{"paper_id":"b95c6b26-3272-418d-9318-c4c349e36def","body_text":"Effect of tetrabromobisphenol A (TBBPA) on early implantation using the three-dimensional spheroid model with human endometrial cell line, Ishikawa | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effect of tetrabromobisphenol A (TBBPA) on early implantation using the three-dimensional spheroid model with human endometrial cell line, Ishikawa Myoungjoo KOO, Inyoung KANG, Jin Hyun JUN, Jaewang Lee This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3013348/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Aug, 2023 Read the published version in Environmental Sciences Europe → Version 1 posted 8 You are reading this latest preprint version Abstract Background Tetrabromobisphenol A (TBBPA) has endocrine-disrupting effect and has been widely used as brominated flame retardant in industrial products. The endocrine-disrupting chemical has effects on female reproduction such as infertility, hormone imbalance, and endometriosis. In Korea, the problems of infertility and the decreasing birth rate are concerns. Exposure to EDCs might have a harmful effect on childbearing-age women. This study aimed to investigate the effect of TBBPA on infertility, particularly on uterus endometrium early implantation events. Results The expression of mRNA was analyzed by qRT-PCR. The mRNA expression of TBBPA on Ishikawa cells, IL-6, IL-1β, TNF- α, and LIF were significantly increased. Moreover, the outgrowth area in the TBBPA group has significantly decreased compared with the control. On the other hand, TBBPA had a slight effect on protein level and attachment rate. Conclusions In this study, TBBPA occurred inflammatory environmental milieu in mRNA expression. The increase of inflammation-related cytokines in the endometrium can disrupt the implantation of the embryo. Also, the TBBPA disrupted the outgrowth of spheroids on the endometrium, however, the protein levels and attachment rate were comparable to the control group. The effect of TBBPA on implantation events should be further clarified. Implantation Endocrine disrupting chemicals (EDCs) Tetrabromobisphenol A Ishikawa cells Jeg-3 cells Infertility Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Female infertility refers to the inability of a woman to conceive or carry a pregnancy to its full term. There can be many causes of female infertility, including problems with ovulation, fallopian tube or uterus, and hormone imbalance. (National Health Insurance Service, 2007–2015),(Yun et al., 2022 ) The birth rate in South Korea has been dramatically declining for decades. According to a report published by the Korea Society of Obstetrics and Gynecology, the infertility rate in Korea was estimated to be around 10% in 2019 which is higher than the global average of 8%. (Healthcare big data system, 2022) and over the decades in South Korea, then the number of assisted reproductive technology (ART) patients have been continuously increased.(Lee et al., 2017 ) (National Health Insurance Service, 2007–2015) Endocrine-disrupting chemicals (EDCs) are substances that interfere with the function of the endocrine system in humans and animals. (Kabir et al., 2015 ) The endocrine system regulates body function, especially reproduction, and the hormone cycle. (Berger et al., 2016 )(Berger A, 2015) EDCs can block the effects of natural hormones with their structures like hormones, leading to altered hormone levels and disrupted physiological homeostasis. (Guarnotta et al., 2022 ), (Yilmaz et al., 2020 ) They can bind to sex-hormone receptors and inhibit or activate the production of hormones such as estrogen and progesterone (Brehm and Flaws, 2019 ). Eventually, the hormone pathway is interfered by the action of EDCs.(Mlynarcikova and Scsukova, 2020 ) Bisphenol A (BPA) is an industrial chemical used in the production of plastic and epoxy resin. (Vandenberg et al., 2007 ) It is commonly found in polycarbonate plastics which are used in water bottles, food containers, and canned foods. (Yaguchi, 2019 ) BPA is known to be an EDC that can bind to estrogen receptors in the human endocrine system. Some studies have reported that exposure to BPA leads to reproductive disorders, developmental disorders, and impaired immune function. (Konieczna et al., 2015 ), (Fan et al., 2021 ) Tetrabromobisphenol- A (TBBPA) is a member of the bisphenol family, which means it is structurally similar to bisphenol A (BPA). Considered a general and perpetual contaminant in the environment, TBBPA is widely used in brominated flame retardants (BFRs) in industrial products. (Sunday et al., 2022 ). BFRs contain bromine atoms that can prevent the spread of fire. (Ma et al., 2015 ) TBBPA has been shown to have the potential to EDCs, it may interfere with hormones in the body but has not been clarified yet. (Zhou et al., 2020 ), (Reed et al., 2022 ), (Vasiljevic and Harner, 2021 ). TBBPA is the highest production of BFRs, estimated to around 60% of the total BFR market. Even though the worldwide use of TBBPA and its endocrine-disrupting effects have been considered serious as potential health risks for humans and the environment (Dong et al., 2021 ), the Asia market registered the highest consumption of TBBPA (den Braver-Sewradj et al., 2020 ). TBBPA has effect on uterine leiomyoma proliferation (Liu et al., 2022 ) and this can lead to infertility. TBBPA has been detected in Korean human serum, ranging at 0.05 ~ 75 ng/g lipid weight. (Kim and Oh, 2014 ) Nevertheless, the effects of TBBPA on the implantation events has not been fully clarified. Therefore, we aimed to investigate the effect of TBBPA on early human implantation using a 3D spheroid culture model. 2. Materials and Methods 2.1. Cell culture The endometrial epithelial-like Ishikawa cell line and human trophoblastic Jeg-3 cell line were cultured in Dulbecco’s modified Eagle medium (DMEM, Welgene, Gyeongsan, Korea) supplemented with 10% fetal bovine serum (FBS, Gibco, Waltham, MA, USA) and 1% penicillin & streptomycin (P/S, Lonza, Morristown, NJ, USA). Cells were cultured under standard conditions (37℃, 5% CO 2 ). Ishikawa cells were used to represent the human endometrium, which is the place where implantation occurs. Jeg-3 cells were used to represent the human embryo because of inner cell mass surrounded by trophoblastic cells. Tetrabromobisphenol-A (TBBPA, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in DMSO (Sigma-Aldrich) and treated on Ishikawa cells to find the effect of TBBPA on endometrial cells. 2.2. Cell viability assay The cell viability assay was performed to find the cytotoxic dose of TBBPA. Ishikawa cells and Jeg-3 cells were detached using 0.25% Trypsin/EDTA (Gibco) and suspended to seed in 96-well plates at a density of 1x10 3 /well. The cells were exposed to 0.01 µM ~ 100 µM TBBPA for 24hrs or 48 hours. The viability of the cells was evaluated by using Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan). After incubating with different concentrations of TBBPA, CCK-8 reagent was added to each well and incubated for 1 hour at 37°C. The cell viability was detected at the 450 nm absorbance using MultiskanGO (ThermoFisher, Waltham, MA, USA). The groups were composed of control and TBBPA groups. The control group was cultured with 1% FBS and containing 0.1% DMSO in DMEM. 2.3. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) Ishikawa cells and Jeg-3 cells were detached using 0.25% Trypsin/EDTA and seeded in a 6-well plate (SPL, Pocheon, Korea) at a density of 1x10 5 /well. Ishikawa cells were treated with TBBPA concentration at 0.1 µM, 1 µM, and 10 µM for 48 hours. For the comparison test of 2D versus 3D spheroids about Jeg-3, 1x10 5 cells per well were seeded for the 2D group, and one hundred Jeg-3 spheroids were used for 3D groups. To detect the mRNA expression in 2D versus 3D spheroids, interleukin 1 alpha (IL-1α), interleukin 1 beta (IL-1β), integrin alpha V (ITGaV), and leukemia inhibitory factor (LIF) were used. Total RNA from cells and spheroids were extracted by TRIzol (Invitrogen, Waltham, MA, USA). For real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis, 500 ng of mRNA was converted to complementary DNA using PrimeScript™ 1st strand cDNA Synthesis Kit (Takara, Kusatsu, Japan). RT-qPCR was performed using SYBR green reagent (Meridian Bioscience, Cincinnati, OH, USA) and primer (BIONEER, Daejeon, Korea). Each experiment was performed at least triplicated and repeated more than three times. For inflammation targets, interleukin 6 (IL-6) (Bio-Rad, Hercules, CA, USA), IL-1β and tumor necrosis factor-α (TNF- α) were used. Furthermore, ITGαV and LIF were used for attachment targets. All Data were normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The sequence of a primer used in this study is summarized in Table 1 . The PCR method was as follows: Hold stage at 95℃ for 15 mins and PCR cycle of denaturation at 95℃ for 30 seconds, annealing at 61℃ for 30 seconds, and extension at 72℃ for 30 seconds. Table 1 Primer sequence Genes Primer Sequence Product size(bp) GenBank accession number Annealing Temperature(℃) IL-1α F: CTG AAG GAG ATG CCT GAG ATA C 383 NM_000575.5 61 R: GAA CTG TCA ACA CTG CAC AAG IL-1β F: TAA AGA GAG CTG TAC CCA GAG A 217 NM_000576.3 R: AAG TGA GTA GGA GAG GTG AGA G TNF-α F: CTC CTC ACC CAC ACC ATC AG 134 NM000594.4 R: ATA GAT GGG CTC ATA CCA GGG ITGαV F: AAT CTT CCA ATT GAG GAT ATC AC 140 NM_002210.5 R: AAA ACA GCC AGT AGC AAC AAT LIF F: CCA ACG TGA CGG ACT TCC C 82 NM000981.4 R: TAC ACG ACT ATG CGG TAC AG GAPDH F: GGA GCG AGA TCC CTC CAA AA 197 NM_002046.7 R: GGC TGT TGT CAT ACT TCT CA 2.4. Western blot analysis To quantify the level of protein, western blot analysis was performed by using cell lysates. Ishikawa cells were seeded in 6-well plates at a density of 1x10 5 cells/well. Total protein was extracted by RIPA lysis buffer (ThermoFisher) with proteinase inhibitor (ThermoFisher). Total protein concentration was conducted by BCA assay (ThermoFisher). Fifteen micrograms of protein were used for experiments. Each sample was separated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. Blocking with 5% skim milk for 2 hours at room temperature, then the membranes were incubated with primary antibody against Interleukin- 6 (1: 800) (Abcam, Cambridge, UK), TNF-α (1: 500) (Abcam), LIF (1: 200) (Santacruz, Dallas, TX, USA) and GAPDH (1: 5,000) (Santacruz) overnight at 4℃ on orbital shaker. The membranes were washed three times using Tris-buffered saline with 1% Tween 20. For Interleukin-6, TNF-α and LIF were incubated with anti-rabbit horseradish peroxidase-conjugated IgG secondary antibody for 2 hours at room temperature. GAPDH was incubated with anti-mouse horseradish peroxidase-conjugated IgG secondary antibody for 2 hours at room temperature. The proteins on the membranes were analyzed by the iBright CL750 imaging system (Applied Biosystems, Waltham, MA, USA). The data was measured using ImageJ software (NIH, Bethesda, MD, USA). 2.5. Attachment assay Ishikawa cells were cultured for 48 hours in a T25 flask with or without TBBPA treatment. Then the cells were detached using 0.25% Trypsin/EDTA and seeded in a 12-well plate at a density of 1x10 5 cultured with DMEM containing 10% FBS. After 8 hours, the media was exchanged to each experiment media. For control groups and TBBPA groups, DMEM with 1% FBS was used. Formation of Jeg-3 spheroid using hanging drop method at a density of 500 cells per spheroid for 48 hours in DMEM supplemented with 10% FBS rotated at 45 RPM in a shaking incubator as shown in Fig. 5 A. 150 ~ 300 µm size spheroids were used for this experiment. After harvest the spheroids, there are seeded on the Ishikawa cells treated with TBBPA. Attachment rate was observed at 5, 10, 20, 30 min, 1, 2, 4, and 24 hours. The attachment assay used in this study was illustrated in Fig. 1 A. 2.6. Outgrowth Assay For finding the effect of TBBPA on spheroid outgrowth. Ishikawa cells were treated with TBBPA for 48 hours in a T25 flask. Then detached using 0.25% Trypsin/EDTA and seeded on a 12-well plate at a density of 5x10 4 in DMEM with 10% FBS. After 8 hours, the media was exchanged to 1% FBS in DMEM for control groups and TBBPA groups. And the cells of negative control groups were exchanged to DMEM without FBS. Spheroids were seeded on the Ishikawa cells after the media was changed and incubated for 72 hours under standard conditions (37℃, 5% CO 2 ). Using EVOS M500 imaging system (ThermoFisher) for taking photos of outgrowth areas with magnification 40X. Then the outgrowth area was measured using Image J software (NIH). The outgrowth assay in this study was illustrated in Fig. 1 A. 2.7 Statistical analysis All experiments were performed in at least triplicates. All values were represented as ± standard error of the mean (SEM). Attachment rate data were analyzed by the Chi-square test. And the outgrowth area data were analyzed by the t-test and the difference in group P value less than 0.001 was considered statistically significant. Other results were analyzed by one-way analysis of variance (one-way ANOVA) with Tukey’s post hoc multiple comparison tests. The difference in an experiment P value of less than 0.05 was considered statistically significant. The Jeg-3 2D vs 3D mRNA expression was analyzed by t-test and the difference in the experiment P value of less than 0.05 was considered statistically significant. 3. Results 3.1. The expression of Jeg-3 cells 2D vs Jeg-3 spheroids. To investigate the difference between 2D culture and 3D culture, the expression of mRNA markers related to attachment (ITGaV, LIF) and inflammation (IL-1α, IL-1β) was evaluated using qRT-PCR. The mRNA expression of IL-1α was increased in the 3D culture group compared to the 2D culture group. (Fig. 1 B). The mRNA expressions of ITGaV, LIF, and IL-1β in the 2D culture group were comparable to the 3D cultured group. Based on this, we applied three-dimensional spheroid model to imitate in vivo condition. 3.2. The effects of TBBPA on the viability of Ishikawa and Jeg-3 cells. To find the concentration that has cytotoxicity on cell TBBPA for 24 hours and 48 hours, TBBPA was treated at a concentration of 0.1, 1, 10, and 100 µM. The highest dose of TBBPA for 24 hours and 48 hours has decreased cell viability in both Ishikawa cells and Jeg-3 cells. The concentration of TBBPA at 0.1, 1, 10 µM did not affect cell viability in both Ishikawa cells and Jeg-3 cells for 24 hours (Fig. 2 A, 2 C) and 48 hours (Fig. 2 B, 2 D). Since there was no statistical difference among TBBPA groups except 100 µM, the cells were treated with 0.1, 1, and 10 µM for next steps. 3.3. The gene expression of Ishikawa cells treated with TBBPA. To investigate the effect of TBBPA on Ishikawa cells, the mRNA expression was detected using the qPT-PCR. The mRNA expression of Ishikawa cells treated with TBBPA showed increasing in inflammation markers. In inflammation relates genes, especially, the mRNA expression of IL-6, IL-1β, and TNF-α were significantly increased in TBBPA-treated groups. (Fig. 3 A) In attachment relates genes, LIF in TBBPA groups were significantly increased compared to the control groups. (Fig. 3 B) This result means TBBPA has effects on inflammation and attachment of endometrium cells on mRNA levels. 3.4. The protein expression of Ishikawa cells treated with TBBPA. To identify the protein level of TBBPA treated on Ishikawa cells, inflammation and attachment relates proteins, LIF, TNF- α, and IL-6 were detected. In the previous experiment, the mRNA expression of LIF, TNF- α, and IL-6 were elevated in TBBPA-treated groups. Identifying mRNA elevation led to an increase the protein levels, Western blot assay was used. The proteins were slightly increased in TBBPA groups at all groups but not statistically. (Fig. 4 A, 4 B) 3.5. The attachment rate of Jeg-3 spheroid on Ishikawa cells treated with TBBPA. As in the previous experiment, the attachment relates marker, LIF was increased in mRNA levels and protein levels refers that TBBPA might enhance the attachment rate of spheroids. To identify the attachment rate of spheroids on Ishikawa cells treated with TBBPA, attachment assays were performed. (Fig. 5 A) Early attachment rates (0 ~ 30 minutes) were slightly higher in the TBBPA groups (10 µM) than the control groups. After 90 minutes, the attachment rate was no significant statistical difference among experiment groups which means attachment rate might involve only early attachment rates. (Fig. 5 B) 3.6. The outgrowth assay of Jeg-3 spheroid on Ishikawa cells treated with TBBPA. To find the effect of TBBPA on outgrowth, an outgrowth assay was conducted on Jeg-3 spheroids seeded on the Ishikawa cells as shown in Fig. 6 A. TBBPA was treated with a concentration of 10 µM on Ishikawa cells. In TBBPA groups, the outgrowth area significantly decreased. (P < 0.0001) (Fig. 6 B) This result means TBBPA negatively influences the outgrowth area even though the attachment-related protein levels were comparable. 4. Discussion In the present study, we investigated whether TBBPA has deleterious effects on the implantation and invasion process using the three-dimensional spheroid cell culture method. Finding the potential risks and giving evidence of TBBPA on female infertility, it is essential to investigate the toxicity of TBBPA in the endometrium and trophoblastic spheroid which represented embryo in this study. TBBPA induced inflammation milieu , decreased the expression of the mRNA level, and reduced outgrown area even though its protein and attachment rate were comparable to the control groups in this study. However, the potential mechanism of TBBPA has not been clarified in recent studies. Further studies to find the toxicity and the harmful mechanisms of EDCs on the female reproduction system should be conducted. For the in vitro spheroid models, 2D cultured systems were used generally but it has the limitation that does not fully represent physiological human cells, 3D spheroids can mimic human in vivo cell conditions much better than 2D culture systems. (Stojanovska et al., 2022 ) The main concern of these 3D culture models in this study is that they are made with a cancer cell line. The cancer cells do not represent the natural cells and leading to different results compared to the result of primary cells. In this study, the 2D cultured system and 3D culture system showed different mRNA expressions in inflammation relates genes such as IL-1β. The difference in mRNA expression can represent that using the 3D cultured system in the study possibly showed the actual environment in the human body. In this study, TBBPA has not affected on cytotoxicity in the human endometrium cell line at the concentration of 0.1, 1, 10 µM for 24 hours or 48 hours but 100µM has significantly decreased cell viability. On the aspects of real human endocrine systems and environments, long-term and low doses of EDCs are exposed. However, long-term experiments cannot be reconstructed using cell line experiments. To substitute the low dose and exposure time to EDCs problems (Calafat et al., 2008 ), TBBPA was treated at a high dose for short-term (Strack et al., 2007 ). The mRNA expressions, associated with inflammation and attachment genes were detected in TBBPA-treated human endometrium cells. (Guan et al., 2021 ) When the human embryo is attached and invaded in the endometrium, cytokines related to attachment (Fukui et al., 2021 ) and inflammation are released into the endometrium and embryo. The mRNA expression associated with inflammation and attachment can show EDC’s effects on embryo implantation and invasion. IL-6, TNF-α, and IL-1β which are associated with inflammation, have dramatically increased in TBBPA-treated endometrium cells. This result means TBBPA has harmful effects on embryos and endometrium. The protein level was evaluated using a Western blot, for the primary antibody IL-6, TNF- α, and LIF were used. These targets are increased in mRNA level conducted by qRT-PCR. To investigate whether the mRNA expression leads to the protein level, these targets were chosen for Western blot. In this experiment, TBBPA did not change the protein levels significantly and this result can describe that TBBPA has effects on mRNA expression not on protein level in these targets. However, further studies how TBBPA regulates inflammation and attachment related genes should be clarified to evaluate its toxicity on female reproduction. In the attachment assay, TBBPA treated group showed a slightly higher rate of early implantation compared to the control group. This result can explain the increase of mRNA expression in LIF. LIF is associated with attachment of the spheroid, this can increase the attachment rate of the spheroid, but this cannot represent TBBPA can increase the implantation of the embryo. On the other hand, after 60 minutes TBBPA groups showed a lower attachment rate than control groups. Also, there were no significant differences among the groups. The limitation of this assay is that spheroids are made with cancer cells. High proliferation and adhesion are the main characteristics of the cancer cell line. (Martínez-Reyes and Chandel, 2021 ) For next study, we are evaluating the impacts of TBBPA on mouse primary uterine cells because cancer cells cannot completely imitate the primary cells’ pathophysiology. The outgrowth assay is conducted to check the invasion of embryos on the endometrium, and this implantation process occurs within 72 hours in the human body. Jeg-3 spheroids seeded on TBBPA-treated endometrium cells represent the human endometrium exposed to EDCs and embryo implantation. In the actual human body, the implantation process occurs within 72 hours, so the outgrowth assay was conducted for 72 hours. In this experiment, TBBPA has decreased in the outgrowth area, therefore TBBPA might have negative impacts on implantation process in the human body. In this study, there are several limitations. First, treatment time and concentration of TBBPA. In the natural environment, EDCs are exposed to the human body for a long-term at low concentrations, but cell-based experiments have limitations in conducting experiments while maintaining the cell line over the years. Therefore, it is not possible to fully represent the EDCs exposed in the actual environment on the human body. Second, the characteristic of the cell line used in this study. The cell line used in this experiment is the human endometrial adenocarcinoma cell line and the human choriocarcinoma cell line. Compared to the primary cell line to cancer cell lines, cancer cell lines have high proliferation, adhesion, and differentiation than the primary cell line. Due to these features, the attachment assay might not be matched the actual results. For further study, we strongly suggest that using the primary cell line with real embryo using animal model for the attachment assay. For the last, the effects of EDCs on Jeg-3 spheroids are the limitation of this study. In the physiological human body, when implantation occurs, EDCs are affecting embryos and endometrium. In this experiment, we only treated TBBPA on the Ishikawa cells which a representative model for human endometrium. Studying the effect of EDCs both in the endometrium and spheroid for mimicking embryos may provide a solutions to overcome the limitations of this study. The purpose of this study was to investigate the effects of TBBPA on the female reproduction system, as well as the process of the implantation system. Further research might investigate the effect of TBBPA on implantation, using a primary cell line or mouse embryo and mouse endometrial cell is strongly required. When studying EDCs, it is ideal to treat them at low concentrations for a long-period of time as actual endocrine disruptors, so proceeding with animal experiments possibly showed similar results compared to cell line experiments. 5. Conclusion In this study, TBBPA has effects on mRNA levels of inflammation and attachment such as TNF-a, LIF, and ITGB5. The outgrowth area treated with TBBPA has significantly decreased compared to the control. Collectively, TBBPA has shown to affect the endometrium, further study is required. Declarations Availability of data and materials The data that support the findings of this studyare available from the corresponding author upon reasonable request. Acknowledgements Not applicable. Funding This project was financially supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Republic of Korea (NRF-2018R1D-1A1B07046419 to J.L.), Author information Authors and Affiliations Department of Biomedical Laboratory Sciences, Eulji university 553, Sanseong-daero, Sujeong-gu, Seongnam-si, Gyeonggi-do, Republic of Korea Myoungjoo Koo, Inyoung Kang, Jaewang Lee, Jinhyun Jun Contrubutions Conceptualization: KOO M, KANG I, LEE J; Investigation: KOO M, KANG I; Writing – Original Draft: KOO M, KANG I; Writing – Review & Editing: LEE J, JUN J; Supervision: LEE J, JUN J; Project administration: LEE J, JUN J; Funding acquisition: LEE J. Ethics approval and consent to participate. 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Healthcare Bigdata Hub(2007-2015) Republic of Korea, Seoul. http://opendata.hira.or.kr/op/opc/olapMfrnIntrsDiagBhvInfo.do Accessed 01 June 2023 Healthcare Bigdata Hub (2017-2021) Republic of Korea, Seoul. http://opendata.hira.or.kr/op/opc/olapMfrnIntrsIlnsInfo.do Accessed 01 June 2023 KOSIS(Korean Statistical information Service) Republic of Korea, Seoul, https://kosis.kr/statHtml/statHtml.do?orgId=350&tblId=DT_35007_N002_1&vw_cd=MT_OTITLE&list_id=350_35007_A001&scrId=&seqNo=&lang_mode=ko&obj_var_id=&itm_id=&conn_path=K2&path=%252Fcommon%252Fmeta_onedepth.jsp Accessed 01 June 2023 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 17 Aug, 2023 Read the published version in Environmental Sciences Europe → Version 1 posted Editorial decision: Major revision 11 Jul, 2023 Reviews received at journal 01 Jul, 2023 Reviewers agreed at journal 20 Jun, 2023 Reviewers agreed at journal 14 Jun, 2023 Reviewers invited by journal 11 Jun, 2023 Editor assigned by journal 02 Jun, 2023 Submission checks completed at journal 02 Jun, 2023 First submitted to journal 02 Jun, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-3013348\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":206438438,\"identity\":\"b5f55cff-4986-40eb-9d5e-74bffcd0e316\",\"order_by\":0,\"name\":\"Myoungjoo KOO\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Eulji University\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Myoungjoo\",\"middleName\":\"\",\"lastName\":\"KOO\",\"suffix\":\"\"},{\"id\":206438439,\"identity\":\"dffc04d9-c32c-4131-9baa-374cd3651ea8\",\"order_by\":1,\"name\":\"Inyoung KANG\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Eulji University\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Inyoung\",\"middleName\":\"\",\"lastName\":\"KANG\",\"suffix\":\"\"},{\"id\":206438440,\"identity\":\"5abae192-8b3d-4f7a-acfd-896633fcd7ab\",\"order_by\":2,\"name\":\"Jin Hyun JUN\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Eulji University\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Jin\",\"middleName\":\"Hyun\",\"lastName\":\"JUN\",\"suffix\":\"\"},{\"id\":206438441,\"identity\":\"3deef486-29ae-4dd3-96a8-d7a9a5ba9a4f\",\"order_by\":3,\"name\":\"Jaewang Lee\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYLACCSDmBzESCkjRItkA0mJAik0GB8AkMSqPnz38wqLGxt74/OrEDw8MGOT5xQ4Q0HImL81C4lha4rYbbzdLAB1mOHN2An4tZgdyzAwk2A4nmN04uwGkJcHgNiEt598Atfz7b2884+zmH8RpuZFj/ECy7QDjBv7ebcTZYn/jjRmDZF9y4owbvNssEgwkCPtFsj/H+LPENzt7/v6zm2/+qLCR55cmoAUI2KRBUckgAVYpQVA5CDB//ACi+A8QpXoUjIJRMApGIAAAKIRGDLMFBCoAAAAASUVORK5CYII=\",\"orcid\":\"\",\"institution\":\"Eulji University\",\"correspondingAuthor\":true,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Jaewang\",\"middleName\":\"\",\"lastName\":\"Lee\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2023-06-02 07:59:32\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-3013348/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-3013348/v1\",\"draftVersion\":[],\"editorialEvents\":[{\"content\":\"https://doi.org/10.1186/s12302-023-00780-x\",\"type\":\"published\",\"date\":\"2023-08-17T22:00:47+00:00\"}],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":38098575,\"identity\":\"9aafee78-3206-4968-9f98-80737f6329dc\",\"added_by\":\"auto\",\"created_at\":\"2023-06-06 14:17:26\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1712746,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eExperimental design and difference of mRNA expression between 2D culture and 3D culture of Jeg-3. \\u003c/strong\\u003eThe mRNA expression of Jeg-3 2D culture and 3D culture (\\u003cem\\u003eITGaV, LIF, IL-1α and IL-1β\\u003c/em\\u003e) (n\\u0026gt;3) One-way analysis of variance; Tukey’s multiple comparison test (p\\u0026lt;0.05)\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Experimental design for this study. (B) Jeg-3 2D cultured and 3D culture mRNA expression.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3013348/v1/1ac9166ab03024ab7e9ab18e.png\"},{\"id\":38098576,\"identity\":\"173e4b0d-5047-403e-95d4-1ebf2cd173c2\",\"added_by\":\"auto\",\"created_at\":\"2023-06-06 14:17:26\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":929628,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eThe\\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003e \\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003eCell viability of Ishikawa and Jeg-3 by Cell Counting Kit (CCK8) assay. \\u003c/strong\\u003eCell count kit 8 (CCK-8) assay was conducted to determine cell cytotoxicity. Ishikawa cells and Jeg-3 cells were treated with TBBPA for 24/48 h. (n\\u0026gt;3) One-way analysis of variance; Tukey’s multiple comparison tests, a vs b vs c vs d (p\\u0026lt;0.05). (A) Ishikawa cell line TBBPA 0.1 μM ~ 100 μM treated for 24h. (B) Ishikawa cell line TBBPA 0.1 μM ~ 100 μM treated for 48 h. (C) Jeg3 cell line TBBPA 0.1 μM ~ 100 μM treated 24h. (D) Jeg3 cell line TBBPA 0.1 μM ~ 100 μM treated 48h.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3013348/v1/2baaee427d01dd61aace8dd4.png\"},{\"id\":38099550,\"identity\":\"acba14b0-d2e1-4829-a922-0d5a11d11ca2\",\"added_by\":\"auto\",\"created_at\":\"2023-06-06 14:25:26\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":831378,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eThe mRNA expression related to inflammation and attachment in Ishikawa cells. The mRNA markers related to inflammation were assessed by qRT-PCR. \\u003c/strong\\u003eThe mRNA expression of Ishikawa cells treated with TBBPA (0.1 μM~ 10 μM) for 48h. The mRNA expressions were normalized with \\u003cem\\u003eGAPDH.\\u003c/em\\u003e (n\\u0026gt;3) One-way analysis of variance; Tukey’s multiple comparison tests (p\\u0026lt;0.05) (A\\u003cem\\u003e) IL-6, IL-1β, \\u003c/em\\u003eand\\u003cem\\u003e TNF-α\\u003c/em\\u003e mRNA expression of Ishikawa cells treated with TBBPA for 48h. (B) \\u003cem\\u003eITGαV \\u003c/em\\u003eand \\u003cem\\u003eLIF \\u003c/em\\u003emRNA expression of Ishikawa cells treated TBBPA for 48h.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3013348/v1/35e187e742321e3cd2a4807f.png\"},{\"id\":38098577,\"identity\":\"0a58025a-8731-48de-9cf3-10ee56e4041f\",\"added_by\":\"auto\",\"created_at\":\"2023-06-06 14:17:26\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":529412,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eThe protein level of Ishikawa cells treated with TBBPA. The protein level that has increased in the qRT-PCR. \\u003c/strong\\u003eAll targets normalized with GAPDH. Ishikawa cells were treated with TBBPA for 48 hours and 20µg/mL were used for Western blot assay. (A)The protein level of Ishikawa cell treated with TBBPA (LIF, TNF-a, IL-6) (n\\u0026gt;3) One-way analysis of variance; Tukey’s multiple comparison tests (p\\u0026lt;0.05) (B)The Western blot band of GAPDH, TNF-a, LIF, and IL-6.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3013348/v1/23ac0ef2ee0f8a5b043a3b5e.png\"},{\"id\":38098578,\"identity\":\"a66f7e51-e7d4-4a22-8636-d3bb680a340b\",\"added_by\":\"auto\",\"created_at\":\"2023-06-06 14:17:26\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1788501,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eThe attachment assay of Jeg3 spheroid on Tetrabrombisphenol A treated Ishikawa cells. \\u003c/strong\\u003e(A) The morphology of Ishikawa cells, Jeg-3 spheroid, and Jeg-3 Spheroid on Ishikawa cells treated with TBBPA. (B) Attachment rate (%) (n\\u0026gt;65) T-test;(p\\u0026lt;0.001). The morphology of Ishikawa cells and Jeg-3 spheroids were taken by the EVOS imaging system. The attachment assay was performed to detect the Jeg-3 spheroids attachment rate.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3013348/v1/da7424148ab41b4cd750da46.png\"},{\"id\":38098580,\"identity\":\"dd46670d-d120-4b15-a1e9-6339952098e7\",\"added_by\":\"auto\",\"created_at\":\"2023-06-06 14:17:26\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1387585,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eThe outgrowth area of Jeg3 spheroid on TBBPA-treated Ishikawa cells\\u003c/strong\\u003e. (A) The morphology and area of Jeg-3 spheroid outgrowth. (B) The outgrowth area. (n=45 control group, n=63 TBBPA group, n=42 negative control group) One-way analysis of variance; Tukey’s multiple comparison tests (p\\u0026lt;0.0001) The outgrowth area was detected and calculated by the ImageJ system. The relative area was measured compared to the control group.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3013348/v1/6ddcedad51fe3a52254847c6.png\"},{\"id\":44736281,\"identity\":\"f0258edf-ac23-4f4f-8e33-d1625560d966\",\"added_by\":\"auto\",\"created_at\":\"2023-10-16 22:29:49\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":1802435,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3013348/v1/5ae14291-44af-48f1-ae49-c9e8cd36b285.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Effect of tetrabromobisphenol A (TBBPA) on early implantation using the three-dimensional spheroid model with human endometrial cell line, Ishikawa\",\"fulltext\":[{\"header\":\"1. Introduction\",\"content\":\"\\u003cp\\u003eFemale infertility refers to the inability of a woman to conceive or carry a pregnancy to its full term. There can be many causes of female infertility, including problems with ovulation, fallopian tube or uterus, and hormone imbalance. (National Health Insurance Service, 2007\\u0026ndash;2015),(Yun et al., \\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e) The birth rate in South Korea has been dramatically declining for decades. According to a report published by the Korea Society of Obstetrics and Gynecology, the infertility rate in Korea was estimated to be around 10% in 2019 which is higher than the global average of 8%. (Healthcare big data system, 2022) and over the decades in South Korea, then the number of assisted reproductive technology (ART) patients have been continuously increased.(Lee et al., \\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e) (National Health Insurance Service, 2007\\u0026ndash;2015)\\u003c/p\\u003e \\u003cp\\u003eEndocrine-disrupting chemicals (EDCs) are substances that interfere with the function of the endocrine system in humans and animals. (Kabir et al., \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e) The endocrine system regulates body function, especially reproduction, and the hormone cycle. (Berger et al., \\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e)(Berger A, 2015) EDCs can block the effects of natural hormones with their structures like hormones, leading to altered hormone levels and disrupted physiological homeostasis. (Guarnotta et al., \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e), (Yilmaz et al., \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e) They can bind to sex-hormone receptors and inhibit or activate the production of hormones such as estrogen and progesterone (Brehm and Flaws, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). Eventually, the hormone pathway is interfered by the action of EDCs.(Mlynarcikova and Scsukova, \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e)\\u003c/p\\u003e \\u003cp\\u003eBisphenol A (BPA) is an industrial chemical used in the production of plastic and epoxy resin. (Vandenberg et al., \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e2007\\u003c/span\\u003e) It is commonly found in polycarbonate plastics which are used in water bottles, food containers, and canned foods. (Yaguchi, \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e) BPA is known to be an EDC that can bind to estrogen receptors in the human endocrine system. Some studies have reported that exposure to BPA leads to reproductive disorders, developmental disorders, and impaired immune function. (Konieczna et al., \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e), (Fan et al., \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e) Tetrabromobisphenol- A (TBBPA) is a member of the bisphenol family, which means it is structurally similar to bisphenol A (BPA). Considered a general and perpetual contaminant in the environment, TBBPA is widely used in brominated flame retardants (BFRs) in industrial products. (Sunday et al., \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). BFRs contain bromine atoms that can prevent the spread of fire. (Ma et al., \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e) TBBPA has been shown to have the potential to EDCs, it may interfere with hormones in the body but has not been clarified yet. (Zhou et al., \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e), (Reed et al., \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e), (Vasiljevic and Harner, \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). TBBPA is the highest production of BFRs, estimated to around 60% of the total BFR market. Even though the worldwide use of TBBPA and its endocrine-disrupting effects have been considered serious as potential health risks for humans and the environment (Dong et al., \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e), the Asia market registered the highest consumption of TBBPA (den Braver-Sewradj et al., \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). TBBPA has effect on uterine leiomyoma proliferation (Liu et al., \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e) and this can lead to infertility. TBBPA has been detected in Korean human serum, ranging at 0.05\\u0026thinsp;~\\u0026thinsp;75 ng/g lipid weight. (Kim and Oh, \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e) Nevertheless, the effects of TBBPA on the implantation events has not been fully clarified. Therefore, we aimed to investigate the effect of TBBPA on early human implantation using a 3D spheroid culture model.\\u003c/p\\u003e\"},{\"header\":\"2. Materials and Methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.1. Cell culture\\u003c/h2\\u003e \\u003cp\\u003eThe endometrial epithelial-like Ishikawa cell line and human trophoblastic Jeg-3 cell line were cultured in Dulbecco\\u0026rsquo;s modified Eagle medium (DMEM, Welgene, Gyeongsan, Korea) supplemented with 10% fetal bovine serum (FBS, Gibco, Waltham, MA, USA) and 1% penicillin \\u0026amp; streptomycin (P/S, Lonza, Morristown, NJ, USA). Cells were cultured under standard conditions (37℃, 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e). Ishikawa cells were used to represent the human endometrium, which is the place where implantation occurs. Jeg-3 cells were used to represent the human embryo because of inner cell mass surrounded by trophoblastic cells. Tetrabromobisphenol-A (TBBPA, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in DMSO (Sigma-Aldrich) and treated on Ishikawa cells to find the effect of TBBPA on endometrial cells.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.2. Cell viability assay\\u003c/h2\\u003e \\u003cp\\u003eThe cell viability assay was performed to find the cytotoxic dose of TBBPA. Ishikawa cells and Jeg-3 cells were detached using 0.25% Trypsin/EDTA (Gibco) and suspended to seed in 96-well plates at a density of 1x10\\u003csup\\u003e3\\u003c/sup\\u003e/well. The cells were exposed to 0.01 \\u0026micro;M\\u0026thinsp;~\\u0026thinsp;100 \\u0026micro;M TBBPA for 24hrs or 48 hours. The viability of the cells was evaluated by using Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan). After incubating with different concentrations of TBBPA, CCK-8 reagent was added to each well and incubated for 1 hour at 37\\u0026deg;C. The cell viability was detected at the 450 nm absorbance using MultiskanGO (ThermoFisher, Waltham, MA, USA). The groups were composed of control and TBBPA groups. The control group was cultured with 1% FBS and containing 0.1% DMSO in DMEM.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.3. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)\\u003c/h2\\u003e \\u003cp\\u003eIshikawa cells and Jeg-3 cells were detached using 0.25% Trypsin/EDTA and seeded in a 6-well plate (SPL, Pocheon, Korea) at a density of 1x10\\u003csup\\u003e5\\u003c/sup\\u003e /well. Ishikawa cells were treated with TBBPA concentration at 0.1 \\u0026micro;M, 1 \\u0026micro;M, and 10 \\u0026micro;M for 48 hours. For the comparison test of 2D versus 3D spheroids about Jeg-3, 1x10\\u003csup\\u003e5\\u003c/sup\\u003e cells per well were seeded for the 2D group, and one hundred Jeg-3 spheroids were used for 3D groups. To detect the mRNA expression in 2D versus 3D spheroids, interleukin 1 alpha (IL-1α), interleukin 1 beta (IL-1β), integrin alpha V (ITGaV), and leukemia inhibitory factor (LIF) were used. Total RNA from cells and spheroids were extracted by TRIzol (Invitrogen, Waltham, MA, USA). For real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis, 500 ng of mRNA was converted to complementary DNA using PrimeScript\\u0026trade; 1st strand cDNA Synthesis Kit (Takara, Kusatsu, Japan). RT-qPCR was performed using SYBR green reagent (Meridian Bioscience, Cincinnati, OH, USA) and primer (BIONEER, Daejeon, Korea). Each experiment was performed at least triplicated and repeated more than three times. For inflammation targets, interleukin 6 (IL-6) (Bio-Rad, Hercules, CA, USA), IL-1β and tumor necrosis factor-α (TNF- α) were used. Furthermore, ITGαV and LIF were used for attachment targets. All Data were normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The sequence of a primer used in this study is summarized in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e. The PCR method was as follows: Hold stage at 95℃ for 15 mins and PCR cycle of denaturation at 95℃ for 30 seconds, annealing at 61℃ for 30 seconds, and extension at 72℃ for 30 seconds.\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003ePrimer sequence\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"5\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eGenes\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003ePrimer Sequence\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eProduct size(bp)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eGenBank accession number\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eAnnealing \\u003c/p\\u003e \\u003cp\\u003eTemperature(℃)\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eIL-1α\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eF: CTG AAG GAG ATG CCT GAG ATA C\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003e383\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003eNM_000575.5\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\" morerows=\\\"11\\\" rowspan=\\\"12\\\"\\u003e \\u003cp\\u003e61\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eR: GAA CTG TCA ACA CTG CAC AAG\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eIL-1β\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eF: TAA AGA GAG CTG TAC CCA GAG A\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003e217\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003eNM_000576.3\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eR: AAG TGA GTA GGA GAG GTG AGA G\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eTNF-α\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eF: CTC CTC ACC CAC ACC ATC AG\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003e134\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003eNM000594.4\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eR: ATA GAT GGG CTC ATA CCA GGG\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eITGαV\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eF: AAT CTT CCA ATT GAG GAT ATC AC\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003e140\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003eNM_002210.5\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eR: AAA ACA GCC AGT AGC AAC AAT\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eLIF\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eF: CCA ACG TGA CGG ACT TCC C\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003e82\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003eNM000981.4\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eR: TAC ACG ACT ATG CGG TAC AG\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eGAPDH\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eF: GGA GCG AGA TCC CTC CAA AA\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003e197\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003eNM_002046.7\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eR: GGC TGT TGT CAT ACT TCT CA\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.4. Western blot analysis\\u003c/h2\\u003e \\u003cp\\u003eTo quantify the level of protein, western blot analysis was performed by using cell lysates. Ishikawa cells were seeded in 6-well plates at a density of 1x10\\u003csup\\u003e5\\u003c/sup\\u003e cells/well. Total protein was extracted by RIPA lysis buffer (ThermoFisher) with proteinase inhibitor (ThermoFisher). Total protein concentration was conducted by BCA assay (ThermoFisher). Fifteen micrograms of protein were used for experiments. Each sample was separated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. Blocking with 5% skim milk for 2 hours at room temperature, then the membranes were incubated with primary antibody against Interleukin- 6 (1: 800) (Abcam, Cambridge, UK), TNF-α (1: 500) (Abcam), LIF (1: 200) (Santacruz, Dallas, TX, USA) and GAPDH (1: 5,000) (Santacruz) overnight at 4℃ on orbital shaker. The membranes were washed three times using Tris-buffered saline with 1% Tween 20. For Interleukin-6, TNF-α and LIF were incubated with anti-rabbit horseradish peroxidase-conjugated IgG secondary antibody for 2 hours at room temperature. GAPDH was incubated with anti-mouse horseradish peroxidase-conjugated IgG secondary antibody for 2 hours at room temperature. The proteins on the membranes were analyzed by the iBright CL750 imaging system (Applied Biosystems, Waltham, MA, USA). The data was measured using ImageJ software (NIH, Bethesda, MD, USA).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.5. Attachment assay\\u003c/h2\\u003e \\u003cp\\u003eIshikawa cells were cultured for 48 hours in a T25 flask with or without TBBPA treatment. Then the cells were detached using 0.25% Trypsin/EDTA and seeded in a 12-well plate at a density of 1x10\\u003csup\\u003e5\\u003c/sup\\u003e cultured with DMEM containing 10% FBS. After 8 hours, the media was exchanged to each experiment media. For control groups and TBBPA groups, DMEM with 1% FBS was used. Formation of Jeg-3 spheroid using hanging drop method at a density of 500 cells per spheroid for 48 hours in DMEM supplemented with 10% FBS rotated at 45 RPM in a shaking incubator as shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA. 150\\u0026thinsp;~\\u0026thinsp;300 \\u0026micro;m size spheroids were used for this experiment. After harvest the spheroids, there are seeded on the Ishikawa cells treated with TBBPA. Attachment rate was observed at 5, 10, 20, 30 min, 1, 2, 4, and 24 hours. The attachment assay used in this study was illustrated in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.6. Outgrowth Assay\\u003c/h2\\u003e \\u003cp\\u003eFor finding the effect of TBBPA on spheroid outgrowth. Ishikawa cells were treated with TBBPA for 48 hours in a T25 flask. Then detached using 0.25% Trypsin/EDTA and seeded on a 12-well plate at a density of 5x10\\u003csup\\u003e4\\u003c/sup\\u003e in DMEM with 10% FBS. After 8 hours, the media was exchanged to 1% FBS in DMEM for control groups and TBBPA groups. And the cells of negative control groups were exchanged to DMEM without FBS. Spheroids were seeded on the Ishikawa cells after the media was changed and incubated for 72 hours under standard conditions (37℃, 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e). Using EVOS M500 imaging system (ThermoFisher) for taking photos of outgrowth areas with magnification 40X. Then the outgrowth area was measured using Image J software (NIH). The outgrowth assay in this study was illustrated in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec9\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.7 Statistical analysis\\u003c/h2\\u003e \\u003cp\\u003eAll experiments were performed in at least triplicates. All values were represented as \\u0026plusmn;\\u0026thinsp;standard error of the mean (SEM). Attachment rate data were analyzed by the Chi-square test. And the outgrowth area data were analyzed by the t-test and the difference in group P value less than 0.001 was considered statistically significant. Other results were analyzed by one-way analysis of variance (one-way ANOVA) with Tukey\\u0026rsquo;s post hoc multiple comparison tests. The difference in an experiment P value of less than 0.05 was considered statistically significant. The Jeg-3 2D vs 3D mRNA expression was analyzed by t-test and the difference in the experiment P value of less than 0.05 was considered statistically significant.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"3. Results\",\"content\":\"\\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.1. The expression of Jeg-3 cells 2D vs Jeg-3 spheroids.\\u003c/h2\\u003e \\u003cp\\u003eTo investigate the difference between 2D culture and 3D culture, the expression of mRNA markers related to attachment (ITGaV, LIF) and inflammation (IL-1α, IL-1β) was evaluated using qRT-PCR. The mRNA expression of IL-1α was increased in the 3D culture group compared to the 2D culture group. (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB). The mRNA expressions of ITGaV, LIF, and IL-1β in the 2D culture group were comparable to the 3D cultured group. Based on this, we applied three-dimensional spheroid model to imitate \\u003cem\\u003ein vivo\\u003c/em\\u003e condition.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.2. The effects of TBBPA on the viability of Ishikawa and Jeg-3 cells.\\u003c/h2\\u003e \\u003cp\\u003eTo find the concentration that has cytotoxicity on cell TBBPA for 24 hours and 48 hours, TBBPA was treated at a concentration of 0.1, 1, 10, and 100 \\u0026micro;M. The highest dose of TBBPA for 24 hours and 48 hours has decreased cell viability in both Ishikawa cells and Jeg-3 cells. The concentration of TBBPA at 0.1, 1, 10 \\u0026micro;M did not affect cell viability in both Ishikawa cells and Jeg-3 cells for 24 hours (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA, \\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC) and 48 hours (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB, \\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD). Since there was no statistical difference among TBBPA groups except 100 \\u0026micro;M, the cells were treated with 0.1, 1, and 10 \\u0026micro;M for next steps.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.3. The gene expression of Ishikawa cells treated with TBBPA.\\u003c/h2\\u003e \\u003cp\\u003eTo investigate the effect of TBBPA on Ishikawa cells, the mRNA expression was detected using the qPT-PCR. The mRNA expression of Ishikawa cells treated with TBBPA showed increasing in inflammation markers. In inflammation relates genes, especially, the mRNA expression of IL-6, IL-1β, and TNF-α were significantly increased in TBBPA-treated groups. (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA) In attachment relates genes, LIF in TBBPA groups were significantly increased compared to the control groups. (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB) This result means TBBPA has effects on inflammation and attachment of endometrium cells on mRNA levels.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.4. The protein expression of Ishikawa cells treated with TBBPA.\\u003c/h2\\u003e \\u003cp\\u003eTo identify the protein level of TBBPA treated on Ishikawa cells, inflammation and attachment relates proteins, LIF, TNF- α, and IL-6 were detected. In the previous experiment, the mRNA expression of LIF, TNF- α, and IL-6 were elevated in TBBPA-treated groups. Identifying mRNA elevation led to an increase the protein levels, Western blot assay was used. The proteins were slightly increased in TBBPA groups at all groups but not statistically. (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA, \\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB)\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.5. The attachment rate of Jeg-3 spheroid on Ishikawa cells treated with TBBPA.\\u003c/h2\\u003e \\u003cp\\u003eAs in the previous experiment, the attachment relates marker, LIF was increased in mRNA levels and protein levels refers that TBBPA might enhance the attachment rate of spheroids. To identify the attachment rate of spheroids on Ishikawa cells treated with TBBPA, attachment assays were performed. (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA) Early attachment rates (0\\u0026thinsp;~\\u0026thinsp;30 minutes) were slightly higher in the TBBPA groups (10 \\u0026micro;M) than the control groups. After 90 minutes, the attachment rate was no significant statistical difference among experiment groups which means attachment rate might involve only early attachment rates. (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eB)\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.6. The outgrowth assay of Jeg-3 spheroid on Ishikawa cells treated with TBBPA.\\u003c/h2\\u003e \\u003cp\\u003eTo find the effect of TBBPA on outgrowth, an outgrowth assay was conducted on Jeg-3 spheroids seeded on the Ishikawa cells as shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eA. TBBPA was treated with a concentration of 10 \\u0026micro;M on Ishikawa cells. In TBBPA groups, the outgrowth area significantly decreased. (P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.0001) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eB) This result means TBBPA negatively influences the outgrowth area even though the attachment-related protein levels were comparable.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"4. Discussion\",\"content\":\"\\u003cp\\u003eIn the present study, we investigated whether TBBPA has deleterious effects on the implantation and invasion process using the three-dimensional spheroid cell culture method. Finding the potential risks and giving evidence of TBBPA on female infertility, it is essential to investigate the toxicity of TBBPA in the endometrium and trophoblastic spheroid which represented embryo in this study. TBBPA induced inflammation \\u003cem\\u003emilieu\\u003c/em\\u003e, decreased the expression of the mRNA level, and reduced outgrown area even though its protein and attachment rate were comparable to the control groups in this study. However, the potential mechanism of TBBPA has not been clarified in recent studies. Further studies to find the toxicity and the harmful mechanisms of EDCs on the female reproduction system should be conducted.\\u003c/p\\u003e \\u003cp\\u003eFor the \\u003cem\\u003ein vitro\\u003c/em\\u003e spheroid models, 2D cultured systems were used generally but it has the limitation that does not fully represent physiological human cells, 3D spheroids can mimic human \\u003cem\\u003ein vivo\\u003c/em\\u003e cell conditions much better than 2D culture systems. (Stojanovska et al., \\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e) The main concern of these 3D culture models in this study is that they are made with a cancer cell line. The cancer cells do not represent the natural cells and leading to different results compared to the result of primary cells. In this study, the 2D cultured system and 3D culture system showed different mRNA expressions in inflammation relates genes such as IL-1β. The difference in mRNA expression can represent that using the 3D cultured system in the study possibly showed the actual environment in the human body.\\u003c/p\\u003e \\u003cp\\u003eIn this study, TBBPA has not affected on cytotoxicity in the human endometrium cell line at the concentration of 0.1, 1, 10 \\u0026micro;M for 24 hours or 48 hours but 100\\u0026micro;M has significantly decreased cell viability. On the aspects of real human endocrine systems and environments, long-term and low doses of EDCs are exposed. However, long-term experiments cannot be reconstructed using cell line experiments. To substitute the low dose and exposure time to EDCs problems (Calafat et al., \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e2008\\u003c/span\\u003e), TBBPA was treated at a high dose for short-term (Strack et al., \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e2007\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eThe mRNA expressions, associated with inflammation and attachment genes were detected in TBBPA-treated human endometrium cells. (Guan et al., \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e) When the human embryo is attached and invaded in the endometrium, cytokines related to attachment (Fukui et al., \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e) and inflammation are released into the endometrium and embryo. The mRNA expression associated with inflammation and attachment can show EDC\\u0026rsquo;s effects on embryo implantation and invasion. IL-6, TNF-α, and IL-1β which are associated with inflammation, have dramatically increased in TBBPA-treated endometrium cells. This result means TBBPA has harmful effects on embryos and endometrium. The protein level was evaluated using a Western blot, for the primary antibody IL-6, TNF- α, and LIF were used. These targets are increased in mRNA level conducted by qRT-PCR. To investigate whether the mRNA expression leads to the protein level, these targets were chosen for Western blot. In this experiment, TBBPA did not change the protein levels significantly and this result can describe that TBBPA has effects on mRNA expression not on protein level in these targets. However, further studies how TBBPA regulates inflammation and attachment related genes should be clarified to evaluate its toxicity on female reproduction.\\u003c/p\\u003e \\u003cp\\u003eIn the attachment assay, TBBPA treated group showed a slightly higher rate of early implantation compared to the control group. This result can explain the increase of mRNA expression in LIF. LIF is associated with attachment of the spheroid, this can increase the attachment rate of the spheroid, but this cannot represent TBBPA can increase the implantation of the embryo. On the other hand, after 60 minutes TBBPA groups showed a lower attachment rate than control groups. Also, there were no significant differences among the groups. The limitation of this assay is that spheroids are made with cancer cells. High proliferation and adhesion are the main characteristics of the cancer cell line. (Mart\\u0026iacute;nez-Reyes and Chandel, \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e) For next study, we are evaluating the impacts of TBBPA on mouse primary uterine cells because cancer cells cannot completely imitate the primary cells\\u0026rsquo; pathophysiology.\\u003c/p\\u003e \\u003cp\\u003eThe outgrowth assay is conducted to check the invasion of embryos on the endometrium, and this implantation process occurs within 72 hours in the human body. Jeg-3 spheroids seeded on TBBPA-treated endometrium cells represent the human endometrium exposed to EDCs and embryo implantation. In the actual human body, the implantation process occurs within 72 hours, so the outgrowth assay was conducted for 72 hours. In this experiment, TBBPA has decreased in the outgrowth area, therefore TBBPA might have negative impacts on implantation process in the human body.\\u003c/p\\u003e \\u003cp\\u003eIn this study, there are several limitations. First, treatment time and concentration of TBBPA. In the natural environment, EDCs are exposed to the human body for a long-term at low concentrations, but cell-based experiments have limitations in conducting experiments while maintaining the cell line over the years. Therefore, it is not possible to fully represent the EDCs exposed in the actual environment on the human body. Second, the characteristic of the cell line used in this study. The cell line used in this experiment is the human endometrial adenocarcinoma cell line and the human choriocarcinoma cell line. Compared to the primary cell line to cancer cell lines, cancer cell lines have high proliferation, adhesion, and differentiation than the primary cell line. Due to these features, the attachment assay might not be matched the actual results. For further study, we strongly suggest that using the primary cell line with real embryo using animal model for the attachment assay. For the last, the effects of EDCs on Jeg-3 spheroids are the limitation of this study. In the physiological human body, when implantation occurs, EDCs are affecting embryos and endometrium. In this experiment, we only treated TBBPA on the Ishikawa cells which a representative model for human endometrium. Studying the effect of EDCs both in the endometrium and spheroid for mimicking embryos may provide a solutions to overcome the limitations of this study.\\u003c/p\\u003e \\u003cp\\u003eThe purpose of this study was to investigate the effects of TBBPA on the female reproduction system, as well as the process of the implantation system. Further research might investigate the effect of TBBPA on implantation, using a primary cell line or mouse embryo and mouse endometrial cell is strongly required. When studying EDCs, it is ideal to treat them at low concentrations for a long-period of time as actual endocrine disruptors, so proceeding with animal experiments possibly showed similar results compared to cell line experiments.\\u003c/p\\u003e\"},{\"header\":\"5. Conclusion\",\"content\":\"\\u003cp\\u003eIn this study, TBBPA has effects on mRNA levels of inflammation and attachment such as TNF-a, LIF, and ITGB5. The outgrowth area treated with TBBPA has significantly decreased compared to the control. Collectively, TBBPA has shown to affect the endometrium, further study is required.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAvailability of data and materials\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe data\\u0026nbsp;that support the findings of this studyare available from the corresponding author upon reasonable request.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis project was financially supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Republic of Korea (NRF-2018R1D-1A1B07046419 to J.L.),\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor information\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthors and Affiliations\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eDepartment of Biomedical Laboratory Sciences, Eulji university\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e553, Sanseong-daero, Sujeong-gu, Seongnam-si, Gyeonggi-do, Republic of Korea\\u003c/p\\u003e\\n\\u003cp\\u003eMyoungjoo Koo, Inyoung Kang, Jaewang Lee, Jinhyun Jun\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eContrubutions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eConceptualization: KOO M, KANG I, LEE J; Investigation: KOO M, KANG I; Writing \\u0026ndash; Original Draft: KOO M, KANG I; Writing \\u0026ndash; Review \\u0026amp; Editing: LEE J, JUN J; Supervision: LEE J, JUN J; Project administration: LEE J, JUN J; Funding acquisition: LEE J.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthics approval and consent to participate.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent for publication\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll authors agreed to publish the paper.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare that they have no competing interests,\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConflict of interest\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors have no conflict of interest to declare.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n \\u003cli\\u003eBerger, M H, Messore, M, Pastuszak, A W, Ramasamy, R, (2016). Association Between Infertility and Sexual Dysfunction in Men and Women. Sex Med Rev 4:353-365.\\u003c/li\\u003e\\n \\u003cli\\u003eBrehm, E, Flaws, J A, (2019). Transgenerational Effects of Endocrine-Disrupting Chemicals on Male and Female Reproduction. Endocrinology 160:1421-1435.\\u003c/li\\u003e\\n \\u003cli\\u003eCalafat, A M, Ye, X, Wong, L Y, Reidy, J A, Needham, L L, (2008). Exposure of the U.S. population to bisphenol A and 4-tertiary-octylphenol: 2003-2004. Environ Health Perspect 116:39-44.\\u003c/li\\u003e\\n \\u003cli\\u003eden Braver-Sewradj, S P, van Spronsen, R, Hessel, E V S, (2020). Substitution of bisphenol A: a review of the carcinogenicity, reproductive toxicity, and endocrine disruption potential of alternative substances. Crit Rev Toxicol 50:128-147.\\u003c/li\\u003e\\n \\u003cli\\u003eDong, M, Li, Y, Zhu, M, Qin, Z, (2021). Tetrabromobisphenol A: a neurotoxicant or not? Environ Sci Pollut Res Int 28:54466-54476.\\u003c/li\\u003e\\n \\u003cli\\u003eFan, H, Fernando, S R, Jiang, L, Wang, Z, Kodithuwakku, S P, Wong, C K C, Ng, E H Y, Yeung, W S B, Lee, K F, (2021). Bisphenol A Analogues Suppress Spheroid Attachment on Human Endometrial Epithelial Cells through Modulation of Steroid Hormone Receptors Signaling Pathway. Cells 10:\\u003c/li\\u003e\\n \\u003cli\\u003eFukui, Y, Hirota, Y, Saito-Fujita, T, Aikawa, S, Hiraoka, T, Kaku, T, Hirata, T, Akaeda, S, Matsuo, M, Shimizu-Hirota, R, Takeda, N, Ikawa, M, Osuga, Y, (2021). Uterine Epithelial LIF Receptors Contribute to Implantation Chamber Formation in Blastocyst Attachment. Endocrinology 162:\\u003c/li\\u003e\\n \\u003cli\\u003eGuan, G, Su, H, Wei, X, Zheng, Y, Jin, X, (2021). The promotion of tetrabromobisphenol A exposure on Ishikawa cells proliferation and pivotal role of ubiquitin-mediated I\\u0026kappa;B\\u0026apos; degradation. Ecotoxicol Environ Saf 207:111254.\\u003c/li\\u003e\\n \\u003cli\\u003eGuarnotta, V, Amodei, R, Frasca, F, Aversa, A, Giordano, C, (2022). Impact of Chemical Endocrine Disruptors and Hormone Modulators on the Endocrine System. Int J Mol Sci 23:\\u003c/li\\u003e\\n \\u003cli\\u003eKabir, E R, Rahman, M S, Rahman, I, (2015). A review on endocrine disruptors and their possible impacts on human health. Environ Toxicol Pharmacol 40:241-258.\\u003c/li\\u003e\\n \\u003cli\\u003eKim, U J, Oh, J E, (2014). Tetrabromobisphenol A and hexabromocyclododecane flame retardants in infant-mother paired serum samples, and their relationships with thyroid hormones and environmental factors. Environ Pollut 184:193-200.\\u003c/li\\u003e\\n \\u003cli\\u003eKonieczna, A, Rutkowska, A, Rachoń, D, (2015). Health risk of exposure to Bisphenol A (BPA). Rocz Panstw Zakl Hig 66:5-11.\\u003c/li\\u003e\\n \\u003cli\\u003eLee, G H, Song, H J, Choi, Y M, Han, H D, (2017). The status of assisted reproductive technology in Korea in 2012. Clin Exp Reprod Med 44:47-51.\\u003c/li\\u003e\\n \\u003cli\\u003eLiu, J, Yu, L, Castro, L, Yan, Y, Clayton, N P, Bushel, P, Flagler, N D, Scappini, E, Dixon, D, (2022). Short-term tetrabromobisphenol A exposure promotes fibrosis of human uterine fibroid cells in a 3D culture system through TGF-beta signaling. Faseb j 36:e22101.\\u003c/li\\u003e\\n \\u003cli\\u003eMa, M, Crump, D, Farmahin, R, Kennedy, S W, (2015). Comparing the effects of tetrabromobisphenol-A, bisphenol A, and their potential replacement alternatives, TBBPA-bis(2,3-dibromopropyl ether) and bisphenol S, on cell viability and messenger ribonucleic acid expression in chicken embryonic hepatocytes. Environ Toxicol Chem 34:391-401.\\u003c/li\\u003e\\n \\u003cli\\u003eMart\\u0026iacute;nez-Reyes, I, Chandel, N S, (2021). Cancer metabolism: looking forward. Nat Rev Cancer 21:669-680.\\u003c/li\\u003e\\n \\u003cli\\u003eMlynarcikova, A B, Scsukova, S, (2020). Effect of selected bisphenol derivatives on nuclear receptor expression in ovarian cell line COV434. Endocr Regul 54:275-283.\\u003c/li\\u003e\\n \\u003cli\\u003eReed, J M, Spinelli, P, Falcone, S, He, M, Goeke, C M, Susiarjo, M, (2022). Evaluating the Effects of BPA and TBBPA Exposure on Pregnancy Loss and Maternal-Fetal Immune Cells in Mice. Environ Health Perspect 130:37010.\\u003c/li\\u003e\\n \\u003cli\\u003eStojanovska, V, Arnold, S, Bauer, M, Voss, H, Fest, S, Zenclussen, A C, (2022). Characterization of Three-Dimensional Trophoblast Spheroids: An Alternative Model to Study the Physiological Properties of the Placental Unit. Cells 11:\\u003c/li\\u003e\\n \\u003cli\\u003eStrack, S, Detzel, T, Wahl, M, Kuch, B, Krug, H F, (2007). Cytotoxicity of TBBPA and effects on proliferation, cell cycle and MAPK pathways in mammalian cells. Chemosphere 67:S405-411.\\u003c/li\\u003e\\n \\u003cli\\u003eSunday, O E, Bin, H, Guanghua, M, Yao, C, Zhengjia, Z, Xian, Q, Xiangyang, W, Weiwei, F, (2022). Review of the environmental occurrence, analytical techniques, degradation and toxicity of TBBPA and its derivatives. Environ Res 206:112594.\\u003c/li\\u003e\\n \\u003cli\\u003eVandenberg, L N, Hauser, R, Marcus, M, Olea, N, Welshons, W V, (2007). Human exposure to bisphenol A (BPA). Reprod Toxicol 24:139-177.\\u003c/li\\u003e\\n \\u003cli\\u003eVasiljevic, T, Harner, T, (2021). Bisphenol A and its analogues in outdoor and indoor air: Properties, sources and global levels. Sci Total Environ 789:148013.\\u003c/li\\u003e\\n \\u003cli\\u003eYaguchi, T, (2019). The endocrine disruptor bisphenol A promotes nuclear ERR\\u0026gamma; translocation, facilitating cell proliferation of Grade I endometrial cancer cells via EGF-dependent and EGF-independent pathways. Mol Cell Biochem 452:41-50.\\u003c/li\\u003e\\n \\u003cli\\u003eYilmaz, B, Terekeci, H, Sandal, S, Kelestimur, F, (2020). Endocrine disrupting chemicals: exposure, effects on human health, mechanism of action, models for testing and strategies for prevention. Rev Endocr Metab Disord 21:127-147.\\u003c/li\\u003e\\n \\u003cli\\u003eYun, J, Kim, C Y, Son, S H, Bae, C W, Choi, Y S, Chung, S H, (2022). Birth Rate Transition in the Republic of Korea: Trends and Prospects. J Korean Med Sci 37:e304.\\u003c/li\\u003e\\n \\u003cli\\u003eZhou, H, Yin, N, Faiola, F, (2020). Tetrabromobisphenol A (TBBPA): A controversial environmental pollutant. J Environ Sci (China) 97:54-66.\\u003c/li\\u003e\\n \\u003cli\\u003eHealthcare Bigdata Hub(2007-2015) Republic of Korea, Seoul. http://opendata.hira.or.kr/op/opc/olapMfrnIntrsDiagBhvInfo.do Accessed 01 June 2023\\u003c/li\\u003e\\n \\u003cli\\u003eHealthcare Bigdata Hub (2017-2021) Republic of Korea, Seoul. http://opendata.hira.or.kr/op/opc/olapMfrnIntrsIlnsInfo.do Accessed 01 June 2023\\u003c/li\\u003e\\n \\u003cli\\u003eKOSIS(Korean Statistical information Service) Republic of Korea, Seoul, https://kosis.kr/statHtml/statHtml.do?orgId=350\\u0026amp;tblId=DT_35007_N002_1\\u0026amp;vw_cd=MT_OTITLE\\u0026amp;list_id=350_35007_A001\\u0026amp;scrId=\\u0026amp;seqNo=\\u0026amp;lang_mode=ko\\u0026amp;obj_var_id=\\u0026amp;itm_id=\\u0026amp;conn_path=K2\\u0026amp;path=%252Fcommon%252Fmeta_onedepth.jsp Accessed 01 June 2023\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"environmental-sciences-europe\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"eseu\",\"sideBox\":\"Learn more about [Environmental Sciences Europe](http://enveurope.springeropen.com)\",\"snPcode\":\"12302\",\"submissionUrl\":\"https://submission.nature.com/new-submission/12302/3\",\"title\":\"Environmental Sciences Europe\",\"twitterHandle\":\"@SpringerOpen\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Implantation, Endocrine disrupting chemicals (EDCs), Tetrabromobisphenol A, Ishikawa cells, Jeg-3 cells, Infertility\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-3013348/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-3013348/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003ch2\\u003eBackground\\u003c/h2\\u003e \\u003cp\\u003eTetrabromobisphenol A (TBBPA) has endocrine-disrupting effect and has been widely used as brominated flame retardant in industrial products. The endocrine-disrupting chemical has effects on female reproduction such as infertility, hormone imbalance, and endometriosis. In Korea, the problems of infertility and the decreasing birth rate are concerns. Exposure to EDCs might have a harmful effect on childbearing-age women. This study aimed to investigate the effect of TBBPA on infertility, particularly on uterus endometrium early implantation events.\\u003c/p\\u003e\\u003ch2\\u003eResults\\u003c/h2\\u003e \\u003cp\\u003eThe expression of mRNA was analyzed by qRT-PCR. The mRNA expression of TBBPA on Ishikawa cells, IL-6, IL-1β, TNF- α, and LIF were significantly increased. Moreover, the outgrowth area in the TBBPA group has significantly decreased compared with the control. On the other hand, TBBPA had a slight effect on protein level and attachment rate.\\u003c/p\\u003e\\u003ch2\\u003eConclusions\\u003c/h2\\u003e \\u003cp\\u003eIn this study, TBBPA occurred inflammatory environmental \\u003cem\\u003emilieu\\u003c/em\\u003e in mRNA expression. The increase of inflammation-related cytokines in the endometrium can disrupt the implantation of the embryo. Also, the TBBPA disrupted the outgrowth of spheroids on the endometrium, however, the protein levels and attachment rate were comparable to the control group. The effect of TBBPA on implantation events should be further clarified.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Effect of tetrabromobisphenol A (TBBPA) on early implantation using the three-dimensional spheroid model with human endometrial cell line, Ishikawa\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2023-06-06 14:17:21\",\"doi\":\"10.21203/rs.3.rs-3013348/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Major revision\",\"date\":\"2023-07-11T18:05:18+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2023-07-01T04:25:26+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"2c184e2c-7a67-4ce9-a6c4-88260076c19d\",\"date\":\"2023-06-20T08:17:32+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"a20fb0f4-d649-45de-9ceb-82266d1a0864\",\"date\":\"2023-06-14T07:11:47+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2023-06-11T17:38:18+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2023-06-03T03:07:02+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2023-06-03T03:07:01+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Environmental Sciences Europe\",\"date\":\"2023-06-02T07:54:13+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"environmental-sciences-europe\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"eseu\",\"sideBox\":\"Learn more about [Environmental Sciences Europe](http://enveurope.springeropen.com)\",\"snPcode\":\"12302\",\"submissionUrl\":\"https://submission.nature.com/new-submission/12302/3\",\"title\":\"Environmental Sciences Europe\",\"twitterHandle\":\"@SpringerOpen\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"19412d8c-e209-48d2-bdaf-20384d5d174e\",\"owner\":[],\"postedDate\":\"June 6th, 2023\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2023-10-16T22:21:23+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-3013348\",\"link\":\"https://doi.org/10.1186/s12302-023-00780-x\",\"journal\":{\"identity\":\"environmental-sciences-europe\",\"isVorOnly\":false,\"title\":\"Environmental Sciences Europe\"},\"publishedOn\":\"2023-08-17 22:00:47\",\"publishedOnDateReadable\":\"August 17th, 2023\"},\"versionCreatedAt\":\"2023-06-06 14:17:21\",\"video\":\"\",\"vorDoi\":\"10.1186/s12302-023-00780-x\",\"vorDoiUrl\":\"https://doi.org/10.1186/s12302-023-00780-x\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-3013348\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-3013348\",\"identity\":\"rs-3013348\",\"version\":[\"v1\"]},\"buildId\":\"7rjqhiLT3MXkJMwkYKINL\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC0","license_restricted":false}