Bulgarelli Daiane L, Vireque Alessandra A, Pitangui-Molina Caroline P, Silva-de-Sá Marcos F, de Sá Rosa-E-Silva Ana Carolina J
Department of Obstetrics and Gynecology,School of Medicine of Ribeirão Preto,University of São Paulo,Ribeirão Preto,SP,Brazil.
Zygote. 2017 Apr;25(2):222-230. doi: 10.1017/S0967199416000381. Epub 2017 Jan 10.
This study aimed to evaluate the embryo development competence, the nuclear maturation and the viability of germinal vesicle (GV) and metaphase II (MII) oocytes vitrified by the Cryotop method. Cumulus-oocyte complexes were derived from bovine ovaries and three experiments were conducted. In Experiment 1, GV oocytes were vitrified and underwent in vitro maturation (IVM) or not and their nuclear maturation was assessed by orcein staining. In Experiment 2, GV oocytes and MII oocytes were vitrified or not and the viability was assessed by calcein/ethidium homodimer-1 staining. In Experiment 3, MII oocytes matured before or after vitrification were submitted to in vitro fertilization (IVF) and parthenogenetic activation (PA) in order to evaluate embryo development. No difference was found for the nuclear maturation rate in the GV group (50%) and the GV control group (67%; P = 0.23) and for viability rate (56%; 77%; P = 0.055, respectively). However, in the MII group (27%) viability was significantly lower than that of the MII control group (84%; P < 0.0001). The cleavage rate by IVF and PA was similar in the GV group and the MII group. In contrast, vitrified MII oocytes showed no capacity for blastocyst development after IVF or PA and vitrified GV oocytes were able to develop to blastocysts only after PA, but not after IVF. In conclusion, oocyte vitrification by the Cryotop method reduced the capacity for embryo development. Vitrification of GV oocytes, however, did not influence the capacity of meiotic nuclear maturation and they exhibited higher viability following vitrification at the MII stage.
本研究旨在评估采用Cryotop法玻璃化冷冻的生发泡（GV）期和减数分裂中期II（MII）期卵母细胞的胚胎发育能力、核成熟情况及活力。卵丘-卵母细胞复合体取自牛卵巢，并进行了三个实验。在实验1中，GV期卵母细胞经玻璃化冷冻后进行或不进行体外成熟（IVM），并通过地衣红染色评估其核成熟情况。在实验2中，GV期卵母细胞和MII期卵母细胞经或未经玻璃化冷冻，通过钙黄绿素/乙锭同二聚体-1染色评估其活力。在实验3中，对玻璃化冷冻前或后的MII期卵母细胞进行体外受精（IVF）和孤雌激活（PA），以评估胚胎发育情况。GV组（50%）和GV对照组（67%；P = 0.23）的核成熟率以及活力率（分别为56%；77%；P = 0.055）均未发现差异。然而，MII组（27%）的活力显著低于MII对照组（84%；P < 0.0001）。GV组和MII组通过IVF和PA的卵裂率相似。相比之下，玻璃化冷冻的MII期卵母细胞在IVF或PA后均无囊胚发育能力，而玻璃化冷冻的GV期卵母细胞仅在PA后能发育至囊胚，IVF后则不能。总之，采用Cryotop法进行卵母细胞玻璃化冷冻会降低胚胎发育能力。然而，GV期卵母细胞的玻璃化冷冻并不影响减数分裂核成熟能力，且它们在MII期玻璃化冷冻后表现出更高的活力。
