Transverse and vertical incisions affect the viability of in vitro-produced embryos submitted to a simplified microsurgery approach

Author(s)

Minozzo, Oliveira Andressa ; Vieira, Marsico Thamiris ; Jose, Sudano Mateus

• Document type: Journal article
• Source: Theriogenology; v. 226, p. 8-pg., 2024-07-02.

Abstract

Integrating in vitro embryo production with embryonic microsurgery facilitates the generation of monozygotic twins. However, despite their potential benefits, these methods have not been widely adopted in commercial settings because of their substantial costs. Hence, there is a need to streamline the bisection procedure while ensuring efficient production of viable demi-embryos. In this study, we investigated the impact of different orientations of microsurgical incisions in relation to inner cell mass on embryonic development, morphology, viability, and expression of cell fate protein markers using a simplified microsurgery approach. Ovaries were transported from the slaughterhouse to the laboratory and aspirated to obtain oocytes that were selected and subjected to in vitro embryo production. The selected expanded blastocysts (n = 204) underwent microsurgery. The blastocysts were immobilized to facilitate incision using an adapted microblade, yielding demi-embryos (vertical incision) and viable embryonic fragments (transverse incision). The structures were then re-cultured for 12 h. Viability was assessed by measuring the re-expansion rate after re-culture, followed by immunofluorescence analysis of proteins (CDX2 and NANOG) and apoptosis analysis using terminal deoxynucleotyl transferase dUTP nick end-labeling (TUNEL). Microsurgically derived embryos exhibited remarkable plasticity, as evidenced by a slight reduction (P < 0.05) in the re-expansion rate (transverse 64.2 % and vertical 57.2 %) compared to that of the control group (blastocysts without microsurgery) (86.7 %). They also demonstrated the ability of morphological reconstitution after culturing. Despite the anticipated decrease (P < 0.05) in the total number of cells and embryo volume, microsurgery did not result in a significant increase (P > 0.05) in the number of apoptotic cells. Furthermore, microsurgery led to higher (P < 0.05) expression of markers associated with pluripotency, indicating its efficiency in preserving regenerative capacity. Moreover, microsurgery, whether followed by immunosurgery or not, made the isolation of embryonic cells easier. In conclusion, both transverse and vertical microsurgery incisions enabled the production of identical demi-embryos and served as tools for isolating embryonic cells without compromising the resumption of development and the apoptotic index. (AU)

• FAPESP's process: 21/01446-9 - Multi-user Equipment approved in grant 18/21496-8: Inverted Microscope Equipped with Fluorescence and Differential interference Contrast (DIC)
• Grantee: Mateus José Sudano
• Support Opportunities: Multi-user Equipment Program
• FAPESP's process: 22/02139-5 - Transcriptional profiles of embryo competence
• Grantee: Andressa Minozzo Oliveira
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• FAPESP's process: 18/21496-8 - Pathways for embryo-derived pregnancy
• Grantee: Mateus José Sudano
• Support Opportunities: Research Grants - Young Investigators Grants
• FAPESP's process: 19/17796-9 - Simultaneous biopsy and lipid profile of embryo inner cell mass and trofectoderm
• Grantee: Thamiris Vieira Marsico
• Support Opportunities: Scholarships in Brazil - Doctorate