Homeobox genes for embryo implantation: From mouse to human.

Blastocyst attachment to the luminal epithelium is followed by the development of the post‐implantation uterus, in which stromal cells surrounding the implanting blastocyst undergo extensive proliferation and differentiation into morphologically and functionally distinct cells types; this process is also called decidualization. 1 As mentioned above, many homeobox genes, such as Hoxa10 , Hoxa11 , Msx1 , and so on, are dynamically expressed in the uterus during implantation. Some of these homeobox genes, especially Hoxa10 and Hoxa11 , are persistently expressed in the post‐implantation uterus, suggesting important roles in the development of the post‐implantation uterus. In pregnant mouse uterus, the expression of Hoxa10 is first detectable in the epithelial cells on Day 1.5. It shifts to the stroma underlying the epithelium on Day 4, increases in the stroma surrounding the embryo with the onset of the attachment reaction at midnight of Day 4, and is further enhanced on Day 5 and beyond. By Day 6, the Hoxa10 is strongly expression throughout the whole stroma. 41 , 42 This spatiotemporal expression of Hoxa10 implies an important role during decidualization, which is evidenced by decreased decidualization in response to artificial stimuli in the Hoxa10 −/− mice. 41 Furthermore, dysregulation of cyclin D3 and loss of region‐specific expression of CDK4 and CDK6 has been shown in the decidual bed of Hoxa10 −/− female mice, 70 , 71 , 72 and overexpression of cyclin D3 can improve decidualization defects in Hoxa10 −/− mice. 73 Beyond that, the cell cycle inhibitors p15 and the negative cell cycle regulators cyclins G1 and G2 are all abnormally induced in Hoxa10 −/− mice. 74 , 75 More recently, Gao et al 76 suggest that FoxM1 and cyclin D3, as the downstream targets of Hoxa10 , play crucial roles in normal regional decidualization. All these results suggest that Hoxa10 may be at the control point of cell cycle progression and cellular differentiation during decidualization. Furthermore, Hoxa10 deficiency compromises natural killer cell differentiation and alters expression of region‐specific genes such as Gdf10 , Snail2 , Hgf and others, during decidualization. 77 Collectively, Hoxa10 influences a host of genes necessary for normal decidual development. In humans, HOXA10 is highly expressed in the endometrium cell during the mid‐secretory phase of the menstrual cycle, in which the stroma initiates decidual differentiation, suggesting an essential role of HOXA10 during decidualization. In fact, HOXA10 gene are reported to regulate the expression of the decidualization marker IGFBP‐1. 78 In addition, there is also evidence that HOXA10 plays an essential role in decidualization in humans through regulating the expression of the cell cycle inhibitor P57, and interleukins IL‐11 and IL‐15 during steroid hormone‐mediated decidualization of human endometrial stromal cells in vitro . 79 , 80 Although the more severe phenotype in Hoxa11 −/− mice prevents us from examining the function of Hoxa11 during decidualization, overlapping expression patterns of Hoxa10 and Hoxa11 were also observed in mouse decidua, suggesting a similar role in the process of decidualization. 1 , 6 , 14 All these results suggest that Hoxa10 and Hoxa11 play crucial roles in the decidualization in both mice and humans. In contrast to Hoxa10 / Hoxa11 , the Msx genes have been shown to be strictly silenced during decidualization, suggesting that Msx genes may be dispensable during development of the post‐implantation uterus. 3 , 64 Beyond that, there are no reports showing that the other homeobox genes are critical for decidualization. Nevertheless, it is clear that precisely regulated homeobox genes are essential for normal uterine decidualization.

HBW designed this review and agreed the analysis plan. BH and ZLN collected literature and sorted out the information. SBK and JHL wrote the original draft of this review, with all other authors provideding comments. HBW acts as guarantor. All authors read and approved the final manuscript.

As described above, a well‐orchestrated temporal and spatial expression pattern of homeobox genes is essential for implantation in both mice and humans. Any alterations in the regulation of homeobox gene expression in the developing female reproductive tracts or the adult uterus may lead to disorders of reproductive function. 14 , 85 , 86 Therefore, studies on the roles of the homeobox genes during implantation will provide information to help us prevent and treat infertility. The best‐known example is that humans are easily exposed to a wide variety of chemicals that have profound and lasting effects on development of female reproductive tracts. These chemicals influence reproductive competence by altering the expression of the homeobox genes necessary for development of female reproductive tracts, such as the HOX genes. 87 , 88 , 89 For example, perinatal exposure of humans to DES produces uterine, cervical, and oviductal malformations by altering the expression of HOXA9‐11 genes, and this exposure may lead to permanent alteration of gene expression in the adult. 39 , 88 A persistent abnormality of HOXA10 may be one of the main causes of infertility. These results suggest that keeping mothers and newborns away from exposure to such chemicals is one of the best ways to prevent infertility. Another example of the role of homeobox genes in infertility is endometriosis, which is considered to be a chronic, recurrent and progressive disease. 6 , 14 , 85 On one hand, infertile patients with endometriosis do not show a mid‐secretory rise in HOXA10 and HOXA11 expression, which normally occurs in each menstrual cycle. 49 , 90 This could explain why the endometrium of the endometriosis patient is less receptive to implantation. On the other hand, HOXA10 is reported to be expressed in human peritoneal, ovarian and lung endometriosis, as well as rectosigmoid endometriosis. 91 This ectopic expression of HOXA10 in endometriotic lesions outside the normal domain raises the supposition that HOXA10 might be necessary for “de novo” development of endometrial tissue, which normally occurs in the development of uterus during embryogenesis. 6 , 85 These results may provide new insight into the etiology of endometriosis, about which we know little, and could be helpful in the treatment of the pathology. In addition, there is also evidence that reduced expression of Msx1 in human endometrial tissue is linked to infertility. 67 However, whether other homeobox genes are involved in infertility and whether the abnormal expression of homeobox genes in infertile patients is a defect inherent in the endometrium or secondary to the endometriosis is still unknown, and needs to be explored in the future.

Implantation is the gateway to further embryonic development and is therefore considered to be the critical event during the pregnancy, involving the first physical and physiological interaction between the embryo and uterus. 1 , 14 Clinically, disrupted endometrial receptivity and blastocysts of poor quality also largely account for low pregnancy success rates in assisted reproductive technique programs. 2 , 4 , 5 Therefore, it becomes more and more important for us to understand the molecular mechanisms of implantation. Despite recent progress in elucidating the roles of Msx genes in uterus receptivity and blastocyst diapause, and previous studies on the Hox genes that have greatly increased our knowledge on implantation, the roles of the homeobox genes encoding homeobox transcription factors are far from clear. For example, regarding the molecular mechanisms underlying their functions, we still need to understand whether the other homeobox genes apart from Hox and Msx genes are involved and what roles they play during implantation. One cause of this lack of clarity is the fact that homeobox genes form a superfamily of regulatory genes, which can be divided into different families, each with many different clusters. It is difficult to confirm the function of each gene in the short term. Genome‐wide deletion of the homeobox genes results in embryonic lethality or developmental defects of female reproductive tracts, which limits further research on their roles in implantation. Fortunately, the widely used Cre‐Loxp transgenic mouse models provide a feasible strategy to further explore the roles of homeobox genes during implantation. Recent studies on the role of Msx genes in implantation are excellent examples of the application of conditional knockout mouse models. In fact, one of our purposes in conducting mouse uterus research is to portray the complexity of the human endometrium, given the impossibility of genetically manipulating human uteri. As mentioned above, the expression pattern similarities of homeobox genes in mouse and human, together with the aberrant expression patterns in female infertility, suggest the conserved roles of these genes, which made it feasible to translate research findings in mouse models to humans. In conclusion, subsequent studies will identify other homeobox genes and their target genes to further illuminate the complex regulatory network that is critical for implantation in mouse and human, which ultimately will provide information for the diagnosis and treatment of the female‐related infertility.

As previously described, precisely regulated homeobox genes are essential for implantation in both mice and humans, but few regulators of homeobox gene expression have been identified so far. Sex steroids, which are secreted periodically during each reproductive cycle, have been investigated in studies of the regulation of the homeobox genes. The major steroids that specify implantation are the ovarian steroids E2 and P4, which regulate uterine growth and differentiation. 1 , 2 , 4 , 5 With techniqual advances and the application of genetically engineered mouse models, many genes necessary for implantation, such as cytokines, growth factors and so on, have been shown to be induced and regulated by E2, P4 or both, 3 and expression of the homeobox genes seems also to be directly or indirectly regulated by these 2 hormones in mice and humans. The periodical expression pattern of the homeobox genes in the uterus during peri‐implantation in mice and the menstrual cycle in humans suggests the regulatory roles of E2 and P4, 6 , 45 but direct evidence for such regulatory roles comes from studies in mouse models. Specifically, Hoxa10 expression in the adult uterus is strongly activated by progesterone and the progesterone receptor antagonist RU486 is able to block this induction, but is repressed by estrogen in a protein synthesis independent manner. 39 Correspondingly, decreased expression of Hoxa10 has been shown in progesterone receptor null mice. 81 Furthermore, analysis of adjacent Hoxa genes reveals that Hoxa9 and Hoxa11 are also activated in a collinear fashion by progesterone. 39 These results suggest that the regulation of Hox gene expression in the adult uterus by ovarian steroids is a property related to position within the cluster, mediated by the direct action of estrogen and progesterone receptors upon these genes. Beyond that, the expression of Hoxa10 / HOXA10 and Hoxa11 / HOXA11 in developing female reproductive tracts is also regulated by hormonal factors in both mice and humans, as evidenced by the repression of Hoxa10 / HOXA10 and Hoxa11 / HOXA11 when developing female reproductive tracts were exposed to the synthetic estrogen diethylstilbestrol (DES) during reproductive tract morphogenesis. 82 , 83 , 84 Apart from the Hox/HOX genes, Msx genes, another homeobox gene family that profoundly influences receptivity and implantation in mice, are not obviously regulated by these hormones in ovariectomized mouse models. 3 Even so, the persistent expression of Msx1 in the delayed implantation uterus and rapid loss of expression following a single injection of estrogen suggests that the expression of Msx1 is repressed by estrogen. 68 In fact, rapid Lif induction by E2 is responsible for the loss of Msx1 in the delayed implantation uterus because E2 failed to downregulate Msx1 expression in Lif −/− uteri. In addition, Msx1 expression was downregulated if P4 treatment was combined with Lif, suggesting a direct regulatory role of Lif on Msx1 . 59 , 64 , 68 , 69 All these results suggest that E2 regulates the expression of Msx1 in an indirect manner at peri‐implantation. Although the way in which homeobox genes are precisely regulated remains largely unknown, the existing evidence is sufficient to demonstrate that the ovarian hormones induce and regulate the expression of homeobox genes directly or indirectly during peri‐implantation.

Embryo implantation involves the first physical and physiological interaction between the embryo and uterus, which determines the success of post‐implantation conceptus development and term pregnancy outcome. As the gateway to further embryonic development, successful implantation depends on the proper development of uterus to a receptive state and the synchronized development of blastocyst to a state of implantation competency. 2 , 4 , 5 Initially, the adult uterus undergoes proliferation and differentiation in specific uterine cell types to render the uterus receptive to blastocyst implantation. 2 , 5 Uterine receptivity is defined as a condition in which the uterus is suitable for embryo implantation. The results from blastocyst transfer experiments suggest that the uterus is not constantly receptive to blastocysts; its receptivity lasts only for a limited time, which is defined as the “implantation window”. 13 In fact, uterine sensitivity to implantation‐competent blastocysts is classically divided into 3 stages: pre‐receptive, receptive and refractory phases. During the pre‐receptive stage, the uterus is suitable for embryo development but not ready for implantation, while during the receptive stage, the uterus can initiate implantation when there are competent blastocysts. However, during the refractory stage, implantation‐competent blastocysts cannot implant into the uterus and the uterus is even hostile to blastocyst survival. 1 It is generally accepted that the uterus is a remarkable organ which is periodically regulated by ovarian estrogen and progesterone. This periodic event is usually called the menstrual cycle and estrous cycle in humans and mice, respectively. In humans, the receptive phase can be defined based on the menstrual cycle: the first 7 days of the secretory phase of the menstrual cycle is considered as the pre‐receptive stage, days 7‐10 after ovulation is the receptive stage, and the rest of the secretory phase is defined as the non‐receptive stage. However, in mice the receptive phase is difficult to determine based on the estrous cycle, because it is short (~4 days) and often irregular. Therefore, it is usually defined based on pregnancy: the uterus on Days 1‐3 (Day 1 = vaginal plug) of pregnancy is conventionally considered to be in the pre‐receptive phase in mice, in which the uterine epithelium undergoes proliferation stimulated by preovulatory estrogen. On Day 4 of pregnancy, the uterus becomes fully receptive following the priming actions of ovarian progesterone and pre‐implantation estrogen, as a result, the epithelium begins to differentiate, accompanied by extensive proliferation of stromal cells. However, by late Day 5 the uterus is refractory to initiation of implantation. 1 , 2 , 5 , 14 At the same time, the fertilized egg undergoes several rounds of division to form the blastocyst. The blastocyst then attains a state of implantation competency which is known as blastocyst activation. In mice, pre‐implantation embryos can be suspended at the blastocyst stage without further initiation of attachment reaction during lactation, which is known as delayed implantation or embryonic diapause. 4 , 15 , 16 , 17 In addition, embryonic diapause can be induced experimentally through ovariectomy on Day 4 before pre‐implantation estrogen secretion and then with daily injections of progesterone from Day 5, which can be terminated by a single injection of estrogen. 4 , 18 The delayed implantation mouse model makes it possible for us to explore blastocyst activation in mice. However, whether embryo diapause occurs in humans is not known. Evidence from embryo transfer experiments suggests that implantation occurs during a limited time span when blastocyst competency is superimposed on the receptive state of the uterus, known as the “implantation window”. 1 , 13 Any disturbance in the “implantation window” will cause implantation failure or defective implantation, and abnormal implantation can generate a range of adverse ripple effects, such as defective decidualization and placentation, eventually leading to a poor pregnancy outcome. 14 With advancing techniques and the application of genetically engineered mouse models, the molecular and cellular events that confer uterine receptivity and blastocyst competency have been extensively explored. A wide range of regulatory molecules, such as adhesion molecules, growth factors, cytokines and transcription factors, have been identified. Under the influence of ovarian estrogen and progesterone, the molecular signalling network consisting of these regulatory molecules elaborately orchestrate a successful implantation. 1 , 2 , 3 , 4 , 5 As summarized in Table  1 , the homeobox transcription factors, especially Hox and Msx genes, are reported to be essential for implantation in mice and humans. Homeobox genes implicated in embryo implantation: from mouse to human

It is well known that the beginning of a new life starts with the union of an egg and sperm through the process of fertilization in mammals, which naturally happens in the reproductive tract of adult females. The fertilized egg then undergoes several rounds of mitosis to form a competent blastocyst. Simultaneously, the adult uterus undergoes proliferation and differentiation into specific uterine cell types to render the uterus receptive for blastocyst implantation. 1 , 2 With the advance of gene expression studies and the application of genetically engineered mouse models, the cellular and molecular events of implantation have been extensively explored. Like many developmental processes, numerous transcription factors are known to participate in orchestrating this process directed by ovarian estrogen (E2) and progesterone (P4) in a spatiotemporal manner. 3 , 4 , 5 Among a range of identified transcription factors, the homeobox transcription factors, which attracted widespread attention because of their critical role during embryonic development, have been broadly investigated in early pregnancy, such as during implantation and decidualization. Homeobox genes are a family of regulatory genes coding for specific nuclear proteins that act as transcription factors. 6 , 7 They are characterized by sharing a homeobox sequence, a highly conserved 183‐nucleotide sequence that encodes a 61‐amino‐acid domain, termed the homeodomain (HD), which is responsible for the recognition and binding of sequence‐specific DNA motifs. 8 , 9 The homeobox genes, initially identified in Drosophila , can be divided into different families in mammals, such as Hox, Msx, Emx, Hmx and others. 9 Previous studies have revealed that homeobox transcription factors encoded by the homeobox genes play important roles during various developmental and pathophysiological processes, including embryogenesis, organogenesis, tumorigenesis, and so on. 6 , 7 , 10 , 11 Since implantation is a complicated but precisely orchestrated physiological process similar to embryogenesis and tumorigenesis, the homeobox transcription factors are likely to control the dynamic expression of the implantation‐related genes. 6 In fact, evidence from transgenic mouse models and human studies support the view that the homeobox transcription factors play essential roles in both the development of uterus and embryo implantation. 6 , 12 This review aims to illustrate progress in understanding the pathophysiological role of homeobox transcription factors, especially those encoded by the Hox and Msx genes, during the process of implantation.

None.

This review article was supported in part by National Key R&D program of China (2017YFC1001402) and the National Natural Science Foundation (81330017 and 81490744).