P-394 Neurotrophin-4 supplementation during human secondary follicle in-vitro-culture supports morphologically normal blastocyst formation

Abstract

Abstract Study question Can matrix-free culture system supplemented with Neurotrophic factor 4 (NT4) improve human follicular development and achieve mature and fertile oocytes in vitro? Summary answer NT4 promotes the growth, steroid hormone production of human secondary follicles cultured in vitro (including post- and pre-pubertal patients), thereby yielding mature and fertile oocytes. What is known already Reconstituting folliculogenesis in vitro play vital roles in reproductive biology research, fertility preservation and reproductive toxicity testing. However, fertile oocyte from in-vitro-grown (IVG) human follicle remains unachieved. It has been demonstrated that NT4 promotes in-vitro survival, growth, and maturation of mice secondary follicles. NT4 and its receptors have been detected in human ovaries. However, if the advance proved in mice model is effective to replicate in human has not been reported. Study design, size, duration Discarded ovarian tissues after ovarian tissue cryopreservation (OTC) were collected from 6 patients aged from 11 to 21 years old who underwent unilateral oophorectomy for fertility preservation, after obtaining written informed consent. Isolated secondary follicles were cultured in vitro with or without NT4 in a matrix-free system for 4-6 weeks individually. Participants/materials, setting, methods Secondary follicles isolated from each patient were randomly assigned to control or NT4 group, followed by incubation on the ultra-low attachment microplate individually. Follicle growth and survival were assessed by microscopy. Anti-Müllerian hormone (AMH), estradiol and progesterone levels were quantified in the medium. Oocyte marker expression was evaluated by DEAD box polypeptide 4 (Ddx4) staining. Oocyte mature and fertile potential were assessed by in-vitro maturation and intracytoplasmic sperm injection (ICSI) with donated sperm, separately. Main results and the role of chance In control condition, isolated follicles survived for 4-6 weeks with increased diameters over time (P < 0.05), reaching a terminal diameter of 967.00 ± 41.1 μm with the confirmed induction of steroidogenesis and expression of widely accepted oocyte markers (DDX4). Three out of twelve (25%) alive follicles were matured successfully in vitro, most of which produced morphologically normal MII oocytes with sizes of 120 ± 2.1 μm. The diameter and steroid hormone production were significantly higher in the group cultured with NT4 than in the control group (P < 0.05). An increased efficiency of MII oocyte production in the NT4 group was also observed, while the difference was not statistically significant. MII oocyte obtained from the control group showed abnormal fertilization after ICSI. In contrast, MII oocyte acquired from the NT4 group progressed to available blastocyst. Limitations, reasons for caution The population included were all patients with thalassemia. Whether this culture system is effective for patients with other diseases is unknown. Limited by the small sample, the effect of NT4 on maturation competence needs further confirmation. Oocytes obtained have not been quantified with ploidy status and epigenetic signatures. Wider implications of the findings Surplus ovarian tissue after OTC, which is otherwise discarded, may serve as an additional precious source of fertile oocytes for fertility preservation, even for pre-pubertal girls, without a threat of tumor reintroduction. The system described here will provide a powerful research tool for reproductive biology research and reproductive toxicity testing. Trial registration number not applicable