Decreased ANGPTL4 impairs endometrial angiogenesis during peri‐implantation period in patients with recurrent implantation failure

Abstract Insufficient endometrial angiogenesis during peri‐implantation impairs endometrial receptivity (ER), which contributes to recurrent implantation failure (RIF) during in vitro fertilization and embryo transfer (IVF‐ET). Angiopoietin‐like protein 4 (ANGPTL4) acts as a multifunctional secretory protein and is involved in the regulation of lipid metabolism and angiogenesis in various tissues including the endometrium. Herein, we found decreased ANGPTL4 expression in endometrial tissue and serum during peri‐implantation period in 18 RIF‐affected women with elevated uterine arterial impedance (UAI) compared with the pregnancy controls. ANGPTL4 and peroxisome proliferator‐activated receptor gamma (PPARγ) expression were up‐regulated upon decidualization on human endometrial stromal cells (HESCs). Rosiglitazone promoted the expression of ANGPTL4 in HESCs and human umbilical vein endothelial cells (HUVECs) via PPARγ. ANGPTL4 promoted the proliferation, migration and angiogenesis of HUVECs in vitro. Our results suggest that decreased abundance of ANGPTL4 in endometrial tissues impairs the endometrial receptivity via restraining endometrial angiogenesis during decidualization; while rosiglitazone‐induced ANGPTL4 up‐regulation in hESCs and HUVECs through PPARγ. Therefore, ANGPTL4 could be a potential therapeutic approach for some RIF‐affected women with elevated UAI.


| INTRODUC TI ON
Embryo implantation is a key process for continuous pregnancy.
About 1 in 5 pregnancies end in an early pregnancy loss, with aneuploidy being a significant single factor of early pregnancy failure, while about 50% of early pregnancy loss is due to implantation failure. 1 In addition, implantation failure remains a major limiting factor for assisted reproductive technology (ART). The continuous pregnancy rate is 20-30% in the therapy of in vitro fertilization and embryo transfer (IVF-ET) relatively, possibly less effective than normal human pregnancy. 2 In spite of the application of pre-implantation genetic diagnosis (PGD) and the transference of a single euploid blastocyst of good quality, the cumulative pregnancy rate after three in vitro fertilization (IVF) cycles has only improved to 40-55%. 3 Thus, recurrent implantation failure (RIF) due to impaired endometrial receptivity still remains a challenging and frustrating problem in IVF. 4 There are no strict criteria defined for RIF yet, but general consensus refers to a failure to achieve clinical pregnancy after transferring no less than four embryos of good quality in at least three fresh or frozen cycles in women under the age of 40. 5,6,7 Impaired endometrial receptivity (ER) is considered to be the leading cause of implantation failure in IVF. 8 The synchronization of decidualization and vascular processes during the window of implantation (WOI) period is essential for the development of a receptive endometrial environment for embryo implantation. 9 Decidualization of human endometrial stromal cells (hESCs) involves obvious morphological and functional differentiation, which includes edema of endometrial stromal, proliferation and differentiation of stromal cells, leucocyte invasion, vascular remodelling, enhancement of vascular permeability and angiogenesis. 10 Insufficient endometrial angiogenesis during WOI inevitably affect the establishment of appropriate endometrial receptivity, leading to embryo implantation failure. 11 Angiopoietin-like protein 4 (ANGPTL4) functions as a multifunctional secretory protein and is involved in the regulation of lipid metabolism, glucose homoeostasis, inflammation, angiogenesis and vascular permeability . 12 The transcriptional regulation of ANGPTL4 can be modulated by a number of factors including glucocorticoids, free fatty acids, peroxisome proliferator-activated receptors (PPARs) agonists, transforming growth factor-β (TGF-β) and hypoxia inducible factor-1α (HIF-1α). [12][13][14] Moreover, it has been reported that the activation of PPARγ promotes the expression of ANGPTL4 in trophoblast, thereby enhances placental angiogenesis. 15 However, the role of ANGPTL4 in endometrial angiogenesis during decidualization has not been reported yet.
Herein, we have detected decreasing ANGPTL4 abundance in serum and endometrial tissues of RIF-affected women with elevated uterine arterial impedance (UAI) during peri-implantation period. Moreover, we explored the effect of rosiglitazone (a PPARγ agonist) on the expression of ANGPTL4. The detailed functions of ANGPTL4 involved in decidualization were further conducted in immortal hESCs and human umbilical vein endothelial cells (HUVECs) cell lines.

| Experimental subjects
All patients in the study were recruited from the Center for

| Collection of endometrial tissues and blood samples
Peripheral blood and endometrial biopsies were obtained from 36 recruited patients on Day LH + 7. The follicle growth was traced by ultrasound monitoring (Prosound, Aloka, Japan) and urine luteinizing hormone (LH) detection in a natural cycle. The day on which a dominant follicle reached 18 mm, together with positive urinary LH was defined to be the day of LH 0; the window phase for implantation was defined as the day of LH + 7. Serum was taken after centrifugation of peripheral blood to determine the concentrations of ANGPTL4

| Immunohistochemistry
Localization of PPARγ and ANGPTL4 in endometrial tissues was performed by immunohistochemistry (IHC). In brief, the fixed endometrial paraffin sections obtained from the RIF and control groups were cut in 4μm section and treated with dimethylbenzene and alcohol.
Then the sections incubated in citric acid antigen retrieval buffer were heated in a microwave oven.

| Cell culture, in vitro decidualization and treatments
The immortalized HESC line, a kindly gift from Professor Haibin Wang of Xiamen University in China, has underwent the process of immortalization by transfection of telomerase (hTERT) using a retroviral system using pA317 hTERT-expressing cell line and is karyotypically, morphologically and phenotypically similar to the primary parent cells. 16

| Real-time PCR
According to the manufacturer's instructions, total RNA was ex- to ACTB were calculated and differences were quantified using the 2 -⊿⊿Ct method. 17

| Western blot analysis
Total protein was extracted using RIPA buffer (Beyotime, Shanghai,

| Enzyme-linked immunosorbent assay
According to the manufacturer's protocol, the concentrations of ANGPTL4 in the serum and culture medium were measured using an enzyme immunoassay kit (R&D systems, MN, USA) with a detecting range from 1.25 to 80 ng/mL. The serum and culture medium were performed a 2-fold dilution before detection. Each sample was run in duplicate.

| Small interfering RNA and transfection
Specific small interfering RNA (siRNA) was obtained from Biotend

| Cell proliferation assays in HUVECs
Cell proliferation ability was determined using the Cell Counting

| Wound-healing assays
HUVECs were incubated with DMEM/F12 supplemented with 10% charcoal-stripped FBS in six-well plates. After siRNA-mediated knockdown of ANGPTL4, HUVECs were treated with 10 μM rosiglitazone or not. The control groups were treated with 10 μM rosiglitazone or 100 nM rhANGPTL4 after transfecting with scrambled siRNA. A wound was made using a sterilized 200 µL pipette tip.
The scratch area between two sides of the wound was pictured and measured immediately (0 h) and again at 24 h and 48 h after wounding using the Image J 1.46r. Wound closure rates were calculated by subtracting the area of the wound at 24h or 48 h from that of the 0 h time point.

| Tube formation assay
Tube formation assay was performed to observing angiogenesis as follows: growth factor reduced Matrigel (BD Biosciences, USA) was

| Statistical analysis
All data were analysed by SPSS statistical software (IBM 25.0, USA).
The values are shown as the means ± SD. Differences among the groups were compared for significance using paired or unpaired student's t test or one-way ANOVA. Correlation between the variables was performed using Pearson analysis. All experiments were repeated at least three times. Differences between groups were considered as statistically significant at P < .05. uterine arterial pulsatility index (PI), uterine arterial resistance index (RI) and uterine artery S/D, were significantly higher in the RIF group than those in the control group (P < .05) ( Table 1).

| Decreased abundance of ANGPTL4 in endometrial tissues and serum of RIF patients
Immunochemical staining of endometrial tissue showed that both PPARγ and ANGPTL4 were present in endometrial cavity epithelium, glandular epithelium, vascular endothelium and endometrial stromal cells ( Figure 1A). We found that the mRNA levels of ANGPTL4 and PPARγ in the RIF group (n = 18) in endometrial tissues were significantly lower than those in the control group (n = 18).
Meanwhile, mRNA levels of endometrial decidualization biomarkers including IGFBP1and PRL were significantly reduced, indicating impaired decidualization in the RIF group ( Figure 1B). In addition, protein levels of PPARγ and ANGPTL4 were significantly reduced in the RIF group compared with the control group ( Figure 1C, D).
Consistently, the concentrations of ANGPTL4 in the serum of the RIF group were significantly lower than those of the control group ( Figure 1E). Furthermore, correlation analysis showed a positive correlation between the abundance of ANGPTL4 and PPARγ in all subjects (n = 36) (Figure 1F), indicating a functional interaction between PPARγ and ANGPTL4 in vivo.

| PPARγ and ANGPTL4 both increased upon decidualization of hESCs in vitro
To analyse the expression of PPARγ and ANGPTL4 during decidualization, we used cAMP and P 4 to induce decidualization on hESCs in vitro. These cells were larger and plumper than those untreated on day 4 ( Figure 2A). Meanwhile, the mRNA levels of the decidualization markers IGFBP1 and PRL increased significantly, indicating that decidualization was successfully performed in vitro ( Figure 2B). During decidualization, the abundance of PPARγ and ANGPTL4 mRNA was significantly up-regulated compared to untreated cells ( Figure 2C).
Furthermore, western blotting demonstrated that both PPARγ and ANGPTL4 protein abundance increased after decidualization ( Figure 2D). Meanwhile, increased ANGPTL4 in culture medium was detected by ELISA on the 4th day of decidualization ( Figure 2E).

| PPARγ mediates rosiglitazone-induced upregulation of ANGPTL4 in hESCs and HUVECs
To analyse the role of the PPARγ agonist rosiglitazone in the expres-

| ANGPTL4 mediates rosiglitazone-induced HUVECs proliferation
HUVECs were transfected with small interfering RNA for 48 h, the abundance of ANGPTL4 was significantly reduced regardless of mRNA levels, protein levels or concentrations in cell culture medium ( Figure 5A-C). The effect of ANGPTL4 on HUVECs proliferation was detected by CCK-8 assay. Both rosiglitazone and rhANGPTL4 significantly promoted cell proliferation, while the effect of rosiglitazone was partially counteracted by the siRNA-mediated knockdown of ANGPTL4 ( Figure 5D). Taken together, these results demonstrate that ANGPTL4 plays an important role in HUVEC proliferation induced by the PPARγ agonist.

| ANGPTL4 is important for rosiglitazoneinduced HUVECs migration
Wound-healing assays were conducted in HUVECs to explore the role of ANGPTL4 and rosiglitazone in cell migration. Cell migration was compared at 24 and 48 h. Rosiglitazone and rhANGPTL4 had similar effects on the migration of HUVECs, while depletion of ANGPTL4 revoked the effect of rosiglitazone ( Figure 6A, B). These observations indicated that PPARγ agonist-induced cell migration is at least partially related to ANGPLT4.

| ANGPTL4 is involved in rosiglitazone-induced angiogenesis
Tube formation assay was performed to investigate the role of rosigl-

| D ISCUSS I ON
Recurrent implantation failure is generally considered to be closely related to embryo quality and abnormal endometrial receptivity. 19 The UAI is closely related to the blood supply of endometrium and is a useful tool for identifying impaired uterine circulation, which plays an important role in the establishment of endometrial receptivity. 20,21 In addition, previous studies have reported uterine perfusion could elevate UAI and impair uterine receptivity, leading to recurrent pregnancy loss (RPL). 20 Uterine artery blood flow increases progressively during the luteal phase, with the highest flow encountered in the period that temporally coincides with the implantation window. 22 Accordingly, we conducted uterine arterial blood flow measuring on women undergoing IVF and noted that the UAI of some RIF patients was relatively elevated. Notably, previous studies have found that the uterine artery PI and subendometrial blood flow RI were higher in RIF patients than controls, meanwhile the waveform pattern of uterine artery altered in patients reporting RIF. 23 However, in the current study, the recruited women who PRL mRNA levels in peri-implantation endometrial tissues, suggesting impaired decidualization in RIF patients. Insufficient endometrial angiogenesis is associated with increased resistance to endometrial blood flow from uterine artery. Previous study revealed a strong link between endometrial vascularity and pregnancy rate, and inadequate endometrial vascularization was associated with pregnancy loss. 26 Thus, the synchronicity of decidualization and vascularization process is essential for the establishment of endometrial receptivity.
ANGPTL4 is located on chromosome 19p13.3 and consisted of seven exons which encode a 406-amino-acid secretory glycoprotein with a molecular mass of 45-65 kDa. 13 Many studies have illustrated that ANGPTL4 is a multifunctional factor involved in lipid metabolism, wound healing and angiogenesis. 27 while COX2-derived prostaglandins target the VEGF system, which is important for uterine angiogenesis during implantation and decidualization. 38 In addition, endometrial angiogenesis during pregnancy facilitates embryo implantation and improves pregnancy success rate. 23 In the current study, to further explore the role of ANGPTL4 in angiogenesis, cell proliferation assay, wound-healing assays and tube formation assay were performed on HUVECs after siRNA-mediated knockdown of ANGPTL4. The results showed that ANGPTL4 can enhance the ability of cell proliferation, migration and angiogenesis on HUVECs.
In summary, we found that ANGPTL4 abundance in endometrial tissues and serum were reduced in RIF women with elevated UAI, which was significantly correlated with PPARγ expression.