Impact of Depot Medroxyprogesterone (DMPA) on Human Vaginal Leukocytes

and HIV-1 Target Cells

Andrea Ries Thurman MDNeelima Chandra PhD

(PI) Gustavo F Doncel MD PhD

23 JULY 2012

Background

• Depot Medroxyprogesterone Acetate (DMPA):– Highly effective long acting reversible

contraceptive• Prevents Unintended/Mistimed Pregnancies

– Only one absolute contraindication– High usage in developing world• World Contraceptive Use 2009, UN Population Division

– Sole P-only injectable in the US

Relevance: DMPA and HIV Acquisition

• Observational studies AOR 1.73 – 3.90 for increased HIV acquisition with DMPA use

• Ungchusak K et al. J AIDS Hum Retrovirol 1996• Martin HL et al. J Infect Dis 1998• Baeten JM et al. AIDS 2007• Morrison CS et al. AIDS 2010

• Sero-discordant couples: Injectable use ↑↑ HIV-1 acquisition and transmission (AHR 2 – 3)

• Heffron et al. Lancet Infect Dis 2012

• 2012 WHO meeting re-consider medical eligibility criteria for DMPA in women at high risk of HIV-1

• Category 1*

Epithelial Thinning• Cell layers, height in µm– Dramatic Atrophy with P/DMPA in NHP models

• Marx PA et al. Nat Med 1996• Smith SM et al. J InfectDis 2000• Jiang Y et al. J Med Primatol 2009• Pal R et al. Virology 2009• Salle B et al. J Infect Dis 2010• Veazey RS et al. Nat Med 2003

– Humans• Bahamondes L et al. Contraception 2000• Mauck CK et al. Contraception 1999• Miller L et al. ObstetGynecol 2000• Ildgruben AK et al. ObstetGynecol 2003

Epithelial Integrity• Other markers of epithelial integrity• E Cadherin and Zona Occludin 1 (ZO-1)• Epithelial Cell Proliferation (Ki67)• E Cadherin density altered by vaginal estrogen

therapy• Gorodeski GI. Endocrinology 2007

Alteration of MucosalImmune Cells

• Small cohorts support exogenous progesterone alters mucosal environment

– Wieser F et al. Fertil Steril 2001– Ildgruben AK et al. Obstet Gynecol 2003

• Most data on endogenous and exogenous hormones and alteration of mucosal inflammatory/immune cell populations in humans from endometrium

– Pudney et al. Biol Reprod 2005– Wira et al Am J Reprod Immunol 2011

Objective

• Determine if exogenous and endogenous progesterone alters epithelial integrity and the cellular inflammatory status of the vaginal mucosa

• Secondary analysis– Mauck CK et al. Contraception 1999 Jul;60(1):15-24.

Methods15 women (BTL, no hormonal contraception)

Luteal PhaseDays 22 - 26(Confirmed Serum P)

Follicular PhaseDays 8 - 12(Serum E2 and P)

12 weeks ± 1 week post DMPA

Endpoints• Epithelial integrity– Epithelial height in µm (reticles) and # cell layers– Epithelial proliferation (Ki67 cells)– Intracellular adhesion proteins E Cadherin and ZO-1

(Integrated Optical Density - IOD)

• Immune cell populations by IHC– CD45, CD3, CD4, CD8, CD1a, CD68 bearing leukocytes– HLA-DR, CCR5 (markers of activation and proliferation )

Results

• Demographic data (N = 15)– Mean age 35.9 (± 3.1) years-old– Mean weight 158.5 (± 25.0) pounds– Mean menstrual cycle length 29.0 (± 1.4) days– All with previous pregnancy– Mean gravidity 2.7 (± 1.1)

• Follicular versus luteal phase– All endpoints NS

Epithelial Thickness& Proliferation

Epithelial characteristics

Mean ± standard deviation Median (25th, 75th quantile)

Paired T test P valueWilcoxan Signed Rank P Value

Follicular Luteal DMPA Follicular vs DMPA

Luteal vs DMPA

Thickness (µm) 307.8 ± 96.8 269.6 ± 80.1 272.3 ± 94.6 0.33 0.94

# Cell layers 31.2 ± 8.1 28.2 ± 7.2 28.9 ± 6.5 0.42 0.79

Ki-67 35.7 (19.3, 64.3)

17.1 (9.0, 68.4)

72.6 (43.2, 126.9)

0.004 0.005

Intracellular Adhesion Proteins (NS)

Epithelial adhesion proteins

Median integrated optical density (IOD)per stained field (X 106)

(25th, 75th percentile, quantile)*

Wilcoxon signedrank P value

Follicular Luteal DMPA Follicular vs DMPA

Luteal vs DMPA

E Cadherin 3.5(0.4, 4.7)

1.6 (0.7, 8.1)

1.6(1.0, 3.2)

0.64 0.42

ZO-1 0.1 (0.03, 0.93)

0.2 (0.04, 0.65)

0.2 (0.04, 0.3)

0.38 0.22

Luteal DMPA

ZO-1

E Cadherin

Follicular

Immune Cells(Significant Increase with DMPA)

Phenotype vaginal

epithelial immune cells

Median cell count (cells/mm2) Wilcoxon signed rank P value

Follicular Phase

Luteal Phase DMPA Treatment

Follicular vs DMPA

Luteal vs DMPA

CD45 70.6 73.5 156.9 0.03 0.22

CD3 40.0 45.9 76.5 0.08 0.04

CD8 28.1 29.1 52.6 0.15 0.01

CD1a 26.4 27.6 33.9 0.13 1.00

CD68 1.5 3.3 7.2 0.01 0.07

Activated Immune Cells (Significant Increase with DMPA)

Phenotype of Immune Cells in the Vaginal

Epithelium

Median Cell Count (cells/mm2)(25th, 75th percentile, quantile)

Wilcoxon Signed Rank P value

Follicular Luteal DMPA Follicular vs DMPA

Luteal vs DMPA

CCR5 (LP) 0.2 (0, 0.5) 0.2 (0, 0.6) 0.8 (0, 1.2) 0.09 0.05

HLA-DR 97.4(81.0, 120.0)

82.4 (46.5, 102.6)

113.9 (85.1, 184.3)

0.02 0.35

Follicular Luteal DMPA

H I

CD3

CD8

CCR5

HLA-DR

CONCLUSIONS

• Epithelial Thinning (thickness, #cell layers) NS• Epithelial Integrity (E Cadherin, ZO-1, Ki67)

NS• DMPA use resulted in significant increase in:–CD45, CD3, CD8, CD68 leukocytes–CCR5 (LP), HLA-DR, markers

AcknowledgmentsCONRAD Microbicide Development Laboratory

Gustavo F Doncel MD PhD (Principal Investigator)Neelima Chandra PhDSharon Anderson PhDNazita Yousefieh PhD

Nancy GonyeaDept of Biostatistics Eastern Virginia Medical School

Tina D Cunningham PhD

CONRAD Clinical TeamChristine Mauck MD MPH

Epithelial Thickness& Proliferation

Epithelial characteristics

Mean ± standard deviation Median (25th, 75th quantile)

Paired T test P valueWilcoxan Signed Rank P Value

Follicular Luteal DMPA Follicular vs DMPA

Luteal vs DMPA

Thickness (µm) 307.8 ± 96.8 269.6 ± 80.1 272.3 ± 94.6 0.33 0.94

# Cell layers 31.2 ± 8.1 28.2 ± 7.2 28.9 ± 6.5 0.42 0.79

Ki-67+ 35.7 (19.3, 64.3)

17.1 (9.0, 68.4)

72.6 (43.2, 126.9)

0.004 0.005

Intracellular Adhesion Proteins (NS)

Epithelial adhesion proteins

Median integrated optical density (IOD)per stained field (X 106)

(25th, 75th percentile, quantile)*

Wilcoxon signedrank P value

Follicular Luteal DMPA Follicular vs DMPA

Luteal vs DMPA

E Cadherin 3.5(0.4, 4.7)

1.6 (0.7, 8.1)

1.6(1.0, 3.2)

0.64 0.42

ZO-1 0.1 (0.03, 0.93)

0.2 (0.04, 0.65)

0.2 (0.04, 0.3)

0.38 0.22

Luteal DMPA

ZO-1

E Cadherin

Follicular

Immune Cells(Significant Increase with DMPA)

Phenotype vaginal

epithelial immune cells

Median cell count (cells/mm2) Wilcoxon signed rank P value

Follicular Phase Luteal Phase DMPA Treatment

Follicular vs DMPA

Luteal vs DMPA

CD45 70.6 (37.5, 159.6)

73.5 (31.7, 120.3)

156.9 (112.4, 277.3)

0.03 0.22

CD3 40.0 (23.8, 60.0)

45.9 (11.3, 70.7)

76.5 (47.8, 117.9)

0.08 0.04

CD4 (LP) 0 (0, 0) 0 (max 11.6) 0 (max 9.3) NA NA

CD8 28.1 (18.4, 44.9)

29.1 (9.3, 54.4)

52.6 (40.9, 85.6)

0.15 0.01

CD1a 26.4 (16.7, 60.9)

27.6 (17.8, 41.7)

33.9 (26.0, 54.9)

0.13 1.00

CD68 1.5 (0, 5.1) 3.3 (0, 5.4) 7.2 (3.9, 22.0) 0.01 0.07

Activated Immune Cells (Significant Increase with DMPA)

Phenotype of Immune Cells in the Vaginal

Epithelium

Median Cell Count (cells/mm2)(25th, 75th percentile, quantile)

Wilcoxon Signed Rank P value

Follicular Luteal DMPA Follicular vs DMPA

Luteal vs DMPA

CCR5 (LP) 0.2 (0, 0.5) 0.2 (0, 0.6) 0.8 (0, 1.2) 0.09 0.05

HLA-DR 97.4(81.0, 120.0)

82.4 (46.5, 102.6)

113.9 (85.1, 184.3)

0.02 0.35

Follicular Luteal DMPA

H I

CD3

CD8

CCR5

HLA-DR