High stretch cycling inhibits the morphological and biological decidual process in human endometrial stromal cells

Abstract Purpose Subendometrial myometrium exerts wave‐like activity throughout the menstrual cycle, and uterine peristalsis is markedly reduced during the implantation phase. We hypothesized that abnormal uterine peristalsis has an adverse effect on the endometrial decidualization process. We conducted an in vitro culture experiment to investigate the effect of cyclic stretch on the morphological and biological endometrial decidual process. Methods Primary human endometrial stromal cells (HESCs) were isolated from hysterectomy specimens and incubated with or without 8‐bromo‐cyclic adenosine monophosphate (8‐br‐cAMP) and medroxyprogesterone acetate (MPA) for 3 days. After decidualization, cultures were continued for 24 hours with or without cyclic stretch using a computer‐operated cell tension system. Results Cyclic stretch significantly repressed expression of decidual markers including insulin‐like growth factor‐binding protein 1 (IGFBP1), prolactin (PRL), forkhead box O1 (FOXO1), and WNT4 on decidualized HESCs. In addition, cyclic stretch of decidualized HESCs affected the decidual morphological phenotype to an elongated shape. The alternation of F‐actin localization in decidualized HESCs was not observed in response to cyclic stretch. Conclusions These data suggest that cyclic stretch inhibits the morphological and biological decidual process of HESCs. Our findings imply that uterine abnormal contractions during the implantation period impair endometrial decidualization and contribute to infertility.


| INTRODUC TI ON
Successful pregnancy requires coordination of three interdependent processes: embryo development, endometrial differentiation, and placenta formation. Decidualization is a morphological and biological transformation of human endometrial stromal cells (HESCs) into decidual cells, and it is essential for blastocyst implantation and subsequent formation of the placenta. 1,2 The most characteristic morphological alteration in the decidual process is the drastic transformation of the endometrial stromal fibroblasts into secretory epithelioid decidual cells. 3 At the molecular level, decidual transformation involves extensive reprogramming of many cell functions mediated by the activation of various key transcription factors. The convergence of these transcription factors on promoters of key genes drives the expression and secretion of major decidual factors including prolactin (PRL), WNT4, and insulin-like growth factor-binding protein 1 (IGFBP1). 3,4 Impairment of the decidualizing process is increasingly linked to a variety of pregnancy disorders including infertility, recurrent implantation failure, recurrent miscarriages, preeclampsia, intrauterine growth restriction, endometriosis, and endometrial malignancy. [5][6][7][8] It has been reported that subendometrial myometrium exerts wavelike activity (uterine peristalsis) throughout the menstrual cycle, 9 and uterine peristalsis is markedly reduced during the implantation phase.
The unique feature of uterine peristalsis may facilitate implantation of the embryo to the endometrium. A clinical study reported that none of the enrolled patients with intramural myomas in a high-frequency peristalsis group achieved pregnancy, whereas one-third of patients in the low-frequency peristalsis group became pregnant. 10 Furthermore, myomectomy reduced the frequency of uterine peristalsis in patients who had exhibited an abnormally high-frequency of peristalsis prior to surgery. In addition, myomectomy increased the pregnancy rate in patients who had exhibited high-frequency peristalsis. 11 These observations suggest that abnormal uterine peristalsis during the implantation period influences endometrial functions and contributes to infertility. Harada et al 12 found that endometrial peristalsis elevates IGFBP-1 levels and promotes the decidualization of HESCs. The effect of physiological stretching conditions had been already investigated in that study. However, the effect of abnormal stretching conditions on HESCs is unclear. Therefore, we hypothesized that abnormal uterine peristalsis has an adverse effect on the endometrial decidual process.
We conducted an in vitro culture experiment to investigate the effect of cyclic stretch on the morphological and biological endometrial decidualization process.

| Tissue collection and isolation of HESCs
Human endometrium was collected from patients undergoing hysterectomy for uterine fibroids at Saitama Medical University Hospital.
Institutional Review Board approval (17-085) was obtained for this research project. All women had regular menstrual cycles and were not receiving hormonal treatment at the time of surgery. Informed consent was obtained from all patients before tissue collection. The age of participants was 44.5 ± 4.9. All the endometrial tissues were obtained from women in proliferative phase. HESCs were isolated as previously described. 6,[13][14][15] Harvested HESCs were cultured in maintenance medium of DMEM/F-12 (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% dextran-coated charcoal-treated FBS supplemented with 2 μg/mL insulin from bovine pancreas (Sigma-Aldrich, St. Louis, MO, USA), 1 × 10 −9 M beta-estradiol (Sigma-Aldrich), 1% antibioticantimycotic solution (Thermo Fisher Scientific), and 1% L-glutamine solution (Thermo Fisher Scientific) according to the previous study. 6

| The aspect ratio
Because cell elongation is a morphologic indicator of abnormal cyclic stretch, we determined the aspect ratio of each cell's major axis of cell length over the minor axis of cell width (with an aspect ratio of 1.0 yielding a circle) for the HESCs in culture using ImageJ software.
The shape indexes were calculated from 20 cells from each field to quantify morphologic changes.

| Prolactin measurement
The concentration of PRL was measured by the electrogenerated chemiluminescence immunoassay (ECLIA) method using ECLusys Prolactin III reagent (Roche Diagnostics, Basel, Switzerland) and Cobas 6000 (Roche Diagnostics). HESC culture media was collected on day three of decidualization for PRL measurement.

| Total RNA extraction and quantitative realtime reverse transcriptase PCR
Total RNA extraction from HESCs was performed using miRNeasy Mini Kit (Qiagen, Hilden, Germany). The reverse transcription for synthesis of cDNA from extracted total RNA was performed using BioScript reverse transcriptase (Bioline, London, UK). PRL, IGFBP1, forkhead box O1 (FOXO1), and WNT4 mRNA expression were analyzed by real-time reverse transcriptase PCR (RT-qPCR).

RT-qPCR was performed with PowerUP SYBR Green PCR Master
Mix (Thermo Fisher Scientific) and the PikoReal 96 Real-Time PCR system (Thermo Fisher Scientific). Primer sequences for each gene are shown in Table 1. The mRNA expression levels relative to those of GAPDH were calculated by the 2 −ΔΔCt method. 16

| Statistical analysis
PRL or RT-qPCR analysis was conducted on six sets of measurements. Statistical analyses were performed by two-tailed Student's t test for comparisons within two groups. All graphs with error ranges denote mean ± standard error of the mean (SEM). A P-value of <.05 was considered significant.

| Cyclic stretch inhibits morphological change and expression of decidual markers on decidualized HESCs
In the absence of hormonal treatment with or without cyclic stretch, confluent primary HESCs have a spindle-shaped fibroblast-like appearance via light microscopy. In agreement with our previous study, 8 treatment of decidualizing HESCs with 8-br-cAMP and MPA without cyclic stretch resulted in typical morphology of decidual cells characterized by the presence of larger and rounder cells with larger nuclei and abundant cytoplasm. By contrast, cyclic stretch of decidualized HESCs failed to show typical decidual transformation and instead showed an elongated shape ( Figure 1A). The aspect ratio of the major axis (length) over the minor axis (width) of each cell was used to quantify the changes in cell morphology upon decidualization in the presence or absence of cyclic stretch. For non-decidualized HESCs, cyclic stretch had no effect on the aspect ratio. By contrast, the aspect ratio of decidualized HESCs with cyclic stretch was significantly higher, indicating the relative abundance of spindle-shaped cells compared with decidualized HESCs without cyclic stretch ( Figure 1B)

| Alteration of F-actin localization and cytoskeleton of decidualized of HESCs with cyclic stretch
A previous study reported that the actin-based cytoskeleton contributes to both functional and morphological endometrial decidualization. 17 Therefore, we investigated the subcellular distribution of actin stress fibers in non-decidualized and decidualized HESCs treated with or without to cyclic stretch using Acti-stain™ 555 phalloidin. Well-stretched F-actin was distributed throughout the cytoplasm in undifferentiated HESCs during both stationary and cyclic stretch conditions. In response to decidual stimulation, The aspect ratio of cell major axes (lengths) over minor axes (widths), with an aspect ratio of 1.0 yielding a circle, was determined for the HESC culture system by using ImageJ software. The shape indexes were calculated from 20 cells from each field of cells to quantify morphologic changes. Data shown are mean ± SEM of five biological repeats. ***P < .001 (B)

F I G U R E 2
The effect of cyclic stretch on the secretion for PRL of decidualized HESCs. HESCs were treated with or without 8-bromo-cAMP and 10 −6 M MPA as decidual stimulation for three days. Then decidualized and non-decidualized HESCs were cultured with or without cyclic stretch for 24 h. These data represent the mean of PRL protein concentration in the supernatant, normalized by the total protein content. Data are shown as mean ± SEM of five individuals. **P < .01 switch). 18 It is possible that high stretch cycling may inhibit this cadherin switch. However, further studies need to clarify this mechanism of impairment for decidualization. The alteration of F-actin localization in decidualized HESCs was lost in response to cyclic stretch. These observations together suggest that cyclic stretch inhibits the morphological and functional process of decidualization. The cytoskeleton plays a critical role in various cellular processes including migration. 21 The organization and plasticity of the cytoskeleton are characterized primarily by forces generated by actin-myosin interactions. 17 Previous studies demonstrated that F-actin was localized in the periphery of decidualized HESCs. By contrast, well-stretched F-actin was distributed throughout the cytoplasm in non-decidualized HESCs. 21,22 These observations are consistent with our present microscopic findings. Furthermore, the present study demonstrated that cyclic stretch reversed the distribution of F-action in decidualized HESCs. Interestingly, it has been reported that in women with endometriosis, the endometrium has reduced decidualization capacity compared with that of women without endometriosis. 23 Therefore, it is possible that abnormal uterine contractions may actively contribute to sustaining the mesenchymal phenotype, retaining cell motility of HESCs against decidualization stimulation, and establishing endometriosis lesions.
In conclusion, the present study demonstrates that cyclic stretch inhibits the morphological and functional decidual process of HESCs.
Our findings suggest that uterine abnormal contractions during the implantation period of fertilization impair endometrial decidualization and contribute to infertility.