Official Title: Hypotaurine Supplementation in Freezing and Preparation Media Improves Human Sperm DeoxyriboNucleic Acid (DNA) and Fertilizing Ability
Trial Snapshot
- Phase
- Not Applicable
- Status
- Completed
- Sponsor
- Enrollment
- 33
- Primary Endpoint
- chromatin packaging labelled using aniline blue and chromomycin A3
Study Overview
Brief Summary
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.
Detailed Description
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 Design
- Study Type
- Observational
- Observational Model
- Cohort
- Time Perspective
- Prospective
Eligibility Criteria
- Ages
- 18 Years to — (Adult, Older Adult)
- Sex
- Male
- Accepts Healthy Volunteers
- No
Inclusion Criteria
- Not provided
Exclusion Criteria
- Not provided
Arms & Interventions
Control arm, "H-"
semen treated without hypotaurine supplementation in density gradient centrifugation, washing and cryopreservation media
Experimental arm, "H+"
semen treated with a 50mM hypotaurine supplementation in density gradient centrifugation, washing and cryopreservation media
Intervention: Hypotaurine : antioxidant and osmoregulator (Other)
Outcomes
Primary Outcomes
chromatin packaging labelled using aniline blue and chromomycin A3
Time Frame: Day 0
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
DNA fragmentation using TUNEL assay
Time Frame: Day 0
Comparison after a cycle of freezing-thawing for both conditions, with or without hypotaurine supplementation of DNA fragmentation using TUNEL assay
DNA oxidation assessed by 8-OHdG immunodetections
Time Frame: Day 0
Comparison after a cycle of freezing-thawing for both conditions, with or without hypotaurine supplementation of DNA oxidation assessed by 8-OHdG immunodetections
vacuoles presence in the spermatozoa head using Motile Sperm Organelle Morphology Examination (MSOME)
Time Frame: Day 0
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)
Secondary Outcomes
- motility total and progressive(Day 0)
- vitality using Eosin Nigrosin(Day 0)
- markers of PKA-dependent capacitation intracellular signaling pathway assessing western blot(Day 0)
- integrity of the acrosome using Fluorescein IsoThioCyanate-Pisum Sativum Agglutinin (FITC-PSA) labelling(Day 0)