Regenerative therapies to treat infertility of endometrial origin: from basic to clinics

Diploma a la investigación sanitaria con perspectiva de género, Conselleria de Sanitat de la Comunitat Valenciana

Concurso nacional de Divulgación Científica #HiloTesis 2024, CRUE Universidades

INTRODUCTION The proper functionality of the innermost layer of the uterus, known as the endometrium, is crucial for establishing and maintaining pregnancy. Endometrial pathologies, such as Asherman syndrome (AS) and endometrial atrophy (EA), may arise independently or as a consequence of other conditions or medical interventions. Patients with these disorders often exhibit inadequate endometrial lining and functionality, which typically hinders embryo implantation and elevates the risk of miscarriage. Conventional therapies have demonstrated limited efficacy in addressing these challenges. Consequently, innovative regenerative approaches, encompassing cellular therapies, acellular components like platelet-rich plasma (PRP), and bioengineering tools such as extracellular matrix (ECM)-derived hydrogels, have emerged to enhance reproductive outcomes. HYPOTHESIS AND OBJECTIVES This doctoral thesis proposes the feasibility of various biotechnological options to deal with endometrial pathologies causing infertility, such as AS/EA. The research objectives pursued are as follows: (1)To assess the efficacy of a combination of specific growth factors (GF) loaded in a decellularized ECMderived hydrogel (EndoECM) in treating a preclinical model of endometrial damage. (2)To explore the potential of human umbilical cord (UC) blood derivatives as regenerative therapies in female reproductive medicine. (3)To investigate the applicability of UC-PRP as a treatment for a preclinical model of endometrial damage. (4)To critically evaluate the current state-of-the-art of biotechnological and regenerative therapies employed to clinically treat endometrial pathologies. DESIGN OF THE STUDIES (1)To establish the endometrial damage model, 70% ethanol was injected into the uterine horns of mice. Four days later, mice were treated either with (a) EndoECM, (b) EndoECM loaded with GF (EndoECM+GF) or (c) an innocuous saline solution (control). After two weeks, endometrial regeneration and fertility restoration were evaluated by assessing the number of glands, endometrial area, cell proliferation, neaoangiogenesis, reduction of collagen deposition, and fertility restoration. (2)A comprehensive literature review examined various applications of UC blood derivatives, analyzing the principal molecules involved in tissue regeneration, and conducted a thorough review of published findings regarding the application of UC blood derivatives within the female reproductive tract. (3)The regenerative effect of UC-PRP on the endometrium was then investigated. UC blood was obtained from women in childbirth and processed to obtain UC-PRP. Then, it was characterized. The murine model of endometrial damage was established as described in (1), and mice were intrauterine injected with (a) UC-PRP, (b) EndoECM+UC-PRP, or (c) saline solution (control). Endometrial regeneration, fertility outcomes and immunocompatibility were evaluated two weeks following treatment administration. (4)A systematic search of full-text articles available in Pubmed and Embase was conducted between January 2000 and September 2023. This manuscript evaluated the current status of various cellular, acellular and bioengineered therapies, spanning from preclinical (in vitro and in vivo) to clinical stages. Additionally, it discussed how ongoing research is laying the groundwork for future advancements in personalized medicine. RESULTS AND DISCUSSION (1)Proteomic functional enrichment analysis of EndoECM revealed its involvement in ECM interactions and regenerative processes. In the murine model, EndoECM alone failed to fully mitigate the damage, whereas the combined therapy (EndoECM+GF) led to increased number of endometrial glands, augmented endometrial thickness, improved cell proliferative index and neovascularization, reduced collagen deposition, and restored pregnancy rates. (2)UC blood contains various stem cell types, along with a diverse GF and extracellular vesicles released into the bloodstream. Both UC-PRP and UC serum can be similarly derived from UC blood. They all have potential applications in regenerative reproductive medicine ranging from treating ovarian, vaginal, or oviductal conditions to managing endometrial pathologies such as AS/EA. Further investigation is warranted to ascertain the comparative efficacy of these treatments, with components like UC-PRP expected to play a significant role in the future of regenerative medicine. (3)The commercially closed system effectively concentrated platelet and GF. In vitro release kinetics assays demonstrated sustained release of GF by both UC-PRP and EndoECM+UC-PRP. In the murine model, both treatments were immunotolerated and significantly promoted regeneration of the damaged endometrium, evidenced by increased endometrial area, neoangiogenesis, cell proliferation, gland density, and the upregulation in the expression of specific genes such as AKT serine/threonine kinase 1 (Akt1), vascular endothelial growth factor (VEGF), and angiogenin. Lower collagen deposition compared to non-treated uterine horns was observed. Additionally, UC-PRP treatment restored pregnancy rates in the mouse model. (4)A total of 164 articles were included in the systematic review. In the treatment of endometrial pathologies, cell therapies predominantly involved the use of bone marrow and UC mesenchymal stem cells. Acellular therapies such as PRP or extracellular vesicles are gaining popularity. Additionally, bioengineering approaches utilizing hydrogels derived from ECM, or synthetic biosimilars are on the rise. Combined therapies targeting multiple aspects of tissue repair and regeneration are still undergoing preclinical testing but have shown promising translational potential. CONCLUSIONS Finally, drawing upon our findings and the existing literature, one can conclude that the discrepancies experienced in conventional treatments for endometrial pathologies such as AS/EA stimulate the exploration of innovative biotechnological therapies. These include the utilization of bioengineered EndoECM and the application of UC derivatives such as UC-PRP, which demonstrate promising preclinical outcomes. Upon validation in clinical practice, these approaches may offer viable alternatives for the clinical management of AS/EA.

Ministerio de Ciencia, Innovación y Universidades (FPU19/04850)

Programa Fulbright

Instituto de Salud Carlos III (PI21/00305)

3 - Salut i Benestar