Growth differentiation factor 9 and cumulus cell supplementation in in vitro maturation culture media enhances the viability of human blastocysts / 대한생식의학회지

OBJECTIVE: In vitro maturation (IVM) of immature oocytes can be useful for some infertile patients. In IVM programs, the rates of embryo formation and pregnancy are low. Therefore, it is essential to recognize the main factors involved in regulating oocyte maturation in vitro. The purpose of this study was to investigate the effects of growth differentiation factor 9 (GDF9) and cumulus cell (CC) supplementation in IVM medium on the rates of embryo formation and viability of human blastocysts.METHODS: A total of 80 germinal vesicle oocytes from stimulated cycles underwent an IVM program. The oocytes were divided into four groups, where group I consisted of IVM media only and served as the control, group II consisted of IVM+CCs, group III consisted of IVM+GDF9 (200 ng/mL), and group IV consisted of IVM+CCs+GDF9 (200 ng/mL). Intracytoplasmic sperm injection was performed on the IVM oocytes, and the cleavage embryos that were generated were vitrified. Following thawing, the embryos were cultured for 3 additional days, and the viability rates of the developed blastocysts were determined.RESULTS: The maturation rate of the oocytes did not differ significantly across the four groups. The fertilization rate in group II was significantly higher than that in the control group (76.5% vs. 46.2%). Embryo formation was significantly more frequent in all experimental groups than in the control group, while blastocyst formation did not show significant differences in the three experimental groups compared to the control. The mean viability rates in groups II, III, and IV were 58.16%, 55.91%, and 55.95%, respectively, versus 37.78% in the control group (p<0.05).CONCLUSION: Supplementation of IVM culture media with GDF9 and CCs enhanced the fertilization, embryo formation, and viability rates of blastocysts generated from vitrified cleavage embryos.

Sperm parameters, protamine deficiency, and apoptosis in total globozoospermia

Globozoospermia is a severe form of teratozoospermia [incidence < 0.1%] in infertile men that is characterized by round headed sperm and acrosomeless in semen. To compare the semen parameters, protamine deficiency, and apoptosis in ejaculated spermatozoa between globozoospermic and normozoospermic men. Thirty six semen samples were divided into two groups including 15 infertile men with total globozoospermic [> 90% round-headed sperm] and 21 healthy donors with normal spermograms as controls. Semen analysis was performed according to World Health Organization criteria [2010]. Sperm protamine deficiency was assessed using Chromomycin A3 [CMA3] staining and the rate of apoptotic spermatozoa was evaluated with TUNEL assay. Sperm concentration, motility, and normal morphology in globozoospermic men were significantly decreased compared with controls [p<0.05]. The rate of CMA3-reacted spermatozoa [CMA3+] in globozoospermic men was higher than controls [65.93 +/- 11.77 vs. 21.24 +/- 7.37, respectively, p<0.0001]. The rate of apoptotic spermatozoa [TUNEL positive] were significantly increased in globozoospermic cases with respect to the controls [17.60 +/- 10.72 and 5.95 +/- 3.02, respectively, p<0.0001]. There was no significant correlation between sperm protamine deficiency and apoptosis in globozoospermic men. Globozoospermic samples contain a higher proportion of spermatozoa with abnormal chromatin packaging and DNA fragmentation than normozoospermic samples. Therefore, in addition to absence of acrosome in the spermatozoa of globozoospermic patients, the high percentage of spermatozoa with immature chromatin and apoptotic marker may be considered as the other etiologies of infertility in these patients

Developmental competence of immature oocytes aspirated from antral follicles in patients with gynecological diseases

In vitro maturation [IVM] of immature oocytes collected from ovary has been proposed for fertility preservation. In addition, quality of oocytes post IVM is one of the factors determining its developmental competence. By using the non-invasive Polscope system, both meiotic spindle [MS] and zona pellucida [ZP] can be assessed in living oocytes. The aim was to investigate the developmental potential of immature oocytes retrieved from ovarian tissue after IVM, as a method for fertility preservation, in patients with gynecological diseases. The ovarian cortex from 26 patients with malignant and benign diseases [21-45 years old], were obtained directly from collaborating hospitals, and transported to the IVF center on ice. In total 61 immature oocytes were aspirated, of which 18 [29.5%] were degenerated and discarded. The remaining 43 [70.5%] healthy oocytes were cultured in IVM culture media for 48 hr. The rate of maturity was assessed, and the ZP birefringence and MS were imaged with Polscope technology. Overall 43 immature oocytes underwent IVM technology, of which 30.2% reached viable metaphase II [MII] oocytes. The ovarian tissues of 9 [34.6%] women were lacking oocytes at any stage. During polarized light microscopy examination, MS could be visualized only in one of the MII oocytes, but high ZP birefringence's were observed in the majority of the oocytes post IVM [61.5%]. Oocytes maturation post IVM from unstimulated ovaries showed a good developmental competence in gynecologic patients. Further studies should be performed to advance the oocyte maturation program, such as co-culture system, for fertility preservation

Does women's age influence zona pellucida birefringence of metaphase II oocytes in in-vitro maturation program?

In vitro maturation [IVM] is a promising treatment option for certain infertile women. Nowadays, with the aid of PolScope, it has become possible to evaluate zona pellucida [ZP] characteristics as a parameter of oocyte quality. Moreover, quality of oocytes can be influenced by many factors, such as patient's age. The PolScope system is a non-invasive technique to assess birefringent structures such as the meiotic spindle and ZP in living oocytes. The aim was to determine the influence of the woman's age on ZP birefringence, a sign of oocyte quality, and morphology of in-vitro matured human oocytes using non-invasive polarized light [PolScope] microscopy. ZP birefringence and morphology were determined in 105 retrieved oocytes from 58 women undergoing ICSI in two age groups [>/= 30 years and <30 years]. The immature oocytes were selected and after IVM, the quality of metaphase II [MII] oocytes was assessed. The oocytes abnormalities were classified as intracytoplasmic and extracytoplasmic abnormalities. Oocyte maturation rates were significantly reduced in >/= 30 year's women [56%] in comparison with other age group [80.7%]. In addition, the ZP birefringence was significantly higher in MII oocytes in the younger group compared with the older group [76.2% vs.38.1%; p=0.00]. Following morphologic assessment, the rates of oocytes with extracytoplasmic [p=0.02] and both abnormalities [extra- and intracytoplasmic] [p=0.01] were higher in aged versus the younger women. There was a positive relationship between advanced maternal age with decreased ZP birefringence and oocyte morphological quality in in-vitro matured human oocytes