Department for Reproductive Medicine, Ghent University Hospital,De Pintelaan 185, 9000 Ghent, Belgium.
Hum Reprod. 2014 Jan;29(1):29-40. doi: 10.1093/humrep/det404. Epub 2013 Nov 11.
What are the precise patterns of calcium oscillations during the fertilization of human oocytes matured either in vivo or in vitro or aged in vitro and what is the effect of cryopreservation?
Human oocytes matured in vivo exhibit a specific pattern of calcium oscillations, which is affected by in vitro maturation, in vitro ageing and cryopreservation.
Oscillations in cytoplasmic calcium concentration are crucial for oocyte activation and further embryonic development. While several studies have described in detail the calcium oscillation pattern during fertilization in animal models, studies with human oocytes are scarce.
STUDY DESIGN, SIZE, DURATION: This was a laboratory-based study using human MII oocytes matured in vivo or in vitro either fresh or after cryopreservation with slow freezing or vitrification. Altogether, 205 human oocytes were included in the analysis.
PARTICIPANTS/MATERIALS, SETTING, METHODS: In vivo and in vitro matured human oocytes were used for this research either fresh or following vitrification/warming (V/W) and slow freezing/thawing (F/T). Human oocytes were obtained following written informed consent from patients undergoing ovarian hyperstimulation. For the calcium pattern analysis, oocytes were loaded with the ratiometric calcium indicator fluorescent dye Fura-2. Following ICSI using sperm from a single donor, intracellular calcium was measured for 16 h at 37°C under 6% CO(2). The calcium oscillation parameters were calculated for all intact oocytes that showed calcium oscillations and were analyzed using the Mann-Whitney U-test.
Human in vivo MII oocytes display a specific pattern of calcium oscillations following ICSI. This pattern is significantly affected by in vitro ageing, with the calcium oscillations occurring over a longer period of time and with a lower frequency, shorter duration and higher amplitude (P < 0.05). In vitro matured oocytes from the GV and MI stage exhibit a different pattern of calcium oscillations with calcium transients being of lower frequency and shorter duration compared with in vivo matured MII. In MI oocytes that reached the MII stage within 3 h the calcium oscillations additionally appear over a longer period of time (P < 0.05). In vivo MII oocytes show a different calcium oscillation pattern following V/W with calcium oscillations occurring over a longer period of time, with a higher amplitude and a lower frequency (P < 0.05). In vitro matured oocytes, either from the GV or the MI stage, also display an altered pattern of calcium oscillations after V/W and the parameters that were similarly affected in all these oocyte groups are the frequency and the amplitude of the calcium transients. Slow freezing/thawing differentially affects the calcium oscillation pattern of in vitro matured and in vitro aged oocytes.
LIMITATIONS, REASONS FOR CAUTION: The relationship between a specific pattern of calcium oscillations and subsequent human embryonic development could not be evaluated since the calcium indicator used and the high-intensity excitation light impair development. Furthermore, all oocytes were derived from stimulated cycles and immature oocytes were denuded prior to in vitro maturation.
Our data show for the first time how calcium signalling during human fertilization is affected by oocyte in vitro maturation, in vitro ageing as well as V/W and slow freezing/thawing. The analysis of calcium oscillations could be used as an oocyte quality indicator to evaluate in vitro culture and cryopreservation techniques of human oocytes.
STUDY FUNDING/COMPETING INTEREST(S): This work was supported by a clinical research mandate from the Flemish Foundation of Scientific Research (FWO-Vlaanderen, FWO09/ASP/063) to F.V.M, a fundamental clinical research mandate from the FWO-Vlaanderen (FWO05/FKM/001) to P.D.S and a Ghent University grant (KAN-BOF E/01321/01) to B.H. The authors have no conflict of interest to declare.
体内成熟、体外成熟或体外老化以及冷冻保存的人类卵母细胞受精时钙振荡的确切模式是什么？
体内成熟的人类卵母细胞表现出特定的钙振荡模式，这种模式受体外成熟、体外老化和冷冻保存的影响。
细胞质钙离子浓度的振荡对于卵母细胞的激活和进一步的胚胎发育至关重要。虽然有几项研究详细描述了动物模型中受精过程中的钙振荡模式，但关于人类卵母细胞的研究却很少。
研究设计、大小、持续时间：这是一项基于实验室的研究，使用体内或体外成熟的人类 MII 卵母细胞，无论是新鲜的还是经过慢速冷冻或玻璃化冷冻保存的。总共分析了 205 个人类卵母细胞。
参与者/材料、设置、方法：本研究使用体内和体外成熟的人类卵母细胞，无论是新鲜的还是经过玻璃化/变暖（V/W）和慢速冷冻/解冻（F/T）处理的。人类卵母细胞是从接受卵巢刺激的患者中获得的，并获得书面知情同意。为了进行钙模式分析，将卵母细胞用比率钙指示剂荧光染料 Fura-2 加载。在使用来自单个供体的精子进行 ICSI 后，在 37°C 下于 6% CO2 下测量 16 小时的细胞内钙。使用 Mann-Whitney U 检验计算所有显示钙振荡的完整卵母细胞的钙振荡参数，并进行分析。
人类体内 MII 卵母细胞在 ICSI 后显示出特定的钙振荡模式。这种模式受到体外老化的显著影响，钙振荡发生的时间更长，频率更低，持续时间更短，幅度更高（P <0.05）。来自 GV 和 MI 期的体外成熟卵母细胞表现出不同的钙振荡模式，钙瞬变的频率和持续时间较低与体内成熟的 MII 相比。在 3 小时内达到 MII 期的 MI 卵母细胞，钙振荡另外发生的时间更长（P <0.05）。体内成熟的 MII 卵母细胞在 V/W 后显示出不同的钙振荡模式，钙振荡发生的时间更长，幅度更高，频率更低（P <0.05）。来自 GV 或 MI 期的体外成熟卵母细胞也显示出 V/W 后钙振荡模式的改变，所有这些卵母细胞群体中受类似影响的参数是钙瞬变的频率和幅度。慢速冷冻/解冻对体外成熟和体外老化卵母细胞的钙振荡模式有不同的影响。
局限性、谨慎的原因：由于使用的钙指示剂和高强度激发光会损害发育，因此无法评估特定的钙振荡模式与随后的人类胚胎发育之间的关系。此外，所有卵母细胞均来自刺激周期，在体外成熟前去除了不成熟的卵母细胞。
我们的数据首次显示了体外成熟、体外老化以及 V/W 和慢速冷冻/解冻过程中人类受精期间钙信号传导是如何受到影响的。钙振荡的分析可作为卵母细胞质量指标，用于评估人类卵母细胞的体外培养和冷冻保存技术。
研究资金/利益冲突：这项工作得到了佛兰芒科学研究基金会（FWO-Vlaanderen，FWO09/ASP/063）的临床研究授权、佛兰芒科学研究基金会（FWO05/FKM/001）的基础临床研究授权以及根特大学的资助（KAN-BOF E/01321/01）到 B.H. 作者没有利益冲突需要声明。
