Effect of melatonin and caffeine supplementation in post-thawing asthenozoospermic sperm quality cryopreserved by slow freezing and vitrification methods

• Grant number: 19/22138-0
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: February 01, 2020
• End date: December 31, 2020
• Field of knowledge: Health Sciences - Medicine - Surgery
• Principal Investigator: Jorge Hallak
• Grantee: Ana Clara Monteiro Barduchi
• Host Institution: Hospital das Clínicas da Faculdade de Medicina da USP (HCFMUSP). Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil

Abstract

Male infertility is multifactorial and involve congenital and acquired factors and, therefore, accurate diagnosis must include the evaluation of sex hormones, basic seminal analysis and sperm function tests. Asthenozoospermia is characterized by progressive sperm motility below 32% and is a frequent condition of seminal samples from men with infertility complaints. Thus, the aim of this study is to compare sperm survival rates after slow freezing and vitrification with or without caffeine and melatonin supplementation to describe the effects of each technique on post-thawing sperm quality in samples present asthenozoospermia in the initial analysis. In addition, this work will be carried out with the Men's Health Group of the Institute of Advanced Studies at the University of São Paulo (IEA-USP) in collaboration with Dr. Raúl Sanchez, a researcher in sperm vitrification at the La Frontera (Temuco-Chile), and will contribute to the creation and discussion of public policies for the preservation of male fertility to the low-income population, and may be applied to the country's Unified Health System.For this, this prospective study used 30 asthenozoospermic seminal samples from patients aged from 19 to 45 years in the routine of the Androscience Laboratory and HCFMUSP. Then, the samples were cryopreserved by low freezing and vitrification methods without supplement or with 2mM of melatonin. After thawing, the samples were analyzed or supplemented with 2 mM of caffeine. At the end of the experiments, we obtained 4 groups: Post-thaw samples without supplementation or supplemented with melatonin and caffeine, by low freezing or vitrification methods. Seminal parameters for count and motility, analyzed by WHO criteria, mitochondrial activity, by diaminobenzidine staining, evaluation of DNA fragmentation rate, by the SCSA® method, and dosage of radical oxygen species (ROS) levels, by the luminescence method, pre-cryopreservation and post-thaw were carried out with or without supplementation. The data were analyzed by the paired Student's t-test and by one-way analysisof variance with repeated measurements, followed by the Holm-Sidak post-test, adopting an alpha of 5%.