Z32 - Hormonal Regulation of Lymphatics in Shaping Ovarian Tissue Architecture

Friday, October 10, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

CP

Cristina Pokhrel, BS

Graduate Student
Virginia Polytechnic Institute and State University
Blacksburg, Virginia, United States

Last Author(s)

Monet Roberts

Assistant Professor
Virginia Tech, United States

Co-Author(s)

TP

Tamalika Paul

Graduate Student
Virginia Polytechnic Institute and State University, United States
CE

Carley Elliot

Graduate Student
Virginia Polytechnic Institute and State University, United States
EV

Eli Vlaisavljevich, Ph.D.

Associate Professor
Virginia Tech
Blacksburg, Virginia, United States
IA

Irving Allen

Assistant Head for Research Support, Professor
Virginia Polytechnic Institute and State University, United States

Introduction: : Polycystic Ovary Syndrome (PCOS) is an endocrine disorder affecting approximately 10% of women of reproductive age. PCOS is characterized by hormonal imbalance, chronic inflammation, extracellular matrix remodeling, and tissue dysfunction. Prior evidence in pregnancy links hormonal changes in modulating lymphatic vessel integrity and function in tissue homeostasis in reproductive organs associated, particularly the ovaries. Despite the role of the lymphatic system in maintaining fluid balance throughout the body and evidence in physiological reproductive function, the role of hormonal dysregulation and how it affects lymphatic function in PCOS remains poorly understood. Here, we aim to explore the cellular basis of lymphatic dysfunction under hormone-driven conditions, using a model that mimics the ovarian microenvironment in PCOS.

Materials and

Methods: : Human lymphatic endothelial cells (hLECs) were cultured in endothelial growth medium under standard conditions (37°C, 5% CO₂). Cells were treated with 10 nM 17β-estradiol and 10 nM dihydrotestosterone (DHT) every 24 hours for 72 hours. Co-cultures with human ovarian epithelial cells were established using 8.0 μm transwell inserts. Following hormone treatment, cells were challenged with 100 nM insulin and 25 mM glucose for 24 hours to mimic PCOS-like hyperglycemic stress. hLECs were also treated with conditioned media from hormone-treated ovarian cells. Cell viability was assessed via Live/Dead assay and imaged using fluorescence microscopy. Monolayer integrity and permeability were evaluated using a FITC-dextran transwell assay. For tissue analyses, adult female rats pre-treated with estrogen and progesterone (provided by our collaborators in the Allen laboratory at Virginia Tech) were used. Ovaries were then harvested and fixed in 4% paraformaldehyde, cryopreserved in 30% sucrose, embedded in OCT, and cryosectioned at 12 μm. Immunohistochemistry was performed using antibodies against LYVE-1, CD31, and podoplanin, followed by fluorescent secondary antibodies and DAPI nuclear staining prior to confocal microscopy imaging.

Results, Conclusions, and Discussions:: The exposure to sex hormones via estrogen altered the hLECs morphology and function, specifically increasing the aspect ratio and spacing between cells compared to control conditions. In the ovarian tissues, lymphatic vessels appeared to undergo subtle architectural changes, with observations of some intussusceptive-like folding and occasional looped structures. This study offers foundational insights into hormone-lymphatic interactions in ovarian homeostasis on a cellular and tissue level and lays the groundwork for understanding lymphatics as an underexplored component of reproductive disorders. Here, we show that hormonal dysregulation alters ovarian tissue morphology observed by eye. In vitro, lymphatic endothelial cell morphology and function were altered, suggesting a potential link between hormonal imbalance and lymphatic remodeling within the reproductive tissues. This may help identify new pathways contributing to the pathogenesis of PCOS, and it also opens avenues for targeting lymphatic signaling in reproductive and endocrine disorders in future therapeutic interventions.

Acknowledgements and/or References (Optional): :