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The effect of human sperm chromatin maturity on ICSI outcomes

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Abstract

Because sperm chromatin may play a key role in reproductive success, we verify the associations between sperm chromatin abnormalities, embryo development and the ability to achieve pregnancy. The evaluation of sperm chromatin maturity using aniline blue (AB), chromomycin A3 (CMA3) and toluidine blue (TB) staining were carried out in group of males from infertile couples that underwent ICSI. Low levels of sperm chromatin abnormalities (< 16%) were found in most subjects (> 50%). A higher percentage of TB-positive sperm cells were discovered in the men from couples who achieved ≤ 50% fertilized oocytes compared to men who achieved > 50%. No significant differences were discovered by the applied tests between the men from couples who achieved ≤ 50% and those who achieved > 50% high-quality embryos on the 3rd or 5th day after fertilization, nor between the men from couples who achieved pregnancy and those who failed. The sperm chromatin maturity did not correlate with the ICSI results. However, the ROC analysis revealed a significant predictive value of TB-positive spermatozoa only for fertilization. Therefore, the TB assay can be considered as a useful test for the prediction of fertilization. Our findings suggest that the level of sperm chromatin abnormalities of the examined men was not clinically significant. No found associations between sperm chromatin maturity and embryo development and the ability to achieve pregnancy. We could not exclude the effects of the repairing processes in the fertilized oocyte. The use of complementary tests that verify the status of the sperm chromatin seems justified.

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Acknowledgements

This study was supported by the Pomeranian Medical University, Szczecin, Poland Grants No. WNoZ-322-04/S/2016 and No. FSN-322-5/2016.

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Authors and Affiliations

  1. Department of Histology and Developmental Biology, Pomeranian Medical University, Zolnierska 48, 71-210, Szczecin, Poland

    Kamil Gill, Aleksandra Rosiak, Joanna Jakubik, Anna Kazienko, Maria Laszczynska & Malgorzata Piasecka

  2. Department of Animal Reproduction, Biotechnology and Environmental Hygiene, West Pomeranian University of Technology, 71-270, Szczecin, Poland

    Dariusz Gaczarzewicz

  3. Department of Gynecology and Procreative Health, Pomeranian Medical University, 71-210, Szczecin, Poland

    Rafal Kurzawa

  4. VitroLive Fertility Clinic, 70-001, Szczecin, Poland

    Aleksandra Rosiak & Rafal Kurzawa

  5. Department of Genetics, Faculty of Biology, University of Szczecin, 71-412, Szczecin, Poland

    Anna Rymaszewska

  6. Department of Nursing, Pomeranian Medical University, 71-210, Szczecin, Poland

    Elzbieta Grochans

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  1. Kamil Gill
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Correspondence to Malgorzata Piasecka.

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13577_2018_203_MOESM1_ESM.tif

Supplemental Fig. 1a–c. Non-invasive morphological evaluation of human embryo after intracytoplasmic sperm injection. Fertilized oocyte assessed within the first 24 h (a), embryo development on the 3rd day (b) and on the 5th day (c). The embryo was rated as follows: 2PN – zygote with visible female and male pronuclei containing nucleoli; 8A1 – a top-quality embryo with eight symmetrical blastomeres without cytoplasmic defects and fragmentation (according to Bączkowski et al. [43]); AA – a top-quality embryo with blastocoel filling and a well-developed inner cell mass and trophectoderm (according to Gardner et al. [42]). Original microphotographs obtained from infertile couples treated with ICSI; scale bar = 25 µm. (TIFF 3731 kb)

13577_2018_203_MOESM2_ESM.tif

Supplemental Fig. 2. Dynamics of the chromatin-remodeling process during spermatogenesis and epididymal sperm maturation. Chromatin remodeling is associated with chromosome reorganization, compaction and stabilization. Its condensation is mediated by protamination, characterized by the massive incorporation of protamines into the minor groove of DNA. Protamination is a complex process and is involved in the replacement of the following: the somatic histones by testicular-specific histones (Ht) (1), Ht by transition proteins (TP) (2) and TP by protamines (3). The chemical modifications of histone peptides and the formation of DNA strand breaks by topoisomerase facilitate the incorporation of these proteins to the DNA strand. Stabilization of the chromatin structure is mediated by the binding of zinc ions to the protamines at the end of the spermiogenesis (4). The depletion of these ions from the protamines takes place during epididymal sperm maturation (5). The mature sperm chromatin contains ≤ 15% of testicular histones. Asterisks indicate cytochemical tests that have been applied to reveal nuclear sperm compaction abnormalities at the different stages of nuclear remodeling; aniline blue staining (AB test) is able to detect sperm cells with excessive presence of histones, the TUNEL test (terminal deoxynucleotidyl transferase dUTP nick end labeling) reveals persisting DNA strand breaks, chromomycin A3 staining (CMA3 test) is used to display spermatozoa with protamine deficiency, and toluidine blue (TB test) allows for the discrimination between sperm cells with immature chromatin structure and normal chromatin. According to Carrell et al. [47], Ward [50], Oliva and Castillo [49] and Francis et al. [48] with own modifications. (TIFF 39105 kb)

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Supplemental Fig. 3. Predicted embryo development after fertilization with DNA-damaged sperm cell (details in the text). BER – base excision repair, NER – nucleotide excision repair, MMR – DNA mismatch repair, TDP 1 and 2 – tyrosyl-DNA phosphodiesterase 1 and 2. (TIFF 42007 kb)

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Gill, K., Rosiak, A., Gaczarzewicz, D. et al. The effect of human sperm chromatin maturity on ICSI outcomes. Human Cell 31, 220–231 (2018). https://doi.org/10.1007/s13577-018-0203-4

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