Recent studies of genetic dysfunction in pelvic organ prolapse: The role of collagen defects

Review Article

Recent studies of genetic dysfunction in pelvic organ prolapse: The roleof collagen defects

Veronica F. LIM,1 John K. KHOO,1 Vivien WONG1 and Kate H. MOORE1,2

1Pelvic Floor Unit, St George Hospital/University of New South Wales, Sydney, and 2Department of Urogynaecology,St George Hospital/University of New South Wales, Kogarah, NSW, Australia

Gynaecologists are becoming increasingly aware that women with a family history of prolapse are at an increased risk ofprolapse refractory to treatment. In the past five years, several genetic mutations have been shown to correlate withincreased prolapse susceptibility. These mutations can result in disordered collagen metabolism, which weaken the fascialsupport of the pelvic organs. This review examines the contemporary evidence regarding the role of collagen in prolapse.

Key words: collagen, genetic influences, immunohistochemistry, pelvic organ prolapse.

Introduction

Pelvic organ prolapse (POP) is the herniation of viscerainto the vagina, generally associated with weakness of thesupporting structures. Prolapse is a common condition.Recent Australian data show that the lifetime risk of awoman having surgical treatment for prolapse in thiscountry is 19%.1 Prolapse is likely to become moreprevalent as our population ages, so a review of newevidence regarding the aetiology of this condition is timely.Aside from the traditional risk factors such as age,

parity, vaginal childbirth and increased intra-abdominalpressure (ie obesity, chronic cough and constipation)2, theconcept of a genetic tendency towards prolapse isbecoming more apparent. For example, women with apositive family history of POP are more likely to developprolapse compared to women with no family history (OR2.58, 95% CI 2.12–3.15).3 Furthermore, twin studies haveidentified that genetic factors contribute about 43% of thevariation in liability to prolapse.4

Most gynaecologists are aware of the link betweenconnective tissue disorders and prolapse. For example,women who have collagen-associated disorders such asEhlers–Danlos syndrome and Marfan syndrome have anincreased prevalence of prolapse. Such women tend tohave more severe prolapse symptoms and are more likelyto get recurrence after vaginal repair.5–8 Women with a

history of haemorrhoids, varicose veins, hernias andabdominal striae (conditions thought to be related tocollagen deficiency) are also more likely to developsymptomatic prolapse.9–11 Conversely, women with pro-lapse are significantly more likely to develop hernias com-pared with controls (31.6% vs 5%, n = 120, P = 0.002).11

There is strong evidence that collagen imbalancecontributes to the pathogenesis of hernias.12

Changes in collagen metabolism may lead to weakenedfascia, which plays an essential role in the support of thepelvic viscera.13 For an overview of the ligamentous layers inthe pelvis, see Figure 1, which describes the three imaginaryplanes, based on the level of support. Defects or decreasedvaginal wall resistance in each level may cause differentorgans to prolapse.17 Over the past five years, studies haveidentified several genetic variants (or polymorphisms)implicated in prolapse. These genetic variations affectcollagen synthesis and remodelling of the extracellularmatrix (ECM). This review aims to summarise recentfindings regarding the role of collagen dysfunction and itsrelation to genetic aberrations in the pathogenesis of POP.

Materials and Methods

Literature searches were performed primarily usingMEDLINE from 1996 to 2013. The MeSH terms ‘pelvicorgan prolapse’, ‘uterine prolapse’, ‘cystocele’, ‘rectocele’,uterosacral ligament’, ‘cardinal ligament’, ‘immunohisto-chemistry’, ‘collagen’, ‘matrix metalloproteinase’, ‘connectivetissue’, ‘extracellular matrix’, ‘fibroblast’, ‘polymorphism’,‘genetic predisposition’ and ‘fascia’ were used and combined.Hand-searching of reference lists of relevant main journalarticles was done to increase the search pool. A total of 30articles were identified between 1996 and 2013. Only studies

Correspondence: Prof Kate H. Moore, Department ofUrogynaecology, St George Hospital/UNSW, First floor,Pitney clinical sciences building, Kogarah, NSW 2217,Australia. Email: [email protected]

Received 12 August 2013; accepted 16 November 2013.

198 © 2014 The Royal Australian and New Zealand College of Obstetricians and Gynaecologists

Australian and New Zealand Journal of Obstetrics and Gynaecology 2014; 54: 198–205 DOI: 10.1111/ajo.12169

Th e Australian and New Zealand Journal of Obstetrics and Gynaecology

that focussed on prolapse (not urinary stress incontinence)were examined. Prolapse was defined as ≥ Stage 2 on POPQassessment.18 Studies that were included investigated thesignificance of collagen changes and controlled forconfounding variables (parity, age, menopause, smoking andBMI) during statistical analysis. Studies that included controlsamples taken from patients with gynaecological malignancy,leiomyoma or endometriosis were excluded as these were notconsidered true ‘control’ patients. Therefore, a total of 22studies were included.

Collagen composition

The connective tissue of the supporting ligaments andvaginal wall is composed of ECM. Within this matrix,there is a predominance of fibrillar components (collagen

and elastin), compared to nonfibrillar elements (pro-teoglycans, hyaluron and glycoproteins). Analysis of thecardinal ligaments reveals that 70–80% consists ofcollagen.19 Collagen is thought to be one of the maindeterminants of biomechanical strength. There areapproximately 28 types of collagen found in the humanbody, but the main subtypes in the pelvic floor are types Iand III. Whilst collagen I confers higher tensile strengthdue to its longer and thicker fibres, collagen III is moreprevalent as it provides tissue elasticity and flexibilitywithin the pelvis. Type III collagen is also the first type ofcollagen involved in wound repair and fibrosis, before it isreplaced by collagen I.20–22 Several studies have comparedthe amount of total and individual subtypes of collagen inthe supporting ligaments and vaginal tissue of women withPOP to a control group (see Table 1).19,23–36

Figure 1 Current anatomical considerations for prolapse. Level 1: The upper 1/3 of the vagina is suspended to the pelvic wall by theuterosacral and cardinal ligaments, condensations of endopelvic fascia. These ligaments provide apical support for both the uterus andupper third of vagina. Originally thought to be purely ligamentous, these structures are actually complexes of vascular and hypogastricnerves enveloped by connective tissue. Tissue resilience decreases with age-related atrophic change and injury.14–16 Defect: Uterineprolapse, or vaginal vault prolapse (in hysterectomy) Level 2: The middle 1/3 of the vagina is attached laterally to the arcus tendineousfasciae pelvis (ATFP). Anteriorly, vaginal wall is attached to the bladder via the pubocervical fascia. Defect: Cystocele (protrusion ofbladder). Posteriorly, vaginal wall is attached to rectum via the rectovaginal fascia. Defect: Rectocele (protrusion of rectum) Level 3: Thelower 1/3 of the vagina is fused to the surrounding perineal membrane and perineal body. Defect: Urinary stress incontinence.

© 2014 The Royal Australian and New Zealand College of Obstetricians and Gynaecologists 199

Genetic dysfunction in pelvic organ prolapse

Studies of collagen composition in uterosacral/cardinal ligament

There were 8 studies that specifically analysed collagencomponents within this level 1 structure. In the uterosacraland cardinal ligaments of women with prolapse, moststudies revealed decreased total collagen, but increasedcollagen III/I ratio. As collagen III is more flexible,increased collagen III/collagen I ratio contributes to tissuelaxity and prolapse susceptibility. However, conflictingresults have arisen, partly due to the use of differentmethods of collagen assessment. Due to its molecularstructure, collagen is notoriously difficult to quantify.21

Most of the studies analysed in this review utilisedimmunohistochemistry, a method that quantifies theamount of collagen through visual evaluation. Thesubjectivity of this method, compounded by relativelysmall sample sizes within the published articles, has led tovariable findings. For example, Kokcu et al.,23 foundincreased total collagen content in the uterosacralligaments, but did not clarify the subtype of collageninvolved. Two other studies found no difference in

collagen content between women with prolapse and thecontrol group.24,25 These studies quantified collagen viahydroxyproline assay – hydroxyproline is a component ofcollagen and is thus an indirect measure of collagen levels.This assay is not collagen specific and is not entirelyreliable in unpurified tissue samples, with results differingdepending on assessment methods.37

Studies of collagen composition in vaginal wall

Findings in vaginal wall biopsies are also conflicting(Table 1).30–34 Recently, Zhou et al.,30 found decreasedcollagen III expression (and reduced tissue elasticity) in full-thickness samples of vaginal tissue from patients withprolapse. Previous biomechanical study has alsodemonstrated that women with prolapse have stiffer vaginaltissues.38 In contrast, Moalli et al.,31 found increasedcollagen III in the subepithelial layers of the vaginal wall.These inconsistencies may arise from the following: (i)different methods used to assess collagen quality; and (ii)different biopsy sites from which the collagen samples were

Table 1 Collagen analysis biopsy specimens of supporting ligaments and vaginal tissue in women with and without POP (in the order ofdiscussion of text)

Study (author,year, ref#)

Target populationand sample size Tissue analysed Analytic method

Findings: women with POP vscontrol

Kokcu 200223 24 POP women 21controls

Uterosacral ligaments Histological analysis ↑collagen

Phillips 200524 14 POP women 14controls

Uterosacral ligaments Hydroxyproline assay No difference in hydroxyprolinelevels

Suzme, 200725 14 POP women 12controls

Uterosacral ligaments Hydroxyproline assay No difference in hydroxyprolinelevels

Ewies 200319 33 POP women 25controls

Cardinal ligaments Histology;immunohistochemical analysis

↑collagen III

Gabriel 200526 25 POP women 16controls

Uterosacral ligaments Histomorphological andimmunohistochemical analysis

↑ collagen III No change incollagen I

Liapis 200127 32 POP women 28controls

Uterosacral ligamentsParavaginal fascia

Immunohistochemical analysis No change in collagen III

Vulic 201128 46 POP women 49controls

Uterosacral ligaments Immunohistochemical analysis ↓collagen I

Yucel 201329 29 POP women 35controls

Uterosacral ligaments Immunohistochemical analysis ↓collagen I ↑ collagen III

Zhou 201230 17 women withPOP 5 controls

Anterior and posteriorwall

Western blotting ↓collagen III No change incollagen I

Moalli 200531 62 POP women 15controls

Vaginal apex (fullthickness biopsy)

Histological analysis ↑ collagen III

Jackson 199632 8 POP women 10controls

Vaginal epithelium Hydroxyproline assay ↓total collagen No change incollagen III/I ratio

Lin 200733 23 POP women 15controls

Anterior vaginal wall Immunohistochemical analysis ↓collagen III

Mosier 201034 47 POP women 7controls

Anterior vaginal wall Quantitative real-time PCR ↑collagen I ↑ collagen III ↓collagenIII/I ratio

Salman 201035 10 POP women 10controls

Cardinal ligaments Light and electron microscopy Structural alterations in size anddistribution of collagen fibres

Badiou 200836 11 POP women 8controls

Anterior vaginal wallapex

Histological and morphometricanalysis

Structural alterations in size anddistribution of collagen fibres

POP, pelvic organ prolapse; PCR, polymerase chain reaction.


V. F. Lim et al.

obtained. The amount of collagen varies according to thelayer of the vagina biopsied (adventitia, muscularis, laminapropria and superficial stratified squamous epithelium)(Figure 2).31

Collagen morphology

Collagen function is dependent on its morphology, whichcan be assessed by immunohistochemistry staining. Forexample, Salman et al.,35 used light and electronmicroscopy to analyse connective tissue fibres in thecardinal ligaments. He found that in women with POP,the extracellular matrix was less dense, with sparse butthicker distribution of collagen fibres compared tocontrols. Collagen fibrils were also increased in size, withmean diameter of 61.2 � 11.4 nm as opposed to52.5 � 6.1 nm in women without POP (P < 0.001).Similar biopsies of vaginal tissue from women withprolapse showed looser dispersion of fibrils in themuscular layer of the vaginal wall.36 Thus, alterations incollagen morphology appear to be associated withprolapse.

Regulators of collagen

The ECM is under a dynamic state of remodelling, abalance of synthesis and breakdown (Figure 3). Fibroblastsare the main synthesisers of collagen I and III, whilst matrixenzymes called metalloproteinases (MMP) degradecollagen. Collagen I and III are degraded by MMP-1,-2,-8,-9 and -13. Tissue inhibitors of metalloproteinases, knownas TIMP, then inhibit the MMP, achieving a balancingeffect.21,22,39,40 Genes (ie HOXA11 and COL3A1) governthe synthesis of these enzymes (Figure 3).

Figure 2 Layers of the vagina showing tissues obtained frombiopsies of different depths.

Figure 3 Schematic overview of genetic and biochemical factors governing collagen synthesis and degradation.



Changes in collagen may be due to altered fibroblastbehaviour. Fibroblasts are mechanoresponsive, modifyingtheir actin cytoskeleton when stretched. This results inpoor contractility and decreased functionality after stretch,as reviewed by Kerkhof et al.,20 Women with prolapsehave reduced density of fibroblasts in the uterosacralligaments and paravaginal fascia.23 Several studies havealso demonstrated modified fibroblast responses inprolapse.41–44

Several authors have analysed the expression of collagenregulators (MMP and TIMP) (Table 2). Expression ofMMP-1,-2 and -9 is increased in women with prolapse,demonstrating more collagen breakdown with loss of tissueintegrity. Within the published body of literature,conflicting findings have arisen. Unfortunately, authors donot consistently study all the subtypes of MMP, andbiopsy sites also vary considerably between studies.Regardless, these MMP all perform a common functionand are actually quite similar.Collagen is initially degraded into fragments by MMP-

1,-8 and -13 before it is solubilised by MMP-2 and -9.21

The increased expression of any MMP indicatesaccelerated remodelling and collagen degradation. MMPare inhibited by tissue inhibitors of metalloproteinases(TIMP), and decreased TIMP expression correspondswith increased MMP (Figure 3).22 Recent studies haveinvestigated TIMP expression in relation to MMP.46,49

It is not known whether these differences in theextracellular matrix are a cause or a consequence of injury.Increased mechanical load overstretches connective tissue,

inducing extracellular remodelling. This activates MMPand alters both collagen expression and fibroblastbehaviour.50,51 This cause-and-effect relationship isextremely difficult to investigate. The duration of prolapseis often unspecified in the prolapse cohort, and it is possiblethat remodelling of the ECM has occurred in response tothe prolapse itself. The ideal solution, a large longitudinalstudy in which unaffected women are biopsied andmonitored over time for prolapse, is yet to be carried out.Two studies have attempted to address this issue by

looking at paired samples of prolapsed tissue from thevaginal wall and nonprolapsed tissue from the cervixwithin the same woman. Wong et al.,52 usedhydroxyproline assays and found that in women withprolapse, there was significantly reduced collagen content(P = 0.01) in the cervix compared to women withoutprolapse. However, the cervix is a nonsupporting tissueand is not susceptible to the ECM changes induced byprolapse. Therefore, evidence presented by Wong et al. isdifficult to interpret, partly because they did notdifferentiate between women with stress incontinence andPOP in their study groups. Kannan et al.,53 studied asample of women with prolapse, comparing biopsies ofprolapsed vaginal wall tissue to tissue that appeared‘normal’ macroscopically. They found no differencebetween the two types of tissues, both demonstratedcondensed collagen fibres upon histological analysis.Recently, studies linking several genetic mutations to

prolapse have begun to clarify this confusing ‘cause-and-effect’ issue.

Table 2 Analysis of MMP expression in biopsy specimens of supporting ligaments and vaginal wall of women with POP compared tocontrol

Study (author,year, ref #)

Target populationand sample size Tissue analysed Analytic method

Findings: women withPOP vs control

Jackson 199632 8 POP women 10controls

Vaginal epithelium Immunohistochemical analysis ↑ MMP-2 ↑MMP-9

Phillips 200524 14 POP women 14controls

Uterosacral ligaments Gelatin zymography; ELISA No change in MMP-2 Nochange in MMP-9

Moalli 200531 62 POP women 15controls

Vaginal apex (full-thickness biopsy)

Gelatin zymography ↑MMP-9

Choy 200841 15 women withPOP 15 controls

Cardinal ligaments Quantitative real-time PCR mRNA ↑ MMP-2

Strinic 200945 40 women withPOP 14 controls

Uterosacral ligaments Immunohistochemical analysis ↑MMP-1 No change inMMP-2

Liang 201046 19 women withPOP 9 controls

Uterosacral ligaments Immunohistochemical analysis;quantitative real-time PCR mRNA

↑MMP-2 ↓TIMP-2

Budatha201147

53 women withPOP

Anterior vaginal wall apex(vaginal muscularis)

Quantitative real-time PCR, gelatinzymography

↑MMP-9

Dviri 201148 20 women withPOP 20 controls

Uterosacral ligamentsVaginal tissue

Immunohistochemical analysis ↑MMP-1 ↑MMP-9

Vulic 201128 46 women withPOP 49 controls

Uterosacral ligaments Immunohistochemical analysis ↑MMP-1

Liang 201246 27 women withPOP 14 controls

Uterosacral ligaments Immunohistochemical analysis;quantitative real-time PCR mRNA

↑MMP-2 ↓TIMP-2

POP, pelvic organ prolapse; ELISA, enzyme linked immunosorbent assay; PCR, polymerase chain reaction; mRNA, messengerribonucleic acid; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase.


V. F. Lim et al.

Genetic predisposition

This is a new but promising area of research. Connellet al.,54 demonstrated that the development of the supportligaments is coded by the homeobox gene HOXA11.Knockout mice models (genetically modified mice withdeleted HOXA11) are devoid of uterosacral ligaments(Figure 3). In women with POP, expression of HOXA11is decreased and linked to increased levels of MMP-2. It isproposed that HOXA11 regulates the expression of genesinvolved in extracellular matrix metabolism.55

Allen-Brady et al.,56 used genomewide associationanalysis on 2976 women and identified six single nucleotidepolymorphisms (SNP) linked to POP. SNPs are verycommon mutations, occurring when a different nucleotideis substituted for the normal nucleotide in a DNA sequence.This results in a defective protein being produced duringgene transcription and translation. These authors foundthat at least one of these mutations involves the COL181Agene, which is involved in type I collagen regulation.Other genes may be involved in the regulation of collagen

and MMP formation.57–64 Nucleotide polymorphisms inthe COL3A1 genes are associated with prolapse, thussuggesting the role of defective collagen III in the genesis ofprolapse.57–59 Interestingly, type IV Ehlers–Danlossyndrome is due to mutations in the same COL3A1 gene.Patients with this condition are known to have abnormalcollagen III, which causes fragile tissues and poor woundhealing.65 However, an association between nucleotidepolymorphism in COL1A1 (the gene which codes forcollagen I) and prolapse has not yet been proved.60,61

A mutation in the gene expressing MMP is alsoassociated with prolapse.62–64 Chen et al.,62 conducted acase–control study in Taiwanese women with prolapse(n = 92) versus control (n = 152). The authors found thattwo genetic polymorphisms of MMP-9 were significantlyassociated with prolapse risk (OR: 5.41 and 5.77). Thesefindings contradict the results of an Italian study,64 whichfound no difference in MMP-9 expression between theprolapse and the control groups. However, there was apositive association between a single variation of MMP-1and prolapse. These trends support the hypothesis thatincreased extracellular matrix remodelling contributestowards prolapse. It is important to note that currentstudies are limited as they tend to focuss on one particularethnic group at a time. In the future, evaluation of a largevariety of genetic groups may be possible.The evidence that collagen changes are involved in

prolapse continues to grow. Currently, genes investigatingcomponents other than collagen in the extracellular matrixare being investigated. These include genes coding forelastin metabolism and smooth muscle, as reviewed byothers.39,66

Discussion

The future implications of this new knowledge forimproved practice may be considerable. Firstly, with the

availability of genetic testing and counselling, women witha predisposition to prolapse may be able to measure theirincreased risk, although the cost-effectiveness of this needsto be established.Furthermore, with the identification of gene

polymorphisms associated with POP, individuals shouldbe able to undergo genetic screening to stratify their risk ofprolapse. High-risk individuals can be offered lifestyleoptions that will lower the risk, such as the avoidance ofobesity, chronic constipation, heavy-lifting occupationsand prolonged second stage of labour. Interventionaltherapies aiming to reverse ECM changes and fibrosis canbe developed to halt POP progression. This may not be sofar in the distant future, as fibrosis regression in the liver isalready evident following experimental drug therapy.67

The direct cost of surgical treatment of POP in USAalone is more than $1 billion per year.68 This cost willonly increase, as the number of women affected byprolapse is estimated to double within the next thirtyyears.69 Prevention is thus highly desirable.However, it is important to note that the aetiology of

prolapse is multifactorial with many other contributingmechanisms. Firstly, the impact of different racial groupsupon hereditary aspects of collagen metabolism confersconsiderable heterogeneity upon the results obtained.Furthermore, other factors, such as fascial fibre tearing,altered skeletal anatomy, reduced levator ani tone due toneuronal damage and levator ani avulsion followingchildbirth, may also contribute to prolapsedevelopment.2,70,71 Levator avulsion defect is the traumaticmuscle detachment of the puborectalis muscle from itsinsertion on the inferior pubic rami. This injury occurs inup to 36% of women72 and has been shown to be associatedwith prolapse, particularly in the anterior and centralcompartment.70 More recently, levator avulsion defectshave also been found to be strongly associated withrecurrent pelvic organ prolapse after prolapse repair.73–75 Itis likely that the conflicting results in the published literatureon the association between collagen and prolapse aetiologyare complicated by the presence of major anatomicaltrauma related to childbirth, as these factors are often nottaken into account and controlled for during the assessmentof collagen integrity. Potential ways to reduce thisconfounding bias include limiting the study population tonulliparous women only and incorporating diagnosis andassessment for levator avulsion.Further research is required in all directions in order to

integrate our knowledge of the various components ofprolapse aetiology. However, patients and gynaecologistsare becoming increasingly aware that biochemical and/orgenetic defects in collagen metabolism may have a role ina selected subgroup of women with prolapse.

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Recent studies of genetic dysfunction in pelvic organ prolapse: The role of collagen defects