Although it is widely used, slow freezing can induce strong functional and nuclear spermatic alterations reducing the chances of pregnancy. The study objective is to determinate the effects of the combination of hypotaurine supplementation and spermatozoa selection by Density Gradient Centrifugation (DGC) on human sperm functions and DNA quality during a freezing-thawing cycle.
This prospective study was performed on surplus semen after a density gradient centrifugation-frozen-thawing cycle. Samples were obtained from men undergoing routine semen analysis at the Center for Reproductive Medicine. Spermatozoa were selected by density gradient centrifugation, washed and frozen using a programmable device. Each step was performed in parallel with (H+ arm) or without (H- arm) 50mM hypotaurine supplementation. After thawing, investigator team compared for both conditions the total and progressive mobility, vitality, integrity of the acrosome, markers of Protein Kinase A (PKA) dependent capacitation intracellular signaling pathway and nuclear quality by measuring chromatin packaging, DNA fragmentation and oxidation and vacuoles presence in the spermatozoa head.
Study Type
OBSERVATIONAL
Enrollment
33
Hypotaurine has protective effects on sperm motility, capacitation and acrosome reaction and reduces apoptotic markers. Hypotaurine (50mM) was added in density gradient centrifugation, washing and cryopreservation media washing and cryopreservation media before spermatozoa freezing
chromatin packaging labelled using aniline blue and chromomycin A3
Comparison after a cycle of freezing-thawing for both conditions, with or without hypotaurine supplementation of chromatin packaging labelled using aniline blue and chromomycin A3
Time frame: Day 0
DNA fragmentation using TUNEL assay
Comparison after a cycle of freezing-thawing for both conditions, with or without hypotaurine supplementation of DNA fragmentation using TUNEL assay
Time frame: Day 0
DNA oxidation assessed by 8-OHdG immunodetections
Comparison after a cycle of freezing-thawing for both conditions, with or without hypotaurine supplementation of DNA oxidation assessed by 8-OHdG immunodetections
Time frame: Day 0
vacuoles presence in the spermatozoa head using Motile Sperm Organelle Morphology Examination (MSOME)
Comparison after a cycle of freezing-thawing for both conditions, with or without hypotaurine supplementation of vacuoles presence in the spermatozoa head using Motile Sperm Organelle Morphology Examination (MSOME)
Time frame: Day 0
vitality using Eosin Nigrosin
Comparison after a cycle of freezing-thawing for both conditions, with or without hypotaurine supplementation of vitality using Eosin Nigrosin
Time frame: Day 0
motility total and progressive
Comparison after a cycle of freezing-thawing for both conditions, with or without hypotaurine supplementation of motility total and progressive
Time frame: Day 0
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integrity of the acrosome using Fluorescein IsoThioCyanate-Pisum Sativum Agglutinin (FITC-PSA) labelling
Comparison after a cycle of freezing-thawing for both conditions, with or without hypotaurine supplementation of integrity of the acrosome using Fluorescein IsoThioCyanate-Pisum Sativum Agglutinin (FITC-PSA) labelling
Time frame: Day 0
markers of PKA-dependent capacitation intracellular signaling pathway assessing western blot
Comparison after a cycle of freezing-thawing for both conditions, with or without hypotaurine supplementation of markers of PKA-dependent capacitation intracellular signaling pathway assessing western blot
Time frame: Day 0