Efficient production of common marmoset embryos with in vitro fertilization and somatic cell nuclear transfer following optimized hormonal stimulation and laparoscopic oocyte collection.

Marmosets are a valuable model for studying human disorders, but developing specific genetic models requires efficient protocols for embryo genomic manipulation. Although marmoset embryos have been produced in vitro, oocyte retrieval traditionally involves extended recombinant human follicle-stimulating hormone (FSH) treatment (9-10 days), surgical laparotomy and ovarian exposure, limiting repeat procedures. Here we evaluated a simpler, cost-effective ovarian stimulation protocol using porcine pituitary FSH, combined with laparoscopic ovum pick-up (LOPU), for in vitro production of marmoset embryos via in vitro fertilization (IVF) and somatic cell nuclear transfer. A total of 3,922 oocytes were retrieved from 129 LOPU procedures, averaging 30.4 oocytes per LOPU, with 85.5% (26.0 per LOPU) graded as viable. The procedure was safe, with no adverse effects observed in ovaries or fimbria after up to nine LOPU sessions. Most collected oocytes were meiotically immature and matured in vitro before use. Following IVF, approximately 40% of oocytes cleaved, and 40% of cleaved embryos developed to the expanded blastocyst stage. LOPU-derived oocytes also supported somatic cell nuclear transfer, with 69.8% of embryos cleaving and 21.8% forming blastocysts. IVF-derived blastocyst quality was assessed by total cell count, immunodetection of SOX2 (inner cell mass) and CDX2 (trophectoderm), and cryotolerance. Embryo transfer to recipients resulted in successful live births. These findings demonstrate that a simplified pituitary FSH protocol followed by LOPU is an efficient, less invasive and safe method for retrieving developmentally competent marmoset oocytes, offering a promising approach for advancing marmoset-based research in disease modeling and reproductive biotechnology.