Traditional Chinese Medicine, the Zishen Yutai Pill, Ameliorates Precocious Endometrial Maturation Induced by Controlled Ovarian Hyperstimulation and Improves Uterine Receptivity via Upregulation of HOXA10

Qi Gao, Lu Han, Xiumei Li ,and Xia Cai,  2015

https://doi.org/10.1155/2015/317586

Abstract

Controlled ovarian hyperstimulation (COH) is widely used in assisted reproductive technology (ART), but it often leads to precocious maturation of the endometrium such that it impairs embryonic implantation and limits pregnancy rates. Previous studies have shown the traditional Chinese medicine, the Zishen Yutai pill (ZYP), to be effective in treatment of threatened as well as recurrent miscarriages, and it can improve embryonic implantation rates in patients undergoing IVF treatment. In the present study, the ZYP has been found to ameliorate precocious endometrial maturation in a mouse model of different COH. Molecular evaluations, real-time PCR, relative RT-PCR, Western blotting, and immunohistochemistry have indicated that the ZYP increased the expression of HOXA10, an important marker of uterine receptivity. Elevation of HOXA10 led to further upregulation of its target gene, integrin β3, and downregulation of EMX2, two additional markers of uterine receptivity. In this way, the ZYP may mitigate COH-induced precocious maturation of the endometrium and improve uterine receptivity by upregulating HOXA10.

Discussion

COH was developed to induce mature oocyte follicles, increasing the chances of pregnancy. It is the most commonly used protocol in ART [6]. Studies have shown that COH drugs stimulate production of large amounts of sex hormones and disrupt the hormones themselves. This leads to structural and functional changes in the endometrium ad uterine glands and development of the stroma of dyssynchrony. The endometrial implantation window and embryonic development are not synchronized, and this decreases endometrial receptivity and pregnancy rates [4, 5]. Here, 85% ovulation caused pinopode degradation 1-2 days in advance. Endometrial histological development occurs earlier than embryonic development [5]. van Vaerenbergh et al. compared endometrial estrogen and progesterone receptors in endometrial implantation window between natural luteal cycles and COH cycles. The index of COH cycles was found to decline, affecting the action of estrogen and progesterone on endometrial tissue development, synthesis, and secretion and the expression of genes related to endometrial receptivity related genes, all of which in turn affect embryo implantation [6]. Endometrial receptivity is essential to embryo implantation. A variety of cytokines, genes, and proteins bind to specific receptors on the endometrial cell surface. The endometrium can cause a series of changes in the positioning and implantation of the fertilized egg [3]. Changes in COH are closely associated with endometrial adhesion molecules, cytokines, timing of expression in the ultrastructure, interference of endometrial implantation window open, and blastocyst development of synchronization. In this way, it can affect endometrial receptivity [19].

Traditional Chinese medicine (TCM), considered gentle and safe, has long been used to treat infertility and miscarriages [20, 21]. It has also exhibited effectiveness in improving embryonic implantation rates in IVF [22]. In TCM theory, the kidney stores essence (or jing, which is the essence of qi) and dominates reproduction; so adequate kidney jing and strong kidney qi are vital to a successful pregnancy. The ZYP (“zishen” means “tonifying the kidney”; “yutai” means “nourishing fetus”) is a TCM formula that contains 15 herbs and other natural products developed by Professor Yuankai Luo to treat threatened or recurrent miscarriages by tonifying the kidney and increasing yuan qi [7]. Reproduction theory of TCM is used in ART. This work was performed to determine how to increase conception rates by tonifying the kidney and increasing yuan qi in molecular biology.

HOXA10 belongs to the Hox transcription factor family which functions as master regulators in directing embryonic development [22]. HOXA10 is involved in the embryogenesis of uterine epithelium, stroma, and muscle [23]. The expression of HOXA10 in the endometrium exhibits a cyclical pattern and is responsive for fluctuations in estrogen and progesterone levels, with maximal levels appearing during the window of implantation [14]. Targeted deletion of the Hoxa10 gene was found to lead to uterine factor infertility in mice, primarily due to failure of embryonic implantation and aberrant decidualization [24]. Collectively, these studies suggest an essential role for HOXA10 in endometrial differentiation and embryonic implantation. HOXA10 is considered one of the most notable biochemical markers for endometrial receptivity [18]. The final cellular differentiation of the endometrium during the menstrual cycle resembles that observed during embryogenesis to a great extent, and Hox genes play central roles in determining cell fate during these processes. The downregulation of HOXA10 by COH was found to compromise cellular differentiation, causing precocious endometrial maturation.

HOXA10, a DNA-binding transcription factor, regulates the expression of many genes. Among these, integrin 3 and EXM2 also serve as important markers of endometrial receptivity [18]. Integrin 3 is an adhesion molecule involved in early interactions between the embryo and endometrium, and it plays an important role in embryonic implantation [25]. Endometrial expression of integrin 3 displays a cyclical pattern and appears abruptly on cycle day 20 on luminal and glandular epithelial cells, concomitant with the start of the implantation window [26]. HOXA10 directly controls the expression of the integrin 3 gene by binding to a 41 bp 5′-regulatory element [15], and the sex hormones also regulate the expression of endometrial integrin 3 through the HOXA10 pathway [26]. EMX2 is the human homolog of Drosophila EMS (empty spiracles) that contains a DNA-binding homeodomain involved in transcriptional regulation of animal development [27]. Emx2 has also been shown to regulate mammalian reproduction by limiting endometrial cell proliferation without affecting differentiation [28]. HOXA10 represses EMX2 gene expression via a direct interaction with a 150 bp regulatory region [16].

Because advanced endometrial maturation caused by COH contributes in large part to compromised endometrial receptivity and suboptimal pregnancy rates after IVF [3–5], one mechanism by which the ZYP improves endometrial receptivity is alleviation of COH-induced endometrial developmental defects. In support of this hypothesis, data collected using mouse models showed that ZYP in conjunction with GnRHa were able to ameliorate the precocious endometrial maturation and the resulting glandular and stromal dyssynchrony induced by COS to large extent. Treatment with ZYP alone was found to be less effective than ZYP/GnRHa cotreatment, most likely due to the ability of GnRHa to suppress premature surges in LH [6]. In this way, ameliorating the precocious maturation of the endometrium may represent the major mechanism whereby the ZYP improves uterine receptivity and pregnancy rates after IVF, and combined ZYP and GnRHa treatment may be used in ART to improve pregnancy outcome.

COH led to downregulation of HOXA10 expression and this downregulation was alleviated by the ZYP. It is therefore conceivable that the ZYP could act through the promotion of HOXA10 expression in order to maintain proper endometrial differentiation and ameliorate the aforementioned precocious maturation, thereby improving uterine receptivity and implantation rates. These results showed that COH induced downregulation of integrin 3 expression and upregulation of EMX2 expression and that the changes observed for both integrin 3 and EMX2 expression were alleviated by ZYP treatment. This was consistent with the HOXA10 results. Collectively, these results support a model wherein ZYP treatment upregulates the expression of HOXA10. Increased HOXA10 then drives the expression of many critical genes controlling endometrial receptivity, including integrin 3 and EMX2, which improves endometrial development and ameliorates the precocious maturation observed in COH mouse models.

In TCM, the kidney dominates reproduction, so pregnancy rates may be improved by treatments that enhance kidney qi. Many studies have confirmed the effectiveness of kidney-tonifying medicine in improving reproductive function. Li et al. reported in a mouse model that the kidney-tonifying medicine Bushen Zhuyu Decoction was able to increase numbers of ovarian follicles, endometrial glands, and stromal cells and promote endometrial hyperplasia (with the cells arranged in stratiform fashion) and elevate circulating estradiol and progesterone concentrations [29]. Two studies have shown that the use of kidney-tonifying TCM can reverse endometrial loss of leukemia inhibitory factor (LIF) and integrin 3 expression and improve uterine receptivity and pregnancy rates in mouse models of COH [30, 31]. A randomized controlled clinical study revealed that the use of kidney-tonifying medicine during COH cycles improved embryo quality and endometrial receptivity [32]. Hu et al. showed that kidney-tonifying medicine restored the expression of estrogen receptors in vaginal menopause and that it thus may serve as an alternative for hormonal therapy [33]. Kidney-tonifying medicine has also been found to reduce matrix metalloproteinase-2 (MMP-2) expression in endometriosis (presumably by improving the local endocrine environment), so as to reduce the invasion of ectopic endometrium [34]. The current results support the conclusion that kidney-tonifying medicines mitigate the precocious maturation of endometrium induced by COH, most likely via upregulation of the HOXA10 transcription factor. Collectively, multiple lines of evidence strongly suggest that TCM acts through multiple mechanisms to improve reproduction.

In conclusion, the present study showed that the ZYP was able to ameliorate advanced endometrial maturation via upregulation of HOXA10 in a mouse model of COH, thus providing initial molecular understanding of its underlying mechanism. These results also suggested that ZYP treatment could be a reliable approach to improving IVF outcomes.