Fertility Clinical Trial
Official title:
Molecular Diagnostic of Sperm Cryoresistance of Semen Donors and Clinical Outcomes After Artificial Insemination. Optimization of Sperm Bank
Sperm freezing has been employed for decades for male fertility preservation in cases of foreseeable or unexpected loss of fertility to guarantee future paternity, and also as a complement of assisted reproduction techniques. Sperm quality after thawing is highly variable, even among consecutive samples from the same individual, with mean survival rates around 40%. To date, the molecular basis of the adequate resistance or intolerance to freezing/thawing protocols is unknown, and its knowledge can lead to improvement in the selection of the samples to be frozen and also in the adequate supplement of cryopreservation media. Microarray analysis provides a powerful tool to address the molecular explanation beyond this behaviour, yielding results about comparative messenger ribonucleic acid (mRNA)expression under the two different biological conditions: optimal and suboptimal survival. Then, the aim of the investigators' study is to determine the genomic profile of sperm samples depending on their survival resistance to cryopreservation, to determine genes involved in cryodamage sensitivity.
Nested cases and controls design, with donor sperm samples frozen under conventional protocols, categorized depending on their good (GSR, n=20) (less than 20% motility decrease) or bad (BSR, n=20) (more than 20% motility decrease) survival rates. Sperm mRNA was extracted using Trizol protocol, suspended in diethylpyrocarbonate (DEPC)-treated water and frozen at -80 degrees until the microarray experiments were performed. RNAs were analyzed on Agilent Bioanalyzer 2100. Samples were pooled in 4 (5 samples/pool and 4 pools/group). Finally, 8 Agilent One colour Whole genome microarray (44K) were performed with pooled samples, 4 microarrays per group. The results will be evaluate to detect those genes differentially expressed. ;
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