Steroid hormone-treated endometrial organoids enhance implantation of in vitro-produced porcine embryos.

The endometrium is the internal lining of the uterus, consisting of the mucosa and submucosa, and plays a crucial role in female reproductive physiology, significantly affecting menstruation, pregnancy, and related diseases [ 1 ]. Its thickness and structure vary in response to alterations in steroid hormone levels, such as estrogen (E 2 ) and progesterone (P 4 ), and it is involved in both pregnancy and menstruation [ 2 3 4 ]. Functional defects in the endometrium, such as endometriosis, endometritis, and hormonal receptor abnormalities, can result in infertility, miscarriage, and menstrual cycle irregularities, facilitating active research in these areas [ 5 ]. During the proliferative phase of the menstrual cycle, E 2 predominantly regulates various functions [ 6 ], including enhancing endometrial thickness, stimulating cellular proliferation, and facilitating embryo implantation. Additionally, it facilitates the development of novel blood vessels in the endometrium and enhances the functional layer in preparation for pregnancy [ 7 ]. Similarly, P 4 stabilizes the thickened endometrium from the proliferative phase [ 8 ] and facilitates the implantation of a fertilized embryo. Throughout gestation, sustained P 4 secretion preserves the endometrium. However, in the absence of pregnancy, prostaglandin F2 alpha secreted by the uterus causes corpus luteum involution that reduces P 4 levels [ 9 ], resulting in the constriction of arteries supplying the functional layer [ 10 ]. This results in ischemia and cell death in the functional layer, causing endometrial shedding and onset of menstruation [ 11 ]. The interplay of these steroid hormones throughout the menstrual cycle orchestrates dynamic alterations in the endometrium, ensuring readiness for embryo implantation. In the absence of pregnancy, the endometrium sheds during menstruation. Dysregulation of these hormonal signals can cause various menstrual disorders, such as irregular menstruation, endometriosis, and infertility. Blastocyst elongation is crucial for early embryonic development and implantation. In mammals, such as pigs, the transition of the blastocyst from a spherical to a filamentous shape results in a greater surface area that enhances the contact between the embryo and maternal endometrium [ 7 ]. This process is crucial for a successful implantation [ 12 13 ]. However, because blastocyst elongation results from complex uterine interactions, replicating this phenomenon in vitro remains challenging. To address this, researchers are developing artificial uterine systems and embryo development models, with the aim of advancing animal reproductive technologies [ 14 ]. Organoids are three-dimensional (3D) models that can mimic the physiological and pathological properties of biological tissues in vitro , with applications in tissue formation, regeneration, repair, disease detection, and drug discovery [ 15 16 ]. Endometrial epithelial organoids (EEOs) effectively model the biology, function, and disorders of the endometrium and successfully mimic their histological phenotype [ 17 ]. Additionally, human and mouse organoids have been demonstrated to imitate the physiological responses of the endometrial epithelium to hormones and cell type-specific responses within the endometrium [ 18 19 ]. These models were widely used in studies associated with embryonic-maternal interactions, which are crucial for the successful implantation of embryos [ 20 ]. During co-culture, blastocysts are placed near organoids in a growth-supporting culture medium [ 21 ]. The organoids provide a microenvironment that mimics the maternal reproductive tract, enabling researchers to study the interactions between the developing embryos and maternal tissues. These interactions include embryo implantation, trophoblast invasion, and placental development [ 22 ]. By mimicking the maternal environment, organoid co-culture systems can offer valuable insights into embryonic implantation, including molecular signals and cellular interactions [ 23 ]. In this study, we aimed to assess the effects of steroid hormone treatment on the expression of hormone receptors in porcine EEOs under in vivo -mimicking conditions. By characterizing the molecular responses of these organoids to E 2 and P 4 , we aimed to understand how steroid hormones affect endometrial function. Additionally, we explored the interaction between hormone-treated organoids and blastocysts in a co-culture system to assess the potential of this model to simulate early stage embryo-endometrial communication. This study offers novel insights into hormone-regulated processes in the endometrium, with implications for enhancing fertility treatment and reproductive biology research.

Based on our previous study [ 24 ], the endometrium was obtained from nearby slaughterhouses, dissected, washed with phosphate-buffered saline (PBS) supplemented with penicillin-streptomycin, and cut into 1 mm pieces in a 100 mm petri dish. The sample was filtered thrice using a 100 µm strainer, followed by incubation in pre-warmed 0.1% collagenase type I for 2.5 h, with shaking at 15-min intervals. After digestion, the sample was filtered using a 70 µm strainer to remove the tissue and centrifuged for 2 min. The collected cells were washed thrice with PBS, resuspended in pre-warmed Matrigel (Corning, 356234; Corning, USA), and stored overnight at 4°C. Matrigel was plated on a 35 mm petri dish and incubated at 5% CO 2 and 38°C for 20 min. As applied in previous studies [ 1 ], the EEO culture medium was pre-warmed for 20 min at room temperature and subsequently added to the petri dish for EEO culture. In the hormone-treated group, the culture medium was replaced on day 3 with a medium containing β-E 2 (E2758; Sigma-Aldrich, USA), and on day 5, the medium was replaced with one containing both β-E 2 and P 4 (P8783; Sigma-Aldrich). This hormone treatment schedule was designed based on the physiological hormonal changes during the porcine estrous cycle and follows the protocol previously established in our laboratory [ 25 ]. Additionally, the culture medium of the control group was replaced without hormonal supplementation. The cells were passaged on day 7. After removing the culture medium through suction, the Matrigel drop structures were disrupted using PBS, followed by dissociation with trypsin-ethylenediaminetetraacetic acid (0.5%) for 4 min. Subsequently, the dissociated cells were washed with PBS containing 0.1% bovine serum albumin (BSA), centrifuged, and washed twice with PBS. The cells were resuspended in Matrigel and cultured in petri dishes. To characterize EEOs on day 7, immunofluorescence staining was performed using Ki67, vimentin, mucin1, and pan-cytokeratin [ 26 ]. EEOs were fixed with 4% paraformaldehyde, washed with PBS, and permeabilized with 0.5% Triton-X 100 for 30 min. Subsequently, the EEOs were blocked with 5% BSA for 2 h. Primary antibodies, including Ki67 (rabbit polyclonal anti-Ki67 antibody, ab15580; Abcam, UK), vimentin (rabbit monoclonal recombinant anti-vimentin antibody, ab92547; Abcam), mucin1 (recombinant rabbit anti-MUC1 antibody, [EPR1023], ab109185; Abcam), and pan-cytokeratin (mouse recombinant multiclonal antibody, ab86734; Abcam, Korea), were diluted 1:200 in 5% BSA, applied to the EEOs, and incubated at 38°C for 2 h. Subsequently, secondary antibodies diluted at a 1:500 ratio in PBS, including Alexa Fluor 488 goat anti-rabbit IgG ( Ab150077 ; Abcam), Alexa Fluor 568 goat anti-mouse IgG (A11004; Life Technologies, USA), or Alexa Fluor 568 goat anti-rabbit IgG (A11011; Life Technologies), were applied to the EEOs and incubated for 1 h at 38°C. After washing with PBS, the nuclei were stained using VECTASHIELD antifade mounting medium (Vector Laboratories, USA) containing 4′,6-diamidino-2-phenylindole and covered with a coverslip. Additionally, the E 2 receptor alpha antibody (PA1-309; Thermo Fisher Scientific, USA) and P 4 receptor (MA1-410; Thermo Fisher Scientific) were stained using the same protocol. Images were captured using an inverted microscope (DMi8; Leica, Germany) and quantified using the ImageJ software (National Institutes of Health, USA). This experiment followed a previously established protocol [ 25 ]. On day 7, RNA was extracted from the EEOs using TRIzol reagent (TR 118; Molecular Research Center, USA), chloroform (C2432; Sigma-Aldrich), isopropyl alcohol (I9030; Sigma-Aldrich), and glycogen (AM9510; Invitrogen, USA). The RNA concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). Subsequently, cDNA was synthesized using 2× RT Pre-Mix (BIOFACT, Korea), and real-time PCR was performed using the CFX Connect Real-Time PCR System (Bio-Rad Laboratories, USA) and SYBR 2× Real-Time PCR Pre-Mix (BIOFACT). The expression levels of target genes—including glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ), insulin-like growth factor ( IGF ), vascular endothelial growth factor A ( VEGFA) , transforming growth factor beta 1 ( TGFβ1 ), and fibroblast growth factor 7 ( FGF7 )—were normalized to GAPDH as the reference gene. The primer sequences used for these genes were identical to those reported in our previous study [ 25 ]. This experiment followed a previously established protocol [ 27 28 ]. Ovaries obtained from a nearby slaughterhouse were washed in 0.9% saline solution and aspirated using a 10 mL syringe with an 18-gauge needle. The oocytes were selected based on the condition of their cytoplasm and cumulus cells and then washed thrice with Tyrode’s medium containing 0.05% (w/v) polyvinyl alcohol. Subsequently, the oocytes were cultured for one day in an in vitro maturation medium containing 10% (v/v) porcine follicular fluid, 0.91 mM sodium pyruvate, 0.57 mM L-cysteine, 10 ng/mL epidermal growth factor, 1 μg/mL insulin, 10 IU/mL human chorionic gonadotropin, 10 IU/mL pregnant mare serum gonadotropin, and 75 μg/mL kanamycin sulfate. After one day, the oocytes were cultured in a hormone-free medium. After denuding, mature oocytes of good quality were exposed to two pulses of direct current at 120 V/cm for 60 ms in 280 mM mannitol solution containing 0.01 mM CaCl 2 and 0.05 mM MgCl 2 using a BTX 2001 Electro-cell Manipulator (BTX Molecular Delivery Systems, USA) for parthenogenetic activation [ 29 ]. The oocytes were placed in 25 μL drops of porcine zygote medium-3 medium and cultured at 38°C in 5% CO 2 for seven days. To assess the interaction between hormone-treated and control EEOs with blastocysts, a co-culture experiment was performed. Matrigel was incubated in a 4-well dish for 20 min at 38°C in 5% CO 2 . On day 7, the zona pellucida of the blastocysts was removed using pronase, the blastocysts were placed in Medium 199 (12340030; Gibco, USA), and the EEOs were washed with PBS. Subsequently, the blastocysts were positioned outside the Matrigel dome and co-cultured with the EEOs. Blastocyst outgrowth was monitored on days 2–4 of the culture. Data analysis was performed using SPSS version 29 (IBM, USA). All values are presented as the mean ± standard error of the mean, and statistical significance was set at p < 0.05. Data are analyzed using the generalized linear model procedure and one-way analysis of variance, followed by Tukey’s multiple comparison test.

We isolated endometrial cells from porcine uterine tissue obtained from a slaughterhouse to generate organoids, which were cultured in Matrigel droplets for seven days. Initially, the cells formed clusters, and by the second subculture, the 3D spherical structure of the organoids was clearly visible after three day of culture ( Fig. 1 ). To determine the effect of hormones on organoids, the medium was supplemented with or without sex hormones, specifically E 2 on day 3 and both E 2 and P 4 on day 5, with appropriate medium replacement. The successful establishment of organoids demonstrated the robustness of this protocol for generating 3D models of endometrial tissue. EEO, endometrial epithelial organoid. Seven days after organoid establishment, the cells were separated from Matrigel using a cell recovery solution and analyzed for characteristics using organoid-related antibodies. Antibodies associated with cell proliferation (Ki67), structural integrity (vimentin), and epithelial markers (mucin1 and pan-cytokeratin) were positively expressed in each group. These findings demonstrated that the 3D structure of the organoids remained intact after Matrigel separation ( Fig. 2 ). Immunofluorescence images revealed consistent expression patterns across replicates, thereby confirming the reproducibility of the model. Additionally, the structural integrity and marker expression highlight the suitability of organoids for further functional assays. EEO, endometrial epithelial organoid; DAPI, 4′,6-diamidino-2-phenylindole. To mimic the in vivo hormonal response, the medium containing E 2 was replaced on day 3, followed by a medium containing both E 2 and P 4 on day 5. Hormone receptor expression was compared between the experimental and control groups. The results revealed a significant increase in receptor expression for both sex hormones in the hormone-treated group ( p < 0.05, Fig. 3 ). Specifically, the expression of E 2 receptor alpha and P 4 receptor was significantly enhanced, with fluorescence intensity measurements confirming statistical significance. EEO, endometrial epithelial organoid; E 2 , estrogen; P 4 , progesterone; DAPI, 4′,6-diamidino-2-phenylindole. * Significant difference at p < 0.05. Additionally, hormone-related genes were analyzed in each group. E 2 -related genes, such as FGF7 and IGF1 were significantly upregulated in the hormone group compared to those in the control group. Similarly, the P 4 -related genes, such as TGFβ1 and VEGF exhibited significantly higher expression levels in the hormone group than that in the control group ( Fig. 4 ). These findings indicate that EEOs respond to hormones in a manner comparable to the in vivo endometrium, as evidenced by the consistent upregulation of hormone-responsive genes. This highlights the potential of organoids to accurately model endometrial tissue dynamics under hormonal influence. FGF7 , fibroblast growth factor 7; IGF1 , insulin-like growth factor 1; TGFβ1 , transforming growth factor beta 1; VEGF , vascular endothelial growth factor. * Significant difference at p < 0.05. On day 7, steroid hormone-induced morphological alterations in EEOs were assessed by measuring the cell layer thickness. The cell layer thickness of hormone-treated EEOs was significantly greater than that of the control group ( p < 0.05, Fig. 5 ). Detailed image analysis revealed a uniform thickening of the cell layer, indicating enhanced cellular proliferation and structural reorganization. These findings indicate that steroid hormone treatment induces significant structural alterations, thereby enhancing the EEO cell layer thickness and highlighting the dynamic response of organoids to hormonal stimuli, mirroring in vivo -like morphological adaptations. ** Significant difference at p < 0.01. To assess the differences in blastocyst development between control and hormone-treated EEOs, co-culture experiments were conducted. Blastocysts were co-cultured with day 7-harvested EEOs, and their outgrowth was observed from days 2–4. The results demonstrated that the growth rate of blastocysts co-cultured with hormone-treated EEOs was significantly higher than that of the control group ( p < 0.05, Fig. 6 ). Additionally, morphological alterations in blastocysts, such as an increased surface area and cellular outgrowth, were more significant in the hormone-treated group than that in the control group. This indicated that hormone-treated EEOs provide a microenvironment that enhances blastocyst development and outgrowth. Moreover, the co-culture model demonstrated consistent interactions between the blastocysts and organoids, simulating early maternal-embryonic communication. These findings highlight the potential of hormone-treated EEOs to serve as robust in vitro models for studying embryo implantation dynamics. * Significant difference at p < 0.05.

After confirming organoid formation, we assessed the expression levels of E 2 and P 4 receptors in both the control and hormone-treated groups using fluorescence staining. The results revealed an enhanced expression of both receptors in the hormone-treated group. This finding indicates that similar to in vivo conditions [ 30 ], organoids enhance receptor expression in response to hormones, indicating that porcine EEOs may have the potential to mimic physiological functions in vivo . E 2 induces various factors essential for pregnancy preparation, including cell proliferation and nutrient delivery [ 30 ]. We assessed the expression levels of IGF1 [ 31 32 ]—that facilitates cell proliferation, and FGF7 [ 33 34 ]—that is crucial for embryonic development processes, such as cell growth, embryonic development, and morphogenesis. Our results demonstrated that the expression levels of both IGF1 and FGF7 were higher in the hormone-treated group than that in the control group. Additionally, we assessed the gene expression of TGFβ1 [ 35 36 ], which is involved in various cellular functions, such as growth, proliferation, and P 4 -induced apoptosis, and VEGF [ 37 38 ], which is implicated in angiogenesis. Moreover, our results demonstrated enhanced expression of both TGFβ1 and VEGF in the hormone-treated group. These findings indicated that organoids exhibit hormone-responsive gene expression patterns that are similar to those observed in vivo . In vitro development of porcine blastocysts has advanced; however, observing their morphological transition from a spherical to a filamentous form remains challenging [ 39 ]. Consequently, to facilitate embryo elongation various studies have been conducted, including those using 3D alginate hydrogels [ 40 41 ]. In this study, we assessed the potential of organoids as a model for observing embryo elongation and the effects of hormone treatment on the embryos. After co-culturing the embryos with Matrigel, elongation was observed between days 3–4. A comparative analysis between the control and hormone-treated EEO groups revealed an increased elongation rate in the hormone-treated group. These results indicate that organoids and Matrigel offer a viable model for observing embryo elongation and that hormone treatment enhances embryo-organoid interactions [ 42 ]. By mimicking the maternal environment, organoid co-culture systems can provide insights into embryo implantation, including the molecular signals and cellular interactions involved. Co-culture systems can be used to model pregnancy-related disorders, such as preeclampsia or intrauterine growth restriction, and assess their underlying mechanisms [ 43 ]. Organoid blastocyst co-culture systems can be used to screen drugs or compounds for their effects on embryo development and maternal-fetal interactions [ 44 ]. These systems can assess the potential reproductive toxicity of environmental contaminants and pharmaceuticals. In summary, organoid blastocyst co-culture is a valuable tool for studying early embryonic development, maternal-fetal interactions, and reproductive biology in both research and clinical settings. In this study, we explored the response of porcine EEOs to steroid hormone treatment, focusing on hormone receptor expression and the interaction between hormone-treated organoids and blastocysts in a co-culture system. Our findings provided valuable insights into the hormonal regulation of endometrial function and demonstrated the utility of organoid-based models in studying complex reproductive processes. In conclusion, this study demonstrated the responsiveness of porcine EEOs to steroid hormones and their potential to model crucial interactions between the endometrium and blastocysts. Our findings contributed to the growing body of research using organoids for reproductive biology and highlighted the potential of this model system to advance our understanding of endometrial function and embryo implantation. A major contribution of our model is its ability to replicate the rapid elongation phase observed in vivo , which includes significant cell proliferation, differentiation, and tissue organization within a short developmental period. This enables precise manipulation and observation of factors affecting elongation, including gene expression and external signaling [ 45 ]. Therefore, this model offers valuable insights into early embryogenesis and the regulatory mechanisms underlying species-specific developmental patterns. However, our in vitro model has limitations [ 46 47 ]. Although it mimics numerous aspects of in vivo elongation, the absence of a uterine environment indicates that certain external signals and cellular interactions may be lacking, potentially limiting the full elongation and maturation of the embryo. Enhancing the culture medium or incorporating a synthetic or bioengineered matrix to simulate uterine conditions can enhance the physiological relevance of the model. Our findings have potential implications for reproductive biology, specifically in enhancing fertility treatments and embryo culture techniques in both veterinary and human medicine [ 48 ]. Additionally, because of the economic significance of porcine breeding, a deeper understanding of the elongation process can support enhancements in embryo implantation rates and overall reproductive efficiency, which are crucial for livestock industries [ 49 50 ]. Future studies should focus on optimizing the culture environment to better mimic the in vivo uterine environment, potentially using co-culture systems or bioengineered matrices. Additionally, single-cell RNA sequencing of elongated embryos can help identify cell lineage-specific markers and signalling pathways involved in elongation. Moreover, exploring the effects of various growth factors, hormones, and mechanical stimuli on elongation can provide insights into factors that regulate and support embryonic development.