Background Condition mediums have a potential role in oocyte development. In this study, we evaluated the effects of different mediums on the developmental potential of vitrified immature human oocyte after IVM and parthenogenesis by ionomycin.
Methods Immature oocytes were collected from 184 women after vitrification/thawing and maturation, in three types of IVM mediums separately. Finally, 151 IVM MΙΙ oocytes were obtained and randomly divided into six groups and underwent the following intervention. Fresh and vitrified-thawing MΙΙ oocytes were activated after IVM in three conditioned mediums by ionomycin. Mediums included 1) Minimum Essential Medium Alpha (α-MEM) (as control medium), 2) α-MEM supplemented with supernatants of Mesenchyme bone marrow (B.M), 3) α-MEM with ovarian growth factors (O.F). Then, scoring of parthenote embryos was undertaken in accordance with pertinent morphological properties. Moreover, the expression of Bax and Bcl2 were determined in the parthenote embryos.
Result Percentage of the degenerated oocyte, 2–4 cells, 4–8 cells, and 16 cells, was different in the experimental groups. Also, cytoplasmic maturation and blastocyst formation rates were significantly different (p < 0.05) between the control and the other mediums. The highest mRNA expression levels of Bcl2 and Bax genes in parthenotes were observed in the fIVM O.F and vIVM α-MEM mediums, respectively. vIVM, α-MEM and fIVM O.F showed the lowest expression of Bcl2 and Bax genes, respectively.
Conclusion Our findings indicate that the O.F. medium had more potent effects on oocyte growth and cytoplasmic maturation up to the blastocyst stage with the highest expression level of the BCL2 gene and the lowest relative amount of the BAX gene in this medium. The results of the present study have been verified only for parthenogenetically activated embryos, and any positive effect of the environment on the egg/embryo fertilized with sperm requires more extensive studies.
Keywords : IVM · Conditioned medium · Parthenogenesis · Developmental potential · Oocyte activation
In this study, we used 266 human oocytes in the MII phase matured by IVM protocols in three mediums from patients undergoing infertility treatment in the Afzalipour Infertility Center Kerman, Iran. The vitrified immature human oocytes were thawed and randomly cultured in these conditioned mediums for IVM. Afterward, nuclear oocytes maturation was monitored by detailed microscopic valuation for its morphology and was then classified into the oocyte stages of GV, MI, and MII, followed by the assessment of the developmental competence of MII oocytes (matured in conditioned IVM mediums) in these different mediums. The oocyte cytoplasmic maturation was examined by the developmental competence of in vitro matured oocytes through chemical activation and following culture to the blastocyst stage.
Embryo formation of activated oocytes was evaluated 2–7 days after parthenogenesis using ionomycin. In the conditioned medium group, the percentage of degenerated oocytes was 2–4 cells, 4–8 cells and 16 cells. Blastocyst development was evaluated and compared in these groups. Subsequently 151 IVM matured human oocytes (MII) were divided into one of three mediums. They were then parthenogenetically stimulated using ionomycin and 6 (DMAP) in the culture medium and the oocyte development was monitored every day: (1) 50 oocytes in Minimum Essential Medium Alpha (α-MEM) (as control medium); (2) 51 oocytes in α-MEM supplemented with supernatants of mesenchymal stem cells (MSCs) from bone marrow; and (3) 50 oocytes in α-MEM supplemented with ovarian growth factors (Table 1).
Blastocyst formation rates were significantly different (p < 0.001) between control and the other mediums shown in Table 1.
The embryo was categorized by A, B, and C score according to intensity of fragmentation and condition of the blastomeres.
Score A: equal symmetrical blastomeres with minor fragmentation.
Score B: equal blastomeres with moderate fragmentation
Score C: unequal blastomeres with severe fragmentation.
The differences between the number of arrests in 4–8 cells (p = 0/041), score A (4–8) (p = 0/045) were statistically significant (Table2). Primer sequences used for real-time PCR (qPCR) in embryos are listed in Table 3.
In our study, the expression mRNA levels of BAX and BCL2 (which acts as an apoptotic gene in embryo-derived freshand vitrified IVM human oocyte) was significantly different(p ˂ 0.05) in the three-maturation media. The highest expression of BAX gene and the lowest expression levels of BCL2 were observed in vIVM (in α-MEM), while the highest expression levels of BCL2 gene and the lowest relative amounts of BAX were observed in fIVM in O.F groups (Table 3) (Figs. 1, 2).
Oocyte cryopreservation and in vitro maturation of immature oocytes is an important technique and promising method pointed to preserve female fertility in ART [23]. Supplementation or additions to the typical oocyte culture medium seems to be essential for improving the maturation of immature oocytes. In order to attain better results, different variations like culture conditions mediums, hormonal treatment, and monitoring must be checked. Several studies demonstrate that growth factors, cytokines, amino acids, FSH, LH, PMSG, HMG, transferrin, insulin, and estradiol
