The Vitrification Of Human Ovarian Tissue Combined With Retrieval Of Immature Oocytes

Objective:Vitrification appears to offer a quick,easy and inexpensive means of cryopreserving ovarian tissue and studies on human and different animal species have shown promising results.There is nevertheless room for improvement as most current vitrification protocols have been empirically created or adapted from protocols for embryos and oocytes.So far,the only reliable endpoints are pregnancy and birth.The best approach to understand how vitrification can be used to cryopreserve ovarian tissue would probably be to focus on a single protocol.After obtaining consistent results,the protocol could be improved step by step,changing one parameter at a time if necessary.This study is to find the optimal cryoprotectant concentration for vitrification.The objective of our study is to report an additional method of fertility preservation that combines ovarian tissue cryobanking with retrieval of immature oocytes from excised ovarian tissue followed by in vitro maturation(IVM).It is a promising fertility preservation option for women who cannot undergo ovarian stimulation or cannot delay their gonadotoxic cancer treatment. Materials and methods:Forty patients who underwent operation for benign ovarian cysts were enrolled in this study.The excised ovarian tissue was suspended in MOPS medium and transferred to the laboratory.Before dissecting the ovarian cortical tissue for cryopreservation,we aspirated all visible follicles with an 22-gauge syringe needle that was attached to a 5-m L syringe.The aspirates were flushed in Oocyte Washing Medium.Following follicle aspiration, the ovarian cortical tissues were cut into 1mm× 1mm × 5mm pieces,and separated into 4 group according to the different concentration.The retrieved immature oocytes were incubated in four-well culture dishes at 37℃ in an atmosphere of 5% CO2 in air with high humidity.After maturation in culture for 24 hours, the cumulus cells surrounding the oocytes were removed.The mature, metaphase 2 stage(MII) oocytes were cryopreserved by vitrification.Ovarian pieces from control groups were fixed in 10% neutral buffered formalin.We used the Cell strainer as the carrier for three experiment groups the ovarian tissue was cryopreserved with three different cryoprotectant for 1 week.After warm the vitrified tissues we carried out a prospective parallel comparison of the degree of normal morphology,proliferation and apoptosis of human ovarian follicles after freezing and thawing.Oocytes retrieved from the excised ovarian tissue,oocyte maturation rate,and number of oocytes cryopreserved by vitrification.Results:1.Histologic evaluation and follicular population.The normal form of CPA1 group has no significance difference with control group(P>0.05).The proportions of normal follicles in the control and all of experimental group had no statistically significant differences.2.Cell proliferation.The health and proliferative status of stroma and granulosa cells were confirmed by Ki-67 immunostaining.Only the CPA1 group presented a similar proliferation index to fresh tissue3.Cell death.Primordial and primary follicles were TUNEL negative in all the experimental groups.TUNEL-positive cells were found mainly in the ovarian stoma and occasionally in vascular endothelial cell.4.Number of oocytes cryopreserved by vitrification.Immature oocyte retrieval from the excised ovarian tissue was performed in 12 patients.Following IVM the mean maturation rate was 82.7% and a total of 20 IVM mature oocytes were vitrified.Conclusions:1.It is better for saving ovarian tissue when we use low concentration cryoprotectant DMSO combined EG.It obtained significantly better results than the other experimental groups in terms of the proliferation index and the cell death index.2.Combining ovarian tissue cryobanking with retrieval of immature oocytes from excised ovarian tissue followed by IVM is a promising fertility preservation option for women who cannot undergo ovarian stimulation or cannot delay their gonadotoxic cancer treatment.3.Cell strainer as the new carrier for cryobanking human ovarian tissue promotes the development of vitrification cryopreservation of ovarian tissue.