Beneficial effects of hypotaurine supplementation in preparation and freezing media on human sperm cryo-capacitation and DNA quality

Hanae Pons-Rejraji, Solène Vorilhon, Asmaa Difrane, Sandra Dollet, Céline Bourgne, Marc Berger, Laure Chaput, Bruno Pereira, Cyril Bouche, Joël R Drevet, Florence Brugnon, Hanae Pons-Rejraji, Solène Vorilhon, Asmaa Difrane, Sandra Dollet, Céline Bourgne, Marc Berger, Laure Chaput, Bruno Pereira, Cyril Bouche, Joël R Drevet, Florence Brugnon

Abstract

Background: Although widely used, slow freezing considerably modifies the functions of human spermatozoa. Cryopreservation induces nuclear sperm alterations and cryo-capacitation, reducing the chances of pregnancy. Hypotaurine is naturally present in the male and female genital tracts and has capacitating, osmolytic and anti-oxidant properties. The analysis were performed on surplus semen of men with normal (n = 19) or abnormal (n = 14) sperm parameters. Spermatozoa were selected by density gradient centrifugation before slow freezing. For each sample, these steps were performed in parallel with ("H+" arm) or without ("H-" arm) hypotaurine supplementation. After thawing, we measured total and progressive mobility, vitality, acrosome integrity, markers of capacitation signaling pathway and nuclear quality. For the latter, we focused on sperm chromatin packaging, DNA fragmentation and the presence of vacuoles in the sperm nucleus.

Results: Post-thaw spermatozoa selected and frozen in the presence of hypotaurine had a higher vitality (+ 16.7%, p < 0.001), progressive and total motility (+ 39.9% and + 21.6% respectively, p < 0.005) than spermatozoa from the control "H-" arm. Hypotaurine also reduced the non-specific phosphorylation of the capacitation protein markers P110 and P80 (p < 0.01), indicating a decrease in cryo-capacitation. Hypotaurine supplementation reduced chromatin decondensation, measured by chromomycin A3 (- 16.1%, p < 0.05), DNA fragmentation (- 18.7%, p < 0.05) and nuclear vacuolization (- 20.8%, p < 0.05).

Conclusion: Our study is the first to demonstrate beneficial effects of hypotaurine supplementation in preparation and freezing procedures on human spermatozoa sperm fertilization capacity and nucleus quality. Hypotaurine supplementation limited cryo-capacitation, increased the proportion of live and progressively motile spermatozoa and reduces the percentage of spermatozoa showing chromatin decondensation, DNA fragmentation and nuclear vacuolation.

Trial registration: Clinical Trial, NCT04011813 . Registered 19 May 2019 - Retrospectively registered.

Keywords: Acrosome; Capacitation and cryocapacitation; Chromatin packaging; Cryopreservation; DNA fragmentation and oxidation; Density gradient centrifugation; Human spermatozoa.

Conflict of interest statement

JRD is scientific advisor of a US-based biotech company (Celloxess, Princeton, NJ, USA) involved in preventive medicine with a focus on the generation of antioxidant oral supplements.

© 2021. The Author(s).

Figures

Fig. 1
Fig. 1
Experimental study design. Samples were obtained from 33 men undergoing routine semen analysis. Excess semen from each sample was split in two and processed in parallel in two ways: a control arm “H -”: sperm cells were processed by density gradient centrifugation (DGC) without hypotaurine supplementation, and an “H +” arm: spermatozoa were processed by 50 mM hypotaurine supplementation during DGC. Spermatozoa were washed and frozen in high-security straws in the presence of hypotaurine supplemented (H+) or non-hypotaurine supplemented (H-) media. After thawing, the different analyzes were carried out in parallel for both conditions: - (i) Analysis of standard semen parameters (n = 33): Sperm vitality,progressive and total motilities. - (ii) Measurements of fertilization capacity: Evaluation of (cryo-) capacitation terminal markers by measuring tyrosine phosphorylation of P110 and P80 (n = 15) and analysisof acrosome integrity after FITC- PSA labeling (n = 25), and - (iii) Measurements of nuclear quality markers: Chromatin condensation measured by chromomycin A3 (n = 28), DNA fragmentation by TUNEL assay (n = 18), and nuclear vacuolization observed by examination of motile sperm organelle morphology (MSOME, n = 19)
Fig. 2
Fig. 2
Effects of hypotaurine supplementation on sperm fertilization capacity after thawing. Numbers are averaged ± SEM for (A) P110 and P80 (arbitrary units: a.u.) and percent ± SEM for (B) acrosome integrity (%). “H -”: sperm cells were processed by density gradient centrifugation (DGC), washed and frozen without hypotaurine supplementation. “H +”: sperm cells were processed by DGC, washed and frozen in the presence of 50 mM of hypotaurine. After thawing, measurements were performed on (A) the levels of cryo-capacitation (n = 15), and (B) the percentage of altered or reacted (AR) spermatozoa (n = 25). Cryo-capacitation levels were evaluated by measuring the tyrosine phosphorylation of proteins P110 and P80 after thawing under capacitation conditions (37 °C and 5% CO2). Conditions H+ and H- were compared using parametric or non-parametric tests based on statistical distribution and number of subjects (n).** indicates significant difference with p < 0.01 in comparison to the H- condition
Fig. 3
Fig. 3
Effects of hypotaurine supplementation on sperm nuclear quality after thawing. Values are mean ± SEM for the percentage (%) of spermatozoa with (A) decondensed chromatin, (B) fragmented DNA or (C) head vacuoles.”H -”: sperm cells were processed by density gradient centrifugation (DGC), washed and frozen without hypotaurine supplementation. “H +”: sperm cells were processed by DGC, washed and frozen in the presence of 50 mM hypotaurine. The following were measured after thawing: (A) Chromatin packaging: percentage of spermatozoa with decondensed chromatin evaluated by chromomycine A3 (% CMA3 + spermatozoa, n = 28); (B) DNA fragmentation estimated by the TUNEL assay (% TUNEL + spermatozoa, n = 19). (C) nuclear vacuolization estimated by MSOME (% spermatozoa with head vacuoles, n = 19). Conditions H+ and H- were compared using parametric or non-parametric tests according to statistical distribution and the number of subjects (n).* indicates a significant difference with p < 0.05 in comparison to the H- condition
Fig. 4
Fig. 4
Correlations between post thaw spermatozoa parameters. Heat map showing the positive (blue) and negative (red) correlations between the different parameters using Spearman correlations. *indicates a significant correlation with p < 0.05

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