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Article

Regulatory Mechanism of Human Endometrial Stromal Cell Decidualization by Ergothioneine

by
Namika Yoshida
1,
Hiromi Murata
2,
Konomi Ide
1,
Marika Tanaka
1,
Kurumi Mori
1,
Kensuke Futani
1,
Misa Sawachika
1,
Hidetaka Okada
2 and
Susumu Tanaka
1,*
1
Department of Nutrition Science, University of Nagasaki, Siebold, Nagasaki 851-2195, Japan
2
Department of Obstetrics and Gynecology, Kansai Medical University, 2-5-1 Shin-Machi, Hirakata 573-1010, Japan
*
Author to whom correspondence should be addressed.
Nutraceuticals 2025, 5(3), 16; https://doi.org/10.3390/nutraceuticals5030016
Submission received: 1 April 2025 / Revised: 17 June 2025 / Accepted: 26 June 2025 / Published: 1 July 2025
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Abstract

Endometrial stromal cells (EnSCs) undergo decidualization in response to progesterone. Decidualization facilitates spiral artery remodeling, immune tolerance in the endometrium, and fetal cell invasion and placentation—all essential for successful embryo implantation. Therefore, we aimed to investigate whether ergothioneine (EGT) plays a role in reproduction, particularly in decidualization and implantation. In this study, we found that solute carrier family 22 member 4 (SLC22A4), a specific transporter of EGT—a functional food ingredient with strong anti-aging properties—is upregulated in decidualized EnSCs. The effects of EGT were examined using uterine tissues from patients, primary cultured EnSCs, EnSC cell lines, and co-cultures with a fetal cell line. We observed a significant increase in SLC22A4 expression in secretory-phase human uterine tissue, decidualized EnSCs, and EnSC cell lines. We also found that EGT regulates insulin-like growth factor binding protein 1 expression, which promotes placentation. In co-cultures of EnSC and fetal cell lines, EGT upregulated ectonucleoside triphosphate diphosphohydrolase 1 and major histocompatibility complex, class I, G expression in fetal cell lines—both critical for placentation. These findings suggest that EGT is crucial to regulating decidualization and its markers, particularly insulin-like growth factor-binding protein 1, which contributes to placentation.

1. Introduction

Ergothioneine (EGT) is a hydrophilic sulfur-containing amino acid with a molecular weight of 229.3, originally discovered in wheat horn [1]. It is recognized as a functional ingredient with strong antioxidant properties [2]. Solute carrier family 22 member 4 (SLC22A4), a specific transporter for EGT, has been identified [3]. EGT, obtained from mushroom consumption [4], is absorbed through the intestinal tract and transported into cells via SLC22A4 from the bloodstream, contributing to intracellular oxidative stress homeostasis. EGT functions independently of traditional biological defense systems and rapidly reduces hydroxyl radicals, which conventional mechanisms cannot manage [3]. Its properties include anti-aging, anti-inflammatory, and anti-neurodegenerative effects [5].
EGT has been shown to ameliorate hypertension, reduce placental mitochondrion-derived reactive oxygen species (ROS), increase the expression of placental mitochondrion-related enzymes, and improve fetal weight in a rattus preeclampsia model [6,7]. EGT is believed to alleviate preeclampsia—a multifactorial gestational hypertension syndrome associated with placental dysplasia and infertility—via SLC22A4, which is expressed in extravillous trophoblasts (EVTs) and contributes to placental formation [6,7,8]. EGT may also help prevent preeclampsia in humans [9,10,11].
Despite the known significance of EGT, its effect on the maternal endometrial decidua remains unexplored. The human endometrium follows a menstrual cycle of approximately 28 days, comprising menstruation, proliferation, and secretory phases [12]. Endometrial stromal cells (EnSCs), present in the endometrium, undergo decidualization during the secretory phase in response to ovarian progesterone after ovulation, enhancing embryonic receptivity [12]. Improper decidualization can lead to implantation failure and placental insufficiency, resulting in infertility and recurrent miscarriage [13,14]. Progesterone signaling induces decidualization in EnSCs through progesterone receptor (PGR) and increases the production and secretion of prolactin (PRL), insulin-like growth factor-binding protein 1 (IGFBP1), and interleukin-15 (IL15). PRL promotes spiral artery development in the endometrium, while IGFBP1 activates placentation by stimulating EVTs from the embryo [15,16]. Elevated IGFBP1 protein levels are associated with a decreased risk of preeclampsia and improved pregnancy outcomes [17]. IL15 regulates the proliferation and differentiation of uterine-specific natural killer (uNK) cells. Together with EnSCs, uNK cells remodel the spiral artery and enhance decidualization. Furthermore, uNK cells play an intrinsic role in immune tolerance during embryo implantation [18]. Abnormal decidualization in EnSCs can cause implantation failure, miscarriage, gestational hypertension nephropathy, fetal growth restriction, and placenta accreta. SLC22A4 expression has been reported in the human endometrium; however, its expression level is known to be low [19].
Therefore, in this study, we aimed to investigate whether EGT plays a role in reproduction, particularly in decidualization and implantation. We examined SLC22A4 expression and found it to be upregulated during the secretory phase of the human endometrium. We also observed increased SLC22A4 expression in decidualized primary cultured EnSCs and in the EnSC cell line KC02-44D, suggesting that EGT is associated with decidualization. To further investigate the effects of SLC22A4-mediated EGT on decidualization, we used EnSCs and a co-culture system with KC02-44D and EVT cell lines. Notably, decidualization in human EnSCs occurs before implantation, while in experimental animals, it begins only after implantation. This fundamental discrepancy underlines the importance of studying decidualization in human EnSCs. Therefore, we utilized patient-derived EnSCs and established EnSC cell lines to study human endometrial decidualization.

2. Materials and Methods

2.1. Ethical Statement

All personnel conducted this study in compliance with the Declaration of Helsinki and the Ethical Guidelines for Medical Research Involving Human Subjects. Written informed consent was voluntarily obtained from all participants using a consent form approved by the Ethical Review Committee of Kansai Medical University (approval ID: 2006101; approval date: 12 July 2006). Clinical samples included endometrial tissue stored in liquid nitrogen and isolated primary cultured EnSCs from 24 women aged 35–50 years with normal menstrual cycles who underwent hysterectomy for benign gynecological conditions (primarily myomas) (Table 1). The menstrual cycle phase was estimated based on the number of days from the start of menstruation as reported by each patient.

2.2. Isolation of EnSCs

Endometrial tissue was collected in the operating room, transferred to a clean bench, mechanically shredded, and enzymatically digested with collagenase at 37 °C for 2 h. A 40 µm cell strainer was used to separate EnSCs and immune cells, as glandular cells remained on the filter. The separated EnSCs and immune cells were passaged to 75 cm2 adhesive culture flasks (Corning Inc., Corning, NY, USA) and incubated in Dulbecco’s Modified Eagle’s Medium (DMEM; phenol red, 4.5 g/L D-glucose, and L-glutamine; Life Technologies, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS; HyClone, Cytiva, Tokyo, Japan), 10 mM HEPES (Life Technologies), and 1% antibiotics (Nacalai Tesque, Osaka, Japan). The medium was replaced every 3 days. Over approximately 2 weeks, the floating immune cells died, leaving only EnSCs. When EnSCs reached confluence, they were passaged into well plates as required for the experiments. Only one passage was performed, as additional passages can impair decidualization. EnSC purity was confirmed by fluorescent antibody staining using a vimentin antibody, ensuring that more than 99% of cells were vimentin-positive. Confluent EnSCs were then transferred to 24-well plates (Corning) for stimulation.

2.3. Induction of Decidualization in Primary Cultured Human EnSCs

Once the EnSCs in 24-well plates (Corning Inc.) reached confluence, the medium was replaced with DMEM (phenol red-free, as phenol red has estrogen-like effects, and containing 4.5 g/L D-glucose,) (Life Technologies). This was supplemented with 10% charcoal-stripped FBS (CS-FBS; charcoal adsorbs and removes steroid hormones), 10 mM HEPES, 1% antibiotics, and 1% GlutaMAX (Life Technologies), referred to as DMEM/CS-FBS. To induce decidualization (hereafter referred to as “decidualization treatment”), 10−8 M 17β-estradiol (E2; Sigma-Aldrich, St. Louis, MO, USA) and 10−7 M medroxyprogesterone acetate (MPA; Sigma-Aldrich) were added. The medium was replaced with fresh induction medium every 3 days for 12 days. Successful decidualization was confirmed by observing morphological changes from small, spindle-shaped proliferating cells to round, epithelial-like cells under an inverted microscope (ECLIPSE Ts2; Nikon, Tokyo, Japan).

2.4. Culture of EnSC Cell Line KC02-44D

The human EnSC cell line KC02-44D (CVCL_E224) [20] was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in DMEM (phenol red, 4.5 g/L D-glucose, and L-glutamine) containing 10% FBS, 10 mM HEPES, and 1% antibiotics. Confluent cells (1.2 × 106) in 10 cm dishes were passaged to 24-well plates (Corning).

2.5. Decidualization Treatment for KC02-44D Cells

Cells were seeded in 24-well plates (Corning) to reach confluence and then divided into the control and decidualization groups. The control group was incubated with DMEM/CS-FBS for 6 days, with medium changes every 3 days. The decidualization group was stimulated with 10−8 M E2, 10−6 M MPA, and 0.5 mM 8-Bromo-cAMP (Sigma-Aldrich) in DMEM/CS-FBS for 6 days, also with medium changes every 3 days.

2.6. Appropriate Concentration Test of EGT on Decidualization

To determine the appropriate EGT (L-(+)-Ergothioneine, Cayman chemical, Ann Arbor, MI, USA) concentration for use during decidualization treatment in KC02-44D cells, concentration screening was conducted based on previous studies [21]. Reported EGT concentrations in human blood range from 2.3 to 3.0 mg/100 mL (100.3–130.5 µM) [22].

2.7. Cytotoxicity Lactate Dehydrogenase Assay

KC02-44D cells were passaged into 96-well microplates at 500 cells per well. EGT was added at concentrations of 5 µM, 10 µM, 50 µM, or 100 µM to both untreated and decidualization groups. The medium was replaced every 3 days in a carbon dioxide (CO2) incubator (37 °C, 5% CO2). Cytotoxicity was measured using the Cytotoxicity lactate dehydrogenase (LDH) Assay Kit-WST (Dojindo, Kumamoto, Japan). Absorbance at 450 nm was measured using a microplate reader (PerkinElmer Japan, Yokohama, Japan), and LDH release (an indicator of cytotoxicity) under each condition was calculated using the untreated group as a 0% reference.

2.8. Cell Proliferation Assay

KC02-44D cells were passaged into 96-well microplates at 500 cells/well. EGT was added at concentrations of 5 µM, 10 µM, 50 µM, or 100 µM to both untreated and decidualization groups, with the media changed every 3 days. Cell viability was assessed using the Cell Counting Kit-8 (Dojindo). We evaluated whether cell viability differed significantly between untreated and decidualization-only groups, and whether EGT concentration correlated with cell viability.

2.9. Quantitative Polymerase Chain Reaction (qPCR)

The extraction of total ribonucleic acid (RNA) from decidualized and untreated KC02-44D cells (0.4 × 106 cells) was conducted using Sepasol®-RNA I Super G (Nacalai Tesque, Kyoto, Japan). Reverse transcription was performed with 250 ng of total RNA using ReverTra Ace® Quantitative Polymerase Chain Reaction (qPCR) RT Master Mix with genomic DNA (gDNA) Remover (TOYOBO). qPCR was conducted to assess decidualization and the effect of EGT. After preincubation (95 °C, 30 s), amplification was performed using 45 cycles of 95 °C for 5 s and 60 °C for 30 s, followed by a melting curve analysis (95 °C for 10 s, 65 °C for 60 s, and 97 °C for 1 s) to confirm primer specificity. Table 2 lists the gene names and oligonucleotide sequences used in qPCR. Relative gene expression was calculated using the ΔΔCt method with hypoxanthine phosphoribosyltransferase 1 (HPRT1) as the internal control [23].

2.10. Western Blotting Method

After 6 days of treatment, radio-immunoprecipitation assay (RIPA) buffer-soluble protein fractions were extracted from KC02-44D cells using RIPA buffer (Nacalai Tesque). A total of 10 µg of the RIPA buffer-soluble fraction was subjected to SDS-PAGE (153 mA, 35 min) to separate proteins. The separated proteins were transferred to a Clear Blot P+ membrane (ATTO, Tokyo, Japan) using the Transblot SD cell (Bio-Rad, Hercules, CA, USA). After transfer, the membrane was blocked for 2 h at 25 °C with Blocking One (Nacalai Tesque). Following blocking, the membrane was immunoreacted with either rabbit anti-SLC22A4 antibody (1:1000; Aviva Systems Biology, San Diego, CA, USA) or rabbit anti-forkhead box O1 (FOXO1) (C29H4) monoclonal antibody #2880 (1:1000; Cell Signaling Technology, MA, USA) in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) containing 5% Blocking One overnight at 4 °C. On the next day, after washing for 5 min thrice in TBS-T, the membranes were immunoreacted with horseradish peroxidase-conjugated horse anti-rabbit IgG (1:3000; VECTOR PI-1000) in 5% Blocking One/TBS-T for 1 h at 25 °C. After three additional washes (5 min each in TBS-T), Chemi-Lumi One L (Nacalai Tesque) was added and incubated for 1 min. Protein bands were visualized using LAS4000 (GE Healthcare Japan, Tokyo, Japan).
Band intensities were quantified using ImageJ version 1.54 g software (National Institutes of Health, Maryland, MD, USA). Protein levels of SLC22A4 and FOXO1 were normalized to β-actin (ACTB) levels, as previously described [24]. For ACTB detection, a mouse monoclonal anti-β-actin antibody (1:15,000; Merck, Darmstadt, Germany) and HRP-conjugated horse polyclonal anti-mouse IgG (1:3000; VECTOR PI-2000) were used.

2.11. Co-Culture of KC02-44D Cells with BeWo Cells for In Vitro Implantation Model

The BeWo cell line (ATCC Cat. No. CCL-98, RRID: CVCL_0044), a choriocarcinoma-derived line with characteristics similar to the ectodermal layer of human blastocysts, was used for implantation-related studies [25]. BeWo cells were cultured in Ham’s F-12 medium (Life Technologies) containing 1% GlutaMAX (Life Technologies), 1% penicillin–streptomycin (Nacalai Tesque), and 15% FBS (HyClone) in a humidified incubator with 5% CO2 at 37 °C.
A total of 1.6 × 105 BeWo cells were passaged into each well of a 24-well plate and cultured for 6 days with medium changes every 3 days to form spheroids. The in vitro implantation model was used to assess the spreading area of the BeWo spheroids [26]. The spheroids were seeded onto monolayers of untreated, decidualized, or decidualized-with-EGT EnSCs and cultured for 36 h at 37 °C under 5% CO2 prior to imaging. Images were acquired using an ECLIPSE Ts2 inverted microscope (Nikon). The spreading area of the spheroids was quantified using ImageJ software.
RNA was extracted from a separately prepared KC02-44D, BeWo cells, and a co-culture of KC02-44D and BeWo cells for qPCR analysis. In addition, co-cultured cells were fixed and analyzed using immunofluorescence staining.

2.12. Immunofluorescence Staining

After co-culture, KC02-44D and BeWo cells were fixed in formaldehyde for 5 min at 22 °C. After washing with PBS, the cells were immunoreacted with a mouse monoclonal anti-major histocompatibility complex, class I, G (HLA-G) IgG2b antibody (1:1000; Proteintech, Rosemont, IL, USA, RRID: AB_2881816) overnight at 4 °C. On the following day, the cells were immunoreacted with goat anti-rabbit Alexa Fluor 488 antibody (1:3000; #A-11008, Thermo Fisher Scientific, Waltham, MS, USA, RRID: AB_143165). DAPI counterstaining for nuclei was conducted by using DAPI-Fluoromount-G (SouthernBiotech, Birmingham, AL, USA). The stained cells were observed using an ECLIPSE Ts2 inverted microscope (Nikon).

2.13. Enzyme-Linked Immunosorbent Assay (ELISA)

After 6 days of decidualization and/or EGT treatment of KC02-44D cells, culture supernatants representing 3 days of secretion were collected. The concentration of IGFBP1 in the culture media was measured using the Human IGFBP-1 ELISA Kit (Thermo Fisher Scientific).

2.14. Statistical Analysis

A Shapiro–Wilk normality test was conducted to assess the distribution of each dataset. Two-tailed Welch’s t-tests were used for group comparisons, followed by Bonferroni correction to determine the adjusted significance threshold. IBM SPSS Statistics version 29.0 (IBM Corp., Armonk, NY, USA) was used for statistical analyses. A p-value of <0.05 was considered statistically significant.

3. Results

3.1. SLC22A4 Increases in the Secretory Phase of Human Endometrium

qPCR was conducted to check the expression of SLC22A4, which was found to be significantly elevated in the secretory phase (n = 11; after the 15th day of menstruation) compared with the proliferative phase (n = 7) (p < 0.05; Figure 1A).

3.2. Changes in SLC22A4 Expression in Primary Cultured Human EnSCs

To determine whether the increased SLC22A4 expression observed in endometrial tissue was derived from EnSCs, cells were isolated from six patients’ endometrial tissue. The expression level of SLC22A4 was significantly increased in the decidualization treatment group compared with the untreated group (p < 0.05; Figure 1B).

3.3. SLC22A4 Expression in Human EnSC Strain

Since patient-derived EnSCs vary between individuals, the interpretation of subsequent responses to EGT may be difficult. Therefore, we used the established KC02-44D cell line [20] to examine SLC22A4 expression and ensure some degree of reproducibility. The results showed that SLC22A4 mRNA expression was significantly increased in KC02-44D cells following decidualization treatment (p < 0.05; n = 15 per group; Figure 1C). Moreover, the amount of SLC22A4 protein was also significantly increased in response to decidualization (p < 0.05; n = 4 per group; Figure 1D and Figure S1).

3.4. Effect of EGT Concentration on the Markers for Decidualization

To investigate the effect of EGT, KC02-44D cells were used to examine changes in the decidualization markers [27,28,29]. PRL and IL15 were significantly upregulated in the decidualization treatment group compared with the untreated control (p < 0.05); however, no significant changes were found between decidualization alone and decidualization plus EGT (Figure 2).
In contrast, IGFBP1 expression was significantly upregulated in decidualization treated cells compared with untreated cells (p < 0.05) but was significantly reduced in the EGT plus decidualization group compared with the decidualization-only group (p < 0.05; Figure 2).
Given that physiological EGT concentrations in human blood are approximately 100 µM [22], 100 µM EGT was used in subsequent experiments to assess its effects on KC02-44D cells and decidualization.

3.5. Effects of EGT on Cell Viability and Death

To examine whether the observed decrease in IGFBP1 expression following EGT addition during decidualization was related to reduced cell viability or increased cell death, we assessed EGT-induced cytotoxicity. No concentration-dependent increase in dead cells or membrane-damaged cells was observed in either decidualized or untreated groups (Figure 3A).
Changes in cell viability were analyzed and the findings showed that viability was significantly lower in the decidualized group than in untreated cells (p < 0.05; Figure 3B). However, this may reflect limited culture space due to the increased size of decidualized cells, rather than decreased viability. Furthermore, EGT treatment at various concentrations during decidualization did not significantly affect cell viability, indicating that EGT itself does not impair the survival of KC02-44D cells.

3.6. Effect of EGT on Decidualization

The effect of 100 µM EGT on decidualization markers—PRL, IGFBP1, IL15, and heart and neural crest derivatives expressed 2 (HAND2) [27,28,29,30]—was examined using KC02-44D cells. The expression levels of PRL, IL15, HAND2, and SLC22A4 were significantly upregulated in the decidualization group compared with untreated cells (p < 0.05; Figure 4). IGFBP1 expression was also significantly upregulated in the decidualization group vs. the control (p < 0.05). The addition of EGT to the untreated control did not result in significant alterations; however, a significant reduction in IGFBP1 when EGT was added during decidualization was reconfirmed (p < 0.05; Figure 4). FOXO3 expression was also significantly upregulated in the decidualization group vs. the control (p < 0.05). The addition of EGT to the untreated control induced a slight reduction but did not result in significant alterations; however, a significant reduction in FOXO3 was observed when EGT was added during decidualization (p < 0.05).

3.7. Regulation by FOXO1 in Decidualization

Western blotting revealed a significant increase in FOXO1 protein levels upon decidualization. However, when 100 µM EGT was added during decidualization, a decrease in FOXO1 protein was observed compared to decidualization without EGT A and Figure S2. To investigate whether this decrease was due to reduced transcription, translation inhibition, or increased degradation, FOXO1 mRNA expression was examined by qPCR. The transcription was significantly decreased in the EGT + decidualization group compared with decidualization alone, suggesting that EGT suppresses FOXO1 at the transcriptional level, leading to reduced FOXO1 protein (p < 0.05; Figure 5B). Left-right determination factor 2 (LEFTY2), insulin receptor (INSR), and decorin (DCN) are genes regulated by FOXO1 [31]. Upon EGT stimulation, LEFTY2 expression fell below the detection limit. Similarly to FOXO1, LEFTY2, INSR, and DCN expression levels were significantly upregulated by decidualization and decidualization + EGT stimuli compared with the control. However, they were significantly suppressed in the decidualization + EGT group compared with the decidualization-only group (Figure 5).

3.8. EGT Induced Expansion of BeWo Spheroids on In Vitro Implantation Model

To examine the EGT effect on embryo implantation, an in vitro implantation model was performed. In the control group, there was a clear boundary between undifferentiated KC02-44D cells and BeWo spheroids (Figure 6). When EGT was added to undifferentiated KC02-44D cells, BeWo spheroid spreading was similar to the control; however, concentric spreading patterns were observed. With decidualized KC02-44D cells, the BeWo spheroids spread significantly more than in the control group, and the spheroids appeared to integrate with the decidualized cells. The addition of EGT to decidualized KC02-44D cells further enhanced spheroid spreading compared with both the control and decidualization-only groups (Figure 6).

3.9. Effect of EGT on Function of In Vitro Implantation Model

To examine changes in cell function within an in vitro implantation model, samples were prepared from KC02-44D alone, BeWo alone, and co-culture systems that had been cultured for 7.5 days under three conditions: untreated control, decidualization stimulation, and 100 µM EGT added during decidualization. Keratin 7 (KRT7), a specific marker for trophoblasts [32], was not detected in KC02-44D alone under any condition but was detected in BeWo cells alone, with no difference between groups (Figure 7). However, KRT7 expression was upregulated in the co-culture systems, and a significant increase was found in the EGT + decidualization group compared with both the control and decidualization-only groups.
Macrophages increase during embryo implantation, constituting approximately 20–30% of decidual leukocytes in the first trimester of pregnancy [33]. The extracellular balance of ATP and adenosine, regulated by CD39 and CD73, facilitates the macrophage transformations from M1 (proinflammatory) to M2 (anti-inflammatory) phenotypes [34,35]. No difference in ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1) expression (encoding CD39) was observed under any condition in BeWO cells, and ENTPD1 expression was absent or minimal in KC02-44D or BeWO cells (Figure 7). In contrast, ENTPD1 expression was detected in co-culture systems—possibly derived from BeWo cells—and was significantly increased by the presence of decidualized KC02-44D cells, with further upregulation observed when EGT was added during decidualization (Figure 7). The expression of 5′-nucleotidase ecto (NT5E), which encodes CD73, was detected at a steady-state level in KC02-44D cells and under a co-culture condition, with no differences observed under any condition. Since NT5E was not expressed in BeWo cells alone, it is assumed to originate from KC02-44D in the co-culture system (Figure 7).
HLA-G promotes spiral artery remodeling, contributes to maternal–fetal immune tolerance, and supports fetal growth [36,37,38]. HLA-G gene expression was not detected in KC02-44D or BeWo cells alone and showed no differences between individual cultures (Figure 7). However, in co-culture systems, HLA-G gene expression and protein levels—likely derived from BeWo cells—were significantly upregulated following interaction with decidualized KC02-44D cells and further increased when EGT was added during decidualization (Figure 7 and Figure 8).
IGFBP1 expression was significantly upregulated in the decidualization group vs. the control (p < 0.05), and the significant reduction in IGFBP1 expression when EGT was added during decidualization was reconfirmed in both KC02-44D cells and the co-culture condition (p < 0.05; Figure 7). IGFBP1 expression was not detected in BeWo cells.
During embryo implantation, leukemia inhibitory factor (LIF) from preimplantation embryos, endometrial glands, and EnSCs binds to its respective LIF receptors (LIFR), activating the downstream LIF pathway [39]. This activation contributes to embryonic viability, differentiation, and decidual function [40,41]. The gene expression of LIFR is a known indicator of LIF pathway activation, and the activation of STAT3 downstream of LIFRs regulates the expression of stemness effectors such as KLF transcription factor 4 (KLF4), SRY-box transcription factor 2 (SOX2), Nanog homeobox (NANOG), and POU class 5 homeobox 1 (POU5F1) [39]. LIFR was detected at a steady-state level in KC02-44D cells (Figure 7). In contrast, a significant increase in LIFR expression was observed in the decidualization treatment in the co-cultured condition and BeWo cells. However, there was no effect of EGT in either condition (Figure 7). Despite the absence of LIFR changes, downstream genes such as KLF4, NANOG, and POU5F1, except for SOX2, were significantly altered by decidualization stimuli in KC02-44D cells. However, no effect of EGT was observed for these genes in KC02-44D cells (Figure 7). In BeWo cells, SOX2 and POU5F1 were not detected; however, KLF4 and NANOG were significantly regulated by decidualization stimuli. There was no effect of EGT in all genes (Figure 7). The response to decidualization stimuli in BeWO cells is known to be triggered by cAMP, which induces differentiation and fusion [42]. In the co-cultured condition, KLF4, SOX2, NANOG, and POU5F1 were significantly increased by decidualization stimuli; however, there was no effect of EGT (Figure 7).

3.10. Increased IGFBP1 Secretion by EGT

The results from the in vitro implantation model appeared inconsistent with the previously observed decrease in IGFBP1 expression following EGT treatment (Figure 2 and Figure 4). Therefore, we evaluated the secretion of IGFBP1, which is known to directly promote EVT invasion during embryo implantation. IGFBP1 secretion was not increased by EGT alone but was significantly increased in both the decidualization group and the decidualization + EGT group compared with controls (Figure 9). Furthermore, the addition of EGT during decidualization led to a significant upregulation in IGFBP1 secretion compared with decidualization alone (Figure 9), contrary to expectations, as IGFBP1 mRNA expression was reduced under the same conditions (Figure 2 and Figure 4).

4. Discussion

EGT has been shown to improve pregnancy rates in diabetic rats; however, its effects on the endometrial decidua have not been studied [43,44]. The European Food Safety Authority has confirmed that EGT is non-toxic at doses up to 800 mg/kg/day and has approved its use in supplements for pregnant and lactating women, as well as children and infants [45]. SLC22A4, a specific transporter of EGT, is also present in EVT derived from blastocysts [8]. The administration of EGT in the rat preeclampsia model promotes the elevated expression of the peroxisome proliferator-activated receptor-γ coactivator 1α, a key orchestrator of mitochondrial metabolism, and mitochondrial ROS-detoxifying enzymes (uncoupling protein 1, nuclear factor erythroid 2–related factor 2 [Nrf2], and superoxide dismutase 1 and 2) in the placenta via SLC22A4 [7], suggesting that EGT may influence placental development directly. EGT has been shown to improve outcomes in a rat model of preeclampsia [7]; however, its effects on the maternal decidua have not been fully investigated and may be of significance. Our findings—demonstrating the expression of SLC22A4 in EnSCs, its upregulation during decidualization, and EGT’s regulatory effect on IGFBP1 and implantation—make us the first to identify EGT as a previously unrecognized factor critical to implantation and pregnancy maintenance. From the results obtained in an in vitro implantation model using the two cell lines, it is suggested that EVTs and EnSCs cooperate locally to break down an inflammatory signal ATP released from damaged cells during the EVT invasion of the decidua [46], thereby preventing excessive inflammation, which EGT enhances. Furthermore, it has been reported that an increase in extracellular ATP suppresses trophoblast invasion and spiral artery remodeling in rats [47], and an increase in ENTPD1 by EGT may improve these reductions. EGT further increased HLA-G protein levels, which is also increased by interaction with KC02-44D. These allow EGT to significantly affect placentation.
Progesterone binds to PGR in the cytoplasm of EnSCs and, upon nuclear translocation, directly regulates the transcription of PRL and IGFBP1, and induces the transcription of the transcription factors HAND2 and FOXO1 [48]. Induced HAND2 regulates the transcription of PRL and IL15 [12,49], while FOXO1 contributes to the transcriptional regulation of IGFBP1 [50]. Given the lack of change in PGR expression (Figure S3), we hypothesized that FOXO1 was involved in the downregulation of IGFBP1 expression by EGT, and indeed, a decrease in FOXO1 was observed. Similarly, in our study, the decrease in IGFBP1 gene expression can be explained by the decrease in FOXO1 levels, as other genes whose expression is regulated by FOXO1 (LEFTY2, INSR, and DCN) [31] were also altered.
Conversely, a paradoxical increase in IGFBP1 secretion was observed. IGFBP1 secretion is known to be influenced by amino acid balance and nutritional status [51,52]. Specifically, the ratio of branched-chain amino acids (BCAAs) to non-BCAAs and the absolute amount of BCAAs have been indicated to have a role in regulating IGFBP1 secretion [51,52]. EGT, like BCAAs, contains branched molecular structures [53], and it activates mammalian target of rapamycin, a sensor for intracellular amino acid [54]. Through these mechanisms, EGT appears to decrease IGFBP1 expression in EnSCs while simultaneously promoting IGFBP1 secretion due to its amino acid-like properties.
It is known that blood EGT levels decline with age [55], which may contribute to age-related reductions in antioxidant capacity in the endometrium [56]. This antioxidant capacity reduction could impair decidualization and contribute to the development of pregnancy complications and decreased pregnancy rates [56]. In our study, EGT repressed FOXO3 expression. FOXO3 regulates decidualization and apoptosis-related factors, and indeed, the repression of FOXO3 disables the oxidative cell death pathway [57,58]. Therefore, EGT may play an important role in decidualization during early pregnancy by regulating apoptosis via FOXO3 through its antioxidant stress effect.
Once taken up by cells, EGT removes intracellular ROS and maintains oxidative stress homeostasis [2]. NRF2 is shown to mediate antioxidant responses [59]. In a stress-free environment, Kelch-like ECH-associated protein 1 (KEAP1) retains NRF2 in the cytoplasm and NRF2 is subject to constant degradation via ubiquitination and proteasome-dependent proteolysis [60]. Under oxidative stressed conditions, this suppression is lifted and NRF2, which has migrated to the nucleus, activates antioxidant genes via antioxidant response elements (AREs) [60]. In addition to regulating IGFBP1 transcription, FOXO1 also modulates the transcriptions of antioxidant enzymes [61], acting as part of the biological defense against oxidative stress [62]. AREs are present in the regulatory region of the FOXO1 gene, and FOXO1 expression is enhanced by NRF2 nuclear translocation under oxidative stress [63]. In KC02-44D cells, EGT might alleviate oxidative stress that normally occurs during decidualization and inhibit NRF2 nuclear translocation. This suggests that NRF2 is again degraded by KEAP1, leading to the repression of FOXO1 and the subsequent downregulation of IGFBP1 expression.
IGFBP1 promotes EVT migration and invasion [64,65]. It acts locally via integrin α5 and β1 subunits expressed on EVT, making its precise regulation essential for normal placental formation and fetal development [64]. Excessive IGFBP1 expression, however, can lead to overly deep EVT invasion, resulting in placenta accreta, a serious condition where the placenta penetrates the uterine muscle layer, endangering both mother and child [66]. Furthermore, since EnSCs also express integrin α5/β1, IGFBP1 may participate in autocrine negative feedback [67], leading to a thinner decidua. Based on this, EGT may support placentation by regulating IGFBP1 in EnSCs, thereby controlling EVT migration and preventing excessive decidual thinning.

Research Limitations

We identified the expression of SLC22A4, a specific transporter of EGT, in the human endometrium and found that its expression may be derived from EnSCs; however, we have not been able to determine whether other endometrial cells, such as the endometrial glandular epithelium, immune cells, and vascular endothelial cells, also express SLC22A4. This limits our ability to comprehensively investigate the effects of EGT on the entire endometrium. The amount of EGT uptake reaches a plateau, with minimal EGT leaving the cell [21], suggesting that there may be a threshold amount of EGT that can be contained per cell, independent of the level of SLC22A4 expression. Therefore, measuring intracellular EGT concentrations is crucial; however, this was not possible in this study.
While FOXO1 gene expression and protein levels were correlated, IGFBP1 gene expression and secretion levels were inversely related. Changes in intracellular protein levels need to be examined in conjunction with other markers, which were not measured in this study. In addition, changes in expression observed in this study may not necessarily imply changes in function; therefore, functional studies are required to validate the results of this study.

5. Conclusions

We found that SLC22A4, a specific transporter of EGT, is upregulated in decidualized EnSCs and that EGT might regulate IGFBP1 secretion via SLC22A4 on decidualized EnSCs, which contributes to placentation, indicating that EGT might be involved in placentation.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/nutraceuticals5030016/s1, Figure S1. Original images for Figure 1. Figure S2. Original images for Figure 5. Figure S3. Effect of ergothioneine in decidualization on progesterone receptor expression.

Author Contributions

Conceptualization: S.T.; methodology: S.T., H.M. and H.O.; validation: S.T. and N.Y.; formal analysis: S.T., N.Y., M.S., K.F., K.I., M.T. and K.M.; investigation: S.T., N.Y., M.S. and K.F.; resources: S.T., H.M. and H.O.; data curation: S.T. and N.Y.; writing—original draft preparation: S.T. and N.Y.; writing—review and editing: S.T., N.Y., H.M. and H.O.; visualization: S.T. and N.Y.; supervision: H.M. and H.O.; project administration: S.T.; funding acquisition: S.T., H.M. and H.O. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Takeda Science Foundation (2018) and the Yamaguchi Endocrine Research Foundation (2024) awarded to Susumu Tanaka; and JSPS KAKENHI grants awarded to Susumu Tanaka (grant number 25K14871), Hidetaka Okada (grant number 21K09480), and Hiromi Murata (grant number 21K09529 and 24K12591).

Institutional Review Board Statement

This study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board of Kansai Medical University (protocol code: 2006101; 12 July 2006).

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

The data presented in this study are available upon request from the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analysis, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Nutraceuticals 05 00016 g001
Figure 1. SLC22A4 changes in endometrium and endometrial stromal cells. (A) Changes in SLC22A4 expression were examined by qPCR using cDNA obtained from human endometrial tissue (proliferative phase: n = 7; secretory phase: n = 11). Days 0 to 14 were defined as the proliferative phase, and days 15 and beyond as the secretory phase. Each dot represents SLC22A4 data from a different clinical specimen. (B) Primary cultured human endometrial stromal cells were subjected to 6 days of decidualization treatment, and SLC22A4 was examined by qPCR (n = 6 per group). (C) KC02-44D cells were treated for 6 days with decidualization conditions, and SLC22A4 were analyzed by qPCR (n = 15). (D) SLC22A4 and β-actin (ACTB) protein levels were examined in KC02-44D cells following decidualization. Protein levels were normalized to ACTB (n = 4). Each bar indicates the mean; error bars represent standard deviation. SLC22A4: Solute carrier family 22 member 4; HPRT1: Hypoxanthine phosphoribosyltransferase 1. * p < 0.05 by Welch’s t-test vs. proliferative phase or untreated group.
Figure 1. SLC22A4 changes in endometrium and endometrial stromal cells. (A) Changes in SLC22A4 expression were examined by qPCR using cDNA obtained from human endometrial tissue (proliferative phase: n = 7; secretory phase: n = 11). Days 0 to 14 were defined as the proliferative phase, and days 15 and beyond as the secretory phase. Each dot represents SLC22A4 data from a different clinical specimen. (B) Primary cultured human endometrial stromal cells were subjected to 6 days of decidualization treatment, and SLC22A4 was examined by qPCR (n = 6 per group). (C) KC02-44D cells were treated for 6 days with decidualization conditions, and SLC22A4 were analyzed by qPCR (n = 15). (D) SLC22A4 and β-actin (ACTB) protein levels were examined in KC02-44D cells following decidualization. Protein levels were normalized to ACTB (n = 4). Each bar indicates the mean; error bars represent standard deviation. SLC22A4: Solute carrier family 22 member 4; HPRT1: Hypoxanthine phosphoribosyltransferase 1. * p < 0.05 by Welch’s t-test vs. proliferative phase or untreated group.
Nutraceuticals 05 00016 g001
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Figure 2. Effect of ergothioneine concentration against decidualization markers in KC02-44D cells. Preliminary qPCR analyses were conducted using KC02-44D cells exposed to different concentrations of ergothioneine (0, 5, 10, 50, and 100 µM) under decidualization treatment. Each bar represents the mean; error bars represent standard deviation. Each dot represents an independent experiment (n = 4). EGT: Ergothioneine; PRL: Prolactin; IGFBP1: Insulin-like growth factor binding protein 1; IL15: Interleukin-15; HPRT1: Hypoxanthine phosphoribosyltransferase 1. * p < 0.05 by Welch’s t-test with Bonferroni correction vs. decidualization-only group.
Figure 2. Effect of ergothioneine concentration against decidualization markers in KC02-44D cells. Preliminary qPCR analyses were conducted using KC02-44D cells exposed to different concentrations of ergothioneine (0, 5, 10, 50, and 100 µM) under decidualization treatment. Each bar represents the mean; error bars represent standard deviation. Each dot represents an independent experiment (n = 4). EGT: Ergothioneine; PRL: Prolactin; IGFBP1: Insulin-like growth factor binding protein 1; IL15: Interleukin-15; HPRT1: Hypoxanthine phosphoribosyltransferase 1. * p < 0.05 by Welch’s t-test with Bonferroni correction vs. decidualization-only group.
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Figure 3. Effect of ergothioneine on KC02-44D cell viability and death. KC02-44D cells were treated with decidualization conditions and/or ergothioneine (EGT) for 6 days, and cytotoxicity was assessed using the LDH assay (A), while cell viability was measured using the CCK-8 assay (B). Since the amount of LDH (originally present in cells) released into the medium did not change across different EGT concentrations compared to untreated cells, EGT was not expected to increase the number of dead or membrane-damaged cells. Decidualization treatment itself reduced cell viability; however, varying EGT concentrations did not further affect viability. EGT: Ergothioneine; LDH: L-lactate dehydrogenase; CCK-8: Cell Counting Kit-8. *: p < 0.05 vs. non-decidualized group (-EGT), by Welch’s t-test with Bonferroni correction.
Figure 3. Effect of ergothioneine on KC02-44D cell viability and death. KC02-44D cells were treated with decidualization conditions and/or ergothioneine (EGT) for 6 days, and cytotoxicity was assessed using the LDH assay (A), while cell viability was measured using the CCK-8 assay (B). Since the amount of LDH (originally present in cells) released into the medium did not change across different EGT concentrations compared to untreated cells, EGT was not expected to increase the number of dead or membrane-damaged cells. Decidualization treatment itself reduced cell viability; however, varying EGT concentrations did not further affect viability. EGT: Ergothioneine; LDH: L-lactate dehydrogenase; CCK-8: Cell Counting Kit-8. *: p < 0.05 vs. non-decidualized group (-EGT), by Welch’s t-test with Bonferroni correction.
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Figure 4. Effect of ergothioneine on decidualization. KC02-44D cells were treated with decidualization conditions ± ergothioneine (100 µM) for 6 days, and alterations in the marker genes for decidualization were assessed with qPCR. Each dot represents an independent experiment (n = 3). PRL: prolactin; IGFBP1: insulin-like growth factor binding protein 1; IL15: interleukin-15; HAND2: heart and neural crest derivatives expressed 2; SLC22A4: solute carrier family 22 member 4; FOXO3: forkhead box O3; HPRT1: hypoxanthine phosphoribosyltransferase 1. *: p < 0.05 vs. control; + p < 0.05 vs. decidualization group (Welch’s t-test with Bonferroni correction).
Figure 4. Effect of ergothioneine on decidualization. KC02-44D cells were treated with decidualization conditions ± ergothioneine (100 µM) for 6 days, and alterations in the marker genes for decidualization were assessed with qPCR. Each dot represents an independent experiment (n = 3). PRL: prolactin; IGFBP1: insulin-like growth factor binding protein 1; IL15: interleukin-15; HAND2: heart and neural crest derivatives expressed 2; SLC22A4: solute carrier family 22 member 4; FOXO3: forkhead box O3; HPRT1: hypoxanthine phosphoribosyltransferase 1. *: p < 0.05 vs. control; + p < 0.05 vs. decidualization group (Welch’s t-test with Bonferroni correction).
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Figure 5. Ergothioneine suppresses FOXO1 elevation during decidualization. (A) After 6 days of decidualization/ergothioneine (EGT) treatment, RIPA buffer-soluble fractions from KC02-44D cells were prepared and analyzed by SDS-PAGE, followed by membrane transfer, blocking, and the detection of FOXO1 using specific primary and HRP secondary antibodies. Protein levels were detected with chemiluminescence, quantified with ImageJ, and normalized to ACTB levels. (B) qPCR analysis in KC02-44D cells after 6 days of decidualization ± EGT. Each dot represents an independent experiment (n = 3). FOXO1: forkhead box protein O1; ACTB: β-actin; HPRT1: hypoxanthine phosphoribosyltransferase 1; LEFTY2: left-right determination factor 2; INSR: insulin receptor; DCN: decorin. *: p < 0.05 vs. control; + p < 0.05 vs. decidualization-only group (Welch’s t-test with Bonferroni correction).
Figure 5. Ergothioneine suppresses FOXO1 elevation during decidualization. (A) After 6 days of decidualization/ergothioneine (EGT) treatment, RIPA buffer-soluble fractions from KC02-44D cells were prepared and analyzed by SDS-PAGE, followed by membrane transfer, blocking, and the detection of FOXO1 using specific primary and HRP secondary antibodies. Protein levels were detected with chemiluminescence, quantified with ImageJ, and normalized to ACTB levels. (B) qPCR analysis in KC02-44D cells after 6 days of decidualization ± EGT. Each dot represents an independent experiment (n = 3). FOXO1: forkhead box protein O1; ACTB: β-actin; HPRT1: hypoxanthine phosphoribosyltransferase 1; LEFTY2: left-right determination factor 2; INSR: insulin receptor; DCN: decorin. *: p < 0.05 vs. control; + p < 0.05 vs. decidualization-only group (Welch’s t-test with Bonferroni correction).
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Figure 6. Ergothioneine effect on an in vitro implantation model. The upper panel shows a schematic of the in vitro implantation model. KC02-44D cells were treated with decidualization ± ergothioneine for 6 days, followed by co-culture with BeWo spheroids in Ham’s F12 medium with 15% FBS. After 1.5 days, the BeWo spheroid spreading-showing lower images were quantified. The bar graphs show the mean ± SD. Each dot represents an independent BeWo spheroid. * p < 0.05 vs. control; + p < 0.05 vs. decidualization group (Welch’s t-test with Bonferroni correction). EGT: ergothioneine.
Figure 6. Ergothioneine effect on an in vitro implantation model. The upper panel shows a schematic of the in vitro implantation model. KC02-44D cells were treated with decidualization ± ergothioneine for 6 days, followed by co-culture with BeWo spheroids in Ham’s F12 medium with 15% FBS. After 1.5 days, the BeWo spheroid spreading-showing lower images were quantified. The bar graphs show the mean ± SD. Each dot represents an independent BeWo spheroid. * p < 0.05 vs. control; + p < 0.05 vs. decidualization group (Welch’s t-test with Bonferroni correction). EGT: ergothioneine.
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Figure 7. Gene expression analysis after co-culture. Samples included KC02-44D cells, BeWo cells, and co-cultures with/without treatment for 6–7.5 days. KRT7: keratin 7; ENTPD1: ectonucleoside triphosphate diphosphohydrolase 1; NT5E: 5′-nucleotidase ecto; HLA-G: major histocompatibility complex, class I, G; HPRT1: hypoxanthine phosphoribosyltransferase 1; IGFBP1: insulin-like growth factor binding protein 1; LIFR: leukemia inhibitory factor receptor; KLF4: KLF transcription factor 4; SOX2: SRY-box transcription factor 2; NONOG: Nanog homeobox; POU5F1: POU class 5 homeobox 1; C: Control; D: Decidualization; D + E: Decidualization + ergothioneine (100 µM). * p < 0.05 vs. control; + p < 0.05 vs. decidualization group (Welch’s t-test with Bonferroni correction).
Figure 7. Gene expression analysis after co-culture. Samples included KC02-44D cells, BeWo cells, and co-cultures with/without treatment for 6–7.5 days. KRT7: keratin 7; ENTPD1: ectonucleoside triphosphate diphosphohydrolase 1; NT5E: 5′-nucleotidase ecto; HLA-G: major histocompatibility complex, class I, G; HPRT1: hypoxanthine phosphoribosyltransferase 1; IGFBP1: insulin-like growth factor binding protein 1; LIFR: leukemia inhibitory factor receptor; KLF4: KLF transcription factor 4; SOX2: SRY-box transcription factor 2; NONOG: Nanog homeobox; POU5F1: POU class 5 homeobox 1; C: Control; D: Decidualization; D + E: Decidualization + ergothioneine (100 µM). * p < 0.05 vs. control; + p < 0.05 vs. decidualization group (Welch’s t-test with Bonferroni correction).
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Figure 8. HLA-G protein levels in BeWo spheroids with KC02-44D cells. The images show HLA-G immunofluorescence after co-cultures of BeWo spheroids with KC02-44D cells with/without treatment for 6–7.5 days. HLA-G was labeled with Alexa488 (green), and nuclei were stained with DAPI (blue). EGT: ergothioneine.
Figure 8. HLA-G protein levels in BeWo spheroids with KC02-44D cells. The images show HLA-G immunofluorescence after co-cultures of BeWo spheroids with KC02-44D cells with/without treatment for 6–7.5 days. HLA-G was labeled with Alexa488 (green), and nuclei were stained with DAPI (blue). EGT: ergothioneine.
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Figure 9. IGFBP1 secretion in KC02-44D culture media. After 6 days of decidualization ± ergothioneine treatment, IGFBP1 levels in the culture media of KC02-44D cells were calculated. *: p < 0.05 by Welch’s t-test with Bonferroni correction vs. control. +: p < 0.05 by Welch’s t-test with Bonferroni correction vs. decidualization treatment.
Figure 9. IGFBP1 secretion in KC02-44D culture media. After 6 days of decidualization ± ergothioneine treatment, IGFBP1 levels in the culture media of KC02-44D cells were calculated. *: p < 0.05 by Welch’s t-test with Bonferroni correction vs. control. +: p < 0.05 by Welch’s t-test with Bonferroni correction vs. decidualization treatment.
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Table 1. Patients’ information.
Table 1. Patients’ information.
Sample No.OriginsAgeTime of Collection in Menstrual CycleDays Since Last Menstrual Period
1Liquid nitrogen cryopreservation endometrial tissue46Proliferative10
2Liquid nitrogen cryopreservation endometrial tissue36Proliferative9
3Liquid nitrogen cryopreservation endometrial tissue38Mid-secretory20
4Liquid nitrogen cryopreservation endometrial tissue43Proliferative8
5Liquid nitrogen cryopreservation endometrial tissue42Proliferative8
6Liquid nitrogen cryopreservation endometrial tissue44Early-secretory19
7Liquid nitrogen cryopreservation endometrial tissue43Late-secretory28
8Liquid nitrogen cryopreservation endometrial tissue40Proliferative10
9Liquid nitrogen cryopreservation endometrial tissue42Mid-secretory21
10Liquid nitrogen cryopreservation endometrial tissue42Mid-secretory21
11Liquid nitrogen cryopreservation endometrial tissue46Mid-secretory22
12Liquid nitrogen cryopreservation endometrial tissue41Proliferative8
13Liquid nitrogen cryopreservation endometrial tissue42Mid-secretory24
14Liquid nitrogen cryopreservation endometrial tissue39Early-secretory16
15Liquid nitrogen cryopreservation endometrial tissue39Proliferative6
16Liquid nitrogen cryopreservation endometrial tissue48Early-secretory17
17Liquid nitrogen cryopreservation endometrial tissue35Late-secretory25
18Liquid nitrogen cryopreservation endometrial tissue35Mid-secretory24
19Primary cultured endometrial stromal cells50Proliferativeunidentified
20Primary cultured endometrial stromal cells45Mid-secretoryunidentified
21Primary cultured endometrial stromal cells48Late-secretory28
22Primary cultured endometrial stromal cells50Mid-secretory20
23Primary cultured endometrial stromal cells44Late-secretoryunidentified
24Primary cultured endometrial stromal cells43Early-secretory17
Table 2. Oligonucleotide sequences for qPCR.
Table 2. Oligonucleotide sequences for qPCR.
Gene NameGene SymbolOligonucleotide NameSequence
hypoxanthine phosphoribosyltransferase 1HPRT1895F5′-CTAGTTCTGTGGCCATCTGCTTAG-3′
1034R5′-GGGAACTGATAGTCTATAGGCTCATAGTG-3′
solute carrier family 22 member 4SLC22A4836F5′-ATGGGCCAGATCTCCAACTATG-3′
975R5′-TAAGCAAACAGTGGCAGCAG-3′
ProlactinPRL374F5′-ATTCGATAAACGGTATACCCATGGC-3′
623R5′-TTGCTCCTCAATCTCTACAGCTTTG-3′
insulin-like growth factor binding protein 1IGFBP1636F5′-CTATGATGGCTCGAAGGCTC-3′
791R5′-TTCTTGTTGCAGTTTGGCAG-3′
interleukin15IL15165F5′-GTTCACCCCAGTTGCAAAGT-3′
351R5′-CCTCCAGTTCCTCACATTC-3′
heart and neural crest derivatives expressed 2HAND21479F5′-AGAGGAGCTGAACGA-3′
1552R5′-CGTCCGGCCTTTGGTTTTTT-3′
forkhead box O3FOXO31217F5′-TTCCGTTCACGCACCAATTC-3′
1289R5′-ACTCTGTGCTTGCCATGATG-3′
left-right determination factor 2LEFTY21282F5′-TTAGTGCTCCTGTGTGACCTTC-3′
1418R5′-ATCAGCATGCCAGCATTTCC-3′
insulin receptorINSR2730F5′-TGCACAACGTGGTTTTCGTC-3′
2832R5′-ACATTCCCAACATCGCCAAG-3′
DecorinDCN807F5′- ACATCCGCATTGCTGATACC-3′
921R5′- TTCAGGCTAGCTGCATCAAC-3′
forkhead box O1FOXO12336F5′-ATGTGTTGCCCAACCAAAGC-3′
2475R5′-TTGGACTGCTTCTCTCAGTTCC-3′
keratin 7KRT7603F5′-AATTAACCACCGCACAGCTG-3′
677R5′-TTGCTCATGTAGGCAGCATC-3′
ectonucleoside triphosphate diphosphohydrolase 1ENTPD1189F5′-ACCCAGAACAAAGCATTGCC-3′
306R5′-CGCCTGTGTCATTCTCCTTTTC-3′
5′-nucleotidase ectoNT5E708F5′-ACTGGGACATTCGGGTTTTG-3′
827R5′-TCTTTGGAAGGTGGATTGCC-3′
major histocompatibility complex, class I, GHLA-GF5′-GAAGAGGAGACACGGAACACCA-3′
G5′-TCGCAGCCAATCATCCACTGGA-3′
leukemia inhibitory factor receptorLIFR3454F5′-CTCCAGACTCTCCTAGATCCATAGAC-3′
3587R5′-CCACCCTCCTCCATTAGATTTAGG-3′
KLF transcription factor 4KLF4486F5′-TCGGCCAATTTGGGGTTTTG-3′
613R5′-CAGGTGGCTGCCTCATTAATG-3′
SRY-box transcription factor 2SOX21529F5′-CATCACCCACAGCAAATGACAG-3′
1614R5′-AGTTTTCTTGTCGGCATCGC-3′
Nanog homeoboxNANOG582F5′-GCAGATGCAAGAACTCTCCAAC-3′
691R5′-TCGGCCAGTTGTTTTTCTGC-3′
POU class 5 homeobox 1POU5F1592F5′-TCAAGTGATTCTCCTGCCTCAG-3′
687R5′-AGCTTGGCAAATTGCTCGAG-3′
progesterone receptorPGR2484F5′-CCTTTGGAAGGGCTACGAAGT-3′
2593R5′-GAGCTCGACACAACTCCTTTTTG-3′
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MDPI and ACS Style

Yoshida, N.; Murata, H.; Ide, K.; Tanaka, M.; Mori, K.; Futani, K.; Sawachika, M.; Okada, H.; Tanaka, S. Regulatory Mechanism of Human Endometrial Stromal Cell Decidualization by Ergothioneine. Nutraceuticals 2025, 5, 16. https://doi.org/10.3390/nutraceuticals5030016

AMA Style

Yoshida N, Murata H, Ide K, Tanaka M, Mori K, Futani K, Sawachika M, Okada H, Tanaka S. Regulatory Mechanism of Human Endometrial Stromal Cell Decidualization by Ergothioneine. Nutraceuticals. 2025; 5(3):16. https://doi.org/10.3390/nutraceuticals5030016

Chicago/Turabian Style

Yoshida, Namika, Hiromi Murata, Konomi Ide, Marika Tanaka, Kurumi Mori, Kensuke Futani, Misa Sawachika, Hidetaka Okada, and Susumu Tanaka. 2025. "Regulatory Mechanism of Human Endometrial Stromal Cell Decidualization by Ergothioneine" Nutraceuticals 5, no. 3: 16. https://doi.org/10.3390/nutraceuticals5030016

APA Style

Yoshida, N., Murata, H., Ide, K., Tanaka, M., Mori, K., Futani, K., Sawachika, M., Okada, H., & Tanaka, S. (2025). Regulatory Mechanism of Human Endometrial Stromal Cell Decidualization by Ergothioneine. Nutraceuticals, 5(3), 16. https://doi.org/10.3390/nutraceuticals5030016

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