P-405 Nuclear transfer overcomes poor embryo development of in vitro grown mouse oocytes
Authors: Christodoulaki A , He H , Zhou M , De Roo C , Baetens M , Menten B , Van Soom A , Stoop D , Boel A , Heindryckx B 
Published: June 2023

Abstract

Study question
Is the nuclear transfer technology able to overcome the compromised embryonic development of in vitro cultured secondary follicles from young B6D2 mice?

Summary answer
Nuclear transfer technology was able to restore embryonic development to levels similar of in vivo grown oocytes.

What is known already
In vitro follicle culture has been proposed as a strategy for fertility preservation in cancer patients. Studies in human follicle culture remain scarce, due to the low availability of human tissue. Mouse models have been extensively studied to improve follicle maturation and investigate the potential of in vitro grown (IVG) gametes. Despite significant improvements reported over the years, including increased maturation rates, advanced embryonic development and generation of fertile offspring, the quality of IVG oocytes remains inferior compared to their in vivo grown counterparts.

Study design, size, duration
The experimental study was conducted between October 2020 and January 2023, after approval by the Animal Ethics Committee of Ghent University Hospital (ECD no 19/60). In total, 108x 16-days-old B6D2 females were used for secondary follicle isolation and in vitro culture, 81x 8-12-week-old females for collection of in vivo grown oocytes, 4x 9-14-week-old B6D2 males for sperm freezing, 5x 10-24-week-old CD1 vasectomised males and 10x 8-12-weeks-old CD1 females as surrogate mothers for embryo transfer.

Participants/materials, setting, methods
Follicles isolated from 16-days-old B6D2 mice were cultured for 9 days, followed by maturation for 16-18hrs. Mature oocytes were assessed on diameter, spindle morphology, calcium releasing ability, mitochondrial membrane and embryonic developmental potential. In vivo grown oocytes from stimulated mice were used as controls and cytoplasmic donors for nuclear transfer. Spindle (ST) and Pronuclear transfer (PNT) were applied to overcome poor development of IVG embryos, by transferring the spindle/pronuclei of IVG oocytes/zygotes to enucleated controls.

Main results and the role of chance
In total, 1509 secondary follicles were cultured and 73.6% survived to Day 9. From the 719 cumulus oocyte complexes, 364 (50.6%) oocytes matured. Oocyte diameters were significantly smaller between IVG (67.4μm) and controls (73.1μm) (p &lt; 0.001). Spindle staining revealed a similar number of normal spindles between the two groups (71.4% and 82.6% respectively, p = 0.138). Calcium release was significantly lower in IVG oocytes (1.6) vs controls (5.7) (p &lt; 0.001), as well as mitochondrial membrane potential ((0.9) vs (2.2), p &lt; 0.001). These data implicate a cytoplasmic inferiority in IVG oocytes. Following parthenogenetic activation, 2-cell (59.4%) and blastocyst rate (36.3%) in IVG group was significantly lower than control (89.4% and 88.2% respectively, p &lt; 0.001). For this reason, we performed ST, using controls as cytoplasmic recipients for IVG spindles. The 2-cell rate significantly increased to 100% (p &lt; 0.001) in ST oocytes, reaching 100% blastocyst formation. Next, to create bi-paternal embryos, we performed IVF. From the IVG group, 2-cell (40%) and blastocyst (0%) rates were significantly lower than control (94.4% and 83.3%, p &lt; 0.001). Embryo development was restored following PNT (98% 2-cell and 86% blastocyst rate, p &lt; 0.001). Genetic analysis of PNT embryos revealed that 5/6 were chromosomally normal, similar to controls (8/10). Embryo transfer of PNT and control embryos is ongoing.

Limitations, reasons for caution
Both ST and PNT are promising technologies to overcome poor embryonic development of IVG oocytes, but the benefit for human IVG oocytes remains to be investigated, along with the safety of the technique.

Wider implications of the findings
Our results demonstrate that ST and PNT have the potential to restore embryonic developmental competence of IVG oocytes, without increasing chromosomal abnormalities. This technology could be investigated in the future, as a mean to overcome poor quality of IVG oocytes in human.

Trial registration number
Not applicable