Male gamete copies to characterize genome inheritance and generate progenies

Abstract

Male factor infertility accounts for approximately 30% of infertility cases. When spermatozoa are extremely scarce, replicating the male gamete to fertilize a large cohort of oocytes is ideal. Additionally, patients with inherited disorders currently rely on pre-implantation genetic testing (PGT) to select healthy embryos, which raises ethical concerns owing to the generation of multiple embryos to select one healthy conceptus. Therefore, it would be beneficial to decode the genetics of a single sperm cell before conceptus generation. In this study, we demonstrated the feasibility of replicating the sperm genome via androgenesis and selecting the desired gamete before fertilization to preserve a specific paternal genotype, as confirmed by phenotypic observations and genetic testing in a murine model. We achieved satisfactory pre-implantation development rates with replicated male gametes and generated healthy offspring. Specifically, using 8-cell stage androgenetic embryos, a single spermatozoon can yield up to three conceptuses carrying an identical paternal haplotype.

Fig. 1

Timeline—8-cell stage haploid male pseudo-blastomere NT.

Fig. 2

Haploid androgenetic embryos and karyotyping. (a) Eight-cell Haploid androgenetic embryos generated from B6-GFP sperm. (b) The same embryos under fluorescence microscope. (c) Karyotyping demonstrated a consistent haploidy rate at 88.2% throughout early androgenesis from 2-cell to 8-cell stage. (d) Chromosome spread of haploid androgenetic blastomere.

Fig. 3

Hatching blastocyst generated from embryo reconstruction using haploid pseudo-blastomere generated using B6-GFP sperm.

Fig. 4

Pups generated from reconstructed embryos using (a) B6D2F1 2-cell androgenotes, (b). B6D2F1 4-cell androgenotes, (c). B6-GFP 8-cell androgenotes and (d) dead pup with abnormality.

Fig. 5

To confirm the inheritance of the paternal genome, WES was employed to first identify genetic discrepancies between sibling spermatozoa from a wildtype control mouse to validate the sensitivity of the test. We then use the same genotyping method to identify genetic similarities between sibling haploid blastomeres from the same haploid androgenetic embryo. Chromatogram demonstrated: (a) different nucleotide sequence between sibling spermatozoa in a hybrid wildtype mouse, while (b) cloned spermatozoa displayed identical nucleotide sequence.