Effects of Natural Progesterone and Synthetic Progestin on Germ Layer Gene Expression in a Human Embryoid Body Model.

Yoon Young Kim,Chang Suk Suh,Seung-Yup Ku,Hung-Ching Liu,Zev Rosenwaks,Hoon Kim

doi:10.3390/ijms21030769

Abstract

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 10−7 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 p < 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.

Highlights

Natural progesterone (P4), produced from granulosa cells during the luteal phase following ovulation, is an essential hormone for embryo implantation and maintenance of early pregnancy [1]
The Human embryoid bodies (hEBs) derived from human embryonic stem cells (hESCs) were cultured for nine days in suspension and treated with P4 or medroxyprogesterone acetate (MPA) at 10−8 M, 10−7 M, 10−6 M, and 10−5 M for five days
The down-regulated expression of tested genes was not observed when RU486 was added to MPA treatment

Summary

Introduction

Natural progesterone (P4), produced from granulosa cells during the luteal phase following ovulation, is an essential hormone for embryo implantation and maintenance of early pregnancy [1]. During the IVF cycles, gonadotropin-releasing hormone (GnRH) agonists or antagonists are used for the prevention of premature ovulation, and many granulosa cells are aspirated during the oocyte pick-up procedures. For these reasons, progesterone production may be insufficient and P4 supplementation is common practice in IVF cycles [5]. In IVF cycles, an early rise of serum P4 levels on the day of human chorionic gonadotropin (hCG) administration has been reported to be associated with impaired embryo implantation and reduced live birth rate [6]. There are no controlled data in human pregnancy due to ethical limitations of clinical investigations and the absence of a model mimicking human embryonic development [10]

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Methods

Conclusion