MOLECULAR PERTURBATIONS IN THE FEMALE GENITAL TRACT ... · ccna1 flj22447 scgb3a1 alpp nrcam pam...

METHODS Endocervical cytobrush and soft-cup samples were collected from 202 women, pre- and post-initiation (month 1 and month 3) of contraception (DMPA-IM, LNG or IUD), for transcriptomic (fig 1) and proteomic analyses respectively. The mucosal proteome was assessed by label-free tandem mass spectrometry as per Birse et al. [3]. Figure 1: Transcriptome analysis. RNA exome capture beads were used to generate enriched libraries for Illumina based RNA-Seq. Sequenced reads were mapped to GRCh38 human reference genome. Paired analyses, with participant ID (PTID) and visits as input factors, were carried out for each study-arm using DESeq2 tool in R. Gene set enrichment analysis was conducted to identify gene- sets or pathways specifically enriched in one or more study-arm. MOLECULAR PERTURBATIONS IN THE FEMALE GENITAL TRACT INDUCED BY DMPA, COPPER IUD, AND LNG IMPLANT IN THE ECHO TRIAL Prachi Gupta 1 , Sydney A. Nelson 1 , Gregory K. Tharp 1 , Maricianah A. Onono 2 , Gonasangrie Nair 3 , Thesla Palanee-Phillips 4 , Hossaena Ayele 5 , Laura Noël-Romas 5 , Kelly Arnold 6 , Jared Baeten 7 , Jo-Ann Passmore 8 , Adam Burgener 5 , Heather Jaspan 8 , Renee Heffron 7 , Steven E. Bosinger 1 1 Yerkes National Primate Research Center, Atlanta, GA, USA, 2 Kenya Medical Research Institute, Nairobi, Kenya, 3 Desmond Tutu HIV Foundation, Cape Town, South Africa, 4 Wits Reproductive Health and HIV Institute, Johannesburg, South Africa, 5 University of Manitoba, Winnipeg, MB, Canada, 6 University of Michigan, Ann Arbor, MI, USA, 7 University of Washington, Seattle, WA, USA, 8 University of Cape Town, Cape Town, South Africa 1065 CONCLUSIONS • An enhanced perturbation of inflammatory pathways among women using DMPA-IM and LNG was observed. • While the magnitude and/or durability of these changes do not ultimately impact HIV susceptibility, they may have implications for other pathogens or vaginal symptoms. FUTURE DIRECTIONS • Future analyses will integrate proteomic signatures associated with contraceptive initiation with transcriptomic data. • Study the correlation, if any, between the gene expression in seroconverters and the respective contraceptive used. ACKNOWLEDGEMENTS The work was funded by R01 HD089831 from NIAID/NIH awarded to R. H. and H. J. REFERENCES [1] Heﬀron et al. (2012). Lancet Infect Dis. 12, 1:19-26. [2] ECHO Trial Consortium (2019). Lancet. 394, 10195:303-313. [3] Birse et al. (2015). Journal of Virology. 89, 17:8793-8805. [4] Zalenskaya et al. (2018). J Clin Invest. 128,10:4622-4638. [5] Bosinger et al. (2018). Am J Reprod Immunol. 80, 4, e13029. RESULTS Differential expression analysis • Relative to IUD users, DMPA-IM & LNG showed a higher perturbation in vaginal gene-expression. • The differentially expressed genes (DEGs) were mostly independent across the three study-arms. Figure 2: Visualization of fold-changes in DEGs in each study-arm as (A) volcano plots and (B) heatmaps of fold-changes between month 1 and enrollment visits. In the heatmaps, clustering along the x-axis (participant IDs) and y-axis (genes) helps to study the natural grouping in participants with similar gene-expression patterns. The color-scale shows the lowest to highest fold-changes in a blue to red color gradient. Gene Set Enrichment Analysis • Prevalent antiviral signaling pathways from MSigDB (IFNA, IFNG, KEGG RIG-I like and T-cell signaling), showed a similar enrichment across the three study-arms. • Inflammatory response related genes, NFkB target genes and serpins were differentially induced. • Upregulated genes in DMPA users in Zalenskaya et al. [4] were found to be enriched in all three study- arms. Figure 3: Dual enrichment plots of DMPA-IM vs. LNG or IUD for specific gene-sets. Across the study-arms, (A) Hallmark interferon gamma response and (B) KEGG T-cell receptor signaling pathway (TCR) showed similar enrichment, (C) Serpins [5] induced differential enrichment, and (D) upregulated genes in DMPA users from Zalenskaya et al. were shown to be positively enriched in our study. BACKGROUND • Previous studies have shown that with the consistent use of hormonal contraceptives, specifically intramuscular depot medroxyprogesterone acetate (DMPA-IM), women are at a higher risk of HIV acquisition [1]. • To obtain a high-quality scientific evidence of the above, the ECHO trial, a large-scale, randomized, open-label clinical study was conducted. • The results from the ECHO trial showed that women randomized to DMPA-IM, copper intrauterine device (IUD), and the levonorgestrel (LNG) implant experienced similar HIV incidence rates [2]. AIM • The impact of hormonal contraceptives on the female genital tract (FGT) is poorly understood. • Thus, to investigate the molecular perturbations induced in the FGT by initiation of contraception we carried out transcriptomic and proteomic analysis of genital samples, collected from women in the ECHO trial. Mass-spectrometry based proteomic analysis Label-free MS/MS identified 1876 unique proteins across the 403 samples analyzed. A total of 634 (34%) were consistently seen across all batches. Figure 4: Vaginal mucosal proteome coverage. (A) Hierarchical clustering of all proteins showed a modest clustering of samples by IUD and post-contraception timepoint. (B) The top enriched molecular host pathways were identified using DAVID (enrichment score: ES, Padj: P values adjusted using Benjamini-Hochberg correction). These include cell-cell adhesion ( ES=43.7, Padj=2.5E-35 ), Glycosylation/ Gluconeogenesis (ES=32.9, Padj=4.9E-09), protease inhibition (ES=19.4, Padj=9.1E-24), complement pathway activity (ES=14.4, Padj=1.1E-13) and structural activity/keratins (ES= 10.9, P adj=1.4E-03). ANKRD20A8P COL4A1 PKHD1L1 IL33 SPON1 SLIT2 FGFR1 SGCE PEG10 TNC NRCAM CAV1 MFAP3L TNNC1 NPAS3 RGS7BP SLC16A12 FAM81A UGT2B7 COL1A2 SFRP4 LGR5 APOB MUC6 CAPN6 SOBP PGR ALPP C6orf15 SCGB2A2 ADCYAP1R1 ERBB4 SLC47A1 SCGB3A1 CILP MT1M MT1X LRP1B ANKRD20A9P MROH2A ADGRG4 MUC3A OLFM4 TRIM31 NOS2 PADI3 ANKRD20A11P CYP4B1 SYTL5 MEIS2 GSDMC SERPINB11 ARSF CECR2 FOXE1 CASP14 ACHE B3GALT5−AS1 MTNR1A SRRM4 HRNR MIR205HG CHL1 PTPRZ1 AKR1B10 MMP13 CPA3 MS4A2 HDC MT1A MT1H GBP1P1 CXCL11 CXCL10 CCL8 MT1L MT1M HSPA1A MT2A MT1E MT1G MT1X HBG2 CHGA KIR2DL3 KIR2DS4 PAEP RELN SPON1 STC1 SBSPON DLGAP5 DCBLD2 ZWINT ACADL B3GALNT1 SLC16A12 CAPN6 FAM189A2 FAM81A LTBP1 ANO1 COL1A2 FBLN1 SLIT2 FOLH1 ROBO2 SLIT3 SLITRK4 PLXNA4 RGS6 ERBB4 NME5 PTPRD FGFR1 FRAS1 LAMA4 NUS1P2 RIMS1 PON3 UPK1B PCDHA12 HIST1H1A CXCL5 SLC39A8 TMEM260 CCNA1 FLJ22447 SCGB3A1 ALPP NRCAM PAM GPC4 ESPN SLC4A4 CHST6 PDE3A ANKFN1 LTF AGR2 TNC FOXA2 BMPR1B CDH12 DCHS2 MUC5AC MUC5B SLC5A8 TFF3 SLC51B SST DCDC2 CAV1 CHST3 SERPINA5 ADCYAP1R1 SLC47A1 CILP SCGB2A1 SCGB1D2 NPR3 PGR NPAS3 SPTA1 KIR3DL2 NEXN FAP PDGFRA RORB ITGAD NCR1 GNLY NCAM1 KLRD1 SAMD3 TBX21 CTSW PRF1 NPR1 KIR2DL1 KIR2DL4 KLRC2 KLRC1 KRT86 IGHV3−73 IGLV1−47 IGKV2−30 IGLV1−51 MUC12 DPCR1 ANKRD20A11P ADAM23 RP1L1 MUC3A CASP14 ACHE CYP4B1 OLFM4 ARSF FOXE1 HDC MS4A2 CPA3 TPSAB1 CXCL13 MMP13 IGKV2−24 PKHD1L1 SCGB2A2 SFRP4 KRTDAP TGM5 COL1A2 ALPP ERBB4 SCGB2A1 SOX17 ASRGL1 SLIT2 TGFBR3 NRCAM RIMBP2 FAM81A FAM189A2 NPAS3 MNS1 LTF TFF3 PKHD1L1 SCGB1D2 CILP ADCYAP1R1 PGR SLC47A1 MMP1 INHBA TULP2 ANXA8L1 SRPX2 ITGB6 HAPLN3 MMP13 OLFM4 CYP4B1 TRIM31 −2 −1 0 1 2 LNG DMPA-IM IUD Participants Genes ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● 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●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 0.00 2.81 4.00 8.00 12.00 16.00 20.00 −6 −4 −2 −1 0 1 2 4 6 fold change (log ) p value (− log 10 ) ● ● DEGs not significant ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 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significant ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●●● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 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● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● 0.0 2.3 4.0 8.0 12.0 16.0 20.0 −6 −4 −2 −1 0 1 2 4 6 fold change (log ) p value (− log 10 ) ● ● DEGs not significant DMPA-IM LNG IUD A Volcano plots B Heatmaps of fold-changes in DEGs between month 1 and enrollment DEGs: absolute fold-change >2 and FDR <0.05 A IFNG pathway B TCR signaling pathway C Serpins D Zalenskaya gene set Higher at month 1 Higher at enrollment CE (DMPA_IM) Nom p-value: 0.1061 NES: 1.34 CE (Copper_IUD) Nom p-value: 0.3381 NES: 1.15 0.00 0.25 0.50 0.75 0 10000 20000 30000 40000 50000 Rank in Ordered Dataset Higher at month 1 Higher at enrollmen CE (DMPA_IM) Nom p-value: 0.144 NES: 1.28 CE (LNG_Implant) Nom p-value: 0.076 NES: 1.31 0.0 0.2 0.4 0.6 0 10000 20000 30000 40000 50000 Rank in Ordered Dataset Enrichment Score (ES) Higher at month 1 Higher at enrollment CE (DMPA_IM) Nom p-value: 0.0601 NES: 1.35 CE (Copper_IUD) Nom p-value: 0.4773 NES: 1.01 0.0 0.3 0.6 0.9 0 10000 20000 30000 40000 50000 Rank in Ordered Dataset Higher at month 1 Higher at enrollment CE (DMPA_IM) Nom p-value: 0.0656 NES: 1.4 CE (Copper_IUD) Nom p-value: 0.0438 NES: 1.44 0.00 0.25 0.50 0.75 0 10000 20000 30000 40000 50000 Rank in Ordered Dataset Enrichment Score (ES) DMPA-IM vs. IUD DMPA-IM vs. LNG DMPA-IM vs. IUD DMPA-IM vs. IUD Quality Control (FastQC v0.11.8) Raw sequence data GRCh38 reference genome Gene annotations Indexing, mapping and abundance estimation (STAR v2.5.2b) Differential expression analysis design =~ PTID + visit (DESeq2 in R) RNA extraction (Qiagen RNeasy Micro Kit) Quality control (Bioanalyser) Library construction (RNA Exome Kit) Sequencing (Illumina HiSeq 3000) Endocervical cytobrush samples collected at enrollment & month 1 visits Gene Set Enrichment Analysis (GSEA) Visualization of results (ggplot2 in R) DMPA-IM (150 mg/mL) 66 women HIV-seronegative women initiated on contraception LNG (block sizes 15-30) 66 women IUD 56 women Transcriptome analysis workflow IUD

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