Caffeine Exposure Modulates Trophoblast Differentiation and Estradiol Synthesis

TL;DR

Caffeine affects how placental cells develop and produce estradiol, a hormone important for pregnancy, by changing specific cell processes.

Contribution

This study reveals that caffeine modulates trophoblast differentiation and estradiol synthesis via PKA-dependent mechanisms in human placental cells.

Findings

Caffeine increases CYP19A1 mRNA and estradiol production in BeWo cells and placental explants.

Caffeine's effect on CYP19A1 is mediated through PKA signaling, not PKC or MAPK pathways.

Non-cytotoxic caffeine concentrations enhance aromatase expression and estradiol synthesis.

Abstract

Differentiation of villous cytotrophoblasts into syncytiotrophoblasts is essential for placental endocrine function and estradiol production. Caffeine consumption has been linked to altered estradiol levels, but its effects on human trophoblast differentiation remain incompletely understood. This study investigated the effects of caffeine on biochemical differentiation of human trophoblasts using BeWo cells and human placental explants. Cell viability and apoptosis were assessed using CCK-8 and in situ TUNEL assays. Differentiation-associated changes were evaluated by measuring CYP19A1 and its placenta-specific promoter transcript CYP19 I.1, at the mRNA level, while aromatase protein expression and estradiol production were assessed by Western blotting and ELISA, respectively. Exposure to 2 mM caffeine reduced BeWo cell viability, whereas 1 mM caffeine had no detectable effects on cell viability or apoptosis. At non-cytotoxic concentrations, caffeine significantly increased CYP19A1 mRNA expression under both basal and forskolin-stimulated conditions and elevated estradiol production. Similar transcriptional and endocrine responses were observed in human placental explants. Pharmacological inhibition demonstrated that caffeine-induced CYP19A1 transcriptional upregulation was dependent on PKA signaling, but not on PKC or MAPK pathways. These results indicate that caffeine can modulate trophoblast biochemical differentiation via PKA-dependent regulation of placental aromatase expression and estradiol synthesis. While these findings provide mechanistic insight into caffeine-mediated effects on trophoblast endocrine function, their relevance to physiological exposure levels and in vivo placental development warrants cautious interpretation.

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1. Introduction

The human placenta is a transient but essential organ that supports fetal development and maintains pregnancy through hormone production and maternal–fetal exchange [1]. Proper placental function relies on the differentiation of cytotrophoblasts into multinucleated syncytiotrophoblasts (STBs) along the villous pathway [2,3]. STBs produce key hormones, including estradiol, human chorionic gonadotropin, and human placental lactogen, which are critical for fetal growth and maternal adaptation [2,4].

Trophoblast differentiation involves both biochemical and morphological changes. In particular, CYP19A1, which encodes the aromatase enzyme responsible for estrogen biosynthesis, serves as a key molecular marker of biochemical differentiation [3,4,5]. Dysregulation of this process is associated with placental pathologies such as preeclampsia and fetal growth restriction [6,7].

Caffeine (1,3,7-trimethylxanthine) is the most widely consumed psychoactive substance worldwide, with a high prevalence of intake among pregnant individuals [8,9]. During pregnancy, reduced metabolic clearance leads to prolonged maternal caffeine exposure, and caffeine readily crosses the placenta into the fetal circulation [10]. Epidemiological studies have linked maternal caffeine consumption and adverse pregnancy outcomes, including altered placental hormone levels and increased risks of pregnancy complications, particularly when exposure occurs early in gestation [8,11,12]. However, the placental cellular and molecular mechanisms underlying these associations remain poorly understood.

Caffeine is known to inhibit phosphodiesterase (PDE) activity and antagonize adenosine receptors, leading to elevated cyclic adenosine monophosphate (cAMP) levels [13,14]. This is of particular relevance to placental biology, as CYP19A1 expression is regulated by the cAMP/protein kinase A (PKA) pathway, a central driver of trophoblast differentiation and endocrine function [5,15]. Additionally, caffeine has been shown to increase aromatase expression and estradiol production in ovarian granulosa cells [16], suggesting a potential role for caffeine in modulating steroidogenic pathways. Collectively, these findings raise the possibility that caffeine may directly influence trophoblast biochemical differentiation through cAMP-dependent mechanisms.

Based on this background, the present study aimed to investigate whether caffeine influences biochemical differentiation of trophoblasts by assessing its effects on CYP19A1 and the placenta-specific CYP19 I.1 expression in BeWo cells, a well-established model for villous trophoblast differentiation, and in human placental explant cultures. We further sought to define the involvement of intracellular signaling pathways underlying these effects. This study provides mechanistic insight into how caffeine may modulate trophoblast endocrine function through PKA-dependent regulation of placental aromatase expression and estradiol production.

2. Materials and Methods

2.1. Cell Culture and Treatments

BeWo cells (KCLB No. 10098), a human placental choriocarcinoma cell line, were obtained from the Korean Cell Line Bank (Seoul, Republic of Korea). Cells were routinely grown in Ham’s F-12 medium (#LM010-01; Welgene, Gyeongsan, Republic of Korea) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; #S001-01, Welgene), 1% (v/v) penicillin–streptomycin (#15140-122; Gibco, Grand Island, NY, USA), 1 mM L-glutamine (#LS002-01; Welgene, Gyeongsan, Republic of Korea), and 0.37% sodium bicarbonate (#BB002-01; Welgene, Gyeongsan, Republic of Korea). Cultures were maintained at 37 °C in a humidified incubator under 5% CO_2_ and experiments were conducted using cells at passages 25 or lower. Caffeine (C0750, Sigma-Aldrich, St. Louis, MO, USA) was freshly prepared in sterile distilled water and added to the culture medium to achieve final concentrations of 0.3, 1, or 2 mM. Vehicle-treated control cells received an equivalent volume of sterile water. Depending on the experimental design, cells were exposed to caffeine for 24, 48, or 72 h. The caffeine concentrations were chosen based on prior in vitro studies employing placental and trophoblast-derived cell models, in which sub-millimolar to millimolar concentrations were used to evaluate dose-dependent mechanistic effects on trophoblast differentiation and related signaling pathways [17,18]. For signaling pathway inhibition experiments, cells were pre-incubated for 1 h with specific pharmacological inhibitors before caffeine treatment: H89 (10 μM; PKA inhibitor; GC19396, GLPBIO, Montclair, CA, USA), chelerythrine chloride (CE; 0.1 μM; PKC inhibitor; #C2932; Sigma-Aldrich, St. Louis, MO, USA), PD98059 (10 μM; MEK1 inhibitor; P215, Sigma-Aldrich, St. Louis, MO, USA), or SB203580 (10 μM; p38 MAPK inhibitor; #559389; Sigma-Aldrich, St. Louis, MO, USA). Forskolin (FSK; F6886, Sigma-Aldrich, St. Louis, MO, USA), a well-established activator of the cAMP/PKA pathway, was prepared as a 1000× stock solution in ethanol and applied at a final concentration of 25 μM. Co-treatment with FSK and caffeine was performed to evaluate whether caffeine exerts additive or synergistic effects on CYP19A1 expression beyond direct activation of the cAMP/PKA pathway. The concentrations of pharmacological inhibitors were selected based on previous studies [19,20,21,22,23]. Unless otherwise stated, all chemicals were obtained from Sigma-Aldrich.

2.2. Placental Explant Culture

Human term placentas were collected from uncomplicated singleton pregnancies within 30 min after delivery. Immediately after collection, placental tissues were processed for explant culture. Villous tissue samples (approximately 2 × 2 cm) were excised from the maternal side of the placenta and transferred into ice-cold phosphate-buffered saline (PBS). To remove residual maternal blood, tissues were gently agitated and washed twice with fresh PBS. After removal of visible membranes and large blood vessels using sterile instruments, the villous tissue was minced into small fragments measuring approximately 2–3 mm. Explants (approximately 100 mg per well) were placed in 24-well plates containing RPMI-1640 medium (#LM011-01; Welgene, Gyeongsan, Republic of Korea) supplemented with 10% FBS and 1% penicillin–streptomycin. Caffeine was added to the culture medium at a final concentration of 1 mM prior to explant placement. Cultures were maintained at 37 °C in a humidified incubator with 5% CO_2_ for 48 or 72 h. At the end of the incubation period, explants were collected and snap-frozen at −80 °C for subsequent RNA isolation. Conditioned media were harvested and stored at −80 °C until estradiol analysis. This explant culture system preserves endocrine and metabolic functions of term placental tissue and was used to evaluate caffeine-induced changes in placental aromatase expression and estradiol secretion. All procedures involving human samples were approved by the Institutional Review Board of Hanyang University (HYI-17-168-3) and conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants, and donor characteristics are summarized in Table S1.

2.3. Cell Counting Assay

BeWo cells were plated in 24-well culture plates at a density of 2.5 × 10^4^ cells per well and allowed to attach overnight. The medium was subsequently replaced with fresh medium containing caffeine at final concentrations of 0.3, 1, or 2 mM, followed by incubation for 24, 48, or 72 h. Cell viability was evaluated using the Cell Counting Kit-8 (CCK-8; Abbkine, Wuhan, China) in accordance with the manufacturer’s protocol. Briefly, 10 μL of CCK-8 reagent was added to each well and incubated for 2 h at 37 °C. Absorbance was recorded at 450 nm (Optical density, OD_450_) using a microplate reader (Bio-Rad, Hercules, CA, USA). Each treatment condition was assessed in triplicate wells, and all experiments were repeated independently three times.

2.4. TUNEL Assay

Apoptotic cell death was assessed using an In Situ Cell Death Detection Kit (Roche, Mannheim, Germany). BeWo cells were seeded onto glass coverslips (1.2 × 1.2 cm) placed in 24-well plates at a density of 1 × 10^5^ cells per well and cultured overnight to allow attachment. Cells were subsequently treated with caffeine (1 mM) for 48 h to evaluate apoptosis under non-cytotoxic conditions. Following treatment, cells were fixed in cold methanol (−20 °C) for 10 min, permeabilized with 0.25% Triton X-100 in PBS, and incubated with TUNEL reaction mixture for 1 h at 37 °C in accordance with the manufacturer’s protocol. Nuclear counterstaining was performed using Hoechst 33342 (H3570; Invitrogen, Carlsbad, CA, USA). Fluorescent signals were visualized using a Leica fluorescence microscope (Heidelberg, Germany). For each coverslip, images were acquired from eight randomly selected fields at 100× magnification. The apoptotic index (%) was calculated as the percentage of TUNEL-positive nuclei relative to the total number of nuclei. All experiments were conducted in triplicate.

2.5. Quantitative Real-Time PCR

Total RNA was isolated from cells or placental explants using the AccuPrep Universal RNA Extraction Kit (K-3140, Bioneer, Daejeon, Republic of Korea) following the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from 1–2 µg of total RNA using oligo(dT)18 primers and a commercial reverse transcription kit (R5600, GenDEPOT, Katy, TX, USA). Quantitative real-time PCR was performed to quantify total CYP19A1 expression as well as the placenta-specific CYP19 I.1 transcript, which reflects promoter-specific transcriptional activity of the CYP19 gene. Because both transcripts encode the same aromatase protein, promoter-specific regulation was evaluated at the mRNA level. PCR amplification was carried out using SYBR Green (Prime Q-Master Mix, Q-9200, GeNet Bio, Daejeon, Republic of Korea) on a qTOWER3 G real-time PCR system (Analytik Jena, Jena, Germany). Primer sequences were designed using Primer-BLAST (NCBI, Bethesda, MD, USA; https://www.ncbi.nlm.nih.gov/tools/primer-blast/, accessed on 5 January 2024) and are listed as follows: CYP19A1, forward 5′-CGGCCTTGTTCGTATGGTCA-3′ and reverse 5′-CGTCCACATAGCCCGATTCA-3′ (NM_000103.4); CYP19 I.1, forward 5′-GGATCTTCCAGACGTCGCGA-3′ and reverse 5′- CATGGCTTCAGGCACGATGC-3′ (NM_000103.4); GAPDH, forward 5′-CCAAGGAGTAAGACCCCTGG-3′ and reverse 5′-AGGGGAGATTCAGTGTGGTG-3′ (NM_002046.7) as the internal reference. Amplification product sizes were 95, 119, and 127 bp for CYP19A1, CYP19 I.1, and GAPDH, respectively. Thermal cycling conditions consisted of an initial denaturation at 95 °C for 10 min, followed by 40 amplification cycles of 95 °C for 10 s, 58–63 °C for 10 s, and 72 °C for 10 s. Each sample was analyzed in triplicate, and relative transcript levels were calculated using the ^ΔΔ^Ct method with normalization to GAPDH. Data represent mean ± SD from at least four independent experiments.

2.6. Western Blotting

BeWo cells were harvested 48 h after caffeine treatment and lysed directly in Laemmli sample buffer supplemented with 5% β-mercaptoethanol (M7522; Sigma-Aldrich, St. Louis, MO, USA). Protein concentrations were determined using the Bradford assay (Bio-Rad, Hercules, CA, USA), and equal amounts of protein (40–60 μg per lane) were resolved by 10% SDS–polyacrylamide gel electrophoresis. Separated proteins were transferred onto nitrocellulose membranes (GenDEPOT, Barker, TX, USA), which were subsequently blocked in 5% (w/v) skim milk prepared in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) for 2 h at room temperature. Membranes were incubated overnight at 4 °C with a rabbit monoclonal antibody against CYP19A1 (A12238; ABclonal, Woburn, MA, USA; 1:1000), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (NA934; Cytiva, Marlborough, MA, USA) for 2 h at room temperature. Protein bands were detected using an enhanced chemiluminescence reagent (Geneplex, Seoul, Republic of Korea) and visualized with a ChemiDoc XRS+ imaging system (Bio-Rad, Hercules, CA, USA). Membranes were reprobed with an anti-β-Actin antibody (ADI-905-733-100; ENZO Life Sciences, Farmingdale, NY, USA; 1:2000) to confirm equal protein loading. Band intensities were quantified using ImageJ software (v1.54d; NIH, Bethesda, MD, USA) and normalized to β-actin. All experiments were repeated independently at least three times.

2.7. Immunofluorescence Cell Staining

BeWo cells were seeded onto 2 × 2 cm glass coverslips in 24-well plates at a density of 1 × 10^5^ cells/well and cultured in the presence or absence of caffeine (1 mM) or FSK (25 μM) for 48 h. Cells were fixed with cold methanol (−20 °C) for 10 min and washed twice with PBS. Fixed cells were permeabilized and blocked with PBS containing 0.2% Triton X-100 (Sigma, St. Louis, MO, USA) and 1% normal goat serum for 1 h at room temperature. Cells were then incubated overnight at 4 °C with a rabbit monoclonal anti-CYP19A1 antibody (A12238, ABclonal, Woburn, MA, USA; 1:50). After three washes with PBS, cells were incubated for 1 h at room temperature in the dark with Alexa Fluor 594-conjugated goat anti-rabbit IgG (A11012, Invitrogen, Carlsbad, CA, USA; 1:200). Nuclei were counterstained with Hoechst 33342 (H3570, Invitrogen, Carlsbad, CA, USA; 1:3000), and coverslips were mounted using mounting medium (SP15-100, Fisher Scientific, Waltham, MA, USA). FSK treatment served as a positive control for trophoblast differentiation. Fluorescence images were acquired using a fluorescence microscope (Leica, Heidelberg, Germany) at 200× magnification. All experiments were independently repeated three times to ensure reproducibility.

2.8. Measurement of Estradiol

BeWo cells were seeded in 24-well plates at a density of 2.5 × 10^4^ cells/well and allowed to adhere overnight. The culture medium was then replaced with fresh medium containing caffeine, and cells were incubated for an additional 48 h. Conditioned media were collected and stored at −80 °C until analysis. For placental explant cultures, conditioned media were collected after 48 or 72 h of caffeine treatment. Estradiol concentrations in the conditioned media were measured using a commercially available enzyme-linked immunosorbent assay kit (CSB-E05108h; Cusabio Biotech Co., Ltd., Wuhan, China), according to the manufacturer’s instructions. The intra- and inter-assay coefficients of variation were <15%, and the limit of detection was 15 pg/mL under the assay conditions used. Absorbance was measured at 450 nm using a microplate reader (Bio-Rad, Hercules, CA, USA) within 15 min of reaction termination. All samples were analyzed in duplicate, and data were obtained from four independent experiments.

2.9. Data Analysis

All statistical analyses were conducted using IBM SPSS Statistics software (version 21.0; IBM Corp., Armonk, NY, USA). For comparisons involving more than two experimental groups, data were analyzed using the Kruskal–Wallis non-parametric test, followed by Bonferroni-corrected post hoc analyses when appropriate. Pairwise comparisons between two groups were performed using the Mann–Whitney U test. Experimental results are presented as mean ± standard deviation (SD) derived from a minimum of three independent experiments. A p-value of less than 0.05 was considered indicative of statistical significance.

3. Results

3.1. Effects of Caffeine on Cell Viability and Apoptosis in BeWo Cells

BeWo cells were exposed to caffeine at concentrations ranging from 0.3 to 2 mM to evaluate potential cytotoxicity prior to differentiation-related analyses. Based on previous in vitro studies, 1 mM caffeine was selected as a reference concentration for mechanistic analyses, with 0.3 mM and 2 mM included to assess concentration-dependent responses [17,18]. Cell viability was assessed using the CCK-8 assay at 24, 48, and 72 h. Optical density (OD) values at 450 nm increased over time in all experimental groups, indicating progressive cell growth (Figure 1A). Treatment with 2 mM caffeine resulted in a significant reduction in OD values at both 48 and 72 h compared with the control (p < 0.05), indicating reduced cell viability. In contrast, exposure to 0.3 or 1 mM caffeine did not significantly affect cell viability at any time points examined (Figure 1A). To further evaluate whether caffeine induces apoptotic cell death at a non-cytotoxic concentration, apoptosis was assessed by TUNEL staining following treatment with 1 mM caffeine for 48 h. Quantitative analysis revealed no significant difference in the proportion of TUNEL-positive cells between the caffeine-treated and the control groups (Figure 1B). Representative fluorescence images are shown in Figure 1C. Together, these results indicate that caffeine up to 1 mM does not induce detectable cytotoxicity or apoptosis in BeWo cells under the experimental conditions used, whereas higher concentrations (2 mM) reduce cell viability.

3.2. Effects of Caffeine on CYP19A1 and CYP19 I.1 Expression and Estradiol Production in BeWo Cells

The effects of caffeine on trophoblast biochemical differentiation were examined by analyzing the mRNA expression of CYP19A1 and its placenta-specific promoter transcript, CYP19 I.1, following 48 h of treatment. FSK, a well-established activator of the cAMP/PKA pathway and inducer of trophoblast differentiation, was included as a positive control. FSK treatment significantly increased CYP19A1 and CYP19 I.1 mRNA levels by 2.40-fold and 2.44-fold, respectively, compared with the control (p < 0.05; Figure 2A,B). Caffeine treatment also significantly upregulated both transcripts. Exposure to 0.3 and 1 mM caffeine increased CYP19A1 mRNA levels by 1.50- and 2.01-fold, and CYP19 I.1 mRNA levels by 1.83- and 2.25-fold, respectively, relative to the control (p < 0.05; Figure 2A,B). Although differences between caffeine concentrations did not reach statistical significance, both transcripts exhibited a dose-dependent trend, with expression levels at 1 mM caffeine comparable to those induced by FSK. CYP19A1 protein expression was subsequently assessed by Western blot analysis (Figure 2C). FSK increased CYP19A1 protein expression by 2.14-fold relative to the control after 48 h. Caffeine treatment also increased CYP19A1 protein expression, with 1.51- and 1.98-fold elevations observed at 0.3 and 1 mM, respectively. While the difference between caffeine concentrations was not statistically significant, the overall pattern was consistent with the mRNA expression results. Consistent with these findings, estradiol concentrations in the culture medium were significantly increased by FSK (2.56-fold vs. control; p < 0.05) (Figure 2D). Caffeine treatment at 0.3 and 1 mM also significantly enhanced estradiol production by 1.48- and 1.99-fold, respectively (p < 0.05; Figure 2D). Estradiol levels in the 1 mM caffeine-treated group were comparable to those observed following FSK treatment. Immunofluorescence staining further demonstrated an increased number of aromatase-positive cells following 48 h of caffeine treatment, reaching levels comparable to those observed in the FSK-treated group (Figure 2E).

3.3. Effects of Caffeine on FSK-Induced CYP19A1 and CYP19 I.1 Expression in BeWo Cells

To determine whether caffeine modulates cAMP/PKA-mediated induction of placental aromatase, BeWo cells were treated with FSK alone or co-treated with FSK and caffeine (0.3 or 1 mM) for 48 h. Compared with FSK treatment alone, co-treatment with caffeine resulted in a modest but consistent enhancement of FSK-induced CYP19A1 mRNA expression. CYP19A1 mRNA levels increased by 1.30-fold and 1.44-fold in the presence of 0.3 and 1 mM caffeine, respectively (Figure 3A). Similarly, CYP19 I.1 mRNA expression was further elevated by 1.20-fold and 1.66-fold following co-treatment with 0.3 and 1 mM caffeine, respectively (Figure 3B). CYP19A1 protein expression was assessed by Western blotting after 48 h of treatment (Figure 3C). While FSK markedly increased CYP19A1 protein levels compared with the 0 h control, co-treatment with caffeine did not result in a proportional increase beyond that induced by FSK alone. These findings suggest that although caffeine modestly enhances FSK-induced CYP19A1 transcription, this effect is not fully reflected at the protein level under the experimental conditions used.

3.4. Effects of Caffeine on CYP19A1 and CYP19 I.1 Expression and Estradiol Production in Human Placental Explants

To determine whether the effects of caffeine observed in BeWo cells are also detectable in human placental tissue, explants obtained from term placentas were cultured in control medium (CT) or treated with 1 mM caffeine for 48 or 72 h. Caffeine treatment significantly increased the mRNA expression of both CYP19A1 and the placenta-specific promoter transcript CYP19 I.1 at both time points compared with controls (Figure 4A). At 48 h, approximately two-fold increases in both transcripts were observed. At 72 h, expression levels were further elevated, although greater inter-sample variability was noted; nevertheless, the increases remained statistically significant. Estradiol concentrations in the conditioned culture media were also significantly increased following caffeine treatment (Figure 4B). Compared with controls, estradiol levels were elevated by approximately 1.4-fold at 48 h and 2.2-fold at 72 h (p < 0.05 for both). These results demonstrate that caffeine enhances placental aromatase gene expression and estradiol production in human placental explants.

3.5. Effects of PKA, PKC, and MAPK Inhibition on Caffeine-Induced CYP19A1 Expression

To investigate the signaling pathways involved in caffeine-induced CYP19A1 expression, BeWo cells were treated with caffeine or FSK in the presence or absence of pharmacological inhibitors targeting PKA, PKC, or MAPK pathways. FSK treatment significantly increased CYP19A1 mRNA expression by 2.34-fold compared with the control (p < 0.05) (Figure 5A). This induction was markedly attenuated by co-treatment with the PKA inhibitor H89, reducing CYP19A1 expression to near basal levels. Similarly, caffeine-induced upregulation of CYP19A1 (2.32-fold vs. control; p < 0.05) was significantly suppressed by H89 (Figure 5A). In contrast, inhibition of PKC with chelerythrine did not significantly affect caffeine-induced CYP19A1 expression. Likewise, inhibition of MAPK signaling using PD98059 (MEK1 inhibitor) or SB203580 (p38 MAPK inhibitor) did not significantly alter the induction of CYP19A1 expression by caffeine (Figure 5B). Collectively, these findings indicate that caffeine-induced transcriptional activation of CYP19A1 in BeWo cells is primarily mediated through the cAMP/PKA signaling pathway, whereas PKC and MAPK pathways do not appear to play a major role under the experimental conditions used.

4. Discussion

In the present study, we show that caffeine modulates trophoblast biochemical differentiation, at least in part, by enhancing aromatase expression and estradiol synthesis through activation of the cAMP/PKA signaling pathway. Using both BeWo cells and human placental explants, we show that caffeine upregulates the placenta-specific aromatase transcript CYP19 I.1, increases CYP19A1 protein levels, and elevates estradiol production. Together, these findings provide convergent evidence that caffeine can influence trophoblast endocrine function by regulating key molecular pathways associated with biochemical differentiation.

Caffeine exerted concentration-dependent effects on trophoblast viability. While exposure to 2 mM caffeine significantly reduced cell viability, concentrations up to 1 mM did not induce detectable cytotoxicity or apoptosis in BeWo cells under the experimental conditions used. These results are consistent with previous studies reporting cytotoxic or pro-apoptotic effects of high caffeine concentrations (≥2 mM) in various cell types, including placental and non-placental models [24,25,26,27]. In contrast, lower concentrations of caffeine have been reported to exert cell type-specific regulatory effects without overt toxicity [28,29]. Importantly, our data indicate that at non-cytotoxic concentrations, caffeine does not compromise trophoblast survival but instead selectively modulates differentiation-associated molecular pathways. This distinction supports the interpretation that the observed effects reflect regulated cellular responses rather than nonspecific toxicity.

Aromatase, encoded by CYP19A1, plays a central role in placental estradiol biosynthesis and is a defining marker of trophoblast biochemical differentiation. In placental tissue, transcription from the placenta-specific promoter CYP19 I.1 is closely associated with functional differentiation of villous cytotrophoblasts into STBs [30]. In primary trophoblast cultures, expression of CYP19A1 and CYP19 I.1 increases in parallel with spontaneous syncytialization, reaching maximal levels approximately 48 h after plating [5]. FSK, a classical inducer of trophoblast differentiation through elevation of intracellular cAMP, reproduces this transcriptional profile and is therefore widely used as a positive control in trophoblast differentiation studies [19]. In this study, caffeine induced a significant upregulation of CYP19A1 and CYP19 I.1 in BeWo cells with a temporal pattern and magnitude comparable to that elicited by FSK. At 1 mM, caffeine induced CYP19A1 transcript levels that were comparable in magnitude to those observed following FSK treatment, and co-treatment resulted in a modest additional increase at the transcriptional level. These changes were accompanied by increased CYP19A1 protein expression and enhanced estradiol secretion, indicating that transcriptional regulation translated into functional endocrine output. Consistent with our findings, caffeine has also been reported to modulate aromatase expression and estradiol production in other human reproductive cell types, such as ovarian granulosa cells, suggesting involvement of steroidogenic regulatory pathways [31]. Although detailed morphological differentiation was not systematically evaluated in this study, Syncytin-1 mRNA expression, a trophoblast fusion-associated differentiation marker, was significantly increased following caffeine treatment (Supplementary Figure S1). This finding is consistent with caffeine-induced modulation of differentiation-related molecular programs. Together, these observations support the notion that caffeine can influence steroidogenic differentiation programs across distinct reproductive cell types, while the present study extends these findings to human placental trophoblast models. Importantly, similar increases in CYP19A1, CYP19 I.1, and estradiol production were also observed in human placental explants, indicating that caffeine-induced modulation of aromatase expression is not restricted to trophoblast cell lines.

Our pathway analyses identify the cAMP/PKA signaling axis as a critical mediator of caffeine-induced CYP19A1 expression. The absence of strong additive or synergistic effects upon co-treatment with FSK further suggests that caffeine-induced regulation of CYP19A1 predominantly converges on the cAMP/PKA pathway rather than engaging parallel signaling mechanisms. Pharmacological inhibition of PKA effectively abolished the induction of CYP19A1 by caffeine, whereas inhibition of PKC or MAPK signaling had no significant effect. These findings are consistent with the known ability of caffeine to inhibit PDE activity, leading to intracellular cAMP accumulation and downstream activation of PKA [32,33]. PKA signaling is a well-established upstream regulator of trophoblast differentiation, in part through activation of transcription factors such as GCM1 that control expression of STB-associated genes, including CYP19A1 [34,35]. Although GCM1 was not directly examined in the present study, the strong dependence of caffeine-induced aromatase expression on PKA activity strongly supports involvement of this regulatory axis. In contrast, despite reports of caffeine-mediated modulation of PKC or MAPK pathways in other cellular contexts [36,37,38], our data suggest that these pathways do not play a major role in caffeine-driven regulation of aromatase expression in trophoblasts under the conditions examined.

The biological implications of caffeine-induced modulation of trophoblast differentiation warrant careful consideration. Normal placental development requires a finely regulated balance between cytotrophoblast proliferation and differentiation. Alterations in the timing or extent of biochemical differentiation could potentially influence placental endocrine capacity and adaptability. In this context, caffeine-induced enhancement of aromatase expression and estradiol synthesis may represent a mechanism by which trophoblast endocrine function is altered, rather than a direct cytotoxic effect. However, because the present study utilized in vitro trophoblast models and ex vivo explants derived from term placentas, these findings should be interpreted as mechanistic insights rather than as direct evidence of effects occurring during early gestation.

Several limitations of this study should be acknowledged. First, the experimental caffeine concentrations were selected based on prior in vitro studies and were intended to interrogate dose-dependent signaling mechanisms rather than to replicate physiological exposure levels. Second, BeWo cells and term placental explants do not fully recapitulate the dynamic cellular and structural complexity of placental development in vivo, particularly during early pregnancy. In addition, the number of human placental explants analyzed was limited, which may reduce statistical power and increase inter-individual variability. Finally, the precise upstream molecular targets by which caffeine elevates intracellular cAMP levels in trophoblasts remain to be defined. Future studies using complementary in vivo models and earlier gestational tissues will be required to determine how caffeine exposure influences placental development across gestational stages and exposure windows.

In conclusion, this study provides mechanistic insight into how caffeine may modulate trophoblast biochemical differentiation, in part through PKA-dependent regulation of aromatase expression and estradiol synthesis. These findings identify a previously underappreciated molecular mechanism by which caffeine can influence trophoblast endocrine function and highlight trophoblast differentiation as a sensitive cellular process responsive to caffeine exposure.