Factores de origen femenino y masculino relacionados con la fertilidad humana y su aplicación en clínicas de reproducción asistidaproteínas del fluido folicular y genes de compactación del ADN espermático

Zusammenfassung

Infertility affects 15% of couples in reproductive ages, with similar prevalence between developed and developing countries. Fertility prevalence is increasing worldwide due to clinical factors and social habits such as obesity, sexually transmitted diseases, or delayed childbearing. Infertility has been approached considering its origin as female or male, although there is still a significant percentage of cases defined as idiopathic, so there is growing interest in the proper diagnosis of the infertile couple. Within the management of female infertility, many women require in vitro fertilization (IVF) treatments to achieve a full-term pregnancy with a live-born child. In IVF, the recovery of competent cumulus-oocyte complexes (COCs) is important. Considering that competent COCs are achieved mainly by the contribution of cumulus cells and follicular fluid (FF), the study of the composition of this biofluid is interesting. In fact, the oscillation of the total protein concentrations of the FF could affect the competence of the oocyte; likewise, the reactive nitrogen species (RNS) contained in the FF may allow the evaluation of the oxidative damage of the proteins present in the human follicular fluid (FFH). On the other hand, in male infertility, between 30 and 72% of the cases are still diagnosed as idiopathic so more complete diagnostic tests, that allow the evaluation of molecular and genetic factors as possible causes of infertility, have been developed. However, these tests are not routinely used in clinical practice. Also, the expression of these genes (RNA) and the state of DNA protamination are of interest in the study of male infertility. Considering the above, and after a deep literature review exposed in chapter I, this work aims to broach the study of infertility from its female and male aspects to identify simple and non- invasive biomarkers that can be used as predictors of the success of fertility treatments in routine clinics and, in this way, improve efficiency in assisted reproduction cycles. In chapter II, the concentration of total proteins (TP), albumin and globulins, as well as the levels of nitrotyrosine-modified proteins were studied in FFH to assess the degree of oxidative stress. For this, the FFH of 74 women undergoing ICSI treatment (41 control group and 33 infertile women with different diagnoses) was recovered after follicular aspiration and recovery of the COCs. The FFH was centrifuged, filtered, and stored at -20°C until analysis. The concentration of PT, albumin and globulins was determined by the COBAS c501 analytical platform using the automated photometry method - colorimetric test, while the measurement of the level of nitrotyrosine-modified proteins was analyzed with the 3-nitrotyrosine (3-NT) ELISA test. The results showed a constant distribution in the concentrations of PT, albumin, and globulins in the control group, while in the infertile group great variability was observed in this distribution between the different diagnoses of the patients. The highest concentrations of PT (4.92±0.09 g/dl vs. 4.45±0.12 g/dl), albumin (1.84±0.06 g/dl vs. 1.66±0.04 g/dl) and globulins (3.08±0.05 g/dl vs. 2.79±0.09 g/dl) were observed in the control group vs. the infertile group; and, in the infertile group, women with endometriosis and ovarian failure showed lower PT and albumin concentrations than women with other infertility diagnoses. On the other hand, no difference was found in the concentrations of 3-NT between the control group vs infertile women (28.40±1.79 ng/ml vs. 26.83±2.20 ng/ml), nor were differences observed within the group of infertile women based on their diagnoses. In chapter III, polymorphisms in the TNP1, TNP2, PRM1 and PRM2 genes were studied in fertile and infertile Ecuadorian mestizo men. For this, 144 men were included (43 control and 101 infertile group) who underwent a spermiogram and peripheral blood sampling. Peripheral blood DNA extraction, polymerase chain reaction (PCR) and sequencing of these genes were performed. Nine polymorphisms were identified, none of which were related to fertility and would not be a risk factor for male infertility. The identified polymorphisms were rs62180545 in TNP1, rs11640138 and rs56069754 in TNP2, rs201923496 and rs737008 in PRM1, rs749752404, rs1646022, rs2070923 and rs18211 4260 in PRM2. The only polymorphisms not previously described in other populations were rs749752404 and rs182114260 of PRM2. In chapter IV, the levels of protamination/packaging of chromatin and expression of PRM1 and PRM2 (RNA) in spermatozoa from fertile and infertile men were studied. For this, 131 individuals were included (35 control group and 96 infertile patients), who underwent a semen analysis, and seminal sample was subjected to swim up sperm selection technique and divided into aliquots in cryopreservation tubes and stored at -196°C until further processing. The indirect evaluation of chromatin packaging in spermatozoa was performed using the chromomycin A3 (CMA3) assay, while for the evaluation of PRM1 and PRM2 genes expression, RNA extraction, cDNA, and real-time PCR were performed with Power SYBR® Green PCR Master Mix reagent. The BETA-ACTIN (BA) gene was considered as the reference gene. Results showed a higher percentage of CMA3 spermatozoa in the infertile group vs. the control group (15.37 ± 1.00 vs. 12.10 ± 1.01, P = 0.01). Within the control group, CMA3 levels were increased in individuals with type I overweight and obesity compared to normal weight, being these levels as high as those found in the infertile group. Also, within the control group, individuals with varicocele who underwent surgery had lower CMA3 levels compared to individuals who did not undergo surgery. On the other hand, when evaluating the mean expression levels (ΔCT) of PRM1 and PRM2, as well as the PRM1/PRM2 ratio, no differences were found between both experimental groups. When evaluating the influence of body mass index on the results, only differences were found in the PRM1/PRM2 ratio of infertile patients between individuals with normal weight vs. overweight (P=0.001). The results of this work suggest that the concentration of total proteins in the FFH might be considered as a marker within the assisted reproduction treatment; and the analysis of the state of protamination (CMA3) would allow an identification of a possible cause of infertility in men cataloged with idiopathic infertility.