2011; 682: 3–13.
 Keihani S, Myers JB, Hotaling JM. Spermatogenesis: Fertile ground for contributing to recurrent implantation failure? In: Franasiak JM, Scott Jr RT. Recurrent implantation failure: Etiologies and clinical management. Denmark, Cham: Springer International Publishing; 2018. 33–58.
 Kumar K, Deka D, Singh A, Mitra DK, Vanitha BR, Dada R. Predictive value of DNA integrity analysis in idiopathic recurrent pregnancy loss following spontaneous conception. J Assist Reprod Genet 2012; 29: 861–867.
 Coughlan C, Clarke H, Cutting R, Saxton J, Waite S, Ledger W, et al. Sperm DNA fragmentation, recurrent implantation failure and recurrent miscarriage. Asian J Androl 2015; 17: 681–685.
 Imam SN, Shamsi MB, Kumar K, Deka D, Dada R. Idiopathic recurrent pregnancy loss: Role of paternal factors; A pilot study. J Reprod Infertil 2011; 12: 267–276.
 Bareh GM, Jacoby E, Binkley P, Chang TC, Schenken RS, Robinson RD. Sperm deoxyribonucleic acid fragmentation assessment in normozoospermic male partners of couples with unexplained recurrent pregnancy loss: A prospective study. Fertil Steril 2016; 105: 329–336.
 Carlini T, Paoli D, Pelloni M, Faja F, Dal Lago A, Lombardo F, et al. Sperm DNA fragmentation in Italian couples with recurrent pregnancy loss. Reprod Biomed Online 2017; 34: 58–65.
 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: 792–796.
 Zidi-Jrah I, Hajlaoui A, Mougou-Zerelli S, Kammoun M, Meniaoui I, Sallem A, et al. Relationship between sperm aneuploidy, sperm DNA integrity, chromatin packaging, traditional semen parameters, and recurrent pregnancy loss. Fertil Steril 2016; 105: 58–64.
 Feijó CM, Esteves SC. Sperm chromatin dispersion (SCD) test identifies more spermatozoa with DNA damage than the TUNEL assay in men with unexplained infertility. Fertil Steril 2013; 100: S438.
 Tan J, Taskin O, Albert A, Bedaiwy MA. Association between sperm DNA fragmentation and idiopathic recurrent pregnancy loss: A systematic review and metaanalysis. Reprod Biomed Online 2019; 38: 951–960.
 Khadem N, Poorhoseyni A, Jalali M, Akbary A, Heydari ST. Sperm DNA fragmentation in couples with unexplained recurrent spontaneous abortions. Andrologia 2014; 46: 126–130.
 Ribas-Maynou J, Garcia-Peiro A, Fernandez-Encinas A, Amengual MJ, Prada E, Cortes P, et al. Double stranded sperm DNA breaks, measured by comet assay, are associated with unexplained recurrent miscarriage in couples without a female factor. PLoS One 2012; 7: e44679.
 Zhang L, Wang L, Zhang X, Xu G, Zhang W, Wang K, et al. Sperm chromatin integrity may predict future fertility for unexplained recurrent spontaneous abortion patients. Int J Androl 2012; 35: 752–757.
 Agarwal A, Allamaneni SS. Sperm DNA damage assessment: A test whose time has come. Fertil Steril 2005; 84: 850–853.
 Amann RP, Waberski D. Computer-assisted sperm analysis (CASA): Capabilities and potential developments. Theriogenology 2014; 81: 5–17.
 Eisenberg ML, Sapra KJ, Kim SD, Chen Z, Buck Louis GM. 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. Fertil Steril 2017; 108: 613–619.
 Venkatesh S, Thilagavathi J, Kumar K, Deka D, Talwar P, Dada R. Cytogenetic, Y chromosome microdeletion, sperm chromatin and oxidative stress analysis in male partners of couples experiencing recurrent spontaneous abortions. Arch Gynecol Obstet 2011; 284: 1577–1584.
 Enciso M, Sarasa J, Agarwal A, Fernandez JL, Gosalvez J. A two-tailed comet assay for assessing DNA damage in spermatozoa. Reprod Biomed Online 2009; 18: 609– 616.
 Ribas-Maynou J, Garcia-Peiro A, Abad C, Amengual MJ, Navarro J, Benet J. Alkaline and neutral comet assay profiles of sperm DNA damage in clinical groups. Hum Reprod 2012; 27: 652–658.
 Yuan M, Huang L, Leung WT, Wang M, Meng Y, Huang Z, et al. Sperm DNA fragmentation valued by SCSA and its correlation with conventional sperm parameters in male partner of recurrent spontaneous abortion couple. Biosci Trends 2019; 13: 152–159.
 Ruixue W, Hongli Z, Zhihong Z, Rulin D, Dongfeng G, Ruizhi L. The impact of semen quality, occupational exposure to environmental factors and lifestyle on recurrent pregnancy loss. J Assist Reprod Genet 2013; 30: 1513–1518.
 Nabi A, Khalili MA, Halvaei I, Ghasemzadeh J, Zare E. Seminal bacterial contaminations: Probable factor in unexplained recurrent pregnancy loss. Iran J Reprod Med 2013; 11: 925–932.
 Nazari A, Sabeti P, Pourmasumi S. Comparison between sperm parameters and chromatin in recurrent pregnancy loss couples after antioxidant therapy. J Family Med Prim Care 2020; 9: 597–601.
 Marchiani S, Tamburrino L, Benini F, Fanfani L, Dolce R, Rastrelli G, et al. Chromatin protamination and catsper expression in spermatozoa predict clinical outcomes after assisted reproduction programs. Sci Rep 2017; 7: 15122.
 Kazerooni T, Asadi N, Jadid L, Kazerooni M, Ghanadi A, Ghaffarpasand F, et al. Evaluation of sperm’s chromatin quality with acridine orange test, chromomycin A3 and aniline blue staining in couples with unexplained recurrent abortion. J Assist Reprod Genet 2009; 26: 591– 596.
 Ajina T, Ammar O, Haouas Z, Sallem A, Ezzi L, Grissa I, et al. Assessment of human sperm DNA integrity using two cytochemical tests: Acridine orange test and toluidine blue assay. Andrologia 2017; 49: e12765.
 Hotaling J, Carrell DT. Clinical genetic testing for male factor infertility: Current applications and future directions. Andrology 2014; 2: 339–350.
 Poorang Sh, Abdollahi Sh, Anvar Z, Tabei SMB, Namavar Jahromi B, Moein-Vaziri N, et al. The impact of methylenetetrahydrofolate reductase (MTHFR) sperm methylation and variants on semen parameters and the chance of recurrent pregnancy loss in the couple. Clin Lab 2018; 64: 1121–1128.
 Amin-Beidokhti M, Mirfakhraie R, Zare-Karizi S, Karamoddin F. The role of parental microRNA alleles in recurrent pregnancy loss: An association study. Reprod Biomed Online 2017; 34: 325–330.
 Rogenhofer N, Engels L, Bogdanova N, Tuttelmann F, Markoff A, Thaler C. Paternal and maternal carriage of the annexin A5 M2 haplotype are equal risk factors for recurrent pregnancy loss: A pilot study. Fertil Steril 2012; 98: 383–388.
 Tuttelmann F, Ivanov P, Dietzel C, Sofroniou A, Tsvyatkovska TM, Komsa-Penkova RS, et al. Further insights into the role of the annexin A5 M2 haplotype as recurrent pregnancy loss factor, assessing timing of miscarriage and partner risk. Fertil Steril 2013; 100: 1321–1325.
 Asadpor U, Totonchi M, Sabbaghian M, Hoseinifar H, Akhound MR, Zari Moradi S, et al. Ubiquitin-specific protease (USP26) gene alterations associated with male infertility and recurrent pregnancy loss (RPL) in Iranian infertile patients. J Assist Reprod Genet 2013; 30: 923– 931.
 Rogenhofer N, Ott J, Pilatz A, Wolf J, Thaler CJ, Windischbauer L, et al. Unexplained recurrent miscarriages are associated with an aberrant sperm protamine mRNA content. Hum Reprod 2017; 32: 1574–1582.
 Asgari A, Ghahremani S, Saeedi S, Kamrani E. The study of chromosomal abnormalities and heteromorphism in couples with 2 or 3 recurrent abortions in Shahid Beheshti Hospital of Hamedan. Iran J Reprod Med 2013; 11: 201–208.
 Martinez G, Gillois P, Le Mitouard M, Borye R, Esquerre- Lamare C, Satre V, et al. FISH and tips: A large scale analysis of automated versus manual scoring for sperm aneuploidy detection. Basic Clin Androl 2013; 23: 13.
 Bernardini LM, Costa M, Bottazzi C, Gianaroli L, Magli MC, Venturini PL, et al. Sperm aneuploidy and recurrent pregnancy loss. Reprod Biomed Online 2004; 9: 312– 320.
 Carrell DT, Wilcox AL, Lowy L, Peterson CM, Jones KP, Erickson L, et al. Elevated sperm chromosome aneuploidy and apoptosis in patients with unexplained recurrent pregnancy loss. Obstet Gynecol 2003; 101: 1229–1235.
 Ramasamy R, Scovell JM, Kovac JR, Cook PJ, Lamb DJ, Lipshultz LI. Fluorescence in situ hybridization detects increased sperm aneuploidy in men with recurrent pregnancy loss. Fertil Steril 2015; 103: 906–909.
 Neusser M, Rogenhofer N, Durl S, Ochsenkuhn R, Trottmann M, Jurinovic V, et al. Increased chromosome 16 disomy rates in human spermatozoa and recurrent spontaneous abortions. Fertil Steril 2015; 104: 1130–1137.
 Esquerre-Lamare C, Walschaerts M, Chansel Debordeaux L, Moreau J, Bretelle F, Isus F, et al. Sperm aneuploidy and DNA fragmentation in unexplained recurrent pregnancy loss: A multicenter case-control study. Basic Clin Androl 2018; 28: 4.
 Choi DK, Gong IH, Hwang JH, Oh JJ, Hong JY. Detection of Y chromosome microdeletion is valuable in the treatment of patients with nonobstructive azoospermia and oligoasthenoteratozoospermia: Sperm retrieval rate and birth rate. Korean J Urol 2013; 54: 111–116.
 Karaer A, Karaer K, Ozaksit G, Ceylaner S, Percin EF. Y chromosome azoospermia factor region microdeletions and recurrent pregnancy loss. Am J Obstet Gynecol 2008; 199: 662.
 Agarwal S, Agarwal A, Khanna A, Singh K. Microdeletion of Y chromosome as a cause of recurrent pregnancy loss. J Hum Reprod Sci 2015; 8: 159–164.
 Wettasinghe TK, Jayasekara RW, Dissanayake VH. Y chromosome microdeletions are not associated with spontaneous recurrent pregnancy loss in a Sinhalese population in Sri Lanka. Hum Reprod 2010; 25: 3152– 3156.
 Pina-Aguilar RE, Martinez-Garza SG, Kohls G, Vargas- Maciel MA, Vazquez de Lara LG, Gonzalez-Ortega C, et al. Y chromosome microdeletions in Mexican males of couples with idiopathic recurrent pregnancy loss. J Obstet Gynaecol Res 2012; 38: 912–917.
 Ghorbian S, Saliminejad K, Sadeghi MR, Javadi GR, Kamali K, Amirjannati N, et al. The association between Y chromosome microdeletion and recurrent pregnancy loss. Iran Red Crescent Med J 2012; 14: 358–362.