Local oestrogen therapy modulates extracellular matrix and immune response in the vaginal tissue of post‐menopausal women with severe pelvic organ prolapse

Abstract This study investigates the effect of local oestrogen therapy (LET) on the expression of proteins participating in collagen/elastin biogenesis and immune markers in vaginal tissues of post‐menopausal women with severe pelvic organ prolapse (POP). Vaginal biopsies were collected from the anterior vaginal wall of informed and consented 52 post‐menopausal women with severe POP undergoing total hysterectomy. Twenty‐nine of the 52 women were treated with LET (in the form of vaginal oestrogen cream or tablet), while the remaining 23 untreated patients served as the controls. This study was approved by Sinai Health System REB. Vaginal tissue specimens were analysed for gene and protein expression using real‐time RT‐PCR and Luminex assays, protein localization and immune cell infiltration were assessed by immunohistochemistry. Forty‐four cytokines were detected. We found that LET application: (a) significantly increased (P < 0.05) gene and protein expression levels of extracellular matrix (ECM) structural proteins, collagen and elastin, as well as the expression of ECM maturation enzyme BMP1; (b) decreased protein expression level of ECM degradation enzymes MMP1, MMP2 and MMP3 accompanied by an increase in their tissue inhibitors, TIMP1 and TIMP4; (c) significantly increased (P < 0.05) the gene and protein expression levels of 14 vaginal cytokines involved in leucocyte infiltration, which was confirmed by immunohistochemistry. Our results indicate that LET plays an important role in the activation of immune system within the local vaginal environment, limiting the undesirable ECM degradation, which supports the strengthening of vaginal ECM in post‐menopausal women, therefore resisting menopause/age‐related changes and inducing urogenital tract tissue regeneration.


| INTRODUC TI ON
Pelvic organ prolapse (POP) represents a major health issue for women worldwide and affects nearly 50% of the population of post-menopausal women. 1 Multiple risk factors that lead to POP also include race, parity, obesity and chronic conditions (cough, constipation) that result in higher pressure on the pelvic floor. POP is the most common reason for hysterectomy among post-menopausal women. 2,3 Management options for POP include observation, pessary use and urogynaecologic surgery. The lifetime risk for undergoing at least one operation for POP is 11.1%, however, the re-operation rates are as high as 29%. With an ageing population, the demand for POP operations is expected to rise in the future. 2 Local oestrogen therapy (LET) is clinically used as an aid in POP treatment. In particular, as hypoestrogenism is a major risk factor for POP, some expert gynaecologic surgeons recommend pre-and/ or post-operative vaginal oestrogen therapy for post-menopausal women. 3,4 Importantly, the severity of POP symptoms has been shown to increase after menopause, which suggests that hypoestrogenism may be a contributory factor for the development of POP. 5,6 However, there is no consensus in the medical community about the efficacy of prescribing LET to patients with prolapse leading to a difference in patient care. There are few studies available, which directly address the effect of vaginal oestrogen on POP to ascertain the theoretical basis for its therapeutic application. Circulatory levels of oestrogen in females change in a monthly cyclic pattern during reproductive years. At menopause, however, oestrogen levels in the blood decrease significantly from 15-350 pg/mL (menstruating women) to 6.5 pg/mL (post-menopausal women). 7,8 Deficiency in oestrogen following menopause affects reproductive organs, causing vaginal dryness, and reduction in vaginal weight. 9 These vaginal and urinary symptoms are defined as the genitourinary syndrome of menopause (GSM). Oestrogen plays a supportive role in the pelvis by controlling the synthesis and breakdown of collagen. 10 Oestrogen receptors α and β (ESR1/2) are present throughout the lower urinary tract, bladder and vagina; with ESR1 being the predominant isoform. 11 They are expressed in three vaginal layers (epithelium, lamina propria and muscularis), and are responsive to steroid hormones. 12 Oestrogen therapy has been prescribed to treat urinary symptoms related to urogenital atrophy, such as vaginal dryness, irritation or itching, dyspareunia and thin and frail epithelium. 15 ESRs are also found on a variety of immune cells through which estradiol plays an important role in the regulation of the immune system. 13,14 Additionally, studies have reported a connection between the development of POP and abnormalities in connective tissue structure and repair mechanisms, such as in generalized connective tissue disorders like Ehlers Danlos or Marfan syndrome. 16 We have previously shown that the weakened pelvic floor may arise from aberrant biosynthesis and biodegradation of structural proteins of the connective tissue.
POP patients have lower levels of structural proteins, and maturation enzymes, including lysyl oxidase (LOX) family of enzymes, ADAMTS2 (A Disintegrin and Metalloproteinase with Thrombospondin motifs 2, also known as PNP) and bone morphogenetic protein-1 (BMP-1, also known as PCP), and higher degradation activity with an increase in matrix metalloproteases (MMPs) expression and activity and a decrease in tissue inhibitors of MMP (TIMPs). 16,17 Importantly, there is extensive literature affirming that the immune system plays a role in extracellular matrix (ECM) regulation. For instance, mice deficient in T and B cells exhibit decreased total and cross-linked collagen and LOX, and at the same time, increased MMP2, suggesting a role of lymphocytes in ECM remodelling. 18 In pre-menopausal women, hormonal regulation affects the expression of multiple genes regulating immune cell composition in the vagina, and endocrine balance in the mucosal immune system. 19,20 However, this type of regulation is not present in post-menopausal women. Changes in the immune system accompanying ageing and oestrogen deprivation are known as immunosenescence, the process characterized by a decrease in cell-mediated immune function and humoral response 21 which is reflected by a higher incidence of chronic infectious disease in post-menopausal women. 22 Studies have shown that hormone replacement therapy (HRT) in post-menopausal women can improve wound healing, modulate cytokine production and proliferation of immune cells. 23,24 It appears that oestrogen plays an important role in restoring altered immune profile with normal ageing peri-menopausal women, suggesting that preservation of immune function may be supported by systemic HRT. Despite these known effects of oestrogen, not much work has been done to examine the effect of LET on the immune system of patients with POP.
Thus, the objective of this study was to investigate the effect of LET (oestrogen cream or tablet) on the expression of proteins participating in collagen/elastin biogenesis and on modulation of immune response in vaginal tissues of post-menopausal women with severe POP.

| Patient selection
The Research Ethics Board of Mount Sinai Hospital, Toronto approved this study (05-0193-E). Vaginal biopsy samples were collected from post-menopausal women undergoing vaginal hysterectomy for POP following informed consent. All patients had grade 3-4 of prolapse as per POP-Q classification. The POP patients were either receiving local oestrogen therapy (LET group, n = 29) through oestrogen cream (Premarin) or tablets (Vagifem) or were not receiving oestrogen therapy (No-LET control group, n = 23). Premarin cream was taken once daily (0.5 g) for 10 days, then twice weekly continuously until surgery; Vagifem tablet was taken one tablet once daily for 2 weeks (10 μg) then twice weekly until surgery day. Women with a history of gynaecological malignancy, connective tissue disorders were excluded.

| Tissue collection
The tissue biopsy technique used in this study has been described previously. 25 Full-thickness vaginal wall biopsy (approximately 1 cm 2 ) was obtained from the anterior middle portion of the vaginal vault and placed in ice-cold PBS solution. One part of the vaginal biopsy was fixed in formalin for histological and immunohistochemical analyses, the second part was flash-frozen in liquid nitrogen and stored for biochemical assays (RNA extraction for gene expression analysis, proteins extraction for Luminex and enzyme-linked immunosorbent assay [ELISA]). Due to some variability in the size of the sample, sometimes tissue biopsy was used only for one application.

| RNA extraction & reverse transcription
Frozen tissue samples were crushed and total RNA was extracted using Trizol (Gibco, Canada) according to the manufacturer's protocol. RNA samples were purified using RNeasy MiniElute Cleanup Kit (Qiagen, Canada) and treated with 2.5 μL DNase I (Qiagen) to remove genomic DNA contamination. Total RNA was then reverse transcribed to cDNA (1 μg of RNA in 20 μL reaction) using iScript supermix (Bio-Rad, Canada) to produce cDNA at a final concentration of 50 ng/μL.

| Real-time RT-PCR
Five nanograms of cDNA was mixed with SYBR ® green PCR ready mix (Sigma-Aldrich, Canada) and specific primer pairs (Table 1).
Reverse transcription (RT)-PCR was performed with the CFX384 Touch ™ RT-PCR detection system. A no-template control was   The standard and sensitivity of all cytokines used are reported in Table S1. Tissue samples, standards and controls were analysed in duplicates. The plates were read on Bio-Plex ® 200 System instrument with high-throughput fluidics and analysed using the Bio-Plex ManagerTM 5.0.

| Histochemical analysis of total collagen and elastin
Vaginal tissue biopsy samples fixed in formalin were embedded in paraffin, cut into 5 μm slides and stained with a modified Masson's Trichrome stain to measure the total protein content of collagen and elastin. Verhoff's haematoxylin stain was used to stain elastic fibres black and Biebrich scarlet-acid fuchsin was used to stain collagen green-blue. All slides were scanned at 400× magnification, and pictures were imported into Visiopharm NewCast Software

| Immunohistochemistry
Tissue samples were fixed in 10% neutral buffered formalin or 4% paraformaldehyde and embedded in paraffin. Following rehydration and quenching (3% H 2 O 2 ), antigen retrieval was performed by microwaving slides in 10 μM sodium citrate solution for 20 minutes (pH 6). Following the retrieval, slides were blocked using protein blocking solution (DAKO) and then incubated overnight at 4°C with primary antibody (

| Statistical analysis
Statistical analysis was performed with independent sample t test for continuous data and chi-squared test for categorical data. Graphs were presented as mean ± SEM. The level of significance was set at P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***). GraphPad Prism ver-

| Patient demographics
Fifty-two post-menopausal women with severe POP were recruited into the study. Twenty-nine of these women were using LET, while 23 women did not undergo any hormonal therapy (Control, No-LET).
Groups were matched for age (mean age 64 vs 67 years), mean BMI (26.2 vs 26.1) and median parity (2 vs 2). The majority (79%) of the women recruited for this study had Stage III prolapse, with some (21%) with Stage IV prolapse. The duration of LET for the postmenopausal women ranged from 1 to 24 months, with an average of 8 months (Table 3).

| Effect of LET on ECM biosynthesis and biodegradation
Transcript levels of ECM structural and remodelling genes: col- COL4 transcript levels were increased in LET-treated tissues, however the difference did not reach significance (P = 0.076). In addition, there was a significant increase in the expression of ELN gene after treatment with LET ( Figure 1C, P < 0.05). Transcript levels of proteins involved in the deposition and maturation of elastin fibres, FBN1 and FBN2 showed a significant increase, while FBLN5 showed no significant difference between the two study groups ( Figure 1C, P = 0.24). The gene expression level of ECM maturation enzyme BMP1 showed a significant increase (P < 0.05) in LET as compared to No-LET group. In contrast, expression of ADAMTS2 and LOX family members (LOX, LOXL-1,2,3) did not differ significantly between the two groups ( Figure 1D).

| Total Collagen and Elastin content evaluation
The  showed no change in protein expression in response to oestrogen ( Figure 4B).

| Effect of LET on cytokine protein expression
Next, the expression of 45 cytokine proteins was analysed in vaginal tissue samples from post-menopausal POP patients and 44 cytokines were detected ( Figure 5A and Table S2 Figure S2).

| Immunohistochemical assessment of leucocyte infiltration
As the vast majority of chemokines up-regulated by LET are chemoattractants for immune cells, we next investigated whether they can induce the infiltration of leucocytes into human vaginal tissue.
As we suggested, immunohistological staining for pan-leucocyte marker CD45 showed a significant increase in the infiltration of immune cells in vaginal samples from LET group, with majority of cells localized to sub-SSE ( Figure 6A). There were 175 ± 38 vs 77 ± 17 and muscularis (70 ± 7 vs 31 ± 6 CD68+ cells/mm 2 ) ( Figure 6B).

| D ISCUSS I ON
Pelvic organ prolapse is a condition that affects millions of women worldwide and significantly disrupts their quality of life. With the POP incidence rates expected to increase with an ageing population, and the high reoccurrence rates of this condition, 2 it is important to find therapeutic options that will help patients to enhance pelvic support.
It has been established earlier that the strength of pelvic floor depends on the ECM components of connective tissue. 26 Importantly, the symptoms of POP worsen with the decrease in circulating oestrogens, implying that connective tissue of pelvic floor weakens due to an age-related oestrogen deprivation. 27,28 The weak pelvic floor is unable to provide proper support to pelvic organs, leading to the development of POP in older women. 6 Thus, this study aims to examine whether LET influences connective tissue contents of the pelvic floor tissue in post-menopausal women with severe grade POP.
While beneficial effects of post-operative LET has been proven, there is no set guideline for the pre-operative application of LET in POP patients due to insufficient clinical data. 29 The benefits of pre- Previous studies have shown that the immune system supports tissue homeostasis by regulating the expression of ECM components, ECM-modulating enzymes and growth factors. 44 Oestrogen is well known to exert its effect on the immune system through interaction with ESRs present on immune cells. 14,46 Abnormalities in hormonal status (such as deficiency in sex hormone) can make women susceptible to various immunologic impairments. 21 Similarly, the oestrogen deprivation accompanying menopause is known to cause changes in the immune system called immunosenescence. One of the biggest age-related adaptation that accompany immunosenescence is the involution of the thymus, resulting in fewer naïve and functionally deficient T cells, B cells and macrophages produced. [47][48][49] Studies showed that low levels of oestrogen in post-menopausal women can mitigate immune responses and make them susceptible to diseases and infections. 21 There is a systemic increase in pro-inflammatory serum markers, IL-6, IL-1b and TNF-α in women after menopause that act as predictive markers of functional disability, fragility and mortality. 50 This increase causes a continuous stimulation of the immune system resulting in a subclinical inflammatory status known as inflammaging. 51 The effect of increasing age on type 1 (Th2) and type 2 (Th2) cytokines is challenging to study as it can be altered by many factors.
Majority of studies detected increased IFN-γ secretion by activated F I G U R E 6 Infiltration of (A) leucocytes and (B) macrophages into vaginal tissues of post-menopausal women with severe POP. There was a significantly (P < 0.05) higher number of CD45+ leucocytes and CD68+ macrophages (stain brown) in sub-epithelial space (Sub-SSE) and muscularis layer of human vagina in women treated with LET (black bars) as compared to control women (white bars). Magnification 100×; scale bar 100 μm. N = 10/group. A significant difference is indicated by * (P < 0.05) IL-6, 60 and IFN-γ 55 as well as induction of both M1 and M2 phenotypes of macrophage.

| CON CLUS ION
There has been discussion about the efficacy of LET as a potential supplemental agent for post-menopausal POP patients improv-

CO N FLI C T S O F I NTE R E S T
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.