Molecular determinants of human oocyte quality in assisted reproduction

Abstract

Infertility, defined by the World Health Organization (WHO) as the inability to conceive after a year of unprotected sex, is a disease that affects 1 in 9 couples of reproductive age worldwide. Most of these couples will only achieve pregnancy through assisted reproduction techniques. In recent decades, many improvements have been made in this field, but no method has been able to achieve 100% success. There are many variables that could affect the outcome of assisted reproduction cycles, one of the most important is the quality of the woman's oocytes. Maternal age is the most important factor affecting women’s ability to conceive and give birth, since female reproductive aging is associated with reduced oocyte quality; however, the underlying molecular mechanisms remain poorly understood. It is also well established that as woman age increases, her ovarian reserve diminishes. However, the role of ovarian reserve in the decline of oocyte quality with age is currently unknown. The developmental competence of an oocyte is its ability to sustain embryonic development until embryonic genome activation. It is determined by the transcripts (coding and non-coding RNAs) accumulated during oocyte maturation. The lack of transcription during final oocyte maturation suggests that regulation of the genes involved in this process occurs at the post-transcriptional level. Among the possible mechanisms, alternative splicing (AS) of the messenger RNA could be involved. Studying oocyte gene expression and the spliced mRNA isoforms might provide novel information on the molecular mechanisms driving early development, and might be a source of potential biomarkers of oocyte quality. The main objective of this thesis is to explore new possibilities for the identification of oocyte quality biomarkers at the molecular level in order to better understand the oocyte and how its developmental competence can be improved. For this, the results of oocytes from women with different age and ovarian reserve have been compared. In addition, to identify non-invasive biomarkers for oocyte developmental competence, the evaluation of the association between the expression analysis of different aging markers in human cumulus cells (CCs), the age and ovarian reserve and the oocyte maturation rates was conducted. Finally, because many of the oocytes used were vitrified, the effect of vitrification on oocyte developmental competence was analysed by comparing the reproductive outcomes of fresh and vitrified oocytes from the same stimulation cycle. The results suggest an important role for ncRNAs and alternative splicing in human oocyte biology. Age and ovarian reserve have been shown to independently affect the ncRNAs transcriptome of in vivo matured oocytes. These results might provide valuable information for the search of oocyte quality markers, and for the (re)interpretation of existing dataset. On the other hand, differences in transcribed splicing variants can also provide biomarkers of oocyte quality, since the profile of confirmed AS events could determine the specific transcriptome of the mature oocyte. The expression of common somatic aging markers in CCs didn’t show a clear correlation between the analysed genes and age, suggesting that CCs of reproductively old women do not present the typical transcriptome of aged tissues. In addition, when looking at future clinical applications, these markers have not been found useful for the development of non-invasive markers for oocyte developmental competence, since no correlation was observed either with the ovarian reserve or the oocyte maturation rates. Finally, this study showed that oocyte vitrification per se maintained the developmental potential of human oocytes within a reasonable biological range, clinically comparable to fresh oocytes. As a consequence, we established that the main reason for the reported lower clinical results in vitrified cycles has to be attributed to the loss of oocytes during the warming step. This has important repercussions in the clinical practice, as measures can be easily put in place to offset oocyte loss.

La competencia de desarrollo ovocitaria se define como la capacidad del ovocito para mantener el desarrollo embrionario hasta que el embrión activa su propio genoma. Está determinada por los transcritos (ARN codificantes y no codificantes) acumulados durante la maduración de los ovocitos. La falta de transcripción durante la maduración final de los ovocitos sugiere que la regulación de los genes involucrados en este proceso ocurre a nivel postranscripcional. Entre los posibles mecanismos, el “splicing alternativo” del ARN mensajero, podría estar involucrado. El objetivo principal de esta tesis es explorar nuevas posibilidades para la identificación de biomarcadores de calidad de ovocitos a nivel molecular con el fin de comprender mejor el ovocito y cómo se puede mejorar su competencia de desarrollo. Además, para identificar biomarcadores no invasivos de la competencia de desarrollo de ovocitos, se ha evaluado la expresión de diferentes marcadores de envejecimiento somático en células de cúmulos humanos que rodean los ovocitos, y se ha correlacionado con la edad y la reserva ovárica de las mujeres y con las tasas de maduración de los ovocitos. Finalmente, debido a que muchos de los ovocitos utilizados estaban vitrificados, se ha analizado el efecto de la vitrificación sobre la competencia de desarrollo de los ovocitos comparando los resultados reproductivos de ovocitos frescos y vitrificados del mismo ciclo de estimulación. Los resultados sugieren que los ARNs no codificantes y el splicing alternativo representan un papel importante en el proceso de adquisición de la competencia de desarrollo en ovocitos humanos. Estos resultados pueden proporcionar información valiosa para la búsqueda de marcadores de calidad de ovocitos y para la (re)-interpretación de conjuntos de datos existentes. El estudio de la expresión de marcadores de envejecimiento somático en células de cúmulos humanos no ha mostrado una clara correlación entre los genes analizados y la edad, lo que sugiere que las células del cúmulo de mujeres de edad reproductiva avanzada no presentan el transcriptoma típico de los tejidos envejecidos. Finalmente, este estudio ha demostrado que la vitrificación de ovocitos mantiene per se el potencial de desarrollo de los ovocitos humanos dentro de un rango biológico razonable, clínicamente comparable a los ovocitos frescos.