Cryopreservation, Human Spermatozoa Parameters, Hypotaurine Supplementation, Spermatozoa DNA Alterations
Conditions
Keywords
cryopreservation, density gradient centrifugation, DNA fragmentation, oxidation, chromatin packaging, antioxidant hypotaurine supplementation, cryo-capacitation, human spermatozoa fertilizing ability
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.
Interventions
OTHERHypotaurine : antioxidant and osmoregulator
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
Sponsors
University Hospital, Clermont-Ferrand
Université d'Auvergne, Institut National de la Santéet de la Recherche Médicale
Université d'Auvergne, Centre National de la Recherche Scientifique
University Hospital, Clermont-Ferrand, Hematology laboratory
Study design
Observational model
COHORT
Time perspective
PROSPECTIVE
Eligibility
Sex/Gender
MALE
Age
18 Years to No maximum
Healthy volunteers
No
Inclusion criteria
* None
Exclusion criteria
* None
Design outcomes
Primary
| Measure | Time frame | Description |
|---|---|---|
| chromatin packaging labelled using aniline blue and chromomycin A3 | 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 | 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 | 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) | 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
| Measure | Time frame | Description |
|---|---|---|
| vitality using Eosin Nigrosin | Day 0 | Comparison after a cycle of freezing-thawing for both conditions, with or without hypotaurine supplementation of vitality using Eosin Nigrosin |
| motility total and progressive | Day 0 | Comparison after a cycle of freezing-thawing for both conditions, with or without hypotaurine supplementation of motility total and progressive |
| integrity of the acrosome using Fluorescein IsoThioCyanate-Pisum Sativum Agglutinin (FITC-PSA) labelling | Day 0 | 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 |
| markers of PKA-dependent capacitation intracellular signaling pathway assessing western blot | Day 0 | 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 |
Contacts
PRINCIPAL_INVESTIGATORFlorence BRUGNON, MD, PhD
University Hospital, Clermont-Ferrand
Outcome results
None listed