J Clin Med Res

Journal of Clinical Medicine Research, ISSN 1918-3003 print, 1918-3011 online, Open Access

Article copyright, the authors; Journal compilation copyright, J Clin Med Res and Elmer Press Inc

Journal website http://www.jocmr.org

Original Article

Volume 11, Number 11, November 2019, pages 745-759

Histological and Gene Expression Analysis of the Effects of Menopause Status and Hormone Therapy on the Vaginal Introitus and Labia Majora

Figure 1. Urogenital symptoms. Shown are the symptoms that differed significantly across the study cohorts. ANOVA group effects: vaginal dryness (P = 0.003) and difficulty having intercourse (P = 0.02). ANOVA: analysis of variance; Pre-M: pre-menopause; Post-M: post-menopause; HT: hormone therapy.

Figure 2. H&E-stained introitus and labia majora tissues. Histological sections of biopsies obtained from the introitus (a-c, scale bar = 200 µm) and labia majora (d-f, scale bar = 100 µm) representative of the three study cohorts. The same subject was the source for each site pair. (a) and (d): pre-menopause; (b) and (e): post-menopause + HT; (c) and (f): post-menopause. Arrows point to examples of rete ridges or projections of the epithelium into the dermis. Note the reduced thickness of the epithelium and abundance of rete ridges in the post-menopausal group. Lower magnification images were used for the introitus to illustrate the differences across the cohorts more clearly. H&E: hematoxylin/eosin; HT: hormone therapy.

Figure 3. Hierarchical clustering of differentially regulated gene transcripts. (a) and (c) refer to the introitus and (b) and (d) refer to labia majora. (a) and (b) show probe sets differentially regulated in Post-M tissue. Probe sets were filtered for P < 0.01 by t-test for the group comparisons Post-M to Pre-M or Post-M to Post-M + HT. Other data filtering is described in “Methods”. The results of the Post-M + HT to Pre-M comparison is shown but was not used in the data filtering. The data are presented as log₂ fold changes with red indicating up-regulation and blue down-regulation. The color ramps indicate the scales with log₂ values shown. (c) and (d) show the directional consistency of the differential gene expression patterns in the Post-M to Pre-M and Post-M to Post-M + HT comparisons displayed as Venn diagrams. Pre-M: pre-menopause; Post-M: post-menopause; HT: hormone therapy.

Figure 4. Cross-study comparison of differential gene expression patterns associated with atrophy in introitus and vagina (using reprocessed published data [11, 12]). The heat map shows data for probe sets that met the stringent statistical filtering of q < 0.05 in the comparison of the Post-M to Pre-M introitus and also in the vaginal comparison of atrophied baseline vs. ET. All data are expressed as log₂ fold changes of the atrophied versus non-atrophied tissues. Pre-M: pre-menopause; Post-M: post-menopause; ET: estrogen therapy.

**Table 1.** Subject Attributes
Attribute	Pre-M	P value of Pre-M vs. Post-M	Post-M	P value of Post-M vs. Post-M + HT	Post-M + HT
N = 10 per group. Data are mean ± SE. Pre-M: pre-menopause; Post-M: post-menopause; NA: not applicable; SE: standard error; HT: hormone therapy; SHBG: serum hormone binding globulin.
Mean age	30.5 ± 2.1		62.9 ± 1.1		59.5 ± 1.7
Years on HT	NA		NA		15.7 ± 2.2
Clinical atrophy grade (0 - 10)	0	< 0.0001	8.1 ± 0.4	< 0.0001	0.6 ± 0.3
Vaginal pH	4.2 ± 0.1	< 0.0002	5.4 ± 0.1	< 0.0002	4.1 ± 0.1
Labial pH	5.4 ± 0.2	< 0.03	5.9 ± 0.2		5.55 ± 0.1
Estradiol (pg/mL)	61 ± 17	< 0.002	10 ± 2	< 0.0001	164 ± 58
SHBG (nmol/L)	104 ± 22		67 ± 12		82 ± 16
Testosterone (ng/dL)	30 ± 5		20 ± 4	< 0.009	89 ± 19

Table 1. Subject Attributes

Attribute

Pre-M

P value of Pre-M vs. Post-M

Post-M

P value of Post-M vs. Post-M + HT

Post-M + HT

N = 10 per group. Data are mean ± SE. Pre-M: pre-menopause; Post-M: post-menopause; NA: not applicable; SE: standard error; HT: hormone therapy; SHBG: serum hormone binding globulin.

Mean age

30.5 ± 2.1

62.9 ± 1.1

59.5 ± 1.7

Years on HT

15.7 ± 2.2

Clinical atrophy grade (0 - 10)

< 0.0001

8.1 ± 0.4

< 0.0001

0.6 ± 0.3

Vaginal pH

4.2 ± 0.1

< 0.0002

5.4 ± 0.1

< 0.0002

4.1 ± 0.1

Labial pH

5.4 ± 0.2

< 0.03

5.9 ± 0.2

5.55 ± 0.1

Estradiol (pg/mL)

61 ± 17

< 0.002

10 ± 2

< 0.0001

164 ± 58

SHBG (nmol/L)

104 ± 22

67 ± 12

82 ± 16

Testosterone (ng/dL)

30 ± 5

20 ± 4

< 0.009

89 ± 19

**Table 2.** GO Biological Processes Enriched for the Genes Differentially Expressed in Atrophic Post-M Compared to Normal Pre-M Introital Tissue
Up-regulated in atrophic introitus	Down-regulated in atrophic introitus
The online DAVID functional annotation clustering tool was used for GO term enrichment analysis as described in “Methods”. The probe set lists subjected to enrichment analysis were filtered as follows: Post-M vs. Pre-M introitus fold change up or down ≥ 1.2 and t-test P < 0.05 (corresponding to 1,479 up-regulated and 1,008 down-regulated genes with DAVID IDs). For conciseness only the top 6 most significant clusters are shown for the up- and down-regulated gene lists, as less significant clusters tended to have redundant terms. GO: gene ontology; Pre-M: pre-menopause; Post-M: post-menopause.
Blood vessel morphogenesis	1.21 × 10^-11	56	Epithelial cell differentiation	9.34 × 10^-9	50
Response to wounding	4.92 × 10^-11	124	Fatty acid metabolic process	5.75 × 10^-3	31
Cell migration	1.07 × 10^-8	79	Steroid metabolic process	1.48 × 10^-3	23
Actin cytoskeleton organization	1.13 × 10^-9	64	Hormone metabolic process	7.23 × 10^-3	15
Regulation of locomotion	1.47 × 10^-6	43	Pyridine nucleotide metabolic process	5.99 × 10^-3	29
Positive regulation of immune system process	1.07 × 10^-7	85	Monosaccharide metabolic process	1.17 × 10^-2	25

Table 2. GO Biological Processes Enriched for the Genes Differentially Expressed in Atrophic Post-M Compared to Normal Pre-M Introital Tissue

Up-regulated in atrophic introitus

Down-regulated in atrophic introitus

Most significant term in cluster

P value

Number of genes in cluster

Most significant term in cluster

P value

Number of genes in cluster

The online DAVID functional annotation clustering tool was used for GO term enrichment analysis as described in “Methods”. The probe set lists subjected to enrichment analysis were filtered as follows: Post-M vs. Pre-M introitus fold change up or down ≥ 1.2 and t-test P < 0.05 (corresponding to 1,479 up-regulated and 1,008 down-regulated genes with DAVID IDs). For conciseness only the top 6 most significant clusters are shown for the up- and down-regulated gene lists, as less significant clusters tended to have redundant terms. GO: gene ontology; Pre-M: pre-menopause; Post-M: post-menopause.

Blood vessel morphogenesis

1.21 × 10^-11

Epithelial cell differentiation

9.34 × 10^-9

Response to wounding

4.92 × 10^-11

124

Fatty acid metabolic process

5.75 × 10^-3

Cell migration

1.07 × 10^-8

Steroid metabolic process

1.48 × 10^-3

Actin cytoskeleton organization

1.13 × 10^-9

Hormone metabolic process

7.23 × 10^-3

Regulation of locomotion

1.47 × 10^-6

Pyridine nucleotide metabolic process

5.99 × 10^-3

Positive regulation of immune system process

1.07 × 10^-7

Monosaccharide metabolic process

1.17 × 10^-2

**Table 3.** Highly Consistent Genes Differentially Expressed in Both the Introitus and Labia Majora in Atrophic Versus Non-Atrophic Tissues
Probe set ID	Gene symbol	Gene name	Fold change
The gene list was obtained by stringently filtering the gene expression data as follows: 1) Each of the four introitus or labia majora comparisons shown were significant by t-test P < 0.01; and 2) For each of the comparisons differential gene expression was > 1.2-fold up or down. Other generally applied filtering is described in “Methods”. ^aDown-regulated genes. Although not used as a filtering criterion all of the genes shown were significant at q < 0.05 for the Post-M vs. Pre-M comparison. Pre-M: pre-menopause; Post-M: post-menopause; HT: hormone therapy.
11755246_x_at	GREB1	Growth regulating estrogen receptor binding 1^a	-3.85	-3.23	-1.54	-1.49
11739292_at	EHF	ETS homologous factor^a	-1.82	-1.59	-1.28	-1.23
11721579_a_at	DIO2	Iodothyronine deiodinase 2^a	-1.64	-1.85	-1.92	-2.17
11743202_x_at	IGF1	Insulin like growth factor 1^a	-1.61	-1.72	-1.33	-1.47
11741132_a_at	TP63	Tumor protein p63^a	-1.54	-1.45	-1.27	-1.23
11755523_a_at	RBM47	RNA binding motif protein 47^a	-1.43	-1.33	-1.23	-1.30
11729157_a_at	MFAP4	Microfibril associated protein 4	1.94	2.05	1.39	1.37
11715393_a_at	C3	Complement C3	2.94	3.28	1.61	1.82

Table 3. Highly Consistent Genes Differentially Expressed in Both the Introitus and Labia Majora in Atrophic Versus Non-Atrophic Tissues

Probe set ID

Gene symbol

Gene name

Fold change

Introitus

Labia majora

Post-M vs. Pre-M

Post-M vs. Post-M + HT

Post-M vs. Pre-M

Post-M vs. Post-M + HT

The gene list was obtained by stringently filtering the gene expression data as follows: 1) Each of the four introitus or labia majora comparisons shown were significant by t-test P < 0.01; and 2) For each of the comparisons differential gene expression was > 1.2-fold up or down. Other generally applied filtering is described in “Methods”. ^aDown-regulated genes. Although not used as a filtering criterion all of the genes shown were significant at q < 0.05 for the Post-M vs. Pre-M comparison. Pre-M: pre-menopause; Post-M: post-menopause; HT: hormone therapy.

11755246_x_at

GREB1

Growth regulating estrogen receptor binding 1^a

-3.85

-3.23

-1.54

-1.49

11739292_at

EHF

ETS homologous factor^a

-1.82

-1.59

-1.28

-1.23

11721579_a_at

DIO2

Iodothyronine deiodinase 2^a

-1.64

-1.85

-1.92

-2.17

11743202_x_at

IGF1

Insulin like growth factor 1^a

-1.61

-1.72

-1.33

-1.47

11741132_a_at

TP63

Tumor protein p63^a

-1.54

-1.45

-1.27

-1.23

11755523_a_at

RBM47

RNA binding motif protein 47^a

-1.43

-1.33

-1.23

-1.30

11729157_a_at

MFAP4

Microfibril associated protein 4

1.94

2.05

1.39

1.37

11715393_a_at

Complement C3

2.94

3.28

1.61

1.82

**Table 4.** Differential Expression of Collagen-Related Genes in Atrophied Post-Menopausal Introitus Relative to Non-Atrophied Tissues
Probe set ID	Gene symbol	Gene name	Fold change	t-test P values
The gene list was obtained by filtering the gene expression data for probe sets meeting the following criteria: 1) Annotated with the gene ontology biological process term “collagen fibril organization”; 2) Significant by t-test at P < 0.05 for each of the comparisons; and 3) > 10% change in relative expression up or down. Other generally applied probe set filtering is described in “Methods”. ^aDown-regulated genes. Pre-M: pre-menopause; Post-M: post-menopause; HT: hormone therapy.
11715415_a_at	COL3A1	Collagen type III alpha 1 chain^a	-2.22	-2.22	3.48 × 10^-2	2.61 × 10^-4
11746872_a_at	COL1A1	Collagen type I alpha 1 chain^a	-1.72	-1.54	4.48 × 10^-2	6.79 × 10^-4
11758995_at	LOX	Lysyl oxidase^a	-1.45	-1.41	2.06 × 10^-3	1.95 × 10^-4
11739134_a_at	COL5A2	Collagen type V alpha 2 chain^a	-1.41	-1.43	2.82 × 10^-2	7.00 × 10^-5
11753295_x_at	COL1A2	Collagen type I alpha 2 chain^a	-1.18	-1.16	7.76 × 10^-3	1.23 × 10^-3
11758519_s_at	PLOD3	Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3	1.34	1.25	1.74 × 10^-3	1.06 × 10^-2
11715796_s_at	LUM	Lumican	1.82	1.31	4.10 × 10^-4	1.37 × 10^-2
11744168_at	DPT	Dermatopontin	1.93	1.45	9.00 × 10^-6	1.01 × 10^-2
11720606_a_at	SFRP2	Secreted frizzled related protein 2	2.12	1.84	4.65 × 10^-3	2.22 × 10^-2

Table 4. Differential Expression of Collagen-Related Genes in Atrophied Post-Menopausal Introitus Relative to Non-Atrophied Tissues

Probe set ID

Gene symbol

Gene name

Fold change

t-test P values

Post-M vs. Pre-M

Post-M vs. Post-M + HT

Post-M vs. Pre-M

Post-M vs. Post-M + HT

The gene list was obtained by filtering the gene expression data for probe sets meeting the following criteria: 1) Annotated with the gene ontology biological process term “collagen fibril organization”; 2) Significant by t-test at P < 0.05 for each of the comparisons; and 3) > 10% change in relative expression up or down. Other generally applied probe set filtering is described in “Methods”. ^aDown-regulated genes. Pre-M: pre-menopause; Post-M: post-menopause; HT: hormone therapy.

11715415_a_at

COL3A1

Collagen type III alpha 1 chain^a

-2.22

3.48 × 10^-2

2.61 × 10^-4

11746872_a_at

COL1A1

Collagen type I alpha 1 chain^a

-1.72

-1.54

4.48 × 10^-2

6.79 × 10^-4

11758995_at

LOX

Lysyl oxidase^a

-1.45

-1.41

2.06 × 10^-3

1.95 × 10^-4

11739134_a_at

COL5A2

Collagen type V alpha 2 chain^a

-1.41

-1.43

2.82 × 10^-2

7.00 × 10^-5

11753295_x_at

COL1A2

Collagen type I alpha 2 chain^a

-1.18

-1.16

7.76 × 10^-3

1.23 × 10^-3

11758519_s_at

PLOD3

Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3

1.34

1.25

1.74 × 10^-3

1.06 × 10^-2

11715796_s_at

LUM

Lumican

1.82

1.31

4.10 × 10^-4

1.37 × 10^-2

11744168_at

DPT

Dermatopontin

1.93

1.45

9.00 × 10^-6

1.01 × 10^-2

11720606_a_at

SFRP2

Secreted frizzled related protein 2

2.12

1.84

4.65 × 10^-3

2.22 × 10^-2

**Table 5.** GO Biological Processes Enriched for Genes Commonly Regulated in Atrophied Vaginal and Introital Tissues
Most significant GO term in cluster	P value	Genes in cluster(s)
Gene lists from the cross-study comparison presented in Figure 4 were subjected to GO term enrichment analysis as described in “Methods”. The table displays results for the five most highly enriched clusters of GO biological process terms for the up- or down-regulated gene lists and the P value of the most significant term for each cluster. There was substantial overlap of the genes in the first three up-regulated clusters, so they were grouped together for conciseness. GO: gene ontology.
Up-regulated with atrophy
Immunoglobulin mediated immune response	1.81 × 10^-4	ACKR1, ARHGDIB, C1RL, C3AR1, CD74, CFH, CTSS, CXCL16, ENPP2, GALNT2, HLA-DMA, HLA-DPA1, HLA-DRB1, HLA-E, HRH1, INPP5D, ITPKB, LGALS3BP, MYD88, NTRK3, PRKCA, RGS1, SERPING1, SRGN, STAT5B, SWAP70, TAP2, TCIRG1, TNFRSF1A, VSIG4
Antigen processing and presentation	5.11 × 10^-3
Immune effector process	4.02 × 10^-4
Actin cytoskeleton organization	1.04 × 10^-2	ABI2, ARAP3, ARHGDIB, DAAM2, DBN1, EHD2, FGD5, LIMCH1, PACSIN2, PRR5, PTK2, SHROOM4, TLN1
Apoptosis	1.67 × 10^-2	AARS, BAG1, BCL2A1, BMF, CD74, CIB1, DPF2, DYRK2, ERCC3, GATA6, GSPT1, HIP1, HTRA2, IGFBP3, ILK, INPP5D, ITSN1, LITAF, MCF2L, MDM4, MOAP1, MYD88, NEK6, PCGF2, PRKCA, RPS27L, SETX, SLC11A2, SRGN, STAT5B, TNFRSF1A, TNFRSF21
Down-regulated with atrophy
Epithelial cell differentiation	3.00 × 10^-4	DHRS9, HECTD1, KAZN, LCE3D, PGR, PPL, SCEL, SHROOM3, SPRR2A, SPRR3, UPK1A
Regulation of cellular catabolic process	2.69 × 10^-2	ABHD5, HIF1A, ITCH, PPP1CB, PPP1R3C, PTPN3
Phospholipid metabolic process	2.32 × 10^-2	ABHD5, AGPS, ALOX12, CMAS, CWH43, DHRS9, GPAT3, PIGN, PIK3R1, PLAA, PLD1, PRDX6, PTEN, SCD, SPTLC1
Cellular response to insulin stimulus	3.48 × 10^-2	CDKN2B, ERO1A, GAB1, GATM, GLUL, GNG12, HMOX1, KAT2B, LPIN1, ME1, NOD2, PIK3R1, PLD1, PTEN, VLDLR
Polysaccharide biosynthetic process	3.48 × 10^-2	B3GNT2, CMAS, GALNT5, PPP1CB, PPP1R3C, UGDH

Table 5. GO Biological Processes Enriched for Genes Commonly Regulated in Atrophied Vaginal and Introital Tissues

Most significant GO term in cluster

P value

Genes in cluster(s)

Gene lists from the cross-study comparison presented in Figure 4 were subjected to GO term enrichment analysis as described in “Methods”. The table displays results for the five most highly enriched clusters of GO biological process terms for the up- or down-regulated gene lists and the P value of the most significant term for each cluster. There was substantial overlap of the genes in the first three up-regulated clusters, so they were grouped together for conciseness. GO: gene ontology.

Up-regulated with atrophy

Immunoglobulin mediated immune response

1.81 × 10^-4

ACKR1, ARHGDIB, C1RL, C3AR1, CD74, CFH, CTSS, CXCL16, ENPP2, GALNT2, HLA-DMA, HLA-DPA1, HLA-DRB1, HLA-E, HRH1, INPP5D, ITPKB, LGALS3BP, MYD88, NTRK3, PRKCA, RGS1, SERPING1, SRGN, STAT5B, SWAP70, TAP2, TCIRG1, TNFRSF1A, VSIG4

Antigen processing and presentation

5.11 × 10^-3

Immune effector process

4.02 × 10^-4

Actin cytoskeleton organization

1.04 × 10^-2

ABI2, ARAP3, ARHGDIB, DAAM2, DBN1, EHD2, FGD5, LIMCH1, PACSIN2, PRR5, PTK2, SHROOM4, TLN1

Apoptosis

1.67 × 10^-2

AARS, BAG1, BCL2A1, BMF, CD74, CIB1, DPF2, DYRK2, ERCC3, GATA6, GSPT1, HIP1, HTRA2, IGFBP3, ILK, INPP5D, ITSN1, LITAF, MCF2L, MDM4, MOAP1, MYD88, NEK6, PCGF2, PRKCA, RPS27L, SETX, SLC11A2, SRGN, STAT5B, TNFRSF1A, TNFRSF21

Down-regulated with atrophy

Epithelial cell differentiation

3.00 × 10^-4

DHRS9, HECTD1, KAZN, LCE3D, PGR, PPL, SCEL, SHROOM3, SPRR2A, SPRR3, UPK1A

Regulation of cellular catabolic process

2.69 × 10^-2

ABHD5, HIF1A, ITCH, PPP1CB, PPP1R3C, PTPN3

Phospholipid metabolic process

2.32 × 10^-2

ABHD5, AGPS, ALOX12, CMAS, CWH43, DHRS9, GPAT3, PIGN, PIK3R1, PLAA, PLD1, PRDX6, PTEN, SCD, SPTLC1

Cellular response to insulin stimulus

3.48 × 10^-2

CDKN2B, ERO1A, GAB1, GATM, GLUL, GNG12, HMOX1, KAT2B, LPIN1, ME1, NOD2, PIK3R1, PLD1, PTEN, VLDLR

Polysaccharide biosynthetic process

3.48 × 10^-2

B3GNT2, CMAS, GALNT5, PPP1CB, PPP1R3C, UGDH

**Table 6.** The 20 Most Highly Regulated Transcripts in Atrophied Post-Menopausal Introitus Relative to Normal Premenopausal Tissue
Probe set identifiers	Gene symbol	Gene name	Fold change
These gene expression data were filtered for transcripts with q < 0.05 for the introitus Post-M to Pre-M comparison and then the 10 most down- and up-regulated transcripts were selected. Other filtering is described in “Methods”. ^aDown-regulated genes. ^bUp-regulated genes. The results for the introitus Post-M vs. Post-M + HT and the vagina baseline vs. ET (from reprocessed published data [11, 12]) are also shown and colored if P values < 0.05. Pre-M: pre-menopause; Post-M: post-menopause; HT: hormone therapy.
11737529_a_at	1553454_at	RPTN	Repetin^a	-4.55	-4.17	-3.03
11738277_a_at	1554914_at	PLA2G4D	Phospholipase A2 group IVD^a	-4	-3.13	-4.35
11715242_s_at	224328_s_at	LCE3D	Late cornified envelope 3D^a	-4	-2.86	-4.35
11755246_x_at	205862_at	GREB1	Growth regulating estrogen receptor binding 1^a	-3.85	-3.23	-3.03
11741457_a_at	207206_s_at	ALOX12	Arachidonate 12-lipoxygenase, 12S type^a	-3.57	-2.44	-4.55
11742174_x_at	None	LCE3E	Late cornified envelope 3E^a	-3.45	-2.7
11725309_at	207802_at	CRISP3	Cysteine-rich secretory protein 3^a	-3.33	-2.56	-2.86
11744555_at	210272_at	CYP2B7P	Cytochrome P450 family 2 subfamily B member 7, pseudogene^a	-2.94	-2.04	-3.13
11726168_at	228554_at	PGR	Progesterone receptor^a	-2.86	-2.56	-2.17
11748025_s_at	223278_at	GJB2	Gap junction protein beta 2^a	-2.7	-2.56	-1.79
11717123_a_at	201957_at	PPP1R12B	Protein phosphatase 1 regulatory subunit 12B^b	2.56	2.12	2.26
11755106_a_at	209621_s_at	PDLIM3	PDZ and LIM domain 3^b	2.58	2.27	1.74
11757425_s_at	203868_s_at	VCAM1	Vascular cell adhesion molecule 1^b	2.59	1.87	1.65
11716203_a_at	202291_s_at	MGP	Matrix Gla protein^b	2.65	1.8	1.77
11725584_at	223541_at	HAS3	Hyaluronan synthase 3^b	2.82	1.77	2.05
11722995_at	209613_s_at	ADH1B	Alcohol dehydrogenase 1B (class I), beta polypeptide^b	2.9	2.36	1.83
11719671_a_at	203498_at	RCAN2	Regulator of calcineurin 2^b	2.94	1.97	1.07
11756003_x_at	202409_at	IGF2	Insulin like growth factor 2^b	2.97	1.51	1.76
11718904_s_at	204135_at	FILIP1L	Filamin A interacting protein 1 like^b	2.97	2.14	1.4
11715280_s_at	205157_s_at	KRT17	Keratin 17^b	4.29	1.46	1.22

Table 6. The 20 Most Highly Regulated Transcripts in Atrophied Post-Menopausal Introitus Relative to Normal Premenopausal Tissue

Probe set identifiers

Gene symbol

Gene name

Fold change

Introitus

Vagina

HG-U219

HG-U133 Plus 2.0

Post-M vs. Pre-M

Post-M vs. Post-M + HT

Control vs. ET

These gene expression data were filtered for transcripts with q < 0.05 for the introitus Post-M to Pre-M comparison and then the 10 most down- and up-regulated transcripts were selected. Other filtering is described in “Methods”. ^aDown-regulated genes. ^bUp-regulated genes. The results for the introitus Post-M vs. Post-M + HT and the vagina baseline vs. ET (from reprocessed published data [11, 12]) are also shown and colored if P values < 0.05. Pre-M: pre-menopause; Post-M: post-menopause; HT: hormone therapy.

11737529_a_at

1553454_at

RPTN

Repetin^a

-4.55

-4.17

-3.03

11738277_a_at

1554914_at

PLA2G4D

Phospholipase A2 group IVD^a

-4

-3.13

-4.35

11715242_s_at

224328_s_at

LCE3D

Late cornified envelope 3D^a

-4

-2.86

-4.35

11755246_x_at

205862_at

GREB1

Growth regulating estrogen receptor binding 1^a

-3.85

-3.23

-3.03

11741457_a_at

207206_s_at

ALOX12

Arachidonate 12-lipoxygenase, 12S type^a

-3.57

-2.44

-4.55

11742174_x_at

None

LCE3E

Late cornified envelope 3E^a

-3.45

-2.7

11725309_at

207802_at

CRISP3

Cysteine-rich secretory protein 3^a

-3.33

-2.56

-2.86

11744555_at

210272_at

CYP2B7P

Cytochrome P450 family 2 subfamily B member 7, pseudogene^a

-2.94

-2.04

-3.13

11726168_at

228554_at

PGR

Progesterone receptor^a

-2.86

-2.56

-2.17

11748025_s_at

223278_at

GJB2

Gap junction protein beta 2^a

-2.7

-2.56

-1.79

11717123_a_at

201957_at

PPP1R12B

Protein phosphatase 1 regulatory subunit 12B^b

2.56

2.12

2.26

11755106_a_at

209621_s_at

PDLIM3

PDZ and LIM domain 3^b

2.58

2.27

1.74

11757425_s_at

203868_s_at

VCAM1

Vascular cell adhesion molecule 1^b

2.59

1.87

1.65

11716203_a_at

202291_s_at

MGP

Matrix Gla protein^b

2.65

1.8

1.77

11725584_at

223541_at

HAS3

Hyaluronan synthase 3^b

2.82

1.77

2.05

11722995_at

209613_s_at

ADH1B

Alcohol dehydrogenase 1B (class I), beta polypeptide^b

2.9

2.36

1.83

11719671_a_at

203498_at

RCAN2

Regulator of calcineurin 2^b

2.94

1.97

1.07

11756003_x_at

202409_at

IGF2

Insulin like growth factor 2^b

2.97

1.51

1.76

11718904_s_at

204135_at

FILIP1L

Filamin A interacting protein 1 like^b

2.97

2.14

1.4

11715280_s_at

205157_s_at

KRT17

Keratin 17^b

4.29

1.46

1.22