{"paper_id":"fd33b19a-0058-4cc5-b8d8-cbfcedc80031","body_text":"Endometriosis (EMs) is a common, chronic gynecological disease which can happen in women of reproductive age  1 . The prevalence of EMs approximately to be 5%~10%  2 , with a peak from 25 years old to 35 years old  3 , and up to 50% of infertile women  4 . EMs, an estrogen-dependent chronic inflammatory disease, is associated with pelvic pain and infertility  5 . Notedly, in spontaneous pregnancies, endometriosis appears to be a risk factor of miscarriages (almost 80% increased risk  6 ). There were varieties of epidemiological factors and molecular and cellular alterations of EMs had been reported, such as early age at menarche or a long duration of menstrual flows  7 , familial aggregation  8 , increased estrogen receptor (ER) α and β expression  9 - 11 , progesterone resistance  9 , overproduction of prostaglandins, cytokines and chemokines  12 ,  13 , and so on.\nThe reasons for EMs-associated infertility and miscarriage have been completely unknow yet. Pelvic anatomy distortion, endocrine and ovulatory disorders, inflammation and immune disorders, reduced endometrial receptivity, dysfunctional fallopian tubes and impaired sperm transport are the most common reasons for EMs-associated infertility  14 . Macrophages (Mφ) in the abdominal cavity are the main source of cytokines that are either involved in regulating inflammation or released upon injury to the peritoneum  15 ,  16 . Mφ constitutes 50% of peritoneal leukocytes in human. Dysfunctional phenotypes of peritoneal Mφ were observed in women with EMs, characterized by reducing phagocytic capacity and producing more Prostaglandin E2 (PGE 2 ) and a lot of proinflammatory cytokines, which contribute to pelvic inflammation and anatomical abnormalities  17 ,  18 .\nEndometrial receptivity refers to the ability of the maternal endometrium to accept embryo implantation at a specific time. This period is called the “implantation window period”  19 , which is regulated by estrogen and progesterone. These changings of sex hormones prevent menstruation, destruction of the decidualized endometrium, regulate lymphocytes functions, and promote angiogenesis. In the early pregnancy, the accumulation of leukocytes is up to 40% of total decidual cells, and decidual NK (dNK) cell population accounts for about 70% of total tissue immune cells  20 ,  21 . dNK cells, marked by CD56 bright CD16 - KIR + , are poorly cytotoxic and have positive effects in regulating decidualization, placental development and angiogenesis  22 ,  23 . Currently, researches on the role of dNK cells in endometrium of EMs patients are still scarce. However, more and more studies have shown the decreased number and activated cytotoxicity of dNK are closed related to spontaneous abortion  24 - 26 .\nAlthough the current medical therapies (e.g., gonadotropin-releasing hormone agonists (GnRHa), progestins and aromatase inhibitors) are world wildly used to inhibit ectopic endometrium growth by reducing the systemic levels of estrogen, their side effects cannot be ignored  9 . Therefore, the most urgent need is to develop more effective treatments and to save the fertility of patients to the greatest extent. Nowadays, traditional Chinese medicine has also been scientifically proven to be effective  27 . Ginseng is one of the most famous traditional medicinal herbs, which is widely used in Asian countries. Protopanaxadiol (PPD) is one of two metabolites of ginsenoside, the main components extracted from ginseng. There are many reports about PPD used to treat various tumors, cardiovascular diseases  28 ,  29 . Additionally, our previous study has showed that PPD suppresses ER-mediated inhibition of endometriotic cells autophagy, contributing to anti-EMs effects  30 . However, the possible role and mechanism for PPD on infertility and spontaneous abortion of EMs are unknown.\nTherefore, the current study is to investigate whether and how PPD against on infertility and spontaneous abortion of EMs  in vitro  and  in vivo , and provide the potential intervention strategies for fertility remodeling and preservation of patients with EMs.\n\nA group of adult female BALB/C mice aged 6-8 weeks was purchased from Shanghai Jiesijie Experimental Animal Co., Ltd. (China) and was used for this study. They were maintained for 2 weeks at the animal facility before use. The Animal Care and Use Committee of Fudan University approved all the animal protocols. We constructed an intraperitoneal EMs model. On day 0, one-third of all mice were randomly selected as donors, and their uterus horns were removed and cut into fragments smaller than 1 mm 3 . Then those fragments were suspended in sterile saline and injected them into the remains intraperitoneally (for recipient mice, the ratio of the uterus to intraperitoneal injection of mice was 1:2). On the day 4, peritoneal fluids and uterus of Ctrl and/or EMs mice were collected, or the rest of Ctrl and/or EMs mice were randomly mated with healthy male adult mice, respectively, and then the data of pregnancy rate, the number of embryos, and the absorption rate were counted on day 18. From day 4 to day 32, the recipient mice were randomly divided into 3 groups and injected intraperitoneally with vehicle control (0.5% DMSO, every day, Sigma, MA, USA) or 45 mg/kg PPD (every 4 days, Sigma, MA, USA), or injected intramuscularly with 0.5μg GnRHa (every day, Sigma, MA, USA). The dose of PPD at 45 mg/kg was based on our previous study  30 , and the dose of GnRHa at 0.5 μg was modified from a previous research conducted in female rats  31 . On day 32, half of the mice were selected to mate with healthy male adult mice, and then the tissues of uterus were collected, and the data of pregnancy rate, the number of embryos, and the absorption rate were counted on day 46. In the remaining mice, the serum, the EMs-like lesions, peritoneal fluids (PF), uterus, livers, kidneys, and femurs were collected on day 32. The levels of estradiol in the serum were detected by enzyme linked immunosorbent assay (ELISA). The levels of aspartate aminotransferase (AST) and blood urea nitrogen (BUN) in the serum were measured by an automatic biochemical analyzer (Beckman Counter, USA). The serum levels of tartrate resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) were measured. Micro-CT was used to measure the osteoporosis-related markers. The number and weight of EMs-like lesions were counted. Hematoxylin-Eosin (H&E) staining was used to assess the lymphocytes infiltration in the livers and kidneys.\nHuman and mouse antibodies for flow cytometry assays (all antibodies were purchased Biolegend, CA, USA) were used for measurement of cell markers, including APC/Cyanine 7 (APC/Cy7)-conjugated anti-mouse CD45, fluorescein isothiocyanate (FITC)-conjugated anti-mouse F4/80, brilliant violent (BV) 660-conjugated anti-mouse CD11b, BV510-conjugated anti-mouse interferon (IFN)-γ, APC-conjugated anti-mouse interleukin (IL)-12, FITC-conjugated anti-mouse CD3, PE-Cy7-conjugated anti-mouse CD49a, BV510-conjugated anti-human CD14, BV421-conjugated anti-human CD45, APC/Cy7-conjugated anti-human CD45, phycoerythrin/Cyanine 7 (PE/Cy7)-conjugated anti-human IFN-γ, FITC-conjugated anti-human IL-12, BV605-conjugated anti-human CD56, PE/Cy7-conjugated anti-human CD16, PE-conjugated anti-human NKp30, APC-conjugated anti-human Ki67, APC-conjugated anti-human CXC chemokine ligand (CXCL)10, APC/Cy7-conjugated anti-human vascular endothelial growth factor (VEGF), and BV421-conjugated anti-human transforming growth factor (TGF)-β. Isotype IgG antibody (5 μl separately) was used as the control. Human Trustain FcX (Biolegend, CA, USA) was used to block Fc receptors prior to flow cytometry. Subsequently, cells were washed twice and resuspended in PBS for flow cytometry analysis. Samples were analyzed using a CytoFLEX flow cytometer (Beckman Coulter, Inc.) and data were analyzed using FlowJo (version 10.07 (FlowJo LLC).\nThe total RNA was extracted by TRIzol regent (Invitrogen, Carlsbad, CA, USA). Subsequently, the concentration and purity of RNA was quantified by a NanoDrop spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, MA, USA). The PrimeScript RT Reagent Kit (TaKaRa Biotechnology, Co., Ltd., Dalian, China) was utilized to reversely transcribe total RNA to cDNA. Next, qRT-qPCR was performed with SYBR Green PCR Master Mix (TaKaRa Biotechnology). The qRT-PCR primers are listed in  Table S1 . The target mRNA expressions were normalized to  ACTB  or  Actb  expression. All reactions were processed on the Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific, MA, USA). The test results were analyzed using the 2 -ΔΔCt  method.\nBlood samples were collected and then serum was removed after centrifugation for 15 minutes at 1000 ×g. Plasma estrogen (E2) concentrations were measured using a double-antibody sandwich ELISA at room temperature as per the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA). Absorbance was recorded at a dual-wavelength of 450/630 nm. Each plate also contained a standard control (coefficient of variation < 12%).\nThe monocytes cell line, U937, and the human endometrium stromal cell line, HESC, were purchased from the ATCC collection. HESC were pre-treated by RU486 (1 nM or 10 nM; Sigma, MA, USA) for 48h, then treated with or without PPD (40 μM; Sigma, MA, USA) for 48h, and the expression of related genes were detected by qRT-PCR. HESCs and DSCs were treated with or without PPD (40 µM) for 48 h, and the expression of related genes were detected by qRT-PCR. U937 cells were treated with 100 ng/mL phorbol-12-myristate 13-acetate (PMA, Sigma, MA, USA) for 24 h for differentiation and then they were co-cultured with PPD-pretreated HESCs for 48h. These U937 cells were collected to analyze the expression of IL-12, and IFN-γ by FCM.\nTotal RNA was extracted from the samples by Trizol reagent (Invitrogen, CA, USA) separately. Gene expression analysis was conducted by mRNA-seq for the conditions described in the relevant figures. Quality of the total RNA was measured by the Agilent 2100 Bioanalyzer and samples with RNA integrity number (RIN) higher than 7 were used for sequencing. cDNA libraries were generated using the TruSeq Stranded mRNA Library Prep Kit (Illumina, Inc.) according to the manufacturer's instructions. Libraries were size selected using a 6% polyacrylamide gel and purified using the QIAQuick PCR Purification Kit (Qiagen GmbH). Purified libraries were normalized and pooled to create a double stranded cDNA library ready for sequencing. The samples were sequenced on the Illumina MiSeq to render 50 base pair single end reads. The sequencing library was constructed after high-quality RNA was quantified and then sequenced HiSeq X (Illumina, CA, USA) on a 150 bp paired-end run.\nWe applied DESeq2 algorithm  32  to filter the differentially expressed genes, after the significant analysis. The P-value and FDR analysis  33  were subjected to the following criteria: i) Fold Change>2 or < 0.5; ii), P-value<0.05, FDR<0.05. Gene ontology (GO) analysis  34  and pathway analysis have down as reported  35 .\nThe related moleules were submitted to the STRING database (available online: http://string‑db.org) for PPI recognition. The PPI network is further visualized by using the software of Cytoscape 3.7.2. We used to predict potential cellular sensory proteins or target receptors via the Swiss Target Prediction platform ( http://www.swisstargetprediction.ch/ ).\nAll subjects completed informed written consents forms for tissue collection, and the present study was approved by the Human Research Ethics Committee of Obstetrics and Gynecology Institute, Fudan University (Shanghai, China). Decidual tissues (n=19) were obtained from healthy women during early pregnancy (age, 23-35 years old; gestational age, 7-9 weeks) who underwent elective terminations for non‐medical reasons. The decidual tissues were digested and isolated as a previous procedure  36 . Using this method, 98% of cells obtained were vimentin +  cytokeratin (CK)7 -  DSCs and CD45 +  DICs as previously reported  25 . DICs were used for dNK cells isolation, using magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) for  in vitro  experiments. These NK cells were directly treated with PPD (10, 20 or 40 µM, Sigma, MA, USA) or vehicle control (0.1% DMSO, Sigma, MA, USA) for 48h, and the dNK cells were collected to analyze the expression of NKp30, Ki67, CD16, VEGF, TGF-β1, and CXCL-10 by FCM.\nThe isolated femurs were placed in a holder in the supine position. X-ray micro-computed tomographic scanning of the mice was performed using the Skyscan 1076 Scanner. The present research set the energy as 70 KVp and 100 µA with medium image resolution to obtain the best contrast between bone and soft tissues. The volume of interest (200 slices) for trabecular bone parameters was selected at the metaphyseal area located 1.5 mm below the lowest point of the epiphyseal growth plate of proximal tibia extending distally. To determine the cortical bone parameters, 100 slices were analyzed at the diaphyseal area located 2.5 mm from the metaphyseal area.\nEach experiment was conducted at least three times independently. For data with only two groups, Student's t test was used. For data containing more than two groups and fit the normal distribution, an analysis of variance (ANOVA) test was used, followed by Tukey or Bonferroni test for t tests, and the results were presented as mean ± SEM. All analyses were conducted using the SPSS 20.0 Statistical Package for the Social Sciences software. p <0.05 was considered to indicate a statistically significant difference.\n\nChronic pelvic pain and infertility are the most common symptoms of EMs. We firstly constructed the EMs mice model by allotransplantation, flowing the procedure of Figure  1 A. At Day 4, the expression level of interferon (IFN)-γ in Mφ of peritoneal fluid (PF) was measured by FCM, and the mRNA expression levels of  interleukin 6 family cytokine  ( Lif ) and  Insulin-like growth factor-binding protein 1  ( Igfbp1 ) were measured by qRT-PCR. As shown, the levels of IFN-γ in CD45 + F4/80 + /CD11b +  Mφ were significantly increased (Fig.  1 B and  1 C), and the expression of  Lif  and  Igfbp1  were obviously downregulated (Fig.  1 D) in uterine endometrium from mice with EMs. Additionally, these female mice were randomly mated with healthy male mice for another 14 days. At Day 18, we observed poor pregnancy outcomes in the EMs mice group, including lower pregnancy rate and embryo implantation numbers and higher embryo resorption rates, compared with the Ctrl group (Fig.  1 E-G). These data suggest that the EMs mice model with endometrial allotransplantation can reflect the condition (i.e. elevated pelvic inflammation)  in vivo  and impaired fertility of endometriosis.\nTo evaluate the potential therapeutic role of PPD in EMs, PPD (45 mg/Kg, every 4 days) or GnRHa (as a positive control, 0.5 μg, every day) was used to treat the EMs mice model, flowing the procedure of Figure  2 A. Excitingly, the number and weight of ectopic lesions were diminished significantly in the PPD-treated group, as well as the GnRHa-treated group (Fig.  2 B and  2 C). More notedly, PPD did not affect the serum estrogen (E2) concentration, while GnRHa led to the down-regulation of E2 concentration dramatically as reported  37  (Fig.  2 D). Interestedly, treatment with PPD resulted in up-regulation of  Progestogen receptor  ( Pgr ) and down-regulation of  Estrogen receptor 1  ( Esr1 ) and  Esr2  (Fig.  2 E).\nTo further analyze the possible mechanism of PPD on endometrial receptivity, Swiss Target Prediction (http://swisstargetprediction.ch/) was carried out, and the data showed that there were 66 predicted intracellular sensory proteins or target reporters, including androgen receptor (AR), 11-beta-hydroxysteroid dehydrogenase 1 (HSD11B1), nuclear receptor ROR-gamma (RORC), ERα, and ERβ (Table  1 ).\nTo explore the possible mechanism for PPD on the fertility of EMs, RNA-sequence was performed to evaluate the potential effects of PPD on human endometrial stromal cells line (HESCs). As shown, there were 2139 differential up-regulated genes (e.g.,  MMPS ,  IGFBP1 ,  LIF ,  PRLR ) and 2136 down-regulated genes (e.g.,  COL1A1 ,  COL1A2 ,  COL4A1 ,  ESR1 ) in PPD-treated HESCs vs. Ctrl HESCs (Fig.  3 A). The Top 20 of GO function and KEGG pathway enrichment analysis showed that the differential expressed genes were mainly involved in the cellular response to TGF-β stimulus, immune system process, angiogenesis, extracellular matrix-related pathways, focal adhesion and TGF-β signaling pathway to regulate inflammation, cell adhesion (Fig.  3 B and  3 C), and these biological processes were closed related to endometrial receptivity and embryo implantation. Compared with the Ctrl HESCs, PPD-treated HESCs expressed higher levels of MMPs, and lower levels of collagens (Fig.  3 D). As a common inducer of decidualization, MPA plus 17β- estradiol (E2) significantly increased the expression of genes related to endometrial receptivity (e.g.,  BMP2, IGFBP1, HOXA1, PRL, LIF, IHH, PTGS2, WNT4, MMP2 ). Surprisingly, the stimulatory effects of PPD on endometrial receptivity was even more significant (Fig.  3 E). And these effects were further confirmed in EMs mice models (Fig.  3 F). In contrast, GnRHa significantly suppressed endometrial receptivity (Fig.  3 F), suggesting that PPD may promote the endometrial receptivity of EMs.\nThe protein-protein interaction (PPI) network of predicted sensory proteins of PPD (ER-α and ER-β), progesterone receptor (PR), endometrial receptivity-related molecules and collagens were obtained by STRING database and Cytoscape (Fig.  3 G), suggesting that PPD induces the expression of endometrial receptivity-related molecules and collagen possibly by binding and regulating the expression of ESR and or PGR. Additionally, other sensory proteins of PPD were predicted, and involved in the regulation of endometrial receptivity and extracellular matrix (ECM) remodeling, including AR, CYP19A1, HSD11B1, etc. (Fig.  3 H). To explore the potential relationship between PPD and PR, we used RU486, a PR inhibitor, to inhibit the PR expression of HESC, and found that with the increase of RU486 concentration, the inhibition of PR was enhanced. Due to the decrease of PR expression induced by RU486, the promotion effect of PPD on the expression of genes related to endometrial receptivity,  IGFBP1  and  LIF , was significantly weakened. These data suggest that PR should be a potential regulator for PPD to play a regulating role in remolding fertility (Fig.  3 I).\nOwing to the key roles of macrophage in EMs, we further predict the possible role and PPI relationship between predicted sensory proteins and down-steam regulator (ESR1, ESR2 and PGR), and inflammatory cytokines by STRING, and the regulatory network was shown in Figure  4 A. The data indicate that ESR1 and other sensory proteins (e.g., AR, HSD11B1, and RORC) should be an importance regulator for inflammatory cytokines. To further confirm this possible regulatory effect  in vivo , we analyzed the levels of inflammatory cytokines and macrophage differentiation in PF from EMs mice models. As shown, PPD could significantly down-regulate the expression of IFN-γ and IL-12 in Mφ, compared with control and GnRHa groups (Fig.  4 B and  4 C). Similarly, the results in the co-culture model of PMA-pretreated U937 cells and PPD-pretreated HESC echoed these effects, as shown, the expression of IFN-γ and IL-12 were decreased (Fig.  4 D and  4 E).\nAs expected, subsequently, we observed that treatment with PPD significantly increased the pregnancy rate and embryo implantation numbers, and decreased the risk of embryo abortion of EMs mice (Fig.  5 A- 5 D). However, GnRHa could not improve the pregnancy rate, embryo implantation numbers and embryo absorption rates (Fig.  5 A- 5 D). These results indicate that PPD can lead to higher pregnancy rate, more embryos implantation, and lower embryo miscarriage of EMs mice models.\nTo investigate the role and potential mechanism of PPD against EMs-related infertility and miscarriage, Ctrl and PPD-treated human decidual stromal cells (DSC) were collected and analyzed by RNA sequencing (Fig.  6 A). As shown, there were 2553 up-regulated genes (e.g., decidualization-related genes,  LIF  and  IGFBP1 ) and 3372 downregulated genes (e.g., ECM organization/remodeling-related genes,  MMP2 ,  MMP9 ,  COL1A1 ,  COL1A2  and  COL4A1 ) in the PPD-treated group, compared with the Ctrl group (Fig.  6 A). The top 20 of GO function and KEGG pathway enrichment cluster displayed that PPD might be involved in the regulation of integrin-mediated signaling pathway, cell-matrix adhesion, leukocyte migration, angiogenesis, extracellular matrix-related pathways, and focal adhesion (Fig.  6 B and  6 C). Subsequently, the data of  in vitro  and  in vivo  experiments verified these findings, as shown, PPD significantly up-regulated the decidualization with up-regulation of  LIF ,  IGFBP1 ,  MMPs  and or down-regulation of  Collages  in HESCs and pregnant uterus of EMs mice (Fig.  6 D and  6 E).\nOwing to the important role of decidual NK cells in normal pregnancy  24 ,  26 ,  38 , the PPI analysis between predicted sensory proteins (ESR and PGR) of PPD and cytotoxicity-related molecules (e.g., FCGR3A, NCR3) expressed by NK cells and the network was shown in Figure  7 A. The results showed that ESR and PGR might be important intermediate regulators for NK cell (Fig.  7 A). In EMs pregnant mice models, total CD45 +  lymphocytes were significantly decreased in uterus and the decline became even more pronounced after GnRHa treatment (Fig.  7 B). Notably, PPD markedly increased the percentage of total CD45 +  lymphocytes and CD3 - CD49a + NK cells in uterus of EMs pregnant mice (Fig.  7 B). Further analysis showed that PPD could significantly down-regulate the expression cytotoxicity-related molecules (NKp30 and CD16), up-regulated the expression of Ki67, VEGF, TGF-β and CXCL10 of human decidual NK cells  in vitro , especially at the concentration of 40 µM (Fig.  7 C). These data suggest that PPD should promote the proliferation, decrease the cytotoxicity, promotes the angiogenesis and maternal-fetal immune tolerance.\nTo further evaluate the safety of PPD, the bodyweight of the mice was recorded, the function of liver, kidney, and bone evaluation were detected after 28 days for treatment. As a first-line drug for clinical use, GnRHa was also used as a comparison of evaluation. As shown, the bodyweight was no significant difference among the Ctrl, PPD and GnRHa groups (Fig.  8 A). Besides, HE staining and serum biochemical index did not observe the dysfunctions of the kidney and liver among these groups (Fig.  8 B and  8 C). As a common side effect of GnRHa, we did observe the obstacle of bone remodeling in the GnRHa-treated group with high ratio of TRAP to ALP, which was also confirmed by TRAP staining and ALP staining on mouse femurs (Fig.  8 B and  8 C). According to the data of microCT, additionally, the bone mineral density (BMD), bone volume, tissue volume, trabecular thickness, and trabecular number in GnRHa-treated group at day 32 showed there was severe bone loss (Fig.  8 D and  8 E). However, we did not find similar results in the Ctrl and PPD-treated groups (Fig.  8 B- 8 E). These results suggest that PPD should be more safety for treatment of endometriosis compared with GnRHa.\n\nSurgical removal of lesions and hormonal medication, often with severe side effects and variable efficacy, are the most common therapies of EMs  18 . But neither surgery nor drugs can help to reverse infertility of EMs patients effectively. Techniques of assisted reproduction consisting of superovulation with  in vitro  fertilization represent effective treatment alternatives that improve fertility in patients suffering from endometriosis. However, it still has the disadvantages of being expensive and not improving pregnancy loss. There is therefore an urgent need to find new therapy for EMs-related infertility and miscarriage and to restore and remodel their fertility. In current study, we found that PPD exerted anti-EMs activity and had the ability of fertility remodeling by ESR and PGR-mediated regulation of endometrial receptivity and inflammation response of peritoneal Mφ, and preventing pregnancy loss by increasing decidualization-associating genes expression, and promoting proliferation and function regulation dNK cells. Additionally, it almost no side effects on hepatotoxicity, nephrotoxicity and bone loss.\nThere are four main factors that affect the development of EMs, including interacting endocrine, pro-inflammatory, immunologic and proangiogenic processes  39 . Over-expression of estrogen is required in the proliferation of endometriotic lesions. Progesterone exerts as an anti-estrogen effect in the endometrium, in the way by inducing 17β-hydroxysteroid dehydrogenase 2 (HSD17B2) to produce more estrone to weaken the effects of estrogen  40 ,  41 . However, there is extremely low level of PGR in endometriotic tissue that led to progesterone resistance. This is the most important reason why progesterone has been ineffective for treatment of EMs. Here, we found that PPD could minimize the size and weight of ectopic lesions instant of changing the serum levels of E 2  but though downregulating the expression of  ESR1 ,  ESR2 , and upregulating  PGR  expression in eutopic endometrium.\nThe potential health effects of ginsenosides include immunomodulatory, anti-stress, anti-carcinogenic, anti-inflammatory, anti-allergic, anti-diabetic effects, and anti-hypertensive effects as well as anti-atherosclerotic and regulatory effects on blood pressure and metabolism  42 ,  43 . The structure of PPD is similar to steroid hormones, and it may bind to nuclear receptors, such as AR, ERs, glucocorticoid receptor, and PR to act its pharmacological effects  44 - 46 . Using RU486 blocked PR expression, PPD-induced endometrial receptivity genes' up-regulation was impaired, indicating that PR should be one of the potential regulator of PPD. Zhang and his colleagues  47  used fluorescence polarization assay to find that PPD could bind to human ER-α with moderate affinities, in other words, PPD acted as agonists of ER-α. The results of Swiss Target Prediction also suggested that AR, ER-α, ER-β and so on, were potential targets of PPD. Further analysis of PPI networks,  in vitro  and  in vivo  trials showed that PPD-mediated fertility improvement and remodeling of EMs should be dependent on the ERs, PR and other potential cellular sensory proteins. More efforts should be applied to explore the underlying mechanisms.\nIn EMs patients, the chronic pelvic inflammation contributes to the increased risk of infertility  48 , owing to physically blocking the fallopian tubes, dysfunction of uterine tubes, decreasing receptivity of endometrium, and hindering development of the oocyte and embryo  9 . During normal menstrual periods, endometrial stromal cells and glands will undergo periodic changes with changes in estrogen and progesterone. But overproduced estrogen and persisted progesterone resistance in EMs depart from this normal physiological process  49 . The imbalance of estrogen and progesterone leads to not only inflammatory microenvironment, but also impaired endometrial receptivity  50 . The expression of key implantation biomarkers, HOXA10, LIF, IGFBP1, among others, were seen to be disrupted in women with EMs  50 ,  51 . In this study, after the administration of PPD, the decreased endometrial receptivity was reversed. The expressions of endometrial receptivity or decidualization-associated genes were increased significantly before and after embryos implanting. For example, the expressions of  MMP2  and  MMP9  were significantly increased, and the expressions of collagens were significantly decreased after treatment with PPD, both  in vivo  and  in vitro . Additionally, PPD could diminish the percentage of peritoneal Mφ in the total cells of PF  in vivo , and reduce the pro-inflammatory cytokines, IL-12 and IFN-γ produced by macrophages significantly. According to the PPI network, these effects were probably due to the binding to different cellular sensory proteins of PPD. More researches are needed to explore the underlying mechanism. As a result, pregnancy rate and embryo implantation numbers were improved markedly. Interestedly, the embryo resorption rates of EMs mice undergoing the PPD treatment were decreased, suggesting that PPD has a potential therapy value against pregnancy loss induced by EMs.\nDuring the blastocyst implantation, many changes happened at the maternal-fetal interface, for incident, CD56 brigh CD16 low  NK cells were recruited by CXCR4 and CXCL12 secreted by embryonic trophoblast cells toa proper fetomaternal immune tolerance  52 . Uterus NK cells (uNK) are the predominant leukocyte populations in endometrium and uNK increase and contribute to remodeling the uterine arteries during early pregnancy  53 - 55 . Normally speaking, uNK cells have weakly cytotoxicity, unlike peripheral blood NK cells. Increased expressions of natural cytotoxicity receptors, like NKp 30, NKp40, and CD16 are related to many diseases, miscarriage and preeclampsia  56 . Successful implantation and pregnancy establishment largely depend on uNK cells for producing and secreting VEGF, TGF-β, CXCL10, LIF, which lead to the maturation of endometrial blood vessels, trophoblast invasion and successful placental development  57 ,  58 . In the present work, uNK cells from Ctrl EMs mice expressed higher CD16 and NKp30, compared with EMs mice treated by PPD. the population of dNK cells from the PPD group was increased, PPD could induce dNK cells to differentiate in favor of embryo implantation, owing to higher expression of Ki67, VEGF, TGF-β, and CXCL10. The PPI network between cytokines produced by uNK cells and top 15 PPD predicted cellular sensory proteins show that ESR1, ESR2, and PGR were the center of this regulatory network. However, the molecular mechanism is needed to further research.\nGnRHa used to be the first-line treatment, working by substantially suppressing systemic estrogen levels. The side effects of menopause, including bone loss, cannot be ignored  49 . Here, the EMs mice treated by GnRHa 0.5 ug/d*28 days have obvious symptoms of osteoporosis, with increasing number of osteoclasts, decreasing number of osteoblasts, and the result of Micro-CT showed there was an increase of bone loss. These side effects did not show in the PPD-treated EMs mice. In addition, no abnormalities were observed in the H&E staining and biochemical function testing of livers and kidneys. Treatment strategy of PPD in EMs seems to be more safety than GnRHa. Certainly, more researches are needed in the future.\nIn summary, as shown in Fig.  9 , we have proposed a new strategy for the treatment of EMs and EMs-related infertility and miscarriage. From multiple angles, PPD is primary confirmed as a safe and effective compound for the treatment of EMs, including reducing ectopic foci, suppressing inflammation response of peritoneal Mφ, promoting endometrial receptivity and decidualization, and increasing the proportion, tolerance and pro-angiogenesis phenotypes of dNK cells. These effects should be dependent on hormones receptors or sensory proteins (ESRs and PGR), and other cellular sensory proteins. More efforts are needed to find out the underlying molecular mechanism.\n\nSupplementary table S1.\nClick here for additional data file.","source_license":"CC0","license_restricted":false}