Collection, biobanking and characterization of human tubal and uterine fluids for their future use in assisted reproductive technologies

  1. Canha Gouveia, Analuce
Dirigée par:
  1. Jorge E Chavarro Directeur/trice
  2. Rafael Manuel Latorre Reviriego Directeur
  3. María Pilar Coy Fuster Directrice

Université de défendre: Universidad de Murcia

Fecha de defensa: 05 décembre 2019

Jury:
  1. Nicola Bernabò President
  2. Juan Carlos Martínez Soto Secrétaire
  3. Maria Teresa Escamez Martinez Rapporteur
Département:
  1. Anatomía y Anatomía Patológica Comparadas

Type: Thèses

Résumé

The current literature, reviewed in the chapter I, provide increasing evidence about the potential benefits of female reproductive fluids as supplements for embryo culture media in human species. From those evidences, we hypothesize that it is possible to collect, characterize and validate the use of these fluids in the human species, and to store them in a biobank fulfilling all the sanitary conditions and legal requirements for their future use in biomedical research or in clinical trials. In order to assess the viability of this hypothesis, four main objectives were defined in this thesis described in the chapter II. The first one was the development of a procedure for the collection of human oviductal (HOF) and UF in patients and donors (Chapter III). After testing several methods for the collection of human uterine and oviductal fluids, it was found that human OF (HUF) could be collected ex vivo by the method previously described by Carrasco et al 2008 in animal models. However, it was not found any method or device in the market allowing the collection of human UF (HUF) in vivo. For this reason, a novel device for human fluids collection was developed, which has been submitted for patenting at the present. The establishment of a cooperation agreement with the Biobank network in Murcia has allowed to create the first biobank collection of human reproductive fluids, (Chapter IV). In this chapter, the workflow and the methods for manipulation, registration, traceability, transportation and storage of the fluids is described as well as the numbers and types of samples available at Biobanc-Mur for future use. In Chapter V, a set of physico-chemical parameters, currently used for the certifications included in the quality controls (QC) required for the selling of human culture media, were selected, and the methods to assess them were optimized. In chapter VI, a high-throughput proteomic analysis of female reproductive tract fluids during the secretory phase of the menstrual cycle was performed, which constitutes a novel contribution to the knowledge of the oviductal and uterine proteome. The evaluated set of samples, of only 3 individuals, was highly valuable compared to other studies because it was possible to test the reproductive fluids and plasma of the same women, avoiding bias due to individual variability. Previous studies have focused on HUF, but our study demonstrates that HOF is also a rich fluid with essential proteins that could be a target to improve fertilization rates and early embryo development, if used in the culture media. More studies with similar designs and with established standard operating procedures are needed to corroborate our results/hypothesis and find consistent markers in secretions of female reproductive tract. On the other hand, establishing correlations between demographic and lifestyle of donors with their IVF outcomes has great utility for providers. A better understanding of follicular dynamics and ovarian response to gonadotropin stimulation of the donors could optimize the donor's recruitment and IVF treatments going forward. Hence, in chapter VII, demographic and lifestyle characteristics in relation to ovarian response to hyperstimulation among oocyte donors were studied We evaluated a variety of demographic and lifestyle factors in relation to peak E2, total oocyte yield and MII oocyte yield among oocyte donors in Spain, and found evidence that some key and potentially modifiable factors may impact oocyte yields among otherwise young healthy women. Specifically, we found that women with a BMI on the high end of normal BMI, as well as women with a BMI in the overweight range, had a higher yield of MII oocytes. In addition, women who took naps lasting more than 30 minutes had a higher oocyte yield, whereas women who exercised for at least 5 hours every week had a lower oocyte yield than women who did not take naps and women who did not exercise, respectively. In summary, several important milestones were achieved within this thesis: i) the development of a device to collect human uterine fluid in vivo; ii) the creation of the first biobank of human reproductive fluids. The awareness of the population for this biobank and its impact on infertile couples may contribute for more donations from healthy donors; iii) the establishment of the safety ranges for the parameters used to certificate the quality of the human reproductive fluids stored in the biobank; and iv) the epidemiologic and proteomics analysis of human reproductive fluids, which resulting conclusions will allow to improve the knowledge about the reproductive fluids constituents, and to elaborate a method to use them as supplements for the embryo culture media. As a whole, the strategy developed in this thesis could contribute to prevent epigenetic alterations and to improve the health of ART-derived offspring by means of more natural culture systems during the in vitro preimplantational embryo development.