A Microfluidic Culture Model of the Human Reproductive Tract: Screening of Female Reproductive Toxic Chemicals

A Microfluidic Culture Model of the Human Reproductive Tract: Screening of Female Reproductive Toxic Chemicals Shuo Xiao, PhD Assistant Professor Reproductive Health & Toxicology Laboratory Department of Environmental Health Sciences Arnold School of Public Health, University of South Carolina sxiao@mailbox.sc.edu

Conflict of Interest Statement This is no conflict of interest for the work I present today.

Female Reproductive System Fallopian tube Menstrual cycle Ovary Cervix Ovary Follicles Uterus Vagina Infertility IVF

Female Reproductive Tract and Pregnancy Embryo development & transport Fertilization Primordial follicle Corpus Luteum Embryo implantation Primary follicle Secondary follicle Antral follicle Ovulation

Hormone Regulation of Female Reproductive System Highly regulated by pituitary hormones follicle-stimulating hormone (FSH) and luteinizing hormone (LH), and ovarian hormones Estrogen and Progesterone. Targets for endocrine disrupting chemicals (EDCs), pharmaceutical chemicals https://www.woodrufflab.org

Summary of Female Reproductive Toxicity Testing In vivo animal testing as a standard to test chemicals female reproductive toxicity Organ weight, morphology, and histology Vaginal smear and serum hormone levels Ovulation and oocyte production Fertility, pregnancy, litter size, multiple generational reproduction testing In vivo models are time-consuming, costly, and harmful to animals Lack of in vitro models for testing fallopian tube, uterus, and cervix In vitro 2D ovarian cell/follicle cultures do not phenocopy the physicochemical microenvironment and 3D tissue specific architecture Guidelines for Reproductive Toxicity Risk Assessment

Outline I. Using microfluidic system to establish the ex-vivo 28-day menstrual cycle hormone control and female reproductive tract on a chip - Establish II. Screening of female reproductive toxic chemicals - Discover

Organ-on-a-chip using Microfluidic Technology Brain chip Vessel chip Lung chip Heart chip

Clinical Research Only in Males Early testing of chemical toxicity in female is more difficult than in males because: Existence of menstrual cycle and hormone changes Lake of awareness of the importance of gender as a biological variable Ethical issues associated with women who have potential or current pregnancy Drugs have been withdrawn from the market for greater healthy risk in women Considering gender/sex as a biological variable in all NIH-funded research since 2015!

Hypothesis I. Develop ovarian functions in the microfluidic system to mimic human 28-day menstrual cycle hormone control and ovarian function II. Establish ex-vivo female reproductive tract (FRT) on a chip by integrating and inter-connecting multiple reproductive tissues

Microfluidic Platform (MPS) Design Passed media reservoir Tissue Modules Media donor Fluidic interface Tissue module Actuator interface Materials: can be sterilized, non-toxic to cells/follicles, do not bind hormones; Pumping pattern: computer program controlled. Xiao et al, Nat Commu, 2017

Ovary Chip Based on Microfluidic Culture Passed media reservoir Media donor Tissue module Sampling: easy handling and loading, collect the most recent passed media without disturbing a running culture. Xiao et al, Nat Commu, 2017

Ovary Chip Based on Microfluidic Culture Alginate encapsulation Follicle growth in vitro In vitro maturation Ovulation In vitro fertilization Embryo development Embryo transfer Live birth Apply the 3D in vitro follicle growth (IVFG) model in the microfluidic system, which mimics Xiao et al, Repro, 2015 full ovarian cycle (follicular phase, ovulation, luteal phase) Xu et al, Biomaterials, 2006

Follicle Maturation & Ovulation in Microfluidic Culture day 0 day 14 MII oocyte α-tubulin F-actin DAPI day 0 day 14 In vitro ovulation α-tubulin F-actin DAPI Microfluidic platform supports mouse ovarian follicle development and oocyte maturation. Xiao et al, Nat Commu, 2017

Follicle Luteinization upon hcg Stimulation Before hcg hcg 16h 48h 14 days 10 μm nucleus /1 mm 3 70 60 50 40 30 20 10 0 Before hcg 24h after hcg Error bar: Standard deviation; *p<0.05 * 48h after hcg * 14 days after hcg The in vitro luteinization of follicular cells is similar to corpus luteum formation in vivo (cell hypertrophy and differentiation from granulosa cell to luteal cell). Xiao et al, Nat Commu, 2017

Ovarian Hormone Secretion in Microfluidic Culture Follicular phase Luteal phase Estradiol (nm) 45000 40000 35000 30000 25000 20000 15000 10000 5000 Estrodiol Progesterone 80 70 60 50 40 30 20 10 Progesterone (nm) 0 day -14-13 -12-11 -10-9 -8-7 -6-5 -4-3 -2-1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0 Error bar: Standard deviation Microfluidic platform recapitulates human 28-day menstrual cycle hormone control. Xiao et al, Nat Commu, 2017

Is the Female Reproductive Chip Possible? Fallopian tube Ovary Uterus Follicle Cervix Vagina Female reproductive tract (FRT) Hormones drive 28 days menstrual cycle Female reproductive organs are connected with each other to support reproductive functions: 28-day menstrual cycle and pregnancy.

Ex vivo Female Reproductive Tract in Microfluidic system Ovary Fallopian tube Uterus Uterine Cervix Follicle Ovary Fallopian Fallopian tube Uterus Uterine cervix Liver Integrating and inter-connect multiple female reproductive tissues in vitro based on the microfluidic platform. Xiao et al, Nat Commu, 2017

Integrated Female Reproductive Organs-EVATAR Fallopian tube Uterine endometrium Uterine cervix Ovary/follicles Liver Universal spheroids culture media Xiao et al, Nat Commu, 2017

Fallopian Tube Tissue Remains Viable in MPS day 0 Epithelium H&E day 0 Cilia beating day 0 OVGP1 High E2 day 7 day 7 day 7 Low E2 OVGP1: oviductal glycoprotein Microfluidic culture supported human fallopian tube viability, and cilia beating is controlled by dynamic hormone secretion patterns. Xiao et al, Nat Commu, 2017

Recellularized Endometrium Remain viable in MPS Control Endo-H&E Decelled Endo-H&E Recelled Endo-H&E Recelled Endo-Ki67 Recelled Endo-ER Recelled Endo-PR Endo: uterine endometrium ER: estrogen receptor PR: progesterone receptor Decelled: decellularization Recelled: recellularization Decellularization: removing cells from tissue but keeping extracellular matrix (ECM) scaffold; Recellulzrization: Reseeding cells into the decellularized scaffold. Xiao et al, Nat Commu, 2017 Olalekanet & Kim, BOR, 2017

Microfluidic Culture Supports Ectocervix in Response to Hormone from Upstream Ovary Chip day -7 day 0 with E2 peak H&E Ki67 Xiao et al, Nat Commu, 2017 K McKinnon & S Getsios, unpublished

Microfluidic Culture Supports Liver Microtissues Microfluidic culture supported human liver spheroid viability and albumin production throughout 28-day culture period.

Hormone Secretion in Integrated Microfluidic Culture Ovary only in MPS Multiple tissues in MPS Estradiol (pg / ml) 50000 40000 30000 20000 10000 0 Estrodiol Progesterone -14-12 -10-8 -6-4 -2 0 2 4 6 8 10 12 14 80 70 60 50 40 30 20 10 0 Progesterone (ng / ml) Estradiol (pg / ml) 500 400 300 200 100 0 Estradiol Progesterone -14-12 -10-8 -6-4 -2 0 2 4 6 8 10 12 14 7 6 5 4 3 2 1 0 Progesterone (ng / ml) Downstream tissues consumed ovarian secreted hormones and/or integrated tissues changed ovarian hormone expression patterns!

Outline I. Using microfluidic system to establish the ex-vivo 28-day menstrual cycle hormone control and female reproductive tract on a chip - Establish II. Screening of female reproductive toxic chemicals - Discover

Ovarian Toxicity of Doxorubicin (DOX) Control Estradiol Progesterone DOX 200 nm Estradiol (pg/ml) 16000 12000 8000 4000 0 day * Control 200 nm DOX 2 6 10 14 18 22 26 Progesterone (ng/ml) 20 15 10 5 0 day Control 200 nm DOX * 2 6 10 14 18 22 26 *p<0.05 DOX is widely used for chemotherapy; WHO recommended DOX significantly changed the follicle ovarian steroid hormone expression patterns in the microfluidic cultures. Xiao et al, Unpublished, 2017

Ovarian Toxicity of Doxorubicin Diameter (µm) 400 350 300 250 200 150 day Follicle growth 0 nm 2 nm 20 nm 200 nm 0 2 4 6 8 * Estradiol (pg/ml) 25 20 15 10 5 0 day Estradiol secretion 0 nm 2 nm 20 nm 200 nm * 2 4 8 Survival rate (%) 1 0.8 0.6 0.4 0.2 0 day Follicle survival 0 nm 2 nm 20 nm 200 nm * 0 2 4 6 8 Survival rate (%) 150 100 50 0 LC50 of DOX y = 0.4197x + 18.773 0 50 100 150 200 nm DOX dose-dependently inhibited follicle growth, survival, and hormone secretion, DOX has LC50 at 75.48 nm, which is relevant or even lower than the human exposure levels. Xiao et al, Tox Sci, 2017

Ovarian Toxicity of Doxorubicin MII percentage (%) 120 100 80 60 40 20 0 DOX (nm) Oocyte MII percentage * * * * 0 2 20 100 200 Spindle & chromosome abnormality (%) 120 100 80 60 40 20 0 DOX (nm) Spindle and chromosome morphology * * 0 2 20 DOX at low level exposure of 20 nm didn t significantly affect follicle growth and survival, However, DOX significantly increased the percentage of oocytes with abnormal spindle morphology and chromosome misalignment. Xiao et al, Tox Sci, 2017

Take Home Message Fallopian tube Ovary Cervix Follicles Uterus Vagina Ovary chip and female reproductive tract chip Introducing gender/sex to co-cultured cells/tissues; DOX has dose-dependent ovarian toxicity, which increases the risk of infertility during chemotherapy; Low but human-relevant exposure level of DOX disrupts oocyte meiotic maturation(avoid this window for oocyte cryopreservation and donation, and pregnancy).

Reference 1. Xiao, S., Zhang, J, Liu, M., Iwahata, H., Rogers, HB, Woodruff, TK. (2017) Doxorubicin has dose-dependent toxicity on mouse ovarian follicle development, hormone secretion, and oocyte maturation. Toxicological Sciences. Doi:10.1093/toxsci/kfx047. 2. Xiao, S., Coppeta, J., Rogers, H., Woodruff, TK. (2017) A microfluidic culture model of the human reproductive tract and 28- day menstrual cycle. Nature Communications. Nature Communications. Mar 28; 8:14584. 3. Laronda, MM., Rutz, AL., Xiao, S., Whelan, KA., Duncan, FD., Roth, EW., Woodruff, TK., Shah, R. (2017) A bioprosthetic ovary created using 3D printed microporous scaffolds restores ovarian function in sterilized mice. Nature Communications. May 16; 8:15261. 4. Xiao, S., Zhang, J., Romero MM., Smith KN., Shea, LD., Woodruff, TK. (2015) In vitro follicle growth supports human oocyte meiotic maturation. Scientific Reports. 5:17323. 5. Xiao, S., Duncan, FE., Bai, L., Nguyen, CT., Shea, LD., Woodruff, TK. (2015) Size-specific follicle selection improves mouse oocyte reproductive outcomes. Reproduction: Piirep-15-0175. 6. Olalekan, SA., Burdette, JE., Getsios, S., Woodruff, TK., Kim, JJ. Development of a novel human recellularized endometrium that responds to a 28-day hormone treatment. Biology of Reproduction. 95(5):971-81.

Acknowledgements University of South Carolina Xiao Lab Yingzheng Wang, MS Mingjun Liu, MS Megan F. Kopp Draper Labs Brett Isenberg, PhD Jeff Borenstein, PhD Jonathan Coppeta, PhD Northwestern University Kim Lab Susan Olalekan, PhD Sevim Yildiz Arslan, PhD J. Julie Kim, PhD Northwestern University Teresa Woodruff Lab Hunter Rogers, MS Jiyang Zhang, MS Mingjun Liu, BS Monica Laronda, PhD Kelly McKinnon Peter Chen, MS Mingyang Jiang, MS Lu Bai, MS Cat Nugyen, MS Alex Rashedi, BS Jie Zhu, MD Danijela Dokic, MD Beth Sefton, PhD Chanel Arnold-Murray Financial supports: NIEHS/NCATS: UH3TR001207 Subcontract award to S Xiao (UH3TR001207) Arnold School of Public Health Research Fund