Sperm motility in mice with oligo-astheno-teratozoospermia restored by in vivo injection and electroporation of naked mRNA.

Abstract

Oligo-astheno-teratozoospermia (OAT), a recurrent cause of male infertility, is the most frequent disorder of spermatogenesis with a predominantly genetic origin. Patients and mice bearing mutations in the <i>ARMC2</i> gene exhibit reduced sperm concentration, multiple morphological defects, and impaired motility, defining a canonical OAT phenotype. Intracytoplasmic sperm injection (ICSI) is required to treat this condition; however, it is associated with a slightly increased risk of birth defects compared with natural conception, highlighting the need for novel targeted therapies. Here, in vivo testicular injection followed by electroporation of capped, polyadenylated naked messenger RNA (mRNA) was evaluated as a strategy to treat <i>ARMC2</i>-related infertility in mice. mRNAs encoding reporter proteins were used to assess expression efficiency and kinetics using in vivo and in vitro 2D and 3D imaging. Reporter proteins were detected in germ cells for up to three weeks, demonstrating the feasibility of mRNA-based approaches. These results were compared with a non-integrative plasmid Enhanced Episomal Vector, which induced weak and transient expression in spermatogenic cells. Delivery of <i>Armc2</i> mRNA restored morphologically normal and motile sperm in deficient males, capable of producing embryos via in vitro fertilization and ICSI. These findings provide proof-of-concept that mRNA electroporation can restore sperm motility and fertilizing potential, offering a novel strategy to correct monogenic male infertility.