{"paper_id":"a1197dae-250c-41f8-89a8-a7b21131bbf4","body_text":"Koo et al. Environmental Sciences Europe           (2023) 35:69  \nhttps://doi.org/10.1186/s12302-023-00780-x\nRESEARCH\nEffect of tetrabromobisphenol \nA (TBBPA) on early implantation using \nthe three-dimensional spheroid model \nwith human endometrial cell line, Ishikawa\nMyoungjoo Koo1†  , Inyoung Kang1†  , Jin Hyun Jun1   and Jaewang Lee1*   \nAbstract \nBackground Tetrabromobisphenol A (TBBPA) can be characterized as an endocrine-disrupting chemical (EDCs). It \nhas been widely used as a brominated flame retardant in industrial products. EDCs have effects on female reproduc-\ntion leading to issues, such as infertility, hormone imbalance, and endometriosis. In Korea, the problems of infertility \nand decreasing birth rate are of significant concern. Exposure to EDCs might have a harmful effect on female fertility \nby mediating a decrease endometrial receptivity. This study aimed to investigate the effects of TBBPA on infertil-\nity, particularly on early implantation events in the uterine endometrium. Human endometrial adenocarcinoma \nand trophoblastic cell lines were used in this study. The cytotoxicity of TBBPA on Ishikawa cells and Jeg-3 cells \nwas measured using the Cell Counting Kit-8 assay. The mRNA expression was analyzed by reverse transcription-\nquantitative polymerase chain reaction, and protein levels were measured by western blotting. The attachment rate \nwas analyzed using an attachment assay, and the outgrowth area was measured using an outgrowth assay.\nResults The mRNA expression of interleukin (IL)-6, IL-1β, tumor necrosis factor-α, and leukemia inhibitory factor \nwas significantly increased upon treatment of Ishikawa cells by TBBPA. Moreover, the outgrowth area in the TBBPA \ngroup was significantly decreased compared to that in the control. In contrast, TBBPA had a minor effect on protein \nlevels and attachment rates.\nConclusions In this study, TBBPA induced an inflammatory milieu in mRNA expression. An increase in inflammation-\nrelated cytokines in the endometrium can disrupt embryo implantation. TBBPA disrupted the outgrowth of spheroids \nin the endometrium; however, the protein levels and attachment rate were comparable to those in the control group. \nThe effect of TBBPA on implantation events should be elucidated further.\nKeywords Implantation, Endocrine disrupting chemicals (EDCs), Tetrabromobisphenol A, Ishikawa cells, Jeg-3 cells, \nInfertility\nBackground\nFemale infertility refers to the inability of a woman to \nconceive or carry a pregnancy to full term. Many causes \nof female infertility are known, including problems with \novulation, fallopian tube or uterus, and hormone imbal -\nance [1, 2]. According to a report published by the Korea \nSociety of Obstetrics and Gynecology, the infertility rate \nin Korea was estimated to be approximately 10% in 2019, \nOpen Access\n© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which \npermits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the \noriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or \nother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line \nto the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory \nregulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this \nlicence, visit http://creativecommons.org/licenses/by/4.0/.\nEnvironmental Sciences Europe \n†Myoungjoo Koo and Inyoung Kang contributed equally to this work.\n*Correspondence:\nJaewang Lee\nwangjaes@gmail.com\n1 Department of Biomedical Laboratory Sciences, Eulji University, 553, \nSanseong-Daero, Sujeong-Gu, Seongnam-Si, Gyeonggi-Do, Republic \nof Korea\n\nPage 2 of 11Koo et al. Environmental Sciences Europe           (2023) 35:69 \nwhich is higher than the global average of 8% [3]. Addi -\ntionally, over the decades, the number of patients receiv -\ning assisted reproductive technology (ART) for infertility \nhas increased in South Korea [4, 5]. Several causes of \nimplantation failure are known, including uterine fac -\ntors, embryo factors, endometrial receptivity, hormonal \nfactors, and immunological factors. However, the effects \nof endocrine-disrupting chemicals (EDCs) including \ntetrabromobisphenol-A (TBBPA) on the uterine endo -\nmetrium have not been fully investigated [6, 7].\nEDCs interfere with the function of the endocrine sys -\ntem in humans and animals [8]. The endocrine system \nregulates body function, especially reproduction and the \nhormone cycle [9, 10]. EDCs can block the effects of nat -\nural hormones though structures similar to hormones, \nleading to alterations in hormone levels and disruption of \nphysiological homeostasis [11, 12]. They can bind to sex \nhormone receptors and inhibit or activate the production \nof hormones, such as estrogen and progesterone [13]. \nAs a result, the hormone pathway is interfered by EDCs, \nwhich might have an impact on cell signaling [14].\nBisphenol A (BPA) is an industrial chemical used in \nthe production of plastics and epoxy resins [15]. It is \ncommonly found in polycarbonate plastics, which are \nused in water bottles, food containers, and canned foods \n[16]. BPA is an EDC that binds to estrogen receptors \nin the human endocrine system [17]. Exposure to BPA \nleads to reproductive and developmental disorders and \nimpaired immune function [18, 19]. TBBPA is a member \nof the bisphenol family; it is structurally similar to BPA. \nDespite being considered a general and perpetual con -\ntaminant of the environment, TBBPA is a widely used \nindustrial product [20]. Exposure to TBBPA is common \nin everyday life as it is included in plastic materials, and \nit accounts for approximately 60% of the production in \nthe entire brominated flame retardants (BFRs) market, \nthereby accounting for the highest percentage of BFRs \n[21, 22]. In addition, highest consumption of TBBPA has \nbeen recorded in Asia when compared to global con -\nsumption [23] and TBBPA has been detected in serum \nof Korean population, ranging from 0.05 to 75 ng/g lipid \nweight [24]. Unfortunately, the function of TBBPA as an \nEDC has not been fully elucidated. Various studies have \nits effects on endocrine and reproductive systems [25]. \nTBBPA promotes the growth of uterine fibroids, which \nare known to cause recurrent implantation failure (RIF) \n[26–28]. Nevertheless, TBBPA is not regulated, and the \neffect of TBBPA on early human implantation has not \nbeen studied.\nEmbryo implantation is also an important part of \nreproduction, specifically in the process of pregnancy, \nand a major cause of pregnancy loss [29]. It refers to \nthe attachment and invasion of a fertilized embryo into \nthe uterine endometrium. The synchronized receptive \nstate of the uterus and development of the embryo are \ncrucial for the implantation process [30, 31]. Endo -\nmetrial receptivity is maintained for only a few days, \nwhich is defined as the implantation window. Endo -\nmetrial receptivity is regulated by ovarian hormones, \nsuch as estrogen and progesterone. These hormones \ninfluence the expression of attachment and inflamma -\ntion molecules such as leukemia inhibitory factor (LIF), \ninterleukin (IL)-6, and tumor necrosis factor (TNF)-α. \nCurrently, whether TBBPA leads to implantation fail -\nure and decreased endometrial receptivity has not been \nexamined [32, 33]. Therefore, the aim of this study was \nto investigate the harmful effects of TBBPA on the early \nimplantation and outgrowth processes using a three-\ndimensional spheroid cell culture model with Jeg-3 and \nIshikawa cells.\nMaterials and methods\nCell culture\nThe endometrial epithelial-like Ishikawa cell line and \nhuman trophoblastic Jeg-3 cell line were cultured in Dul -\nbecco’s modified Eagle medium (DMEM, Welgene, Gyeo-\nngsan, Korea) supplemented with 10% fetal bovine serum \n(FBS, Gibco, Waltham, MA, USA) and 1% penicillin and \nstreptomycin (P/S, Lonza, Morristown, NJ, USA). Cells \nwere cultured under standard conditions (37 °C, 5%  CO2) \n[34]. Ishikawa cells were used to represent the human \nendometrium, which is the implantation site. Jeg-3 cells \nwere used to represent the human embryo because of the \ninner cell mass surrounded by trophoblastic cells. TBBPA \n(Sigma-Aldrich, St. Louis, MO, USA) was dissolved in \ndimethyl sulfoxide (DMSO, Sigma-Aldrich) and used to \ntreat Ishikawa cells to determine its effect of on endome -\ntrial cells.\nCell viability assay\nA cell viability assay was performed to determine the \ncytotoxic dose of TBBPA. Ishikawa cells and Jeg-3 cells \nwere detached using 0.25% Trypsin/EDTA (Gibco) and \nsuspended to seed in 96-well plates at a density of 1 ×  103/\nwell. The cells were exposed to 0.01–100  μM TBBPA \nfor 24 or 48  h. The viability of the cells was evaluated \nusing Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, \nJapan). After incubating with different concentrations of \nTBBPA, 10 µL of CCK-8 reagent was added to each well, \nfollowed by incubation for 1 h at 37 °C. Cell viability was \ndetected at 450  nm using MultiskanGO (ThermoFisher, \nWaltham, MA, USA) [35, 36]. The cells were divided into \ncontrol and TBBPA groups. The control group was cul -\ntured with 1% FBS containing 0.1% DMSO in DMEM.\n\nPage 3 of 11\nKoo et al. Environmental Sciences Europe           (2023) 35:69 \n \nReverse transcription‑quantitative polymerase chain \nreaction (RT‑qPCR)\nIshikawa and Jeg-3 cells were detached using 0.25% \nTrypsin/EDTA and seeded in a 6-well plate (SPL, \nPocheon, Korea) at a density of 1 ×  105 /well. Ishikawa \ncells were treated with TBBPA at concentrations of 0.1, 1, \nand 10 μM for 48 h. For the comparison of 2D versus 3D \nspheroids of Jeg-3 cells, 1 ×  105 cells per well were seeded \nfor the 2D group, and 100 Jeg-3 spheroids were used \nfor 3D groups. The mRNA expression of IL-1α, IL-1β, \nintegrin alpha V (ITGαV), and LIF was detected in 2D \nand 3D spheroids and compared. Total RNA from cells \nand spheroids was extracted using TRIzol (Invitrogen, \nWaltham, MA, USA). For RT-qPCR analysis, 500  ng of \nmRNA was converted to complementary DNA (cDNA) \nusing the PrimeScript ™ 1st strand cDNA Synthesis Kit \n(Takara, Kusatsu, Japan). RT-qPCR was performed using \nSYBR green reagent (Meridian Bioscience, Cincinnati, \nOH, USA) and primers (BIONEER, Daejeon, Korea). \nEach experiment was performed at least in triplicate \nand was repeated more than three times. For inflamma -\ntion targets, IL-6 (Bio-Rad, Hercules, CA, USA), IL-1β, \nand TNF- α were examined. Furthermore, ITGαV and \nLIF were examined as attachment targets. All data were \nnormalized to that of glyceraldehyde-3-phosphate dehy -\ndrogenase (GAPDH). The primer sequences used in this \nstudy are summarized in Table  1. The PCR method was \nperformed as follows: hold stage at 95 °C for 15 min and \nPCR cycle of denaturation at 95 °C for 30 s, annealing at \n61 °C for 30 s, and extension at 72 °C for 30 s [37].\nWestern blot analysis\nTo quantify the level of protein, western blot analysis was \nperformed using cell lysates. Ishikawa cells were seeded \nin 6-well plates at a density of 1 ×  105 cells/well. Total \nprotein was extracted using radioimmunoprecipitation \nassay lysis buffer (Thermo Fisher) with proteinase inhibi -\ntor (Thermo Fisher). Total protein concentration was \ndetermined by bicinchoninic Acid assay (Thermo Fisher). \nA total of 15 µg of protein was used for the experiments. \nEach sample was separated using 10% sodium dodecyl \nsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) \nand transferred to polyvinylidene fluoride (PVDF) mem -\nbranes. The membranes were blocked with 5% skim milk \nfor 2 h at room temperature and then incubated with pri-\nmary antibody against IL-6 (1:800) (Abcam, Cambridge, \nUK), TNF-α (1:500) (Abcam), LIF (1:200) (Santa Cruz \nBiotechnology, Dallas, TX, USA), and GAPDH (1:5000) \n(Santa Cruz Biotechnology) overnight at 4  °C on an \norbital shaker. The membranes were washed thrice using \nTris-buffered saline with 1% Tween 20. For IL-6, TNF-\nα, and LIF, incubation was performed with anti-rabbit \nhorseradish peroxidase-conjugated immunoglobulin \n(IgG) secondary antibody for 2  h at room temperature. \nFor GAPDH, incubation was performed with anti-mouse \nhorseradish peroxidase-conjugated IgG secondary anti -\nbody for 2  h at room temperature. The proteins on the \nmembranes were analyzed using the iBright CL750 imag-\ning system (Applied Biosystems, Waltham, MA, USA). \nThe data were evaluated using ImageJ software (NIH, \nBethesda, MD, USA).\nAttachment assay\nIshikawa cells were cultured for 48 h in a T25 flask with or \nwithout TBBPA treatment. Then, the cells were detached \nusing 0.25% Trypsin/EDTA and seeded in a 12-well plate \nat a density of 1 ×  105 and cultured with DMEM contain -\ning 10% FBS. After 8  h, the medium was changed for \neach experimental group. DMEM with 1% FBS was used \nfor the control and TBBPA groups. Formation of Jeg-3 \nTable 1 Primer sequence\nGenes Primer sequence Product size (bp) GenBank Accession \nnumber\nAnnealing\nTemperature (℃)\nIL-1α F: CTG AAG GAG ATG CCT GAG ATA C 383 NM_000575.5 61\nR: GAA CTG TCA ACA CTG CAC AAG \nIL-1β F: TAA AGA GAG CTG TAC CCA GAG A 217 NM_000576.3\nR: AAG TGA GTA GGA GAG GTG AGA G\nTNF-α F: CTC CTC ACC CAC ACC ATC AG 134 NM000594.4\nR: ATA GAT GGG CTC ATA CCA GGG \nITGαV F: AAT CTT CCA ATT GAG GAT ATC AC 140 NM_002210.5\nR: AAA ACA GCC AGT AGC AAC AAT \nLIF F: CCA ACG TGA CGG ACT TCC C 82 NM000981.4\nR: TAC ACG ACT ATG CGG TAC AG\nGAPDH F: GGA GCG AGA TCC CTC CAA AA 197 NM_002046.7\nR: GGC TGT TGT CAT ACT TCT CA\n\nPage 4 of 11Koo et al. Environmental Sciences Europe           (2023) 35:69 \nspheroid was performed using hanging drop method at \na density of 500 cells per spheroid for 48  h in DMEM \nsupplemented with 10% FBS and rotation at 45 RPM in \na shaking incubator, as shown in Fig.  5A. Spheroids with \nsizes ranging from 150 to 300 were used for this experi -\nment. After harvesting the spheroids, they were seeded \non Ishikawa cells treated with TBBPA. The attachment \nrates were observed at 5, 10, 20, and 30 min and 1, 2, 4, \nand 24  h [38]. The attachment assay performed in this \nstudy is illustrated in Fig. 1A.\nOutgrowth assay\nTo determine the effect of TBBPA on spheroid out -\ngrowth, Ishikawa cells were treated with TBBPA for 48 h \nin a T25 flask. The cells were then detached using 0.25% \nTrypsin/EDTA and seeded on a 12-well plate at a density \nof 5 ×  104 in DMEM with 10% FBS. After 8 h, the medium \nwas replaced with DMEM supplemented with 1% FBS \nfor the control and TBBPA groups. The cells in the nega -\ntive control groups were replaced with DMEM without \nFBS. Spheroids were seeded on Ishikawa cells after the \nmedium was changed, followed by incubation for 72  h \nunder standard conditions (37 °C, 5%  CO2) [39, 40]. The \nEVOS M500 imaging system (ThermoFisher) was used \nfor capturing photos of outgrowth areas with a magnifi -\ncation of 40X. The outgrowth area was measured using \nthe ImageJ software (NIH). The outgrowth assay in this \nstudy is illustrated in Fig. 1A.\nStatistical analysis\nAll experiments were performed at least in triplicates. All \nvalues are represented as ± standard error of the mean \n(SEM). Attachment rate data were analyzed using the \nchi-square test. The outgrowth area data were analyzed \nusing the t-test, and a difference between groups with a \nP-value of less than 0.001 was considered statistically sig -\nnificant. Other results were analyzed using one-way anal-\nysis of variance (one-way ANOVA) with Tukey’s post-hoc \nmultiple comparison tests. Statistical significance was set \nat P value of less than 0.05. The Jeg-3 2D vs. 3D mRNA \nexpression was analyzed by t-test and a difference with \na P value of less than 0.05 was considered statistically \nsignificant.\nResults\nThe expression of Jeg‑3 cells 2D vs Jeg‑3 spheroids\nTo investigate the difference between 2 and 3D cultures, \nthe expression of mRNA markers related to attachment \nFig. 1 Experimental design and difference of mRNA expression between 2D culture and 3D culture of Jeg-3. The mRNA expression of Jeg-3 2D \nculture and 3D culture using qRT-PCR (ITGaV, LIF , IL-1α and IL-1β) (n > 3) One-way analysis of variance; Tukey’s multiple comparison test (p < 0.05). A \nExperimental design for this study. B Jeg-3 2D cultured and 3D culture mRNA expression. ***P < 0.0001\n\nPage 5 of 11\nKoo et al. Environmental Sciences Europe           (2023) 35:69 \n \n(ITGαV and LIF) and inflammation (IL-1α and IL-1β) \nwas evaluated using qRT-PCR. The mRNA expression of \nIL-1α was increased in the 3D culture group than in the \n2D culture group. (IL-1α, 2D 1.04 ± 0.16 vs 3D 3.68 ± 0.21) \n(Fig. 1B). The mRNA expression levels of ITGαV, LIF, and \nIL-1β in the 2D culture group were comparable to those \nin the 3D culture group. Based on these results, we con -\nfirmed the difference in mRNA expression between 2D \nmodels and 3D models, and 3D models were more suit -\nable for recapitulating the physiological state. Therefore, \nin this study, we applied 3D spheroid model to the fol -\nlowing experiments performed under in vivo conditions.\nThe effects of TBBPA on the viability of Ishikawa and Jeg‑3 \ncells\nTo examine the concentration of TBBPA that demon -\nstrated cytotoxic effects on cells for 24  h and 48  h, the \ncells were treated with TBBPA at concentrations of 0.1, \n1, 10, and 100 μM. The highest dose of TBBPA decreased \nviability of both Ishikawa and Jeg-3 cells at 24 h and 48 h \n(P < 0.0001). The concentrations of 0.1, 1, and 10  μM of \nTBBPA did not affect viability of either Ishikawa cells \nor Jeg-3 cells for 24  h (Fig.  2A, C) or 48  h (Fig.  2B, D). \n(Ishikawa 24  h, control, 1.00 vs TBBPA 0.1  µM, 1.23 vs \nTBBPA 1  µM, 1.12 vs TBBPA 10  µM, 1.04 vs TBBPA \n100  µM, 0.19), (Ishikawa 48  h, control, 1.00 vs TBBPA \n0.1  µM, 0.96 vs TBBPA 1  µM, 0.99 vs TBBPA 10  µM, \n0.97 vs TBBPA 100  µM, 0.14), (Jeg-3 24  h control, 1.00 \nvs TBBPA 0.1 µM, 1.04 vs TBBPA 1 µM, 1.04 vs TBBPA \n10 µM, 1.04 vs TBBPA 100 µM, 0.21), (Jeg-3 48 h, con -\ntrol, 1.00 vs TBBPA 0.1  µM, 1.04 vs TBBPA 1  µM, 1.01 \nvs TBBPA 10  µM, 1.00 vs TBBPA 100  µM, 0.16). Since \nno statistical differences were observed among TBBPA \ngroups except among cells treated with a concentration \nof 100 μM, the cells were treated with 0.1, 1, and 10 μM \nduring the next steps. A cytotoxicity test was performed \nto confirm the absence of cytotoxic effects.\nGene expression in Ishikawa cells treated with TBBPA\nTo investigate the effect of TBBPA on Ishikawa cells, \nmRNA expression was detected using qPT-PCR. \nThe mRNA expression of inflammatory markers was \nincreased in Ishikawa cells treated with TBBPA. Among \ninflammation-related genes, the mRNA expression of \nFig. 2 The cell viability of Ishikawa and Jeg-3 by Cell Counting Kit (CCK8) assay. Cell count kit 8 (CCK-8) assay was conducted to determine cell \ncytotoxicity of TBBPA. Ishikawa cells and Jeg-3 cells were treated with TBBPA for 24/48 h (n > 3). One-way analysis of variance; Tukey’s multiple \ncomparison tests, a vs b vs c vs d vs e (p < 0.05). A Ishikawa cell line TBBPA 0.1–100 μM treated for 24 h. B Ishikawa cell line TBBPA 0.1–100 μM treated \nfor 48 h. C Jeg3 cell line TBBPA 0.1 μM ~ 100 μM treated 24 h. D Jeg3 cell line TBBPA 0.1 μM ~ 100 μM treated 48 h. *P < 0.05, **P < 0.001, ***P < 0.0001\n\nPage 6 of 11Koo et al. Environmental Sciences Europe           (2023) 35:69 \nIL-6, IL-1β, and TNF-α was significantly increased in \nthe TBBPA-treated groups compared to that in the con -\ntrol groups (IL-6, control, 1.001 ± 0.028 vs TBBPA 10 µM, \n1.38 ± 0.05), (IL-1β, control, 1.00 ± 0.03 vs TBBPA 0.1 µM, \n1.73 ± 0.07 vs TBBPA 1  µM, 1.30 ± 0.03) (TNF-α, con -\ntrol 1.00 ± 0.03 vs TBBPA 0.1  µM, 4.09 ± 0.36 vs TBBPA \n1 µM, 4.56 ± 0.25 vs TBBPA 10 µM, 4.72 ± 0.56) (Fig. 3A). \nFor attachment-related genes, the expression of LIF in \nthe TBBPA groups was significantly increased com -\npared to that in the control groups (Fig.  3B; LIF, control, \n1.00 ± 0.03 vs TBBPA 1 µM, 3.29 ± 0.13 vs TBBPA 10 µM, \n4.31 ± 0.15). These findings indicate that TBBPA affects \ninflammation and attachment of endometrium cells, \nwhich was determined based on the effects observed at \nthe mRNA level.\nProtein expression of Ishikawa cells treated with TBBPA\nTo identify the protein levels of TBBPA in Ishikawa cells, \nthe expression of inflammation- and attachment-related \nproteins, namely, LIF, TNF-α, and IL-6 were detected. \nIn the previous experiment, the mRNA expression of \nLIF, TNF-α, and IL-6 was elevated in the TBBPA-treated \ngroups. To identify whether mRNA elevation led to an \nincrease in protein levels, western blot assay was used. \nThe levels of the proteins were slightly increased in all \nTBBPA groups; however, the difference was not statisti -\ncally significant (Fig. 4A, B).\nAttachment rate of Jeg‑3 spheroid on Ishikawa cells \ntreated with TBBPA.\nAs observed in the previous experiment, the expression \nof attachment-related marker LIF was increased at the \nmRNA and protein levels, indicating that TBBPA might \nenhance the attachment rate of spheroids. To determine \nthe attachment rate of spheroids to Ishikawa cells treated \nwith TBBPA, attachment assays were performed (Con -\ntrol vs TBBPA 10  µM, 5  min 0/89 vs 0/93, 10  min 1/89 \nvs 2/93, 20  min 17/89 vs19/93, 30  min 27/89 vs 32/93, \n60 min 80/89 vs 85/93, 90 min 85/89 vs 87/93, 2 h 89/89 \nvs 92/93, 4 h 89/89 vs 92/93, 24 89/89 vs 93/93; Fig.  5A). \nEarly attachment rates (0 ~ 30  min) were slightly higher \nin the TBBPA groups (10 μM) than in the control groups. \nAfter 90  min, the attachment rate was not significantly \ndifferent among the experimental groups (Fig. 5B).\nFig. 3 The mRNA expression related to inflammation \nand attachment in Ishikawa cells. The mRNA markers related \nto inflammation were assessed by qRT-PCR. The mRNA expression \nof Ishikawa cells treated with TBBPA (0.1 μM ~ 10 μM) for 48 h. The \nmRNA expressions were normalized with GAPDH. (n > 3) One-way \nanalysis of variance; Tukey’s multiple comparison tests (p < 0.05) \nA IL-6, IL-1β, and TNF-α mRNA expression of Ishikawa cells treated \nwith TBBPA for 48 h. B ITGαV and LIF mRNA expression of Ishikawa \ncells treated TBBPA for 48 h. *P < 0.05, **P < 0.001, ***P < 0.0001\nFig. 4 The protein level of Ishikawa cells treated with TBBPA. The \nprotein level that has increased in the qRT-PCR. All targets normalized \nwith GAPDH. Ishikawa cells were treated with TBBPA for 48 h \nand 20 µg/mL were used for Western blot assay. A The protein level \nof Ishikawa cell treated with TBBPA (LIF, TNF-a, IL-6) (n > 3) One-way \nanalysis of variance; Tukey’s multiple comparison tests (p < 0.05) B The \nWestern blot band of GAPDH, TNF-a, LIF, and IL-6\n\nPage 7 of 11\nKoo et al. Environmental Sciences Europe           (2023) 35:69 \n \nOutgrowth assay of Jeg‑3 spheroids on Ishikawa cells \ntreated with TBBPA.\nTo determine the effect of TBBPA on outgrowth, an out -\ngrowth assay was conducted by examining Jeg-3 sphe -\nroids seeded on Ishikawa cells, as shown in Fig.  6A. \nIshikawa cells were treated with TBBPA at a concentra -\ntion of 10 μM. In the TBBPA groups, the outgrowth area \nsignificantly decreased (P < 0.0001; control 11.6 ± 0.55 vs \nTBBPA 10 µM 7.38 ± 0.35 vs negative control 6.77 ± 0.36; \nFig.  6B). This result indicates that TBBPA negatively \ninfluenced the outgrowth area even though the attach -\nment-related protein levels were comparable.\nDiscussion\nIn the present study, we investigated whether TBBPA has \ndeleterious effects on the implantation and invasion pro -\ncesses using the three-dimensional spheroid cell culture \nmethod. For examining the potential risks and evidence \nof the effects of TBBPA on female infertility, it is essen -\ntial to investigate the toxicity of TBBPA in the endome -\ntrium and trophoblastic spheroids, which represented \nthe embryo in this study. TBBPA induced inflammation \nmilieu, increased the expression of the IL-6, IL-1β and \nTNF-a mRNA levels, and reduced the outgrown area, \neven though the protein levels and attachment rate were \ncomparable to the control groups in this study. However, \nthe potential mechanism of TBBPA has not yet been \nelucidated.\nFor in vitro spheroid models, 2D cultured systems are \ngenerally used, but they do not fully represent physi -\nological human cells. However, 3D spheroids can mimic \nhuman in vivo cell conditions much better than 2D cul -\nture systems [41]. Differences in data among 2D and 3D \nmodels have been observed [42, 43]. The main concern \nassociated with the use 3D culture models in this study \nwas that they were prepared with a cancer cell line. \nCancer cells do not represent primary non-cancer cells, \nwhich may lead to contrasting results [44]. In this study, \nthe 2D and 3D culture systems showed different mRNA \nexpression patterns of inflammation-related genes, such \nas IL-1β. Only IL-1β showed a significant difference. \nThese results indicate that our 3D and 2D models are dif-\nferent from those used in other studies. In addition, the \ndifferences in mRNA expression showed that the use of \nthe 3D culture system in the study possibly recapitulated \nthe actual physiology of the human body as previously \ndescribed.\nFig. 5 The attachment assay of Jeg3 spheroid on Tetrabrombisphenol A treated Ishikawa cells. A The morphology of Ishikawa cells, Jeg-3 spheroid, \nand Jeg-3 Spheroid on Ishikawa cells treated with TBBPA. B Attachment rate (%) (n > 65) T-test; (p < 0.05). The morphology of Ishikawa cells and Jeg-3 \nspheroids were taken by the EVOS imaging system. The attachment assay was performed to detect the Jeg-3 spheroids attachment rate\n\nPage 8 of 11Koo et al. Environmental Sciences Europe           (2023) 35:69 \nIn this study, TBBPA did not affect viability of the \nhuman endometrial cell line at concentrations of 0.1, \n1, and 10  μM for 24 or 48  h. However, a concentration \nof 100  μM showed significantly decreased cell viability \n(P < 0.0001). To confirm the effect of TBBPA at the cel -\nlular level, the concentration that did not exert cytotoxic-\nity was determined using the CCK-8 assay. With regard \nto the elements concerning authentic human endocrine \nsystems and the environment, exposure to EDCs occurs \nover extended periods and at low concentrations. It is \npreferable to conduct the experiment in an environment \nidentical to the actual setting. However, it is quite diffi -\ncult to maintain a cell line for a long time, and it cannot \nbe reconstructed using cell line experiments. As a refer -\nence, TBBPA treatment was performed at a high dose \nwithout short-term cytotoxicity [45, 46].\nQuantitative RT-PCR was used to examine the effect \nof TBBPA on mRNA expression. To determine the \neffect of TBBPA on the endometrium, the marker asso -\nciated with inflammation and attachment genes was \nexamined [47]. When the human embryo is attached \nand invaded in the endometrium, cytokines related \nto attachment and inflammation are released into the \nendometrium and embryo. For inflammation, IL-6, \nTNF-α, and IL-1α were used, and for attachment, \nLIF and ITGαV, which are released from the embryo, \nwere used [48]. The inflammation marker IL-6 and \nattachment marker LIF interact with each other [49–\n51]. The mRNA expression associated with inflamma -\ntion and attachment can show the effect of EDCs on \nembryo implantation and invasion. The levels of IL-6, \nTNF-α, and IL-1β, which are associated with inflam -\nmation, were remarkably increased in TBBPA-treated \nendometrium cells (P < 0.001). The findings indi -\ncate that TBBPA induced an inflammatory milieu in \nembryos and the endometrium.\nThe protein levels were evaluated using western blot \nand primary antibodies against IL-6, TNF-α, and LIF. \nThe results are reflected by an increase at the mRNA \nlevel. Western blotting was performed to confirm the \nincrease in mRNA expression at the protein level. In this \nexperiment, TBBPA did not change the protein levels sig-\nnificantly. However, the levels of TNF-α in cells treated \nwith 10 µM of TBBPA were slightly increased (P = 0.058), \nindicating that TBBPA has significant effects on mRNA \nexpression and protein levels, but not on these targets \nthat used in this experiment. We did not detect changes \nin protein levels in response to TBBPA exposure. Their \nnon-responsiveness to TBBPA may be attributed to the \nfact that the treatment time might be too short to detect \nresponses in the changes in expression. Further studies \nshould clarify how TBBPA regulates the expression of \ninflammation and attachment-related genes to evaluate \nits toxicity on female reproduction.\nFig. 6 The outgrowth area of Jeg3 spheroid on TBBPA-treated Ishikawa cells. A The morphology and area of Jeg-3 spheroid outgrowth. B The \noutgrowth area. (n = 45 control group, n = 63 TBBPA group, n = 42 negative control group) One-way analysis of variance; Tukey’s multiple comparison \ntests (p < 0.0001). The outgrowth area was detected and calculated by the ImageJ system. The relative area was measured compared to the control \ngroup. ***P < 0.0001\n\nPage 9 of 11\nKoo et al. Environmental Sciences Europe           (2023) 35:69 \n \nAn attachment assay was used to investigate the effect \nof TBBPA on implantation, especially on attachment pro-\ncess. We attempted to mimic the EDC-influenced endo -\nmetrium by treating endometrial cell lines with TBBPA. \nIn the attachment assay, TBBPA-treated group showed \na slightly higher rate of early implantation than the con -\ntrol group. However, after 60  min, the TBBPA groups \nshowed a lower attachment rate than the control groups. \nMoreover, no significant differences were noted between \nthe groups. Because the mRNA expression of LIF was \nincreased in the TBBPA group, it is considered that the \nrate of TBBPA-treated group increased compared to \nthe control group at the early attachment rate. As men -\ntioned above, a limitation of this assay is that spheroids \nare made with cancer cells. High proliferation and adhe -\nsion are the main characteristics of cancer cell lines and \ncan sufficiently affect the attachment of spheroids to \nthe endometrium [52]. For the next study, we are evalu -\nating the impact of TBBPA on mouse primary uterine \ncells because cancer cells cannot completely imitate the \npathophysiology of primary cells.\nThe outgrowth assay was conducted to examine the \ninvasion of embryo into the endometrium. This implan -\ntation process occurs within 72  h in the human body. \nJeg-3 spheroids seeded on TBBPA-treated endometrial \ncells represent the human endometrium exposed to \nEDCs and embryo implantation. Hence, the outgrowth \nassay was conducted for 72  h. This result suggests that \nthe outgrowth area is decreased because TBBPA affects \nmolecules when the embryo attaches to the endome -\ntrium. In this experiment, TBBPA might have a negative \nimpact on the implantation process in the human body.\nThe implantation process follows a series of critical \nsteps, including attachment, invasion, and outgrowth. \nEach of these steps must occur in a coordinated manner \nfor successful implantation to take place. Disruptions in \ncytokine levels can affect the delicate balance required \nduring implantation and may lead to implantation fail -\nure or decreased outgrowth of the embryo. In our study, \nwe examined inflammatory cytokines, including IL-6, \nIL-1b, TNF-a, and LIF, which are important cytokines \nand factors involved in the complex process of implanta -\ntion [53–55]. LIF plays a crucial role in embryo implan -\ntation. It is released from the blastocyst and binds to \nLIFR on endometrial cells, facilitating the attachment \nof the blastocyst to the endometrial lining. Moreover, \nthe interaction of IL-6 and IL6-R with LIF is involved in \nthis process of attachment. By studying the mRNA lev -\nels of these markers, this study provides valuable insights \ninto how TBBPA may affect the expression and regula -\ntion of these important factors during implantation. The \nfact that these markers interact with each other during \nthe implantation process highlights the importance of \nexamining them in conjunction to understand their com-\nbined effects on implantation success. Research in this \narea may provide crucial information about how EDCs \nsuch as TBBPA might impact the process of implanta -\ntion and potentially lead to recurrent implantation fail -\nure. In this study, we found that the outgrowth area was \nsignificantly decreased in the TBBPA group, suggesting \nthat TBBPA exposure may have an adverse impact on the \noutgrowth stage of implantation. Understanding these \nmechanisms can pave the way for the development of tar-\ngeted interventions or treatments to counteract negative \neffects and improve implantation success rates. Overall, \nwe suggest that this is a promising and important study \nthat can contribute significantly to the field of reproduc -\ntive biology and fertility research.\nThis study had several limitations. First, the treatment \ntime and concentration of TBBPA. In the natural envi -\nronment, human bodies are exposed to EDCs for a long \ntime at low concentrations. Cell-based experiments have \nlimitations associated with maintenance of the cell line \nover the years. Therefore, it is not possible to fully reca -\npitulate the exposure of the human body to EDCs found \nin the actual environment. It is expected that experi -\nments on EDC can be carried out through animal experi -\nments. Second, the characteristics of the cell line used \nin this study. The cell lines used in this experiment were \nhuman endometrial adenocarcinoma and human chorio -\ncarcinoma cell lines. When primary cell lines are com -\npared to cancer cell lines, cancer cell lines demonstrate \nhigher proliferation, adhesion, and differentiation than \nthe primary cell line. Due to these features, the attach -\nment assay might not recapitulate the actual results. For \nfurther studies, we strongly suggest using a primary cell \nline with a real embryo and using an animal model for \nthe attachment assay. Finally, the effects of EDCs on Jeg-3 \nspheroids are a limitation of this study. In the physiologi -\ncal human body, when implantation occurs, EDCs affect \nthe embryos and endometrium. In this experiment, we \nonly treated Ishikawa cells with TBBPA, which is a repre-\nsentative model of the human endometrium. Examining \nthe effect of EDCs both in the endometrium and sphe -\nroids used for mimicking embryos may provide a solu -\ntion to overcome the limitations of this study.\nConclusion\nIn this study, the effect of TBBPA on implantation was \nstudied. The the mRNA levels of IL-6, TNF-α, and IL-1β \nincreased significantly. In addition, the outgrowth area \ndecreased significantly in the TBBPA group. Collectively, \nwe confirmed that TBBPA has an effect on implantation, \nespecially on the endometrium.\n\nPage 10 of 11Koo et al. Environmental Sciences Europe           (2023) 35:69 \nAbbreviations\nART   Assisted reproductive technology\nEDCs  Endocrine disrupting chemicals\nBPA  Bisphenol A\nTBBPA  Tetrabromobisphenol A\nBFRs  Brominated flame retardants\nRIF  Recurrent implantation failure\nDMEM  Dulbecco’s modified Eagle medium\nFBS  Fetal bovine serum\nP/S  Penicillin and streptomycin\nDMSO  Dimethyl sulfoxide\nCCK-8  Cell Counting Kit-8\nRT-qPCR  Reverse Transcription-quantitative polymerase chain reaction\nIL-1α  Interleukin 1 alpha\nIL-1β  Interleukin 1 beta\nITGαV  Integrin alpha V\nLIF  Leukemia inhibitory factor\nIL-6  Interleukin-6\nTNF-α  Tumor necrosis factor α\nGAPDH  Glyceraldehyde 3 phosphate dehydrogenase\nBCA  Bicinchoninic acid assay\nSDS  Sodium dodecyl sulfate\nPAGE  Polyacrylamide gel electrophoresis\nPVDF  Polyvinylidene fluoride\nSEM  Standard error of the mean\nAcknowledgements\nNot applicable.\nAuthor contributions\nConceptualization: MK, KI, LJ; investigation: MK, IK; writing—original draft: MK, \nIK; writing—review and editing: JL, JHJ; supervision: JL, JHJ; project administra-\ntion: JL, JHJ; funding acquisition: JL.\nFunding\nThis project was financially supported by the Basic Science Research Program \nthrough the National Research Foundation of Korea (NRF), funded by the \nMinistry of Education, Republic of Korea (NRF-2018R1D-1A1B07046419 to J.L.).\nAvailability of data and materials\nThe data that support the findings of this study are available from the cor-\nresponding author upon reasonable request.\nDeclarations\nEthics approval and consent to participate\nNot applicable.\nConsent for publication\nAll authors agreed to publish the paper.\nCompeting interests\nThe authors declare that they have no competing interests.\nReceived: 2 June 2023   Accepted: 12 August 2023\nReferences\n 1. 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