TJCYR ameliorates embryo implantation dysfunction through PI3K/Akt/eNOS signalling to improve endometrial receptivity

: Backgroud: In our clinical practice, we found that Tiao Jing Cu Yun Recipe (TJCYR), which was composed of Dangshen, Danshen, Danggui, Huangqi, Shudihuang, Bajitian and Yinyanghuo, had obviously enhanced the rate of pregnancy in the women with infertility. Although the application effects are desirable and satisfactory, the therapeutic mechanism of TJCYR remains poorly understood. In this study, we evaluated the effect of TJCYR on embryo implantation dysfunction (EID)-induced damage of endometrial receptivity in mice and investigated the mechanisms underlying the effect. Methods: Pregnant mice were randomly divided into six groups: Control, TJCYR-medium-dose+EID, TJCYR-high-dose+EID. Mifepristone was injected to make EID model. On the eighth day of pregnancy, the mice were sacrificed and the number of uterus-implanted blastocysts was counted. On the fourth day of pregnancy, the serum was to analyze the level of hormone by radioimmunoassay, the uterus was to analyze morphology by H&E and SEM, the combination of immunofluorescence and western blot were to identify the related proteins. Results: Compared with the EID group, the mice treated with high-dose TJCYR had a greater number of implanted sites, so we choose that dose of TJCYR as treatment in the following study. The mice treatment with E, G and K) Protein levels Integrin Integrin LIF OPN uterine tissue by Western blotting (n=3); (C, F, I and L) Quantification of protein levels. Results are expressed as


Abstract:
Backgroud: In our clinical practice, we found that Tiao Jing Cu Yun Recipe (TJCYR), which was composed of Dangshen, Danshen, Danggui, Huangqi, Shudihuang, Bajitian and Yinyanghuo, had obviously enhanced the rate of pregnancy in the women with infertility. Although the application effects are desirable and satisfactory, the therapeutic mechanism of TJCYR remains poorly understood. In this study, we evaluated the effect of TJCYR on embryo implantation dysfunction (EID)-induced damage of endometrial receptivity in mice and investigated the mechanisms underlying the effect.
Methods: Pregnant mice were randomly divided into six groups: Control, EID only, Progesterone (Prog)+EID, TJCYR-low-dose+EID, TJCYR-medium-dose+EID, TJCYR-high-dose+EID. Mifepristone was injected to make EID model. On the eighth day of pregnancy, the mice were sacrificed and the number of uterus-implanted blastocysts was counted. On the fourth day of pregnancy, the serum was to analyze the level of hormone by radioimmunoassay, the uterus was to analyze morphology by H&E and SEM, the combination of immunofluorescence and western blot were to identify the related proteins.
Results: Compared with the EID group, the mice treated with high-dose TJCYR had a greater number of implanted sites, so we choose that dose of TJCYR as treatment in the following study. The mice treatment with TJCYR could significantly enhance the level of P 4 , and inhibite the decrease in the expression of PR that induced by EID. Compared with the EID only, the SEM showed that a marked increase in the number of well-developed pinopodes in the TJCYR treatment group. Except morphological marker, several molecules in relation to pinopodes that could be used as biomarkers. TJCYR abrogated the EID-induced weakened in those biomarkers. Additionally, the vascular density and VEGF were decreased in the EID group, it appeared severe tissue hypoxia, while TJCYR reversed that change. Compared with the control, the p-Akt and p-eNOS were decreased in EID group, accompanied with decline of NO. While TJCYR promoted the activation of Akt and eNOS, to improve the poor microvascular environment of endometrium.
Conclusion: TJCYR has therapeutic potential against poor endometrial receptivity via activation of the PI3K/AKT/eNOS signaling pathway. 4

Background
The incidence of infertility has been one of the health problems worldwide in women [1] , which increase the economic, physical, and emotional burden on approximately 9% of couples worldwide [2] .
Although access to assisted reproductive technology (ART) is abundant in developed countries and has overcome the majority of infertility causes, success rates have stagnated to around 30% [3] . The successful implantation of embryo is a dynamic process that occurs as a result of interplay among the quality of embryo and the status of endometrium. In the recent years, advances in ART allowed for the selection of high quality embryos, but poor reproductive outcomes remained high. It has been confirmed that 2/3 of in vitro fertilization and embryo transfer (IVF-ET) implantation failures were considered to be due to uterus with decreased endometrial receptivity [4] . Endometrial receptivity refers to the finite window in each menstrual cycle in which the endometrium is sufficiently prepared for an implanting blastocyst, therfore improving endometrial receptivity is the key point to rise the pregnancy rate in women with infertility [5][6][7][8] . and blood stasis pattern [12] . In our clinic, we found that TJCYR had a good effect on pregnancy, but the pharmacological mechanism of its action is still under investigation. The aim of this study was to document the beneficial effect of TJCYR on EID-induced damage of endometrial receptivity in the mice and to investigate the mechanism of action. From this point of view, we have strived to find novel candidates from traditional medicinal herbs for improving endometrial receptivity.

Treatments and Establishment of EID
The female mice were mixed with male mice in ratio of 2:1 mating from 19:00 to 07:00 and then checked for the presence of a vaginal plug at 08:00 the next day. Females presenting vaginal plugs (classified as day 1 of pregnancy, Pd1) were randomly divided into six experimental groups (n=20 for each group), including the Control, EID only, Progesterone (Prog) +EID, TJCYR-low-dose+EID, TJCYR-medium-dose+EID and TJCYR-high-dose+EID. In the Control and the EID only group, the mice were given intragastric administration of physiological saline solution once daily for 4 days, the treatment group were given Prog and TJCYR respectively. Mifepriston (0.1mg/mouse) was subcutaneously injected at Pd4 in the morning, to establish the EID model. Mice were sacrificed on Pd4 and Pd8, respectively. The uteri were harvested for the evaluation of endometrial receptivity and the potential mechanism (Figure1A).

Determination of implantation sites number
In one set of our experiment, the gestational mice were sacrificed on Pd8.
On this time, the implantation sites in the uterus were captured easily.
And the number of implantation sites was counted.

Radioimmunoassay for the test of E 2 and P 4
Serum sex-steroid levels were determined from the serum of blood collected on Pd4. Anesthesia was administered using 0.4% sodium pentobarbital. Blood samples were collected from the abdominal aortic under anesthesia. The serum was separated by centrifugation at 3000×g (4℃) for 10 min. Serum was stored at -20℃. The level of E 2 and P 4 were detected using radioimmunoassay, which followed the instructions of the

Scanning electron microscopy (SEM)
The uteri were excised and then cut open along the longitudinal axis. The endometrial tissue was rinsed in saline solution for 1min, fixed in 1.25% (w/v) glutaraldehyde solution and 1% osmic acid at 4℃ for 2h respectively. After several washes in buffer, the samples were dehydrated through graded concentrations of ethanol and subsequently dried in a critical-point drier with carbon dioxide, then mounted onto the specimen holder and coated with gold palladium. Finally, all specimens were observed under the scanning electron microscope (SU8010, Hitachi Instruments, Tokyo, Japan).

Immunofluorescence staining
For immunofluorescence assay, the samples were frozen in OCT in liquid nitrogen and were serially frozen-sectioned at 8 μm thick. Nonspecific

Quantification of NO content
Nitric Oxide (NO) concentration in uteri tissue was measured with the NO assay kit (Beyotime, Shanghai, China) following the user manual.
The quantitative determination of nitrite levels represents NO production.
The level of NO was shown in μM/μg protein.

Western blotting analysis
Uteri tissue protein lysates were separated by 10% SDS-PAGE and then

Statistical analysis
Data were analyzed using SPSS21.0, and results were presented as means ±SEM. Data from the experimental groups were compared by one-way analysis of variance (ANOVA) followed by Tukey's post hoc analysis, and differences were considered significant at P<0.05.

The quality evaluation of TJCYR by HPLC.
HPLC chromatograms of TJCYR crude sample and the standard substance were seen in Figure 1B. Under this separation condition, the index components of herbs can be separated and quantified. It well guaranteed the quality and pharmacodynamics of TJCYR.

TJCYR was able to protect blastocysts implantation
Numbers of blastocyst sites were recorded on Pd8. As our results indicated (Figure 2), the mean number of implanted embryos was markedly lower in EID group than in the control group (P<0.05), which suggested successful model. The medium and high-dose of TJCYR treatment increased the implantation numbers compared with the EID only. By comparison, the high-dose of TJCYR dramatically enhances blastocysts implantation, especially, so we choose that dose of TJCYR as treatment in the following study.  TJCYR-L, the low-dose TJCYR.

TJCYR improved EID-induced endometrium morphological changes.
As shown in the Figure 3A, H&E staining was used to evaluate the pathological changes of endometrium. We discovered that there were little loose endometrial stroma, followed with insufficient glands and vessels in EID only group, which were opposite in mice treated with TJCYR. To further morphologically assess the TJCYR caused changes on EID, we checked the effect of TJCYR on the pinopodes by SEM. As shown in the Figure 3B, in the control group, the sample revealed that the majority of well-developed pinopodes were evenly distributed over the endometrial epithelial surfaces, while EID-induced surfaces only sparsely presented a part of well-developed pinopodes and a few developing pinopodes . However, the restrained expression of pinopodes in the EID group was significantly improved following treatment with TJCYR. The results suggest that TJCYR can improved the morphology and vasculature of endometrium of EID model to a certain extent, which is beneficial to embryo implantation.

The effect of TJCYR on hormones and hormone receptors.
The receptive mouse uterus displays stromal proliferation and epithelial differentiation as the indicator of proper arrangement for blastocyst implantation, which are governed by ovarian hormones, such as: 17β-estradiol (E 2 ) and progesterone (P 4 ). And the primary mediators of these E 2 and P 4 -induced events are their receptors-progesterone receptor (PR) and estrogen receptor (ER). In our results, we found that in the EID only group, the level of P 4 was obviously lower than those in the Control group, and the expression of PR lowered induced by EID . However, the treatment with TJCYR could reverse those changes ( Figure 4) .

TJCYR enhanced the related proteins as the markers of endometrial receptivity.
There are several molecular markers related to the endometrial receptivity, including Integrin αV, Integrin β3, LIF, and OPN. As shown in the Figure   5, we found that the expression of Integrin αV, Integrin β3, LIF, and OPN significantly decreased in EID only group by immunofluorescence and western blot analysis. TJCYR had been found to attenuate the EDI-induced damage through increasing the expression of Integrin αV, Integrin β3, LIF, and OPN. These results were consisted with the changes of pinopodes shown in Figure 3B .

TJCYR.
Angiogenesis is one of important biological events which could be involved in implantation. Tomato Lectin is recognized as the most sensitive vessel marker, the fluorescence intensity could reflect the vascular density. Compared with the control group, we discovered that in the EID only group, the vascular density decreased significantly, which was prevented by TJCYR ( Figure 6A). Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis, which has beneficial effects on endometrial receptivity and plays a key role in the embryonic development of mice. As shown in Figure 6B and C, the treatment of TJCYR obviously inhibited the decrease of VEGF in mice that subjected to EID, which is consistent with the immunofluorescence analysis ( Figure   6A) that showed increased density of vascular.
Hypoxyprobe-1 is the hypoxia marker associated monoclonal and polyclonal antibodies that bind to pimonidazole adducts in hypoxia tissue.
As shown in the Figure 6D, the fluorescence intensity was increased significantly in mice that subjected to EID only, which suggested serious hypoxia in tissue, while, TJCYR could ameliorat hypoxia

Discussion
For successful embryo implantation to be achieved, it is a highly orchestrated process that involves blastocyst-uterine interactions. While blastocyst quality has been extensively studied, equally as important is the preparation of the receptive endometrium, or more precisely, it is endometrial receptivity. By definition, endometrial receptivity is "that period of endometrial maturation during which the trophectoderm of the blastocyst can attach to the endometrial epithelial cells and subsequently proceed to invade the endometrial stroma and vasculature" [13] .Therefore, improving endometrial receptivity is the key point to rise the pregnancy rate.
In mice, the implantation window extends from the morning of Pd4 to the end of Pd5 [14] . The use of mifepristone for both planned and emergency contraception has been widely accepted [15] . And mifepristone played a role in inhibiting development and maturation of endometrium, hence affecting embryo implantation in mice, as well as for terminatin of pregnancy [16] . In our study, we used the EID mice model by treatment with mifepristone, and collected samples between 21:00 and 22:00 on Pd4 (as show in Figure 1A).
The endometrium is a unique tissue which undergoes dramatic and rapid changes throughout the menstrual cycle. It progresses through specific stages in response to ovarian hormones. The receptive mouse uterus displays stromal proliferation and epithelial differentiation as an indicator of proper arrangement for blastocyst implantation [17] , which is governed by ovarian hormones. In another word, increased production of ovarian hormones E 2 and P 4 provides the uterus with a capacity of blastocyst attachment pre-decidualization. Circulating P 4 levels rises on Pd3 due to its production from newly-formed corpus luteum. Elevated level of P 4 takes over E 2 and becomes a dominant ovarian hormone on Pd4, increased P 4 promoted stromal proliferation and inhibited E 2 -induced epithelial proliferation. E 2 and P 4 exerted their respective functions through ER and PR. We found that, compared with EID only, TJCYR could obviously enhance the level of P 4 and the corresponding receptor-PR , while the influence of E 2 and ER were little. We thought the difference of influence on E 2 and P 4 was due to EID model. In our study, mifepristone was prepared for EID model, which has a strong affinity with PR and competitively inhibits the effect of P 4 . So the regulation of P 4 maybe obvious after treatment with TJCYR.
The pinopodes are membrane protrusions on the apical surface of luminal epithelium. The pinopodes play the key role in the initial stage of implantation by promoting attachment of the embryo, because they prevent the cilia from sweeping off the blastocyst as well as promoting withdrawal of uterine fluid and closure of the uterine cavity [5,18] . The pinopodes have been considered as the characteristic morphologic markers to assess endometrial receptivity and to locate the implantation window [16,19] . In our study, the specimens were examined by scanning during the implantation window. There are many isoforms of integrins in mammals, only three isoforms α1β1, α4β1 and αVβ3 are found to be particularly involved in implantation, and αVβ3 is seemingly playing more conspicuous roles than others, especially [20] . The Integrin αVβ3 is a potential receptor for blastocyst attachment and is localized on pinopodes [18] . Some studies revealed that the blockade of the Integrin αVβ3 or lack of Integrin β3 may be related to unexplained infertility [21,22] . The role of Integrins in the implantation process had been reached extensively.
Several studies had identified an association between Integrin expression and female fertility, which suggested the potential use of Integrins as markers of uterine receptivity [23,24] . LIF is a member of the interleukin-6 family of cytokines, which plays a critical role in implantation and pinopodes release secretory vesicles that contain LIF in uterine lumen to enable trophoblast invasion and also affect on immune tolerance during implantation [18,25] . Some studies [26,27] showed that the deletion or mutation of LIF reduced implantation failure. Other studies [28,29] reported that endometrial LIF and receptor were higher around the time of implantation in fertile women compared with unexplained infertile women. Other finding [30] also showed that LIF may maintain the proper development of the endometrium and implantation receptivity by regulating downstream target genes. OPN is one of the co-factors involved in cell adhesion and invasion during the implantation process, and it is an acidic member of the small integrin-binding ligand family of proteins [31] , had been shown to be maximally expressed in the epithelial layer in human, mouse and rabbit uterine. OPN was an important constituent of the uterus during pregnancy [31][32][33] . In this study, the EID group showed histopathological lesions characteristic of pinopodes, as well as low expression of Integrin αV, Integrin β3, LIF, and OPN. While the expression of these biochemical markers were significantly increased in mice treated with TJCYR. These data strongly promised a repairment of TJCYR in endometrial receptivity.
During the window of implantation, a rich vascular network is necessary to supply nutrients and oxygen. Proper endometrial vascular development and maintenance are crucial for successful pregnancy. Vascular endothelial growth factor (VEGF) has a predominant role in successful implantation and maintenance of pregnancy by increasing vascular permeability or forming vascular network. The expression of VEGF and its receptor were confirmed in early pregnancy. It was reported that VEGF knock-out mice do not produce viable offspring [34] . The expression of VEGF increased significantly during implantation windows, which suggested that VEGF promotes angiogenesis and the establishment of capillary network, further improved endometrial receptivity and promoted embryo implantation. Otherwise, deficiency of VEGF may induce the reduction of angiogenesis on implantation site, then lead to miscarry [35] .
In this study, we found that there was a poor vascular network in the mice treated with EID only, accompany with serious hypoxia. Here we found that treatment with TJCYR significantly inhibited decline in density of vessel and expression of VEGF in EID only group, and improved endometrial blood circulation, further prevented the hypoxia. All the data showed that VEGF regulated angiogenesis and built endometrial microenvironment for embryo implantation. As we all know, VEGF is an angiogenesis accelerator, which could activate the PI3K/Akt/eNOS pathway. Here, we found that the expressions of p-Akt and p-eNOS were all reduced in the EID mice compared to the control group, and all of these decreased expressions were restored by the treatment of TJCYR.
And the activated eNOS could produce nitric oxide (NO). NO, as an endogenous vasodilator, could increase the vascular dilatation and permeability at the implantation site, further to provide safeguard for embryo implantation and pregnancy maintenance.

Conclusion
TJCYR can activate the PI3K/AKT/eNOS signaling pathway, promote angiogenesis, and increase the endometrial receptivity, which were beneficial to the implantation of embryos.

Availability of data and materials
The data used to support the findings of this study are available from the corresponding author upon request.

Ethics approval and consent to participate
All experimental protocols were approved by the Animal Care and Use Committee of Shanghai University of Traditional Chinese Medicine (Shanghai, China).

Consent for publication
The manuscript is approved by all authors for publication.