Department of Obstetrics and Gynecology, Seoul National University College of Medicine, Seoul 03080, Korea.
Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University College of Medicine, Seoul 03080, Korea.
Int J Mol Sci. 2020 Jan 24;21(3):769. doi: 10.3390/ijms21030769.
Natural progesterone and synthetic progestin are widely used for the treatment of threatened abortion or in in vitro fertilization (IVF) cycles. This in vitro study aimed to assess whether the treatment with natural progesterone or synthetic progestin influences the germ layer gene expression on the early human embryonic development using human embryonic stem cells (hESCs)-derived embryoid bodies (hEBs) as a surrogate of early stage human embryonic development. Human EBs derived from hESCs were cultured for nine days, and were treated with natural progesterone (P4) or synthetic progestin, medroxyprogesterone acetate (MPA) at 10-7 M for five days. To reverse the effects of treatment, mifepristone (RU486) as progesterone antagonist was added to the hEBs for four days starting one day after the initiation of treatment. Mouse blastocysts (mBLs) were cultured in vitro for 24 h, and P4 or MPA at 10 M was treated for an additional 24 h. The treated embryos were further transferred onto in vitro cultured endometrial cells to evaluate chorionic gonadotropin (CG) expression. To analyze the effects of P4 or MPA, the expression of differentiation genes representing the three germ layers was investigated, GATA-binding factor 4 (GATA4), α-fetoprotein (AFP), hepatocyte nuclear factor (HNF)-3β, hepatocyte nuclear factor (HNF)-4α (endoderm), Brachyury, cardiac actin (cACT) (mesoderm), and Nestin (ectoderm), using quantitative reverse transcription PCR (qRT-PCR) and immunostaining. Significantly lower expressions of HNF-3β, HNF-4α, Brachyury, and Nestin were observed in MPA-treated hEBs (all < 0.05), which was negated by RU486 treatment. This inhibitory effect of MPA was also observed in mouse embryos. Conclusively, the effects of natural progesterone and synthetic progestin may differ in the germ layer gene expression in the hEB model, which suggests that caution is necessary in the use of progestogen.
天然孕酮和合成孕激素广泛用于治疗先兆流产或体外受精（IVF）周期。本体外研究旨在评估使用天然孕酮或合成孕激素是否会影响人胚胎干细胞（hESC）衍生的类胚体（hEB）的早期人类胚胎发育的胚层基因表达，hEB 作为早期人类胚胎发育的替代物。用人胚胎干细胞衍生的类胚体培养 9 天，用 10-7 M 的天然孕酮（P4）或合成孕激素醋酸甲羟孕酮（MPA）处理 5 天。为了逆转治疗效果，在开始治疗后一天，向 hEB 中添加米非司酮（RU486）作为孕酮拮抗剂，持续 4 天。将小鼠囊胚（mBL）在体外培养 24 小时，用 10 M 的 P4 或 MPA 处理 24 小时。用处理后的胚胎进一步转移到体外培养的子宫内膜细胞上，以评估绒毛膜促性腺激素（CG）的表达。为了分析 P4 或 MPA 的作用，使用定量逆转录 PCR（qRT-PCR）和免疫染色法研究了代表三个胚层的分化基因的表达，GATA 结合因子 4（GATA4）、α-胎蛋白（AFP）、肝细胞核因子（HNF）-3β、肝细胞核因子（HNF）-4α（内胚层）、Brachyury、心脏肌动蛋白（cACT）（中胚层）和巢蛋白（Nestin）（外胚层）。在 MPA 处理的 hEB 中，观察到 HNF-3β、HNF-4α、Brachyury 和 Nestin 的表达明显降低（均 < 0.05），RU486 处理可消除这种抑制作用。在小鼠胚胎中也观察到 MPA 的这种抑制作用。总之，天然孕酮和合成孕激素对 hEB 模型中的胚层基因表达的影响可能不同，这表明在使用孕激素时需要谨慎。
