Travaglione, Angela (2025) Innovative technologies for optimizing the in vitro culture of mammalian embryos. [Tesi di dottorato]

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Abstract

Since the birth of Louise Brown in 1978, assisted reproductive technologies (ARTs) have become indispensable in the treatment of infertility. Despite the advancements and success of ARTs, embryos produced in vitro (IVP) exhibit lower developmental competence compared to their in vivo-derived (IVD) counterparts. A critical challenge with IVP embryos lies in the fact that post-fertilization culture conditions can significantly influence embryo quality. Key factors affecting embryo competence include the composition of the culture medium, embryo density (i.e., the number of embryos per culture medium volume), temperature, and gas balance within the incubator. The main goal of in vitro embryo production is to closely mimic the in vivo environment to generate high-quality embryos capable of resulting in viable births. In vivo, embryos develop within confined microenvironments, such as the oviduct and uterus. These natural cavities are rich in maternal factors, secreted by somatic cells of the oviductal (OF) and uterine fluid (UF), as well as embryo-derived signals. These factors, present as soluble molecules or molecular cargos enclosed within extracellular vesicles (EVs), play critical roles in promoting preimplantation embryo development and enhancing developmental competence. Conversely, in vitro embryo culture lacks both the confined environment and the bidirectional communication between embryos and the maternal reproductive tract. As a result, individually cultured embryos often show compromised development. To counteract this, embryos are commonly cultured in groups to enrich the environment with beneficial factors secreted by the embryos themselves. However, the trend in both human and animal ART is shifting toward selecting and transferring a single euploid, competent blastocyst, aided by novel non-invasive embryo quality markers. In human clinical embryology, embryos are often cultured in small numbers per patient, making individual embryo culture an inevitable choice. Individual embryo culture systems could offer advantages over group culture, as they allow for the simultaneous analysis of spent media for metabolomic, proteomic, and non-invasive preimplantation genetic testing (niPGT), which are rapidly emerging technologies. Despite these benefits, individual embryo culture has generally shown poorer outcomes compared to group culture in the same medium volume both in human and animal ART. Against this backdrop, the aim of my PhD thesis is to establish and validate an innovative method to improve the in vitro culture of individual embryos, more closely replicating the natural microenvironment of the female reproductive tract to enhance their developmental competence. Chapter 2 explores the main factors influencing in vitro embryo culture. Specifically, it compares the characteristics and developmental outcomes of embryos produced in vivo and in vitro. It also examines the dynamic physicochemical conditions embryos experience during their journey and development in the maternal tract, as well as the standard in vitro culture conditions. While time-lapse imaging systems for assessing embryo morphokinetics have gained widespread adoption, their utility in reliably identifying the highest quality embryos remains limited. Alternatively, omics-based approaches, which analyze the molecular composition of spent culture media and correlate it with embryonic euploidy, may soon offer a more robust solution. This highlights the need for innovative individual embryo culture systems that avoid the detrimental effects associated with isolated culture, ensuring embryo individuality while supporting the effective application of advanced methodologies. Chapter 3 investigates the development of an individual embryo culture system within a confined environment using extremely reduced culture volumes, aiming to maintain developmental competence comparable to conventional group culture. The developmental competence of blastocysts produced under various culture conditions—conventional group culture (GC), semi-confined group culture (MG), and individual culture (MS)—was evaluated. Parameters such as the number of TUNEL-positive cells, lipid content, mitochondrial function, and mean cell number were analyzed in detail. However, recreating a confined microenvironment for in vitro embryo development represents only one aspect of the conditions embryos experience during their transit through the maternal reproductive tract. Another critical factor is the embryo’s exposure to extracellular vesicles (EVs) derived from the oviduct and uterus. To address this, Chapter 4 examines the combined effects of a confined environment and sequential exposure to oviductal and uterine EVs, exploring their potential synergistic impact on in vitro bovine embryo culture. Developmental competence was characterized in depth under various culture conditions—group culture (GC) and microwell single culture (MS), with and without supplementation of EVs-OF and EVs-UF. Key parameters such as TUNEL-positive cells, lipid content, mitochondrial function, and mean cell number were assessed to elucidate these effects.

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