Transcriptomic profiling analysis of human endometrial stromal cells treated with autologous platelet‐rich plasma

Abstract Purpose To clarify the mechanisms of intrauterine platelet‐rich plasma (PRP) infusion that support embryo implantation in in vitro fertilization treatment. Methods Blood and endometrial samples were collected from four infertile women. Human endometrial stromal cells (HESCs) were cultured and passaged equally into four cell culture dishes in each patient. Two were treated with PRP twice, and the other two were treated with vehicle. Subsequently, two cultures with and without PRP were decidualized with 8‐bromoadenosine 3′,5′‐cyclic AMP and progesterone for 5 days. Results The gene expression in undifferentiated or decidualized HESCs with and without PRP was compared. In the microarray analysis, 381 and 63 differentially expressed genes were detected in undifferentiated and decidualized HESCs, respectively. In the undifferentiated HESCs, PRP was found to promote the gene expression associated with cell growth, tissue regeneration, proinflammatory response, and antibiotic effects. In decidualized HESCs, PRP was found to attenuate the gene expression involved in cell proliferation and inflammation by inhibiting the expression of phosphoinositide 3‐kinase signaling. Conclusions Platelet‐rich plasma regulates the reprogramming of cell proliferation and inflammation depending on menstrual cycle phases in an appropriate manner, suggesting that PRP has the potential to increase endometrial thickness in the proliferative phase and improve immune tolerance in the secretory phase.

into decidual cells via the stimulation of progesterone secreted from the corpus luteum. In this process, the decidualizing endometrium acquires receptivity to a semiallograft or a complete allograft embryo with optimal local angiogenesis and trophoblast invasion at implantation. 4 Ultrasonography is used in clinical in vitro fertilization (IVF) treatment to evaluate whether the endometrium of the patient is stable for implantation. A thin endometrium is reportedly one of the prognostic factors of a low chance of conceiving after embryo transfer. 5 A thin endometrium is primarily the result of intrauterine damage and adhesions after mechanical manipulations, including endometrial curettage and hysteroscopic surgery. The human endometrium is regenerated cyclically with the menstrual cycle because the endometrial stem cells contribute significantly to the proliferation and decidualization of the endometrium. 6,7 Therefore, the main target of treatment for a thin endometrium is the regeneration of mesenchymal stem cells, such as endometrial infusion of granulocyte colony-stimulating factor. 8,9 However, the efficacy of this therapy remains controversial. 10 Platelet-rich plasma (PRP) has been utilized in various clinical areas, including orthopedics, ophthalmology, and dermatology. [11][12][13] In the process of tissue repair, platelets are activated for the purpose of regeneration with inflammation, cellular migration, extracellular matrix remodeling, cell proliferation, differentiation, and angiogenesis. [14][15][16] Platelets also have antimicrobial activity with the secretion of antimicrobial peptides. 17 Thus, PRP is used at the site of tissue injury for repair and pain relief in various pathologies. In gynecology, administration of PRP has been recently introduced to treat impaired endometrial and ovarian functions in infertile women. 18 Many studies have reported the therapeutic effects of intrauterine PRP infusion with increasing endometrial thickness and chance of embryo implantation. [19][20][21][22][23][24][25] Thus, PRP is a widely used treatment for infertile women. However, the mechanisms underlying the improvement in endometrial receptivity remain poorly understood. In this study, the molecular influences of PRP treatment on human endometrial and decidualized cells were studied.  Table S1 presents the patients' characteristics. None of the patients had chronic endometritis (CE) resulting from CD138 immunostaining of the endometrium or abnormal complete blood count data. The diagnosis of CE was defined as five or more CD138positive cells in 10 random areas at ×10 magnification based on our previous studies. 26,27

| Endometrial and blood sampling
All participating subjects provided written informed consent before sample collection. Endometrial sampling was performed using an endometrial suction curette (Pipet Curet; Fuji Medical Corporation) in 7-10 days following the luteinizing hormone surge. On the same day, 60 ml of blood sample was collected slowly using an 18-gauge needle and transferred into a blood transfusion pack containing the anticoagulant citrate phosphate dextrose adenine (CPDA; Terumo Blood Bag CPDA, Terumo Corporation), which was mixed gently and kept in cold storage for 4-6 days until PRP treatment.

| PRP preparation
On the day of cell treatment with PRP, approximately 30 ml of blood was drawn from the blood transfusion pack and processed using a PRP preparation system (SmartPrep® 3; Terumo Bct. Inc.) according to the manufacturer's instructions. The whole blood sample was added to the first chamber, and the centrifuge was activated. The red blood cells and the PRP were separated. The PRP was automatically decanted into the second chamber and concentrated by centrifugation. After platelet-poor plasma was removed, 3 ml of the PRP was collected.
We conducted the experiments to reproduce the clinical protocol for PRP treatment in vitro. 19,20,[23][24][25] Endometrial cells were cultured until they achieved confluency in 75-cm 2 culture flasks at 37°C in 5% carbon dioxide and then passaged equally into four 1-well cell culture dishes (100 × 20 mm). After the HESCs were grown to approximately 80% confluence, a cell-free strip as the cell proliferation zone was created by scratching using a 200-ml pipette tip. Assuming clinical intrauterine PRP infusion, two cell culture dishes were treated and cultured with 1 ml of PRP each for 24 h (media containing 10% of PRP in total) on 1 and 3 days after endometrial scratching. The other two dishes were cultured with media without PRP. The 10% of PRP concentration in the culture media was determined based on previous clinical studies using 1 ml of PRP 19,20,22,23,25 and approximately 10 cm 3 of an average intrauterine volume in adult women with 57 mm of uterine length, 34 mm of width, and 8-10 mm of endometrial thickness. 30 Four days after scratching, two culture dishes with and without PRP treatment were maintained in DMEM/F12 without phenol red (Thermo Fisher Scientific) containing 2% (v/v) DCC-FBS and treated with 0.5 mM 8-bromoadenosine 3′,5′-cyclic adenosine monophosphate (8-bromo-cAMP) and 1 μM progesterone (P4) for decidualization. The other two culture dishes were cultured in media without 8-bromo-cAMP and P4. All culture samples were harvested 9 days after scratching (5 days of decidualization).

| Cell proliferation assay
To confirm the effects of PRP on cell proliferation, the images of primary cultures of HESCs were acquired at 400-fold magnification using an All-in-one Fluorescence Microscope (BZ-X800; KEYENCE Corporation) on the day of scratching, after 1 day of scratching (the day of PRP treatment), and after 3 days of scratching (2 days after PRP treatment). The images were modified into phase contrast images of the cell proliferation zone using Image J 1.53k software (Wayne Rasband and contributors, National Institutes of Health). The number of endometrial cells in the strip was then counted.

| RNA extraction and real-time quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from primary HESC cultures using the RNeasy plus mini kit (QIAGEN). cDNA was generated using the SuperScript II Reverse Transcriptase for RT-qPCR kit (Thermo Fisher Scientific) and performed template quantification using the 7500 fast RT-qPCR system (Thermo Fisher Scientific) with a dye layer, power SYBER Green PCR Master Mix (Thermo Fisher Scientific). RNA input variances were normalized against the levels of the housekeeping gene, L19, which encodes a ribosomal protein. All measurements were performed in duplicate or triplicate.  then subtracted from the sample. Altered transcripts were identified using a comparative method on the baseline transformed intensity values between the "control" and "sample" probes. The genes corresponding to the probes that had a change in intensity exceeding a ratio of 2.0 were considered as genes with a significant differential expression pattern. In parallel, an unpaired t-test with unequal variance (Welch's t-test) including false discovery rate filter was performed on the totality of the probes to compare the means of the two groups of replicates. Probes with p < 0.05 were considered to have significantly different signal value means. Gene ontology (GO) analysis was performed on the sets of probes that were common to both groups. The ontologies with a corrected p < 0.1 were considered significant. Z-score hierarchical clustering heatmaps were created using heatmap analysis software, Heatplus (annHeatmap2) of R package version 3.4.0.

| Statistical analysis
Results are reported as mean ± standard deviation or standard error of the mean. Statistical analysis was performed using one-way analysis of variance followed by the Wilcoxon signed-rank test in the cell proliferation array and the Mann-Whitney U-test in the PCR array of human PI3K-AKT signaling pathway, following normalization of the data with GraphPad Prism 5 (GraphPad Software Inc.). The level of significance was defined as p < 0.05.

| Proliferation of HESCs with or without PRP treatment
To examine cell proliferation of HESCs treated with or without PRP, a cell-free strip as the cell proliferation zone was created by endometrial scratching, and the migrated cells in the strip on 1 and 3 days after endometrial scratching (before and 2 days after PRP treatment, respectively) were counted ( Figure 1). The migrated cells had reached confluency in all strips on the third day after endometrial scratching. In HESCs without PRP treatment, the numbers of cells in the strips on the first and third days after scratching were 97.9 ± 16.3 and 460.0 ± 60.7 cells, respectively. In the HESCs treated with PRP, the cell counts before and after PRP treatment were 79.4 ± 11.9 and 704.4 ± 211.9 cells, respectively.
The number of cells treated with PRP after 3 days was significantly larger than that treated without PRP (p = 0.039). The periphery of the cells that underwent PRP treatment appeared whitish with high cell confluency ( Figure 1A, lower right panel). The growth rate of HESCs treated with PRP was higher than that of HESCs without PRP treatment (8.8 ± 2.1-fold and 4.8 ± 0.9-fold, respectively, p = 0.008; Figure 1B).

| Identification of genes affected by PRP in undifferentiated and decidualized HESCs
In order to identify the genes affected by PRP treatment in undif-  Table S2). Ten genes were identified in both undifferentiated and decidualized HESCs (Table S3) (Table 1). Table 3 shows the top 50 genes downregulated in undifferentiated HESCs upon treatment with PRP.

| PRP regulates the phosphoinositide 3-kinase/AKT signaling pathway in decidualized HESCs
In contrast to undifferentiated HESCs, the impact of PRP was limited with only 63 genes in the decidualized cells (Figure 2A).
PRP treatment identified key genes involved in the PI3K signaling pathway, including SERPINE2 (also referred to as protease nexin-1: PN1) and PTEN as upregulated genes and TLR4, DEPTOR, and

| DISCUSS ION
In this study, the molecular impacts of PRP treatment on the human endometrium in a primary culture were identified first. In the proliferative phase, the exposure of estrogen in the endometrium increases endometrial thickness and the linear growth of endometrial glands and angiogenesis, which are processes that are indispensable for pregnancy. PRP was found to strongly promote PRP also upregulated the genes involved in the beta chain of the T cell receptor (TRBV3-1, TRBV6-5, TRBV7-2, TRBV27, TRBC2, and TRBJ2-3), which is responsible for antigen recognition and activation of cellular immunity during bacterial infection. 40

ACK N O WLE D G E M ENTS
We thank all patients who participated in this study. This study received financial support from TERUMO Corporation, Tokyo, Japan. We also thank the Laboratory of Molecular and Biochemical Research, Biomedical Research Core Facilities, Juntendo University Graduate School of Medicine, Tokyo, Japan, for technical assistance.

CO N FLI C T O F I NTE R E S T
All authors have no conflicts of interest to declare relevant to this study.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The microarray data sets used in the present study were deposited in the Gene Expression Omnibus under the accession number of GSE213873 (https://www. ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=GSE21 3873).

E TH I C A L A PPROVA L
This study was approved by the local ethics committee of Juntendo University, Faculty of Medicine (No. 20-178) and Sugiyama Clinic F I G U R E 3 Platelet-rich plasma (PRP)-affected genes in the PI3K/AKT signaling pathway in decidualized human endometrial stromal cells (HESCs). In decidualized HESCs treated with and without platelet-rich plasma, inclusive real-time quantitative polymerase chain reaction (RTQ-PCR) array analysis of the PI3K/AKT signaling pathway demonstrated 10 significantly upregulated genes and 10 significantly downregulated genes, respectively, using a cutoff value of p < 0.05.

F I G U R E 4
Therapeutic effects of intrauterine platelet-rich plasma (PRP) infusion in proliferative and secretory phases. Intrauterine PRP infusion promotes tissue repair with transient inflammatory response, cell growth and migration, and antibiotic effect in undifferentiated human endometrial stromal cells, whereas cell proliferation and the inflammatory immune response are attenuated during decidualization of the endometrium. Depending on the menstrual cycle phases, PRP can regulate the reprogramming of inflammation and cell proliferation in an appropriate manner to support embryo implantation.
(No. 19-005). All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation and with the Helsinki Declaration of 1964 and its later amendments.

I N FO R M E D CO N S E NT
All recruited women provided written informed consent.

A N I M A L S TU D I E S
This article does not contain any study with animal participants that have been performed by any of the authors.