(Poster 121) Effect of Local Estrogen on Cannabinoid Receptor Expression in the Human Vagina

Objectives: The impact of estrogen on adjacent molecular pathways is poorly understood. One emerging area of interest is the interplay between estrogen and the endocannabinoid system. The endocannabinoid system is comprised of endogenous cannabinoids, such as N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), cannabinoid receptors type-1 (CB1) and type-2 (CB2), and associated enzymes, such as fatty acid amide hydrolase-1 (FAAH-1), which inactivates AEA, and monoacylglycerol lipase (MAGL), which inactivates 2-AG. While most overlapping pathways of sex hormones and cannabinoids regulate cell proliferation, there are also well-established interactions between cannabinoids and estrogen within the hypothalamic-pituitary-gonadal axis. The study's objective was to evaluate the effect of vaginal estrogen on expression of key elements of the endocannabinoid pathway in the vagina.

Methods: This study utilizes preserved vaginal tissue specimens, previously obtained from postmenopausal women undergoing surgery for pelvic organ prolapse (IRB#20-004EX). Control/nonestrogenized patients received 0 weeks of pre-operative topical estrogen. Treatment/estrogenized patients received 2+ weeks of preoperative topical estrogen. Tissue samples were stored at -80°C until undergoing molecular analyses. A subset of specimens were placed in 10% formalin.

RNA isolation was performed following Invitrogen Trizol Reagent RNA Isolation Procedural Guidelines. We measured RNA concentration and A260/A280 (RNA purity) using NanoDrop® Spectophotometry.

We converted 1µg RNA of each sample to cDNA using SuperScript®III First-Strand Synthesis System for RT-PCR protocol.

RT-qPCR was performed on cDNA with quantification by SYBR green fluorescence to determine presence of and relative amounts of CB1, CB2, FAAH, MAGL, and ER-α mRNA. Cycle threshold (CT) values obtained from RT-PCR were used to quantify expression of receptors at the designated locations. We compared relative receptor expression between untreated and treated tissues using the 2^ΔΔCt method and either independent T-tests or Mann-Whitney-U tests depending on data distribution.

Biopsy specimens stored in 10% formalin were fixed into paraffin blocks and sliced into 5µm sections. Fixed slides were de-paraffinized and underwent IHC to localize expression of target proteins. A subset of slides were stained with H&E.

Results: 12 vaginal tissue samples (6 estrogenized, 6 untreated) underwent tissue processing, RNA extraction, conversion of RNA to cDNA, and reverse RT-PCR analysis of various gene targets. When comparing relative mRNA expression of CB1, CB2, FAAH, MAGL, and ER-α between non-estrogenized and estrogenized samples, there were no statistically significant differences (Table1). 2^ΔΔCt values for CB1, CB2, and ER-α were non-normally distributed, therefore, Mann-Whitney-U tests were performed to evaluate for differences between treated and untreated samples. Independent T-tests were used for analysis of FAAH and MAGL 2^ΔΔCt values due to normal distribution.

Conclusions: Short term vaginal estrogen therapy has no significant effect on expression of key regulators of the endocannabinoid pathway in the vagina.

Table1. A)Independent T-test results comparing 2^ΔΔCt values for MAGL and FAAH-1. B)Mann-Whitney-U test results comparing 2^ΔΔCt values for CB1, CB2, and ER-α.