Estudio de pomzp3 y sus polimorfismos como potenciales indicadores de fertilidad de la especie humana

Abstract

Background and objectives: The success of in vitro fertilization (IVF) treatment depends on numerous factors, such as an optimal incubation environment, accurate embryo evaluation, and a receptive endometrium. Single embryo transfer is increasingly common in IVF laboratories to avoid the risk of multiple pregnancies, making the selection of the optimal embryo even more crucial. With the integration of new assisted reproductive techniques, particularly genetic ones, the aim is to reduce embryo and/or gestational loss, thereby advancing the goal of achieving a healthy newborn sooner. In this doctoral thesis, we have investigated the polymorphisms present in the POMZP3 gene and its potential variants within the population of oocyte donors and males undergoing oocyte donation treatment. We aimed to ascertain whether these polymorphisms could correlate with oocyte quality, embryo development, and gestational success, with the ultimate goal of establishing a biomarker with potential application in assisted reproductive technology (ART) cycles within the oocyte donation program. Another objective of our study was to analyse the presence of the protein encoded by the POMZP3 gene and its variants in oocytes, cumulus cells, and the culture medium used during IVFprocedures. Finally, we endeavoredto produce recombinant POM-ZP3 protein in two types of mammalian cells. • Method: Analysis of POMZP3 polymorphisms has been performed by obtaining genomic DNA from samples obtained from buccal swabs by amplification of regions of interest of POMZP3 genomic DNA. The protein study has been conducted using functional immunohistochemical techniques, electrophoresis in SDS-PAGE and Western blot, and transfection of CHO and HEK293 cells with POM-ZP3, to further investigate the functional properties of this protein. The embryonic development data were integrated into an artificial intelligence (AI) system, together with other data related to the reproductive cycle of the patients. • Results: Usingsequencing techniques, we have investigated the polymorphisms of POMZP3 and its variants, identifying 9 isoforms, one of which is not found in homozygosity in the population. This fact could be related to early embryonic death and, thus may serve as a potential biomarker of infertility. Data extracted from embryonic development, morphokinetics, and results of patient cycles with this polymorphism were processed by artificial intelligence, revealing no significant differences between them, except for a slower t2 and slightly faster embryonic divisions in the group where both parents presented the polymorphism in heterozygosity. We have demonstrated in silico the consistent presence of POM-ZP3 throughout all stages of embryonic development up to the morula stage, as well as in cumulus cells, except in the blastocyst stage. The protein encoded by the POMZP3 gene has been identified in human oocytes and oocyte cumulus cells. Additionally, various variants of this protein have been detected. Furthermore, the protein encoded by POMZP3 and its variants have been identified in the culture medium where IVF has occurred. This suggests the potential impact of this protein on the fertilization process. Finally, two recombinant proteins, consisting of canonical and truncated variants of POMZP3, have been successfully produced in mammalian cells. These proteins will allow functional studies to determine their impact on fertilization. Analysis of POMZP3 and its variants may serve as a beneficial non-invasive marker, facilitating improved allocation of oocyte and sperm donors to couples, thereby increasing the success rate of IVF treatments.