Effects of assisted reproductive technologies and reproductive fluids on phenotypical and epigenetics modifications in bos taurus oocytes, preimplantational embryos and offspring

Resumen

Human infertility is a disease that has been growing in large numbers in the past few years. Assisted reproductive technologies (ART) are the solution for many fertility issues in both human and animal world. Everyday new procedures are being developed but data on the consequences of these are not being generated at the same rate as they are being implemented. It is, then, imperative to investigate in this field in order to improve the results and to guarantee the health status of ART resulting offspring. The aim of this thesis was to study both the effect of the techniques as well as the protocols used, covering from the oocyte to the offspring. In Chapter 1, a brief review on the literature was made, where infertility in both humans and animals was approached, as well as the history of ART development, the most used techniques at the moment and lastly, the problems that have been associated with those ART. Chapter 2 summarize the main hypotheses and objectives of this thesis, while Chapter 3 embarked on the study of one of the most used ART: controlled ovarian stimulation (COS). We hypothesized that the methylome of oocytes submitted to hormonal stimulation would be altered. Cow oocytes from unstimulated animals were collected from pre-ovulatory stage and later, the same animals were hormonally stimulated to collect the oocytes as well. We performed single cell whole genome sequencing to those oocytes and results showed that, although the methylome of unstimulated and stimulated oocytes was quite similar globally, differences in methylation of specific CpG islands of imprinted genes and imprinting control regions were detected. Chapter 4 focused on the study of protocols of oocyte maturation in vitro. By adding the natural milieu of the oocyte (follicular fluid), we hypothesized that the oocyte competence would increase. Oocytes were matured in vitro using as supplement bovine follicular fluid (bFF), heat-inactivated bovine follicular fluid (bFFin) and fetal bovine serum (FBS). Results from oocyte competence evaluation showed no difference in nuclear maturation nor cortical granules distribution. However, bFF oocytes showed higher cumulus expansion rate than the other groups. Efficiency of in vitro fertilization of the oocytes showed lower values for bFFin group, but cleavage and blastocyst yield were similar across groups. Both bFF-derived embryos developed faster than FBS embryos and had increased total cell number. Nonetheless, higher quality was not decisive when evaluating embryo survivability post-vitrification. In Chapter 5, embryos were produced in vitro using culture media improved by reproductive fluids (RF), as well as control embryos (standard protocol, with bovine serum albumin, BSA). These embryos were vitrified and stored until warming and transfer to synchronized recipients. A parallel group consisting of artificial inseminated (AI) cows was created using the same bull as IVP embryos. Hormonal concentrations of pregnant recipients were followed during gestation in order to detect possible differences. These differences were more frequent between BSA and AI group, while RF pregnant group had more intermediate values. However, calving issues were more severe in both IVP groups than in AI, as well as neonatal mortality only happened in the IVP groups. In Chapter 6, we studied the calves resulting from chapter 5 and evaluated their growth and haematological status during the first 30 days of life. Only one difference in growth parameters was constantly different, which was the height at withers from AI calves being higher than IVP calves. Haematological parameters showed isolated differences in each day, not being repeated in different days. But all animals showed values that are in accordance to expected normal values. However, it is yet to be understood if these calves will have any difference in their development, as it might be too soon to draw conclusions. Additionally, biopsies were made to these calves in order to detect any change in the imprinting status of genes that are involved in the appearance of overgrowth syndromes. In summary, this thesis has shown that ART are many kilometres away from being the perfect substitute to in vivo embryo production, development and birth. The use of hormonal stimulation that is widely implemented in fertility clinics might be the reason of many alterations found in in vitro produced offspring. The supplementation of maturation medium might also be the key to change the in vitro embryo quality. Offspring derived from in vitro production of embryos might have a tendency to have more difficulties during deliver, but seem particularly similar to their in vivo controls after birth until one month of life. Changes are necessary in current used protocols and studies need to be made to fully understand the effects that ART have on the future offspring.