(366) Cannabinoid exposure duration differentially alters human endometrial epithelial organoid behavior

Zachary L. Bonomo¹; Iebu Devkota¹; Dailin M. Fuego¹; Michael A. Allon^1,2; Dmitri Dozortsev²; Beata Tralik²; Margeaux W. Marbrey³; Xing Fu¹; Constantine A. Simintiras¹

1. School of Animal Sciences, Agricultural Center, Louisiana State University, Baton Rouge, LA 70803, USA.

2. Advanced Fertility Center of Texas, Houston, TX, USA.

3. Division of Reproductive Sciences, Department of Obstetrics & Gynecology, Duke University School of Medicine, Durham, NC, USA

Abstract Text:

Organoids are 3D in vitro cell models that closely replicate key in vivo characteristics, including intra-organoid fluid (IOF) composition, which mirrors the proteomic and metabolomic profiles of the tissue of origin lumen. One advantage of organoids is their ability to maintain both phenotypic and genetic integrity over extended periods, making them valuable for studying cellular behavior and tissue responses. While most cell culture studies focus on acute exposures, our research aimed to explore the effects of varying exposure durations on human endometrial epithelial organoids (EEO). Cannabis is the most used drug among women of reproductive age in the United States, with increasing use during pregnancy. This trend is concerning, as cannabis consumption during pregnancy has been linked to adverse outcomes, including first trimester pregnancy loss. The primary cannabinoids circulating after cannabis use are cannabidiol (CBD) and tetrahydrocannabinol (THC). As such, we used CBD and THC to test our overarching hypothesis that cannabinoid concentration and exposure duration correlate with changes in EEO physiology and behavior. EEO were derived from women with a mean (± SD) age of 39.3 ± 6.7 years and body mass index (± SD) of 23.3 ± 0.4, with no history of endometrial pathology. At an average (± SD) passage of 5 ± 1.7, EEO were treated with 10 nM 17β-estradiol (E2) for 48 h before exposure to one of the following treatments: (a) 6 µM THC, (b) 12 µM THC, (c) 6 µM THC + 6 µM CBD, or (d) vehicle control. Exposure durations were either (a) 6 days (acute), (b) 18 days (mid-term), and (c) 30 days (chronic). EEO were imaged every 48 h after media replenishment, followed by morphological, immunohistochemical, gene expression, and IOF metabolomic analyses. All EEO expressed cannabinoid receptors CB1 and CB2, along with the uterine glandular epithelial marker FOXA2, and both E2 and progesterone receptors following E2 supplementation. Although no differences were observed in EEO area, count, or circularity between treatments, EEO count decreased (P< 0.05) over time, while area and circularity remained unchanged. As merging of EEO over time was not observed, these data suggest selective survival of certain EEO, rather than growth of existing ones. IOF composition analysis revealed differences from the culture medium, indicating selective metabolite consumption and release across the EEO monolayer. Furthermore, culture duration and cannabinoid exposure influenced EEO gene expression and IOF composition independently. These findings demonstrate that exposure duration modulates uterine glandular epithelial secretions and EEO metabolism. Ongoing research is focused on correlating these in vitro results with in vivo data to assess the physiological relevance of these findings and further investigate the impact of cannabis use on reproductive health. This work was approved by the Louisiana State University (LSU) Agricultural Center Institutional Review Board (IRB #23-0046) and supported by the LSU Therapeutic Cannabis Research Committee (PG010126) and the State of Louisiana Board of Regents [LEQSF(2023‐26)‐RD‐A‐03].