(88) Impact of Early Biopsy and Chromatin Accessibility on the Development of Cloned Embryos

Y. Liu¹, E. Grow², I. Viotti Perisse¹, T. Patrick¹, A. Thomas¹, R. Stott¹, B. Cairns³, I. A. Polejaeva¹, K. L. White¹.

1. Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, UT, USA

2. Green Center for Reproductive Biology Sciences, University of Texas Southwestern, Dallas, TX, USA

3. Department of Oncological Sciences, Howard Hughes Medical Institute, University of Utah School of Medicine, Salt Lake City, Utah, USA

Abstract Text:

Somatic Cell Nuclear Transfer (SCNT) is an important tool used in several scientific fields, including regenerative medicine, production of large animal models for human diseases, and reproduction of domestic animals with high genetic value for breeding purposes. However, SCNT efficiency remains low, with only 5-15% of transferred embryos resulting in live births in domestic animals. One possible reason could be abnormal embryonic genome activation (EGA) in SCNT embryos, which leads to incomplete reprogramming during embryo development. Performing a prescreen biopsy on SCNT embryos, to perform an assessment of chromatin accessibility using ATAC-seq, would help identify the EGA-high embryos for embryo transfer. The aim of this study was to develop a method of pre- screening SCNT embryos at an early stage after EGA has occurred, and to investigate the subsequent in vivo development of SCNT embryos following biopsy. Bovine SCNT embryos were generated using our standard protocol and cultured in groups until biopsy. Biopsy of SCNT embryos was performed at the 12-16 cell stage (Day 3), removing 2 blastomeres by aspiration using a beveled pipette, and the biopsied cells processed for single cell ATAC-sequencing (scATAC-seq). Following biopsy, SCNT embryos were cultured individually in the Well of Well system (a microenvironment in which individual embryos are cultured in  microwells within a shared culture medium in a 4-well dish) until Day 6-7, while non-biopsy control SCNT embryos remained in group culture. The cell numbers in the blastocysts at Day 7 were determined via differential staining (11 blastocysts in the biopsy group and 12 in the non-biopsy group). For in vivo developmental assessment, SCNT blastocysts on Day 6, with or without biopsy, were transferred into synchronized recipients on Day 7. In total, 45 biopsied blastocysts and 60 non-biopsied blastocysts from 5 replicates were transferred into 105 recipients. The scATAC-seq data from 34 embryos were analyzed for UMAP plotting using Monocle3, with IVF embryos serving as a reference to classify biopsy samples as EGA-high and EGA-low. All the data were analyzed using a generalized linear model (Jamovi). Total cell numbers and trophectoderm cell numbers in the biopsied-blastocysts were significantly lower than non-biopsied blastocysts (74.6±6.2 and 51.5±5.3 vs. 116±9.8 and 91.8±8.6, P=0.002 and P< 0.001, respectively). However, no differences were observed in the inner cell mass between the two groups (23.2±2.6 vs. 24.6±2.6, P=0.71). There was a significantly greater initial pregnancy rate in the biopsy group than in the non-biopsy group (42.2% (19/45) vs. 18.3% (11/60), P=0.007). Additionally, EGA-high embryos tended to have a higher initial pregnancy rate at Day 40 compared to EGA-low embryos, although the differences were not significant (52.2% (12/23) vs. 36.4% (4/11), P=0.39).  In conclusion, performing a biopsy on SCNT embryos at an early stage (12-16 cell) does not negatively impact their in vitro or in vivo development. Screening SCNT embryos based on chromatin accessibility may help select SCNT embryos with higher developmental potential for subsequent embryo transfer. Further studies will investigate open regions in EGA related gene expression from biopsy samples for pregnant and non-pregnant embryo transfers, as well as those that result in pregnancy loss or successful term development. These insights could help identify key EGA events critical for SCNT embryo development to term.