P-795 Identification of a specific paternal haplotype in the germline and perpetuating progenies by male gamete replication
Authors: Xie P , Aluko A , Cheung S , Rosenwaks Z , Palermo G 
Published: June 2023

Abstract

Study question
Can we identify and replicate a male germ cell genome to generate progenies carrying a specific genotype?

Summary answer
We were able to replicate male genome and identify the androgenote with a specific genotype to generate conceptuses with identical paternal haplotype.

What is known already
For patients with heritable genetic disorders, current treatment relies on preimplantation genetic testing for monogenic disorders (PGT-M) to select unaffected embryos for replacement; however, this can lead to embryo wastage, causing emotional distress. Although genomic editing of gametes is being attempted, CRISPR-based technologies are inaccurate and can lead to loss of heterozygosity in cases where the embryo uses the maternal genome as a template to edit the paternal genome. Therefore, the use of haploid androgenotes may offer a useful tool to select male gametes with genes of interest or a successfully edited genome before the creation of an embryo.

Study design, size, duration
To generate haploid androgenotes, spermatozoa from hemizygous transgenic mice with GFP expression (B6-EGFP x B6D2F1) were used to inseminate enucleated wildtype oocytes. Constructs were cultured to the 8-cell stage. Two pseudo-blastomeres were isolated for genotyping by DNAseq. A blastomere with GFP expression was used as a male gamete. Experiments were repeated using mice with the Fbn1tm1Hcd mutation as a model for Marfan syndrome. Biparental embryos were created to preserve the mutation as a disease model.

Participants/materials, setting, methods
To generate haploid androgenotes, metaphase II oocytes from wildtype B6 mice were enucleated and inseminated using spermatozoa from hemizygous GFP transgenic mice. Haploid androgenetic embryos were cultured in a time-lapse incubator up to the 8-cell stage. Biparental embryos were created by fusing a selected androgenote with an activated oocyte from another cohort. Resulting embryos were genotyped to confirm the paternal haplotype. Experiments were reproduced using spermatozoa from heterozygous Fbn1tm1Hcd-mutant mice.

Main results and the role of chance
In the first set of experiments using spermatozoa from transgenic mice hemizygous of GFP, 200 wildtype oocytes were enucleated and generated 195 (97.5%) ooplasts. Insemination of those ooplasts yielded 168 (86.2%) monopronucleated androgenetic embryos, and subsequently generated 116 (69.0%) 8-cell haploid androgenetic embryos, of which 59 (50.8%) expressed GFP. Up to 2 sibling blastomeres were genotyped individually by DNAseq and confirmed to have an identical paternal haplotype. The remaining blastomeres were used to create biparental embryos and yielded a 97.2% fertilization rate after grafting into 145 oocytes, comparable with the fertilization rate of ICSI controls at 95.6%. Although control zygotes yielded an 88.0% blastulation rate, the experimental embryos yielded a lower blastulation rate of 58.2% (P&lt;0.0001). Albeit with a lower developmental rate, all experimental blastocysts expressed GFP and displayed normal embryo morphokinesis.
For the experiment using spermatozoa from Fbn1tm1Hcd mice, 50 8-cell stage haploid androgenetic embryos were generated. Two individual pseudo-blastomeres from 35 haploid embryos were used for genotyping. The remaining 210 haploid blastomeres were used to construct biparental embryos and yielded 119 (56.7%) blastocysts. PCR on embryo biopsies confirmed that the resulting blastocysts inherited the mutant (n = 63) or wildtype (n = 56) paternal haplotype from the original gamete.

Limitations, reasons for caution
This technique allowed the selection of male gametes with the desired genotype to generate conceptuses with a known haplotype in a reproducible manner. A more prospective experiment is ongoing to simulate clinical application by cryopreserving haploid androgenetic embryos and using only androgenotes with a confirmed genotype to create biparental embryos.

Wider implications of the findings
This approach to utilize gamete substitutes before fertilization ensures the maintenance of rare and unique genetic materials to generate conceptuses with the desired genotype in transgenic mice in disease models. Indeed, once this technique is implemented in humans, genotyped androgenotes can be used to characterize germline heterozygosity.

Trial registration number
N/A