The DNA fragmentation in sperm is associated with reduced outcome in assisted reproduction. Using YoPro1 as the staining dye and flow cytometry and sorting (FACS), the number of spermatozoa with DNA fragmentation can be lowered to 5%. Can the cumulative outcome of ICSI be improved using FACS?
A prospective, randomized, double-blind clinical trial was conducted in 104 infertile couples with male infertility based on abnormal conventional semen analysis results. Cumulative ongoing pregnancy rate was the primary outcome parameter. In 52 cases, semen was processed for ICSI using swim-up. In another 52 cases, spermatozoa with fragmented DNA were removed with FACS.
The cumulative pregnancy rate at 12 weeks of gestation (51.9% versus 46.2%) and live birth rate (42.3% versus 34.6%) were higher and the miscarriage rate was lower (27.8% versus 35.3%) after FACS-sorting as compared with swim-up. An interim analysis scheduled before initiation of the study after 100 cases demonstrated that the aim of a 20% gain in pregnancy rate could not be achieved. For that reason, the prospective study was stopped prematurely.
A trend towards consistently better results was achieved by removing spermatozoa with fragmented DNA. The fragmentation of the DNA in sperm is the end stage of apoptosis. Sorting of spermatozoa may be improved by selecting parameters of processes active more upstream of apoptosis, such as chromatin decondensation.
NCT02166567. June 14, 2014.
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Bonde JP, Ernst E, Jensen TK, Hjollund NH, Kolstad H, Henriksen TB, et al. Relation between semen quality and fertility: a population-based study of 430 first-pregnancy planners. Lancet. 1998;352:1172–7.
Cooper TG, Noonan E, von Eckardstein S, Auger J, Baker HW, Behre HM, et al. World Health Organization reference values for human semen characteristics. Hum Reprod Update. 2010;16:231–45.
De Geyter C, Calhaz-Jorge C, Kupka MS, Wyns C, Mocanu E, Motrenko T, et al. European IVF-monitoring Consortium (EIM) for the European Society of Human Reproduction and Embryology (ESHRE). ART in Europe, 2014: results generated from European registries by ESHRE: The European IVF-monitoring Consortium (EIM) for the European Society of Human Reproduction and Embryology (ESHRE). Hum Reprod. 2018;33:1586–601.
Muratori M, De Geyter C. Chromatin condensation, fragmentation of DNA and differences in the epigenetic signature of infertile men. Best Pract Res Clin Endocrinol Metab. 2018;33(1):117–26.
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;30:120–7.
Simon L, Emery BR, Carrell DT. Review: diagnosis and impact of sperm DNA alterations in assisted reproduction. Best Pract Res Clin Obstet Gynaecol. 2017;44:38–56.
Zini A, Boman JM, Belzile E, Ciampi A. Sperm DNA damage is associated with an increased risk of pregnancy loss after IVF and ICSI: systematic review and meta-analysis. Hum Reprod. 2008;23:2663–8.
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 meta-analysis. Hum Reprod. 2012;27:2908–17.
Taha EA, Ez-Aldin AM, Sayed SK, Ghandour NM, Mostafa T. Effect of smoking on sperm vitality, DNA integrity, seminal oxidative stress, zinc in fertile men. Urology. 2012;80:822–5.
Dupont C, Faure C, Sermondade N, Boubaya M, Eustache F, Clément P, et al. Obesity leads to higher risk of sperm DNA damage in infertile patients. Asian J Androl. 2013;15:622–5.
Roessner C, Paasch U, Kratzsch J, Glander HJ, Grunewald S. Sperm apoptosis signalling in diabetic men. Reprod BioMed Online. 2012;25:292–9.
Said TM, Agarwal A, Zborowski M. Utility of magnetic cell separation as a molecular sperm preparation technique. J Androl. 2008;29:134–42.
Ye H, Huang GN, Gao Y, Liu DY. Relationship between human sperm-hyaluronan binding assay and fertilization rate in conventional in vitro fertilization. Hum Reprod. 2006;21:1545–50.
Gil M, Sar-Shalom V, Melendez Sivira Y, Carreras R, Checa MA. Sperm selection using magnetic activated cell sorting (MACS) in assisted reproduction: a systematic review and meta-analysis. J Assist Reprod Genet. 2013;30:479–85.
McDowell S, Kroon B, Ford E, Hook Y, Glujovsky D, Yazdani A. Advanced sperm selection techniques for assisted reproduction. Cochrane Database Syst Rev. 2014;CD010461.
Ribeiro SC, Sartorius G, Pletscher F, de Geyter M, Zhang H, de Geyter C. Isolation of spermatozoa with low levels of fragmented DNA with the use of flow cytometry and sorting. Fertil Steril. 2013;100:686–94.
De Geyter C, Bals-Pratsch M, Doeren M, Yeung CH, Grunert JH, Bordt J, et al. Human and bovine cervical mucus penetration as a test of sperm function for in-vitro fertilization. Hum Reprod. 1988;3:948–54.
De Geyter C. Assisted reproductive medicine in Switzerland. Swiss Med Wkly. 2012;142:w13569.
Raat H, Landgraf JM, Oostenbrink R, Moll HA, Essink-Bot ML. Reliability and validity of the Infant and Toddler Quality of Life Questionnaire (ITQOL) in a general population and respiratory disease sample. Qual Life Res. 2007;16:445–60.
De Geyter C, Sterthaus O, Miny P, Wenzel F, Lapaire O, De Geyter M, et al. First successful pregnancy in Switzerland after prospective sex determination of the embryo through the separation of X-chromosome bearing spermatozoa. Swiss Med Wkly. 2013;143:w13718.
Aitken RJ, De Iuliis GN. On the possible origins of DNA damage in human spermatozoa. Mol Hum Reprod. 2010;16:3–13.
Muratori M, Marchiani S, Tamburrino L, Cambi M, Lotti F, Natali I, et al. DNA fragmentation in brighter sperm predicts male fertility independently from age and semen parameters. Fertil Steril. 2015;104:582–90.
Ribeiro SC, Muratori M, De Geyter M, De Geyter C. TUNEL labeling with BrdUTP/anti-BrdUTP greatly underestimates the level of sperm DNA fragmentation in semen evaluation. PLoS One. 2017;12:e0181802.
Hammoud SS, Nix DA, Zhang H, Purwar J, Carrell DT, Cairns BR. Distinctive chromatin in human sperm packages genes for embryo development. Nature. 2009;460:473–8.
Brykczynska U, Hisano M, Erkek S, Ramos L, Oakeley EJ, Roloff TC, et al. Repressive and active histone methylation mark distinct promoters in human and mouse spermatozoa. Nat Struct Mol Biol. 2010;17:679–87.
Erkek S, Hisano M, Liang C-Y, Gill M, Murr R, Dieker J, et al. Molecular determinants of nucleosome retention at CpG-rich sequences in mouse spermatozoa. Nat Struct Mol Biol. 2013;20:868–75.
Hammoud SS, Nix DA, Hammoud AO, Gibson M, Cairns BR, Carrell DT. Genome-wide analysis identifies changes in histone retention and epigenetic modifications at developmental and imprinted gene loci in the sperm of infertile men. Hum Reprod. 2011;26:2558–69.
Azpiazu R, Amaral A, Castillo J, Estanyo JM, Guimera’ M, Ballesca’ JL, et al. High-throughput sperm differential proteomics suggests that epigenetic alterations contribute to failed assisted reproduction. Hum Reprod. 2014;29:1225–37.
Ni K, Spiess AN, Schuppe HC, Steger K. The impact of sperm protamine deficiency and sperm DNA damage on human male fertility: a systematic review and meta-analysis. Andrology. 2016;4:789–99.
Muratori M, Tamburrino L, Marchiani S, Cambi M, Olivito B, Azzari C, et al. Investigation on the origin of sperm DNA fragmentation: role of apoptosis. Immaturity and Oxidative Stress Mol Med. 2015;21:109–22.
Manochantr S, Chiamchanya C, Sobhon P. Relationship between chromatin condensation, DNA integrity and quality of ejaculated spermatozoa from infertile men. Andrologia. 2012;44:187–99.
Sakkas D, Urner F, Bizzaro D, Manicardi G, Bianchi PG, Shoukir Y, et al. Sperm nuclear DNA damage and altered chromatin structure: effect on fertilization and embryo development. Hum Reprod. 1998;13(Suppl 4):11–9.
We are grateful to the work of the monitoring group of the Clinical Trial Unit of the Basler University Hospital (www.dkf.unibas.ch) and to Ms. Hanna Flükiger for coordinating all activities related to this study.
This study was funded by the Repronatal Foundation, Basel, Switzerland.
The study was presented to and approved by the local ethics committee (EKBB178/12) and monitored by the clinical trial unit (CTU) of the University Hospital of Basel.
Conflict of interest
The authors declare that they have no conflict of interest.
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De Geyter, C., Gobrecht-Keller, U., Ahler, A. et al. Removal of DNA-fragmented spermatozoa using flow cytometry and sorting does not improve the outcome of intracytoplasmic sperm injection. J Assist Reprod Genet 36, 2079–2086 (2019). https://doi.org/10.1007/s10815-019-01571-1
- DNA fragmentation
- Flow cytometry
- Chromatin remodeling