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 [email protected]

Conflict of Interest Statement

 This is no conflict of interest for the work I present today. Female Reproductive System

FallopianMenstrual tube cycle

Ovary Ovary Follicles Uterus Cervix Vagina

Infertility IVF Female Reproductive Tract and Pregnancy

Fertilization Embryo development & transport

Primordial Corpus follicle Luteum

Primary Ovulation follicle Secondary follicle Antral follicle Embryo implantation 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a biological variable − Ethical issues associated with women who have potential or current pregnancy − Drugs have been withdrawn from the market for greater healthy risk in women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 Follicle growth in vitro In vitro maturation Ovulation encapsulation

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

α-tubulin F-actin DAPI day 0 day 14 In vitro ovulation

• Microfluidic platform supports mouse ovarian follicle development and

oocyte maturation. Xiao et al, Nat Commu, 2017 Follicle Luteinization upon hCG Stimulation

hCG Before hCG 16h 48h 14 days

3 70 60 50 40 30 * 20

nucleus /1 /1 nucleus mm 10 * 0 Before 24h 48h 14 10 μm hCG after after days hCG hCG after hCG Error bar: Standard deviation; *p<0.05

• 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 45000 80 Estrodiol Progesterone 40000 70 ) )

35000 60 nM 30000 50 25000 40 20000 30 15000 Estradiol( nM 10000 20 Progesterone( 5000 10 0 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 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 Fallopian Uterine Uterus Ovary Fallopiantube 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 Uterine Uterine tube endometrium cervix

Ovary/follicles Liver Universal spheroids culture media Xiao et al, Nat Commu, 2017 Fallopian Tube Tissue Remains Viable in MPS

Epithelium H&E Cilia beating OVGP1 day 0 day 0 day 0

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; Xiao et al, Nat Commu, 2017 • Recellulzrization: Reseeding cells into the decellularized scaffold. 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

500 7

50000 80

Estrodiol Progesterone Estradiol Progesterone 70 6 / ml) / ml) / ml) 40000 400

60 / ml) 5 pg ng ng

50 pg 30000 300 4 40 3 20000 30 200

Estradiol ( Estradiol 20 2

10000 ( Estradiol 100 10 1 Progesterone ( Progesterone Progesterone ( Progesterone 0 0 0 0 -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14

• 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 16000 20 Control Control 200 nM DOX 200 nM DOX 12000 15 ng /ml) pg /ml) 8000 10

Estradiol ( 4000 5

* Progesterone ( * 0 0 DOX 200 nM day 2 6 10 14 18 22 26 day 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

Follicle growth Estradiol secretion 400 25 0 nM 0 nM 350 20 2 nM 2 nM 300 20 nM pg /ml) 15 20 nM 200 nM 250 10 200 nM

Diameter (µm) 5 200 * Estradiol ( 150 0 * day 0 2 4 6 8 day 2 4 8 Follicle survival LC50 of DOX 150

1 y = 0.4197x + 18.773 0.8 100 0.6 0 nM 0.4 2 nM 50 20 nM Survival rate (%)

0.2 Survival rate (%) 200 nM 0 * 0 day 0 2 4 6 8 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

Oocyte MII percentage Spindle and chromosome morphology 120 120 100 * 100 * 80 80 60 60 40 40 abnormality (%) abnormality MII percentage(%) 20 20 ** * ** * & Spindle chromosome 0 0 DOX (nM) 0 2 20 100 200 DOX (nM) 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 Follicles Uterus Cervix 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

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 University of South Carolina Draper Labs Northwestern University Danijela Dokic, MD Xiao Lab Brett Isenberg, PhD Kim Lab Beth Sefton, PhD Yingzheng Wang, MS Jeff Borenstein, PhD Susan Olalekan, PhD Chanel Arnold-Murray Mingjun Liu, MS Jonathan Coppeta, PhD Sevim Yildiz Arslan, PhD Megan F. Kopp J. Julie Kim, PhD

Financial supports: NIEHS/NCATS: UH3TR001207 Subcontract award to S Xiao (UH3TR001207) Arnold School of Public Health Research Fund