To overcome the risk of the ovarian hyperstimulation syndrome (OHSS) in patients have polycystic ovarian syndrome (PCOS) and to prepare emergency fertility preservation in patients undergoing anticancer treatment, several researchers have reported IVM of oocytes retrieved from ovaries exposed by only hCG priming. However, the maturation rate and the developmental potential of embryos from IVM oocytes are significantly lower than those of oocytes matured in vivo. Here, we investigated the optimal time point for immature oocyte collection at post hCG only injection for in vitro maturation, in vitro fertilization and blastocyst formation. Immature GV oocytes were collected from 25 days old B6D2F1 female mouse at 12 hr, 14 hr, 16 hr or 24 hr post hCG injection. Oocytes were collected from antral or late secondary follicle by puncturing with 26 G needle. Collected oocytes were cultured in G2 medium with 10% FBS, FSH, estradiol, and hCG for 16 hr in vitro and subjected in vitro fertilization and further embryonic development. To examine follicular maturation, we estimated the numbers of primordial, primary, secondary follicle and antral follicle on ovaries of each time point post hCG. To confirm the optimal time point post hCG injection for collecting immature oocytes, we recovered the oocytes from each time point. There is no difference in the number of oocytes per mice. Oocytes collected at 14 hr post hCG injection were shown higher maturation rate to MII stage and blastocyst formation compare to other three groups (p<0.01). However, there is no difference in the maturation rate on the other three groups. Also, apoptotic signal with TUNEL assay or anti-PARP staining was not change in ovaries from all experimental groups. Granulosa cell proliferation test with anti Ki-67 or anti AMH was not show any difference. According to these results, there are no significant differences in four different time points at 12 hr, 14 hr, 16 hr or 24 hr of collection of immature oocytes in hCG primed mouse. However, oocytes from 14 hr post hCG injection showed higher percentages of maturation rate, in vitro fertilization rate, blastocyst formation.

Since the first case of pregnancy by in vitro matured oocyte was reported (Cha et al., 1991), in vitro maturation (IVM) could be used as an alternative choice for the treatment of infertile women with polycystic ovary syndrome (PCOS) and poor responders to ovarian stimulation and as one of the strategies for fertility preservation (Chian, 2004). Immature oocyte retrieval followed by IVM is a promising potential treatment option, especially for women who are infertile through PCOS. Although the pregnancy and implantation rates of IVM treatment are not as high as conventional IVF treatment, IVM treatment has many advantages for infertile women with PCOS, because this group of patients is extremely sensitive to stimulation with exogenous gonadotropins and is at increased risk of developing ovarian hyperstimulation syndrome (OHSS). Different protocols have been used before immature oocyte retrieval, indicating that there are beneficial effects with FSH or LH priming on oocyte maturation. To date, the clinical pregnancy and implantation rates obtained from IVM treatment in infertile women with PCOS are approximately 30-35% and 10-15% respectively (Chian, 2004). The clinical outcome has substantially improved in recent years with pregnancy rates between 20 and 54% and the postnatal follow-up studies of the children have been reassuring (Suikkari, 2008). Currently, more than 400 healthy infants have been reported with IVM method (Jurema and Nogueira, 2006; Suikkari, 2008). Although good results have been reported by some clinics, IVM has not yet become a mainstream fertility treatment. The most important reason for this is the lower chance of a live birth per treatment compared with conventional IVF. Despite its clinical success, there has been little information about the suitable conditions for human IVM. Therefore, improving developmental competency of immature oocytes continues to be an important concern of most IVM centers. Among many factors which affect efficacy of IVM, culture conditions are believed to be the most important factor, because different culture medium with changes of constituents can affect the oocyte maturation potential and subsequent embryonic development (Trounson et al., 2001). Currently, many different types of commercially available maturation media have been used in clinical IVM. They are commonly supplemented with hormone (recombinant FSH, hCG) and protein sources. Protein component may serve as a nitrogen source and act as a chelator of toxic metal ions and an antioxidant within culture media. In this respect, a development of well defined maturation medium supplemented with an efficient and safe protein source would improve IVM results. We previously reported that developmental competency of immature oocytes (either GV or MI) obtained from stimulated IVF cycles was comparable when matured in vitro with commercial G2 media supplemented by either human follicular fluids (hFF) or human serum albumin (HSA) (Jee et al., 2008). Our results suggest that hFF as a protein supplement for human in vitro maturation can be replaced by highly defined HSA. A development of well defined maturation medium should be continued in the effort to improve IVM results. More research is also needed to determine the roles of specific components and optimal culture conditions required in maturing oocytes. IVM of human oocytes retrieved from antral ovarian follicles is an emerging procedure quickly being incorporated into the realm of assisted reproductive technologies (ART). This new technology has several potential advantages over traditional controlled ovarian hyperstimulation for IVF, such as reduction of costs by minimizing gonadotropin and GnRH analogue use, elimination of OHSS, and simplicity of protocol. IVM of oocytes for ART in human beings still is undergoing refinement but currently is providing efficacy and safety outcome comparable to that of traditional IVF in recent selected studies. Implementing IVM into an established IVF practice is feasible and requires only a few simple adjustments. Crucial to the advancement and optimization of the technology is a better understanding of how to maximize immature oocyte developmental competence and endometrial receptivity.

미성숙의 Germinal Vesicle(GV 단계에서 성숙한 Metaphase II(MII) 단계가 되는 난자성숙 과정은 핵과 세포질의 성숙을 통해 이루어지며, 이를 통해 수정과 배 발달을 할 수 있는 능력을 갖게 된다. GV 난자는 prophase I 단계에 arrest 되어 있다가 meiosis 과정을 거쳐 성숙한 MII로 되는데 이를 조절하는 기작에 대해서는 거의 알려져 있지 않다. 따라서 본 연구는 미성숙 난자와 성숙 난자간의 유전자 발현의 차이

This study was conducted to investigate the effect of vitrification solution(VS) on in vitro developmental competence of immature porcine oocytes. The immature porcine oocytes were exposed to the following vitrification solution, at RT. 1) EFS sol. : 20% ethylene glycol (EG) 3 min, 40% EG + 18%(w/v) Ficoll(MV70, 000) + 0.3 M sucrose 30 sec, 2) GE sol. : 10% glycerol 5 min, 10% G + 20% EG 5 min, 25% G +25% EG 30 sec, 3) EG sol : 1.5M EG 2.5 min, 5.5 M EG + 1.0 M sucrose 30 sec. Oocytes were immediately transferred into 1.0 M, 0.5 M, 0.25 M, 0125 M, 0 M sucrose solution for 2.5 min each at RT. After removal of VS, immature oocytes were matured in vitro and subsequently all oocytes were subjected to IVF followed in vitro culture for 7 days. Maturation rates of oocytes were 38.8%, 44.5%, 22.4% and 57.6%, in EFS, EG, GE and Control, respectively, maturation rates of oocytes in EG and Control was significantly higher than EFS and GE(P<0.01). Fertilization rates of oocytes in Control was significantly higher than other treated groups(P<0.05), but no difference were observed among treated groups. Polyspermic rates were no significant difference among four groups. Cleavage rates of oocytes were 21.9%, 47.1%, 19.0% and 65.9%, in EFS, EG, GE and Control, respectively, cleavage rates of oocytes in EG and Control was significantly higher than EFS and GE(P<0.05), but blastocyst formation rates were no significant difference among four groups. These results suggested that the use of EG solution could be a great challenge for reaching a successful vitrification of immature porcine oocytes.