Mechanism of human chorionic gonadotropin in endometrial receptivity via the miR-126-3p/PI3K/Akt/eNOS axis.

The study was approved by the Ethics Committee of The Reproductive Medicine Center of the First Hospital of Lanzhou University. All animal procedures were in concert with the guidelines for the care and use of laboratory animals of the China Animal Welfare Committee. Considerable effort was made to minimize the animal number and their suffering. The specific‐pathogen‐free adult ICR mice (12 males weighing 29–32 g and 24 females weighing 26–28 g, all aged 8–10 weeks) were provided by Vital River [SCXK (Beijing) 2017–0033, Beijing, China]. The mice were housed in the animal experiment center of The Reproductive Medicine Center of the First Hospital of Lanzhou University at 22–24°C with a relative humidity of 75%–85%, 12 h day‐night cycles, and free access to food and water. All female mice were kept in cages with male mice (ratio 2:1) at 7:00 pm and vaginal plugs were examined at 7:00 am the following day. The presence of vaginal plugs was regarded as evidence of mating, and this day was defined as day 0.5 of pregnancy. The embryo implantation dysfunction (EID) mouse model was induced by intrauterine perfusion of mifepristone via bilateral uterine horns at 9:00 am on day 3.5 of pregnancy. The EID mice were prepared as follows 23 : each mouse was subcutaneously injected with 0.1 ml mifepristone (dissolved in propanediol at 0.08 mg/ml, Hubei Gedian Humanwell Pharmaceutical Co., Ltd, Wuhan, Hubei, China). After anesthetizing the mice with 0.3% pentobarbital sodium (RWD Life Science, Shenzhen, China) on the morning of day 3.5 of pregnancy, we administrated 2.5 μl normal saline into the uterus of some mice through each uterine horn using a microsyringe and similarly injected 2.5 μl hCG into the uterus of EID mice. The overall operation time for a mouse was no more than 20 min and the injection time for unilateral uterine horn was approximately 5 s. At 9:00 pm on the 4th day of pregnancy, all female mice were euthanized by intraperitoneal injection of 100 mg/kg pentobarbital sodium, next the number of mouse uterine embryos was recorded, and later the endometrium was collected for following experimentation. Following the induction of mating and EID in mice, 18 pregnant mice were selected and equally assigned into three groups: normal pregnancy (NP) group (normally treated mice), EID group (EID mice induced by mifepristone), and hCG group (EID mice treated with 2.5 μl hCG). After 4‐day pregnancy, the mice were euthanized via intraperitoneal injection of pentobarbital sodium (≥100 mg/kg) to collect tissues. Next, the tissues were sterilized by immersing in 75% alcohol and transferred to an ultra‐clean bench, and then the uterine horn was quickly removed. The uterine horn with implantation sites was selected, kept on a sterilized dish, and rinsed with sterile ice‐cold phosphate‐buffered saline (PBS) three times. The rinsed uterus was placed on another sterilized dish and the uterine cavity was rinsed with sterile ice‐cold PBS. Thereafter, the flushing fluid was observed under an inverted microscope and we found that the blastocyst had escaped from the zona pellucida at this time. The uterus after washing the uterine cavity was put in another sterilized dish. Later, we used the ophthalmic forceps to clamp unilateral uterine horn and used the treated 4‐gauge needle to gradually squeeze and roll from the proximal fallopian tube to the cervix until the endometrium was completely slipped out in a long strip. Rabbit anti‐mouse CD105 (1:50, ab221675, Abcam, Cambridge, UK) antibody was used as endometrial microvessel density (MVD) markers. After adding the biotinylated secondary antibody (anti‐rabbit), horseradish streptavidin working solution was added and the 2,4‐diaminobutyric acid detection was performed. Microvascular staining in each section was observed under low power lens (×100) and the maximum microvascular staining regions were determined. Three regions with the highest microvessel staining were selected for microvessel counting at high magnification (×400) and the mean value was calculated. Single cells or clusters of brown‐stained endothelial cells would be regarded as one microvessel as long as they were separated from adjacent interstitial cells or connective tissues. The microvascular branch could also be considered an independent microvessel if they were not structurally connected. Neither the formation of lumen structures nor the presence of intraluminal erythrocytes was a criterion for microvascular formation. However, a lumen with a diameter greater than eight erythrocytes or a large vessel on the muscle layer, was not considered as the microvessel. Two sections per animal were examined by two uninformed analysts under a bright field microscope (Olympus, Tokyo, Japan). The mean value of CD105 in each group was used for statistical analysis. Human endometrial epithelial cells (EECs) provided by Mingjing Biology (Shanghai, China) were cultured in the RPMI‐1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific), 100 U/ml streptomycin, and 100 U/ml penicillin in an incubator with 5% CO 2 at 37°C. Once reaching 85% cell confluence, cells were detached with 30% trypsin and passaged. Cells in the exponential growth phase at the 3rd passage were collected for subsequent experiments. The cells were grouped as follows: EEC group, EEC + hCG group (cultured with 20 U/ml hCG), 24 EEC + hCG + inhi‐NC group (based on the EEC + hCG group, cells were transfected with inhibitor‐NC), and EEC + hCG + inhi‐miR group (based on the EEC + hCG group, cells were transfected with miR‐126‐inhibitor). miR‐126‐inhibitor and inhibitor‐NC (miRNA‐inhibitor 50 nM) were designed and synthesized by Yingbiotech (Shanghai, China). Cell transfection was performed utilizing Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) for 48 h. The extraction of total RNA from tissues and cells was conducted using TRIzol reagents (Thermo Fisher Scientific). The measurement of RNA concentration was performed using the NanoDrop‐1000 (Thermo Fisher Scientific). The RNA was reverse‐transcribed into cDNA using ReverAid First‐Strand cDNA kits (Thermo Fisher Scientific) in combination with primers. U6 and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) were used as internal controls to normalize the expression of miR‐126‐3p and PIK3R2, respectively. After the reverse transcription reaction, the levels of miR‐126‐3p and PIK3R2 were detected using the LightCycler 480 SYBR‐Green I Master and LightCycler 480 Real‐Time PCR systems (both from Roche Applied Science, Indianapolis, IN, USA). The relative expression was computed by the 2 −ΔΔCt method. Relevant primer sequences (Table  1 ) were designed and synthesized by Yingbiotech. Primer sequences Note : PIK3R2, phosphoinositol‐3 kinase regulatory subunit 2; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase. Total proteins from tissues and cells were homogenized using radio‐immunoprecipitation assay lysis buffer and the protein concentration was determined by bicinchoninic acid protein reagents (Sangon Biotech, Shanghai, China). The Western blot was conducted as previously described. 25 The primary antibodies were as follows: anti‐CD105 (1:50, ab49228, Abcam), anti‐PIK3R2 (1:1000, ab131067, Abcam), anti‐PI3K p110 α (1:1000, ab40776, Abcam), anti‐p‐PI3K p85 α (1:500, ab182651, Abcam), anti‐Akt (1:1000, ab179463, Abcam), anti‐p‐Akt (1:800, ab38449, Abcam), anti‐eNOS (1:1000, ab252439, Abcam), anti‐p‐eNOS (1:1000, ab215717, Abcam), and anti‐VE Cadherin (1:1000, ab205336, Abcam). Horseradish peroxidase‐labeled goat anti‐rabbit IgG H&L (1:20000, ab97051, Abcam) was the secondary antibody. GAPDH antibody acted as the internal reference. The Bio‐Rad digital image system (Bio‐Rad, Hercules, CA, USA) was used to photograph and analyze the obtained pictures. The gray value of target bands was analyzed using Image J software (NIH, Bethesda, MD, USA). EECs were put into 24‐well plates at 1 × 10 5 cells/well. Firstly, the bioinformatics software Starbase ( http://starbase.sysu.edu.cn/index.php ) predicted the binding sites of miR‐126‐3p and PIK3R2. Later, the complementary binding sequences and mutation sequences of miR‐126‐3p and PIK3R2 were amplified and cloned into the pmiR‐GLO luciferase vectors (Promega, Madison, WI, USA) to construct the PIK3R2‐wild‐type (wt) plasmid and the corresponding PIK3R2‐mutant (mut) plasmid. Thereafter, miR‐126‐3p mimics (miR‐mimics) or miR‐126‐3p negative control (NC; RiboBio, Guangzhou, Guangdong, China) were co‐transfected with the reporter plasmids into EECs using Lipofectamine 2000 (Invitrogen), respectively. After 48 h, luciferase activity was measured using dual‐luciferase assay kits (Promega). The confluent cultured EECs were detached using trypsin and suspended in RPMI‐1640 medium encompassing 10% FBS. The cells (1 × 10 4 /cm 2 ) were introduced into 96‐well plates. Following each treatment, the viable cells and proliferation rate were examined by CCK‐8 (Dojindo, Kumamoto, Japan). 26 CCK‐8 (10 μl) solution was added to each well. After every 24 h, the optical density (OD) value at 450 nm was measured on a microplate reader. The OD value at 450 nm was proportional to the number of viable cells and cell proliferation. Cells were subjected to EdU detection using Cell‐Light EdU Apllo 567 in vitro imaging kits (RiboBio). The cells were firstly introduced into 96‐well plates at 2 × 10 3 cells/well, followed by incubation with 50 μm EdU for 2 h at 37°C. Subsequently, the cells were fixed with 4% formaldehyde for 30 min, soaked in 0.5% Triton X‐100 for 20 min, and then stained with Apollo and Hoechst, followed by observation under a fluorescence microscope (Nikon, Tokyo, Japan). The EdU‐positive cells were calculated as the following formula: EdU addin cells/Hoechst‐stained cells × 100%. Statistical analysis and plotting of data were conducted utilizing SPSS 21.0 (IBM Corp. Armonk, NY, USA) and GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA) software. Independent sample t test was used for comparisons between two groups. Mann–Whitney test was carried out for comparison between two groups. Kruskal‐Wallis test was adopted for data comparison among multiple groups, followed by Dunn's test. The p  < 0.05 indicated statistical significance.

To interrogate the role and regulatory mechanism of hCG in EID mice, we established mouse models of EID according to previous research, and then EID mice were treated with 2.5 μl hCG. Firstly, the statistical results revealed that the EID group had a lower number of uterine embryos than the NP group, whereas the hCG group had a higher number of embryos than the EID group (all p  < 0.05) (Figure  1A ). Subsequently, we detected the levels of cadherin CD144 and CD146 in mouse endometrium by Western blot, which indicated that CD144 and CD146 were significantly lowered in the EID group compared with the NP group, but elevated in the hCG group compared with the EID group (all p  < 0.05) (Figure  1B ). Afterwards, CD105 levels were examined using immunohistochemistry to assess the endometrial MVD, and the results unraveled that CD105 was markedly diminished in the EID group compared with the NP group, indicating that the MVD was significantly decreased; relative to the EID group, CD105 levels were prominently raised in the hCG group (all p  < 0.05) (Figure  1C ). Briefly, the aforesaid results indicated the improving effect of hCG on endometrial receptivity in EID mice. hCG improved endometrial receptivity in EID mice. (A) The number of mouse uterine embryos; (B) Levels of cadherin CD144 and CD146 in the endometrium of mice measured by WB; (C) Expression levels of CD105 determined by immunohistochemistry. N  = 6, data were presented as mean ± standard deviation (SD). Kruskal‐Wallis test was used for comparisons among multiple groups, and Dunn's multiple comparisons test was used for post hoc analysis. * p  < 0.05. PIK3R2 is a potential target gene of miR‐126, which is identified as a suppressor of the PI3K/Akt pathway and can regulate proliferation and apoptosis in multiple cell types. 27 To elucidate the expression of miR‐126‐3p and PIK3R2 in EID mice and the regulatory mechanism of hCG on miR‐126‐3p and PIK3R2, we examined the expression of miR‐126‐3p and PIK3R2 mRNA in the endometrium of mice by RT‐qPCR. The results revealed that the EID group exhibited notably lower miR‐126‐3p expression and higher mRNA level of PIK3R2 than the NP group, while the hCG group exhibited higher miR‐126‐3p expression and lower mRNA level of PIK3R2 than the EID group (all p  < 0.05) (Figure  2A,B ). Additionally, the WB assay indicated that the PIK3R2 protein level was remarkably elevated in EID mice compared with the NP group, but diminished in the hCG group compared with the EID group (all p  < 0.05) (Figure  2C ). Taken together, hCG enhanced miR‐126‐3p expression and inhibited PIK3R2 expression in EID mice. hCG promoted miR‐126‐3p expression and restrained PIK3R2 expression in EID mice. (A) miR‐126‐3p expression detected by RT‐qPCR; (B) mRNA level of PIK3R2 detected by RT‐qPCR; (C) PIK3R2 protein level detected by WB. N  = 6, data were shown as mean ± SD. Kruskal‐Wallis test was applied for comparisons, followed by the Dunn's test. * p  < 0.05. In addition, the targeted binding sites of miR‐126‐3p and PIK3R2 were predicted by Starbase database (Figure  3A ). Subsequent dual‐luciferase assay unveiled that the luciferase activity of EECs co‐transfected with PIK3R2‐wt and miR‐126‐3p mimics plasmids was significantly reduced ( p   0.05) (Figure  3B ), indicating the binding relationship between miR‐126‐3p and PIK3R2. Briefly, PIK3R2 was a downstream target of miR‐126‐3p. miR‐126‐3p targeted PIK3R2. (A) Targeted binding sites between miR‐126‐3p and PIK3R2 predicted by Starbase; (B) Targeted binding of miR‐126‐3p to PIK3R2 validated by the dual‐luciferase assay. Cell experiments were repeated three times. Data were exhibited as mean ± SD. The Mann–Whitney test was performed for comparisons between two groups. * p  < 0.05. To investigate whether hCG promotes EEC proliferation via the miR‐126‐3p/PIK3R2 axis, EECs were cultured in vitro and treated with hCG and miR‐126‐3p inhibitor. RT‐qPCR and WB detection showed that relative to the EEC group, miR‐126‐3p was markedly upregulated and the mRNA and protein levels of PIK3R2 were evidently downregulated in the EEC + hCG group; compared with the EEC + hCG + inhi‐NC group, miR‐126‐3p was clearly lowered and PIK3R2 levels were prominently increased in the EEC + hCG + inhi‐miR group (all p  < 0.05) (Figure  4A–C ). The CCK‐8 and EdU experiments illustrated that cell proliferation was raised in the EEC + hCG group compared with the EEC group, whereas decreased in the EEC + hCG + inhi‐miR group compared with the EEC + hCG + inhi‐NC group (all p  < 0.05) (Figure  4D,E ). The crosstalk between PI3K and Akt/eNOS has been extensively studied and demonstrated to be indispensable in maintaining vascular function. 28 Therefore, we further detected the PI3K/Akt/eNOS axis‐related protein levels by WB, which indicated that the levels of p‐PI3K p85 α/PI3K p110 α, p‐Akt/Akt, and p‐eNOS/eNOS were elevated in the EEC + hCG group compared with the EEC group, but diminished in the EEC + hCG + inhi‐miR group compared with the EEC + hCG + inhi‐NC group (all p  < 0.05) (Figure  4F ). Altogether, hCG promoted EEC proliferation via the miR‐126‐3p/PIK3R2/PI3K/Akt/eNOS axis. hCG facilitated EEC proliferation through the miR‐126‐3p/PIK3R2/PI3K/Akt/eNOS axis. (A) Expression of miR‐126‐3p detected by RT‐qPCR; (B‐C) Expression of PIK3R2 detected by RT‐qPCR and WB; (D) Cell proliferation assessed by CCK‐8; (E) Cell proliferation assessed by EdU assay (red fluorescence indicated proliferative cells; blue fluorescence indicated nuclear staining; and Merge indicated combined picture); (F) Expressions of p‐PI3K p85 α/PI3K p110 α, p‐Akt/Akt, and p‐eNOS/eNOS detected by WB and their ratios calculated. Cell experiment was repeated three times. Data were presented as mean ± SD. Kruskal‐Wallis test was adopted for comparisons among multiple groups, followed by the Dunn's test. * p  < 0.05. Subsequently, we used WB assay to detect PI3K/Akt/eNOS pathway‐related protein levels in mice to further verify the action of hCG in vivo. The results unraveled that the levels of p‐PI3K p85 α/PI3K p110 α, p‐Akt/Akt, and p‐eNOS/eNOS were significantly reduced in the EID group relative to the NP group, but prominently elevated in the hCG group compared with the EID group (all p  < 0.05) (Figure  5 ). In summary, hCG activated the PI3K/Akt/eNOS pathway in EID mice. hCG activated the PI3K/Akt/eNOS pathway in EID mice. WB assay detected the levels of p‐PI3K p85 α, PI3K p110 α, p‐Akt, Akt, p‐eNOS, and eNOS, and the ratios of p‐PI3K p85 α/PI3K p110 α, p‐Akt/Akt, and p‐eNOS/eNOS were calculated. N  = 6, data were expressed as mean ± SD. Kruskal‐Wallis test was used for comparisons among multiple groups, followed by the Dunn's tests. * p  < 0.05.

Approximately 13% of the worldwide population has difficulty in conceiving owing to diverse reasons, and endometrial receptivity and thickness are pivotal in achieving pregnancy. 29 Administration of hCG before embryo transfer exerts a predominant elevation in clinical pregnancy rates. 30 Intrinsically, embryo implantation is a complicated process involving numerous regulators, including several miRNAs. 31 This study probed into the mechanism of hCG in endometrial receptivity through the miR‐126‐3p/PIK3R2/PI3K/Akt/eNOS axis. Cadherins are imperative in the attachment of the blastocyst to the endometrium and this process is known as endometrial receptivity; specifically, CD146 and CD9 are vital adhesion molecules that exert a role in embryo implantation and their levels are downregulated in endometrial stromal cells from infertile women. 32 Equally importantly, reduction in endometrial MVD might result in insufficient microcirculation and changes in the microenvironment that accordingly influence endometrial receptivity by lowering the possibility of blastocyst implantation, and CD105 is a marker for endometrial MVD. 33 Firstly, we established EID mouse models by injecting mifepristone to explore the effect of hCG and then observed that hCG administration increased the uterine embryos and levels of CD144, CD146, and CD105 in EID mice. Consistently, hCG can regulate cytokine generation by endometrial cells to potentiate endometrial receptivity and mediate the trophoblast invasion, embryo adhesion, and immune microenvironment. In brief, our findings indicated that hCG ameliorated endometrial receptivity in EID mice. Based on the stability, sensitivity, conservativeness, and easy availability, extracellular miRNAs are identified to be effective noninvasive biomarkers in assessing endometrial receptivity and embryo viability. 10 miR‐126‐3p, a prototype of an endothelial miRNA, imposes protective functions on endothelial cells and regulates the proliferation of glycogen trophoblasts, which is downregulated in preeclamptic tissues. 34 Moreover, PIK3R2, a target gene of miR‐126, exerts a known effect on endothelial cell signaling and vascular function. 35 Hence, we detected the levels of miR‐126‐3p and PIK3R2 and found that hCG elevated miR‐126‐3p expression and restrained PIK3R2 expression both in EID mice and in vitro EECs. Interestingly, several miRNAs are differentially‐expressed in the endometrium of infertile patients with negative and positive β‐hCG results. 36 Similarly, Menon B et. al showed significantly upregulated miR‐122 in the ovary after hCG treatment. 37 Likewise, consistent with the previous research, 38 our results noted that miR‐126‐3p targeted PIK3R2 and PIK3R2 level was raised after miR‐126‐3p inhibition. Moreover, in vitro studies unveiled that EEC proliferation was enhanced upon hCG treatment but weakened after miR‐126‐3p inhibition. hCG is classically acknowledged for its role in stimulating trophoblast invasion and functional differentiation. 38 Compelling evidence supports that miR‐126 promotes endothelial cell proliferation, migration, survival, and angiogenesis via downregulating PIK3R2 and is related to MVD increase and Akt activation in the placenta. 35 Our findings first revealed the regulation of hCG on miR‐126‐3p and PIK3R2 in EID mice. More notably, the PI3K/Akt/eNOS pathway is inhibited in EID mice and Tiaojing Cuyun Recipe could improve fertility outcomes and endometrial receptivity via activation of this pathway. 39 Herein, we measured the PI3K/Akt/eNOS axis‐related protein levels. The results unveiled that the phosphorylation of PI3K/Akt/eNOS pathway‐associated proteins was enhanced by hCG treatment, but lowered by miR‐126‐3p inhibition. Similarly, miR‐126 overexpression augments NO production of endothelial progenitor cells via activation of the PI3K/Akt/eNOS pathway. 40 Collectively, hCG facilitated EEC proliferation via the miR‐126‐3p/PIK3R2/PI3K/Akt/eNOS axis. Further in vivo studies also supported that hCG activated the PI3K/Akt/eNOS pathway in EID mice. To conclude, the study creatively investigated the regulation of hCG on angiogenesis‐related gene miR‐126‐3p and its target PIK3R2 and the PI3K/Akt/eNOS pathway. This study is valuable for elucidating the miRNA‐regulated processes and identifying hCG‐mediated targets of miR‐126‐3p to enrich and refine the studies about the mechanism of hCG in promoting endometrial angiogenesis and improving endometrial receptivity. However, this study only confirmed that hCG promoted EEC proliferation by activating the PI3K/Akt/eNOS pathway through the miR‐126‐3p/PIK3R2 axis in animal experiments and lacked clinical validation. Likewise, we did not identify the NO expression, a downstream product of the PI3K/Akt/eNOS pathway. The dosage of hCG used in animal experiments was based only on the dosage used in the studies of Chen J et al., and the dosage used in cell experiments was referred to the Shao et al.'s studies. 24 However, we did not investigate the optimal dosage of hCG in vitro and in vivo experiments. Future studies shall explore the optimal dosage of hCG and refine the mechanism of the entire signaling pathway. Moreover, other downstream target genes of miR‐126‐3p and relevant pathways mediated by endometrial receptivity‐related miRNAs are worthwhile investigating.

Endometrial receptivity is conceived as a hormonally limited period during which the endometrial tissue obtains a transient functional status that allows embryo implantation and pregnancy initiation. 1 Embryo implantation can be divided into three stages: apposition, adhesion, and invasion. 2 More in detail, during blastocyst apposition, trophoblast cells could adhere to receptive endometrial epithelial cells, whereas cell adhesion molecules (consisting of selectins, integrins, cadherins, and immunoglobulins), usually glycoproteins, manipulate cell–cell adhesion. 3 Importantly, cadherins are closely related to endometrial receptivity. 4 Receptive endometrium is imperative to successful embryo implantation and the normal implantation process represents the foundation of a healthy pregnancy; by contrast, aberrant receptivity contributes to numerous reproductive problems, including infertility, miscarriage, and preeclampsia. 5 However, understanding of endometrial receptivity remains incomplete to date. Human chorionic gonadotropin (hCG), a complex glycoprotein with approximately 37 kDa in molecular mass, is produced by trophoblast cells, which represents a molecular message released by the pre‐implanting embryo to mediate the implantation site and warrants the timely initiation of nidation process. 6 Interestingly, intrauterine infusion of hCG prior to embryo implantation may improve the chemokine‐induced embryo‐endometrial dialogue and potentiate angiogenesis and immune responses, and hCG infusion before embryo transfer may clinically raise the pregnancy rate in individuals with repeated implantation failure. 7 Meanwhile, hCG might improve endometrial receptivity by upregulating HOXA10, thereby facilitating embryo implantation. 8 The aforementioned studies have indicated the ameliorative effect of hCG on endometrial receptivity. On a separate note, microRNAs (miRNAs) can function as epigenetic regulators of embryo implantation and endometrial receptivity via post‐transcriptional modifications. 9 Moreover, miRNAs exert vital roles in female reproduction by mediating oogenesis, fertilization, implantation, and finally placentation, while dysregulation of miRNAs is implicated in reproductive disorders, including endometriosis and polycystic ovarian syndrome. 10 Moreover, preceding evidence supports that hCG and progesterone could alter endometrial angiogenesis by affecting the expression of VEGF, 11 and meanwhile hormones further alter the distribution of endometrial miRNAs. 12 Several existing studies have reported different functions of miRNAs in uterine receptivity: for instance, miR‐200c may impair uterine receptivity and be applied as a biomarker for clinical applications. 13 miR‐543 is associated with endometrial receptivity and its downregulation might affect embryo implantation. 14 Additionally, miR‐126 regulates the expressions of numerous genes such as Spred‐1, SDF‐1, VCAM‐1, HoxA9, v‐Crk, EGFL‐7, and VEGF, thereby regulating vascular development, neovascularization, and vascular inflammation, 15 and in particular, VEGF expression is correlated with endometrial receptivity. 16 Nevertheless, the association between miR‐126‐3p and endometrial receptivity has not been reported, hence we speculated that hCG may regulate endometrial receptivity through miR‐126‐3p. Intriguingly, miR‐126 directly inhibits the negative regulator of the VEGF pathway by targeting phosphoinositol‐3 kinase regulatory subunit 2 (PIK3R2), thus modulating angiogenic signaling and ensuring vascular integrity. 17 The highly expressed miR‐126‐3p can promote angiogenesis by targeting and inhibiting PIK3R2 and activating the phosphatidylinositol‐3 kinase (PI3K)/protein kinase B (Akt) pathway. 18 PI3K is a dimer composed of a variety of the corresponding regulatory subunit and catalytic subunit, and importantly, excessive accumulation of PI3K p110 α/p85 α is tightly associated with the overgrowth of the fetus. 19 The substantial function of angiogenesis in endometrial maturation has been alluded. 20 More importantly, elevated permeability has changed the functional endometrium and makes it receptive to embryo implantation, 21 and the proper endometrial vascular maintenance and development at embryo implantation is essential for successful pregnancy. 22 Based on these findings, we hypothesized that miR‐126‐3p might regulate endometrial receptivity by targeting the PI3K/Akt/endothelial nitric‐oxide synthase (eNOS) pathway. Herein, this study investigated the action of hCG in endometrial receptivity via the miR‐126‐3p/PI3K/Akt/eNOS axis, with the hope to seek novel and effective diagnostic and therapeutic targets for successful embryo implantation.

All authors declare that there is no conflict of interests in this study.