P-798 Utilization of Haploid Androgenetic Blastomeres to Characterize Male Genomic Inheritance and as a Male Gamete Substitute

Abstract

Abstract Study question Can sperm genomes be replicated by generating androgenetic embryos, and can resulting haploid blastomeres be used as a substitute for male gametes with known genotype? Summary answer Once utilized as male gametes, haploid androgenetic blastomeres can be used for gamete genotyping and can sustain embryo development to live offspring. What is known already Heterozygosity in human spermatozoa may represent a risk to offspring health. This has led to attempts to replicate the gamete genome by generating haploid embryonic stem cells (haESCs) that can be used to typify heterozygosity or as gamete substitutes. Although haESC lines are unstable with a tendency to self-diploidize, utilizing haploid androgenetic cells in the early embryonic stages, such as the blastomere stage, can provide multiple copies of genetically functional pseudo-gametes. Study design, size, duration Haploid androgenetic embryos were generated using spermatozoa from B6 heterozygous mice (B6-EGFP x B6D2F1) and cultured until the 8-cell stage. Individual haploid blastomeres with green fluorescent protein (GFP) expression were fused with activated recipient oocytes. Resulting conceptuses were cultured in a time-lapse system to monitor each stage of preimplantation development. Blastocysts with GFP expression were transferred to pseudo-pregnant surrogates. Pregnancy and offspring reproductive health were evaluated. Piezo-actuated ICSI was performed on unmanipulated oocytes as control. Participants/materials, setting, methods To generate haploid androgenetic embryos, metaphase II oocytes of B6D2F1 mice were treated with cytochalasin B, enucleated under Oosight®, and inseminated by piezo-actuated ICSI. Haploid androgenetic embryos were cultured to the 8-cell stage. Androgenetic blastomeres with GFP expression were coated with inactivated Sendai-virus and grafted to an activated recipient oocyte from another cohort. Cleavage parameters of reconstructed zygotes were compared to controls. Blastocysts were transferred into the uterine cavity of a 2.5-day post-coitus CD-1 surrogate. Main results and the role of chance A total of 421 oocytes were enucleated with a survival rate of 98.6%. The ooplasts underwent ICSI; 351 (84.6%) constructs developed a single male pronucleus 4-6h post-ICSI, and 208 (59.3%) constructs cleaved to the 8-cell stage. Parthenogenic activation by calcium ionophore was successful in 96.4% of the recipient oocytes, confirmed by the appearance of a single female pronucleus. A total of 148 haploid androgenotes with GFP expression were grafted onto corresponding activated oocytes; 145 successfully fused. Up to 2 unused sibling haploid blastomeres per embryo were genotyped using whole exome sequencing by a reference laboratory. A total of 481 untreated oocytes were inseminated, yielding 437 control zygotes. The cleavage of reconstructed embryos into the 2-cell (96.4%), 4-cell (94.3%), and 8-cell stage (91.4%) was comparable to that of control conceptuses (97.5%, 93.8%, and 92.7% respectively). Although morula compaction (80.0%) and blastulation (60.7%) were lower than control (89.2% and 80.8%, P <0.001, respectively), morphokinetic parameters were comparable. A total of 45 blastocysts were transferred into 5 recipient mice; 8 blastocysts implanted (17.8%) in 2 surrogates, yielding 5 live offspring (11.1%) weighing 1.51±0.22g. All live offspring expressed GFP and were confirmed fertile once reaching adulthood. Limitations, reasons for caution Although copies of gamete substitutes were generated that could be genotyped and used to produce live offspring with the desired phenotype in a mouse model, the system needs to be optimized and proven reproducible in humans, specifically in regard to the role of the sperm centrosome. Wider implications of the findings Once our proposed technique is implemented in humans, it may benefit patients to control germline heterozygosity by allowing genotyping of identical copies of the gametes. This technique, when used in conjunction with heritable genomic editing, can obviate the inherently highly condensed sperm genome to provide genome-edited gamete substitutes. Trial registration number N/A