UNIVERSITY OF NEW SOUTH WALES Thesis/ProJect Report Sheet

Surname or Family name: Dietz First name: Hans Peter Other name/s: Abbreviation for degree as given in the University calendar: PhD School: Women's and Children's Health Faculty: Medicine Title: The Female Lower Urinary Tract and Pelvic Floor in Pregnancy and Puerperium

Abstract 350 words maximum:

A prospective observational study was conducted in order to define the effect of pregnancy and childbirth on lower urinary tract and pelvic noor. 200 nulliparous pregnant women were recruited and assessed at 6- 18 weeks gestation, 32- 39 weeks and 2-5 months postpartum. Blood was taken for extraction of DNA and, in a subgroup of 50 women, for relaxin and progesterone serum levels. The study protocol was completed by 169 women (84.5%). Delivery information was available on 183. Main outcome measures were symptoms and signs of stress incontinence and translabial ultrasound parameters of pelvic organ descent on maximal Valsalva manoeuvre, obtained supine and after bladder emptying. Main findings were as follows:

1.) Pelvic organ mobility in young nulliparous women varies markedly, with over Y. of the population showing bladder neck mobility of >=25 mm. 2.) Caucasian ethnicity, increased joint mobility and a family history of incontinence were associated with increased anterior compartment mobility. 3.) Bladder neck mobility did not correlate with symptoms and signs of stress incontinence in pregnancy. 4.) Relaxin and Progesterone serum levels in early pregnancy were not associated with pelvic organ mobility. 5.) Most parameters of pelvic organ descent showed significant increases from early to late pregnancy. 6.) Pelvic organ mobility in pregnancy is associated with delivery mode and length of second stage and is a potential predictor of progress in labour. 7.) Caesarean section is partly protective of postpartum stress incontinence. 8.) Vaginal childbirth, especially vaginal operative delivery, increases pelvic organ mobility in all three compartments. 9.) Women with low antepartum pelvic organ mobility show the most marked increases in mobility after vaginal childbirth. 1O .)Childbirth reduces bladder neck displacement on levator contraction. The main factor affecting levator function seems to be the length of second stage, both active and passive.

Vaginal childbirth significantly alters both the support of pelvic organs and levator muscle function and increases the likelihood of postpartum stress incontinence. It appears possible to characterize a group of women who are unlikely to deliver without operative intervention and/ or more likely to suffer pelvic floor trauma if they do achieve a vaginal delivery.

OeclaraHon relating to disposition of project report/thesis I am tully aware ot the policy ot the University relating to the retention ahd use ot higher degree project reports and theses, namely that the University retains the copies submitted for examination and is free to allow them to be consulted or borrowed. Subject to the provisions of the Copyright Act 1968, the University may issue a project report or thesis in whole or in part, in photost r microfilm or other copying medium. I also th ise the publication by University Microfilms of a 350 word abstract in Dissertation Abstracts International (applicable s only).

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FOR OFFICE USE ONLY Date of completion of requirements for Awar MBT 612.63 38 CER~ICATE!.. O}? ORH~IN.It;I;try

I l1ereby declare tlmt tills .s\thrribs .iou .. · i3 my own wotk a:ml to. tlte best of my knowledge it contains no nuttiil'ialo pt~::vio!tliy ptll.>lis,lmtl oi' wriu~u by unotlter person, nor niaterinl which to a snb;;tmtinl cxt~ut · hru; 'bc•m ai:cP.pt~si tor U1c nward of any other degree or diploma· at UNSW ot ~ny otllr.r to.luc&tiona.l in::titution, c.~cqit where due acknowledgement is mad~ .in the thr.sb. Any c:unuibutiotl· mnde to the rescareh by others, with whom I have·· worked ~~ IJN:JW or ~ls·!whcre, is explicitly ac~owledged in tl1e ·umsin. ·

I also declare that the iute)lcctt•al CL'nl•:n\ 0,1:' !1!13 thc.~iU is the. prod!ir.t of tny own work, except to the ~xtr.ut Uwt assislmt•;c. t1·;,;1l oU,em ·in the proj~ct'.~ design and · =·~,.~· ·~··:.:r ,..,...... ,. !Ko~... ~. l -·· The Female Lower Urinary Tract and Pelvic Floor in Pregnancy and Puerperium

H.P. Dietz MD (Heidelberg) FRANZOG DDU CU

PhD Thesis University of New South Wales Sydney Australia

2003

1 Acknowledgements

• My wife Susanne E.M. Langer, RN RM, and my son Lucas, first and foremost, for their remarkable patience; • Assoc. Prof. Kate Moore and Prof. Michael J. Bennett, University ofNew South Wales, Sydney, Australia, for supervision, help with study planning and methodology and funding support of A$ 1.875.- for a videoprinter; • The Research Foundation of the Royal Australian College of Obstetrics and Gynaecology and Mayne Nickless Health, for funding of this work through a RANZCOG Research Fellowship which on application was extended to 18 months, to a total mlume of A$ 75.000.-; • Dr. Barton Clarke and Prof. S.K. Khoo, Department of Obstetrics and Gynaecology, University of Queensland, for essential funding provided for .the molecular genetic component of this thesis, to a total of A$ 42.300.-; • The Royal Hospital for Women Foundation for funding of A$ 1400.-; • Dr. Peter Warren, Dr. Glenn McNally and the staff of the Imaging Department, Royal Hospital for Women, Sydney, Australia, for the provision of equipment and rooms as well as support with funding of A$ 447.52; • Dr. Anneke B. Steensma, Assoc ProfT.G. Vancaillie and other staff of the Endogynaecology Department, Gynaecology Ward and Junior Medical Staff, Royal Hospital for Women, for help with clinical assessments and ultrasound as well as provision of rooms ani equipment; • Dr. Don Garrett, Department of Endocrinology, Royal Hospital for Women, for serum Progesterone analysis; • Dr. Shane Brown and Lillian Tan, Department of Endocrinology Prince of Wales Hospital, Sydney, Australia, for help with Relaxin ELISA analysis; • Dr. Michael Buckley, Dr. Peter Taylor, George Elakis and Anita Bahar, Molecu­ lar Genetics, SEALS, Prince of Wales Hospital, for help with DNA extraction; • Dr. Georgia Chenevix- Trench, Dr. Mandy Spurdle, Livia Kelemen, Xiaoqing Chen, James Flanagan ani Jeremy Arnold, Queensland Cancer Fund Transgenic Laboratory, Queensland Institute of Medical Research, Brisbane, Australia, for teaching and help with DNA standardization, PCR and DHPLC work. • Prof. Peter Donald Wilson, Department ofWomen's & Children's Health, Dunedin School ofMedicine, University of Otago, Dunedin, New Zealand, for his longstanding support and friendship and his untiring interest in the work contained in this thesis.

2 1 JA~! ZUU4 Lt8BARV

2 List of abbreviations

ANOVA Analysis of Variance ATFP Arcus tendineus Fasciae Pelvis ATLA Arcus tendineus of the levator ani BND Bladder neck descent (vertical) on Valsalva BND-H Bladder neck displacement (horizontal) on Valsalva BSD-RV Vertical distance between bladder neck and inferoposterior border of symphysis pubis at rest BSD-SV Vertical distance between bladder neck and inferoposterior border of symphysis pubis on Valsalva CIS Caesarean Section CT Computed Tomography cv Coefficient of Variation COL1,2,3,5 Collagen 1,2,3,5 genes DHPLC Denaturing high pressure liquid chromatography DNA Deoxyribonucleic acid ELISA Enzyme linked immunosorbent assay ELN Elastin gene FPOP Female Pelvic Organ Prolapse MRI Magnetic Resonance Imaging m-RNA messenger- Ribonuclei acid NVD Normal Vaginal Delivery OR Odds Ratio PCR Polymerase Chain Reaction PNTML Pudendal nerve terminal motor latency POP-Q International Continence Society Prolapse assessment system (quantitative) QIMR Queensland Institute of Medical Research ROT Rotation of proximal urethra on Valsalva

3 RVA-R Retrovesical angle at rest RVA-S Retrovesical angle at stress/ on Valsalva SD Standard deviation SNP Single nucleotide polymorphism

4 Contents

1 Introduction 9

1.1 Stress Urinary Incontinence and pelvic organ prolapse: The natural outcome of pregnancy and vaginal birth? 9 1.2 The extent of the problem: Prevalence of urinary incontinence and female pelvic organ prolapse 11 1.3 The anatomy of stress incontinence and pelvic organ prolapse 13 1.4 What do we know about the aetiology of the problem? 16 1.4.1 Hormonal factors 17 1.4.2 Delivery- related factors 20 1.4.3 Other factors 22 1.5 Is there a genetic basis for female pelvic organ prolapse (FPOP)? 23 1.5.1 Genetic disorders associated with FPOP 24 1.5.2 Ethnic origin as a determinant ofFPOP 25 1.5.3 Collagen biochemistry and biomechanics 26 1.6 Assessment of the genotype of Female Pelvic Organ Prolapse 27 1. 7 Assessment of the phenotype of Female Pelvic Organ Prolapse 29 1.8 Hypotheses 31 2.7.1 First Visit: Early Pregnancy assessment 31 2.7.2 Second Visit: Third trimester assessment 32 2.7.3 Third Visit: Postpartum assessment 33

2 Patients and Methods 35

2.1 Setting and study design 35 2.2 Selection of patient population 37 2.3 Time points 3 8 2.4 Patients 40 2.4.1 Inclusion criteria 41 2.4.2 Exclusion criteria 41 2.5 Clinical methods 42 2.5.1 Interview 42 2.5.2 Family history 43 2.5.3 Joint mobility and striae gravidarum 44 2.5.4 Paper towel testing 45 2.5.5 Ultrasound methodology 46 2.5.5.1 Which outcome parameters should be chosen? 47 2.5.5.2 Assessment methods 49 2.5.5.3 Valsalva manoeuvre 52 2.5.5.4 Pelvic floor muscle(= levator ani) activation 57 2.5.5.5 Intra- and interobserver variability of ultrasound measurements 59 2.5.5.6 Standardization ofValsalva manoeuvre 61

5 2.5.5.7 3D Ultrasound Imaging 63 2.5.6 Other tests considered prior to study commencement 67 2.5.7 Collection of delivery data 68

2.6 Laboratory methods 69 2.6.1 Genetics 69 2.6.1.1 Preparation ofblood sample and DNA extraction 71 2.6.1.2 Identification of known polymorphisms 72 2.6.1.3 Preparation oftemplate DNA and primers 73 2.6.1.4 PCR methodology 75 2.6.1.5 DHPLC methodology 78 2.6.1.6 Sequencing methodology 80 2.6.1.7 Association analysis 81 2.6.2 Relaxin serum levels 82 2.6.3 Progesterone levels 83

2. 7 Statistics 84 2. 7.1 Power calculations, sample size 84 2. 7.2 Descriptive statistics 85 2. 7.3 Analytical statistics 85

3 Results 87

3.1 Validation of ultrasound imaging methodology 87 3 .1.1 Intraobserver variability 87 3 .1.2 Interobserver variability 87 3.2 First trimester assessment 88 3.2.1 Ethnic background 89 3.2.2 History ofphysical activity 91 3.2.3 Symptoms 91 3.2.4 Paper Towel testing 91 3.2.5 Joint hypermobility 92 3.2.6 Ultrasound assessment 93 3.2.6.1 Pelvic organ mobility 94 3.2.6.1.1 The influence of previous pregnancies 97 3.2.6.2 Levator function 98 3.2. 7 Hormones in early pregnancy and pelvic organ mobility 100 3.2.7.1 Relaxin 100 3.2.7.2 Progesterone 101 3.3 Second trimester assessment 103 3.3.1 Symptoms 104 3.3.2 Paper Towel testing 104 3.3.3 Joint hypermobility and striae gravidarum in the 3rd trimester 105 3.3.4 Third trimester ultrasound assessment 106

6 3.3.4.1 Pelvic organ hypermobility 106 3.3.4.2 Levator function 109 3.4 Delivery data 11 0 3.4.1 Pelvic organ mobility as a predictor of Normal Vaginal Delivery 113 3.5 Third visit: Postpartum assessment 116 3.5 .1 Bladder symptoms at the postpartum visit 117 3.5.2 Postpartum Paper Towel testing 118 3.5.3 Postpartum joint hypermobility, striae gravidarum and pelvic girdle pain 119 3.5.4 Ultrasound assessment at the postpartum visit 120 3.5.4.1 Pelvic organ mobility 120 3.5 .4.1.1 Descriptive data 120 3.5.4.1.2 Gestational length and induction of labour 123 3.5.4.1.3 First and second stage oflabour 123 3.5.4.1.4 Birthweight 126 3.5.4.1.5 Delivery mode 126 3.5.4.1.6 Presentation, epidural analgesia and perineal trauma 13 3 3.5.4.2 Levator function 133 3.6 3D Ultrasound imaging 135 3.7 Molecular Genetics 138

4 Discussion 143

4.1 Early Pregnancy assessment 147 4.1.1 Ultrasound data 147 4.1.1.1 Pelvic organ descent 149 4.1.1.1.1 Ethnic background 149 4.1.1.L2 Family History and Joint Hypermobility 150 4.1.1.1.3 Symptoms oflower urinary tract dysfunction 152 4.1.1.1.4 Relaxin and Progesterone serum levels 155 4.1.1.1.5 Mutation detection 156 4.1.1.2 Levator strength 158 4.2. Third trimester assessment 159 4.2.1 Ultrasound data: Pelvic organ descent 159 4.3 Labour and delivery outcome 160 4.3.1 Predictors oflabour and delivery 160 4.4 Postpartum assessment 162 4.4.1 Symptoms 162 4.4.1.1 Prediction of postpartal Stress Incontinence 162 4.4.1.2 Postpartal stress incontinence and delivery mode 163 4.4.1.3 Postpartal stress incontinence and fascial trauma 164 4.4.2 Ultrasound data 165

7 4.4.2.1 Pelvic organ descent 165 4.4.2.2 Levator function 168 4.5 Other results not covered by hypotheses 173 4.5.1 Levator function 173 4.5.2 3D Ultrasound for the identification of pelvic fascial trauma 174

5 Summary and Conclusions 177

5.1 Conclusions 178 5.1.1 Hypothesis 1 178 5 .1.2 Hypothesis 2 178 5.1.3 Hypothesis 3 and 4 179 5.1.4 Hypothesis 5 179 5.1.5 Hypothesis 6 and 7 179 5.1.6 Hypothesis 8 180 5.1.7 Hypothesis 9 181 5.1.8 Hypothesis 10 181 5.1.9 Hypothesis 11 182 5.1.10 Hypothesis 12 182 5.1.11 Hypothesis 13 182 5.1.12 Hypothesis 14 183 5.1.13 Hypothesis 15 185 5.2 Implications for clinical practice 185 5.3 Directions for future research 186 5.3.1 Defining the genetic basis for female pelvic organ prolapse and incontinence 186 5.3.2 Prediction of intervention in labour 187 5.3.3 Prediction and prevention of pelvic floor trauma 189

6 Literature 191 7 Own peer- reviewed publications and awards based on this thesis 207

7.1 Abstracts 207 7.2 Original articles 208 7.3 Awards 208

8 Appendix 209

8.1 Datasheets 209 8.2 Consent forms 212 8.3 Declaration 215 8.4 Test- retest and interrater reliability of the ultrasound assessment of bladder neck mobility 216

8 1 Introduction

1.1 Stress urinary incontinence and pelvic organ prolapse: The natural outcome of pregnancy and vaginal birth?

"The present Caesarean Section rate is entirely unacceptable, however, I do not lmow whether it is too high or too low" Frederick Zuspan

Obstetric practice is based on the belief that vaginal birth is the ideal outcome of most pregnancies. However, this may not be true for a significant number of women who achieve a vaginal delivery, only to "succumb to the sequelae of pelvic organ prolapse, stress urinary incontinence and fecal incontinence that may then precipitate serial surgical procedures and create lifelong disability" (Dainer, 1999). Since womens' life expectancy is increasing, there is more and more demand for health services dealing with the consequences. It has been estimated that more than one in ten women undergo surgery for pelvic organ prolapse or the associated problem of stress urinary incontinence, at some stage in their lives; up to a third end up having several procedures (Olsen et al.,1997). In Australia, numbers may be significantly higher (MacLennan et al., 2000).

From an anthropological perspective, it appears evident that the rapid evolution of man has led to substantial problems regarding the mechanics of childbirth, with upright posture and increased cranial capacity of the offspring being the main conflicting priorities. Man is the only mammal species in whom the infant head fills virtually the whole pelvis, necessitating displacement or deformation of most pelvic organs at the time or parturition (Fisk, 2002). It is therefore not surprising that vaginal childbirth, or even just the attempt at vaginal delivery, frequently gives rise to longterm morbidity.

Epidemiological studies confirm pregnancy and childbirth as the strongest risk factors for the development of pelvic floor dysfunction, with a relative risk of 10.85 determined

9 by the Oxford Family Planning Association study (Mant et al., 1997) for multiparous women. Our patients agree and generally attribute symptoms of stress incontinence and prolapse, the two main entities of pelvic floor morbidity, to childbirth and ageing (Gjorup et al., 1987). This is likely due to the fact that many women first notice stress incontinence during pregnancy. The prevalence of stress incontinence in pregnancy and puerperium varies from 20 to 67% (Mason, 1999) and is most strongly influenced by parity, with primigravid women reporting the lowest rates. Prevalence seems to rise in parallel to the duration of pregnancy (Wijma, 2001): at 16 weeks gestation stress incontinence within the last year was reported by only 3.9% of primigravidae in a Danish cross- sectional population study (Hojberg, 1999). A fall in prevalence is observed postpartum, with rates between 6 and 29% (Mason, 1999).

While clinicians are generally aware of the functional and/ or structural changes that results from pregnancy and vaginal childbirth, most do not discuss the issue with their patients (Drife 1996, Davila, 2001 ). Elective Caesarean Section appears to be the main potential intervention that may improve outcomes, although the extent of any protective effect is debated (MacLennan et al., 2000, Digesu et al., 2001). A recent survey of female obstetricians in the United Kingdom documented that 31% of respondents would choose an elective Caesarean Section to protect themselves from putative pelvic floor trauma (AI Mufti et al., 1997), with similar data recently reported from the USA (Gabbe and Holzman, 2001). This may be an option for subgroups of the population with · superior access to healthcare resources, and clinicians seem to be increasingly prepared to accede to such requests from their patients (Cotzias et al., 2001). However, even if there were proof for the value of such a drastic change in obstetric management derived from randomised controlled trials, the implementation of a policy of elective caesarean childbirth would place a severe burden on healthcare delivery systems even in developed western societies. The issue has triggered a heated debate which is subject to significant bias and a host of nonmedical considerations. The pros and cons of elective caesarean section seem to be in the process of becoming a major political issue in women's health (Bewley and Cockburn, 2002a,b).

10 There may be another way towards improving outcomes rather than establishing a policy of "Caesarean Section for all" (Fisk, 2002). It is evident that certain women are more at risk than others (Brubaker, 1998). Our goal therefore ought to be the estimation of individual risk (Aronson et al., 1995, Brubaker, 2002). At present however, "antepartum markers for the risk ofpelvic floor impairment at birth are still lacking and are much needed" (Jozwik and Jozwik, 2001). The relative weighting of genetic and environmental influences and, eventually, the development of a meaningful risk assessment for the development of female pelvic organ prolapse (FPOP) and urinary incontinence, therefore, are worthwhile long- term goals for research in this field.

It has been predicted that genomic research will result in a shift from disease treatment towards disease prediction and prevention (Fears, 2000). While for urogynaecology this goal may still lie many years in the future, there is no doubt that the time has come to start developing the tools and methods that will allow us to protect high risk women from the negative effects of both congenital connective tissue dysfunction and traumatic childbirth. Those tools may not be those of molecular genetics, at least not in the near future; it may well be that technologies that are already available such as ultrasound imaging could be used to predict individual risk and to reduce pelvic floor morbidity by selecting those women most likely to benefit from preventative intervention.

1.2 The extent of the problem: Prevalence of urinary incontinence and female pelvic organ prolapse

The total prevalence of urinary incontinence in women is thought to be between 15 and 40%, with the highest prevalence figures reached in the elderly. Stress incontinence may occur alone or in combination with urge incontinence, and is generally more prevalent than the latter, especially in younger age groups. It is most common around the time of menopause, with a subsequent decline likely due to lifestyle change, i.e., avoidance ofprovocation (McGrother et al., 2001).

11 The prevalence of pelvic organ prolapse is much harder to define due to the fact that there is no clear distinction between normal and abnormal pelvic organ mobility (Swift and Theofrastous, 2001). Furthermore, milder forms of prolapse are often asymptomatic (Samuelsson et al., 1999); many women with symptomatic prolapse do not seek medical help (Karram and Porter, 2001). It is assumed that there is an inevitable progression from milder forms of prolapse towards the more severe, but this has in fact not been proven. In the Oxford Family Planning study the risk of hospital admission for prolapse was approx. 2 per 1000 person- years of risk, although this study was conducted in women who were at most in their mid-60s at the time of analysis (Mant et al., 1997). Prevalence estimates are made more difficult by the fact that stress incontinence frequently coexists, and symptoms, the reasons for presentation, and decisions in favour of surgical correction do overlap in a large percentage of women. However, in a Swedish cross- sectional population study, the prevalence of (mostly asymptomatic and mild) prolapse was over 30% of 487 women examined (Samuelsson et al., 1999). In 285 women seen at an American menopause clinic, anterior, central and posterior compartment prolapse was seen in 51%, 20% and 27% respectively (Versi et al., 2001). In 1997, over 225.000 women underwent prolapse surgery in the United States alone (Brown et al., 2002).

Regular or severe incontinence may affect close to one million individuals in Australia, most of them women (Millard, 2001). In North America, urinary incontinence is the commonest cause of admission to long-term care ofthe elderly (Drutz and Schulz, 2001). Not surprisingly, it has been estimated that in 1995 urinary incontinence and its consequences cost the taxpayer 26.3 billion US$ in the 65+ age group alone (Gallo et al., 2001). For Australia, the total cost of female urinary incontinence in 1998 was estimated to be over A$ 700 million (Doran et al., 2001). While urge incontinence adds significantly to this problem, especially in the elderly, it is stress incontinence which contributes the larger share of the clinical workload due to the fact that surgical correction is tre main therapeutic option in a large number of women.

12 1.3 The anatomy of stress incontinence and pelvic organ prolapse

The alterations resulting in female pelvic organ prolapse must necessarily be due to macroanatomical changes. To a large degree, this is also true for stress incontinence; anterior vaginal wall prolapse is commonly found in women with stress incontinence, and loss of bladder base support is thought to contribute to the pathophysiology of this condition (Walters and Weber, 2001). Although stress incontinence may occur with normal anatomy, i.e. without defective support structures, the existence of increased anterior vaginal wall mobility is strongly associated with Genuine Stress Incontinence (Dietz et al., 2001b). This finding is in support of the theory that continence is normally preserved because intraabdominal pressure increases are transmitted to the proximal urethra, thereby increasing closure pressures in time of need (Nichols, 1982). In cases of excessive mobility the urethra and bladder neck come to rest below the urogenital diaphragm, i.e., outside the intraabdominal space, with a resultant loss of external urethral compression by increases in intraabdominal pressure.

r

Ischial spine & sacrospinous ligament ---.....:---=-.,...,::\.

Pubocervtcal fascia ---' Rectovaginal fascia --

Figure 1.1: The three levels of fascial support of the vagina according to J.O. De Lancey (De Lancey, 2001; with permission)

13 There are several distinct anatomical structures that may be responsible for excessive bladder neck mobility. In order to organize the complexity of pelvic anatomy into concepts that can be understood more easily, both anteroposterior and craniocaudal systems have been proposed. The author of this thesis feels that such divisions are artificial and arbitrary; nevertheless, they may be useful for teaching purposes. According to John DeLancey, arguably the foremost pelvic floor anatomist of the last two decades, pelvic organ support is organized in three levels. Level I is comprised of the upper paracolpium, the uterosacral ligaments and presumably the upper rectovaginal septum. Level II comprises the central paracolpia, in particular vaginal attachments to the arcus tendineus fasciae pelvis and the obturator fascia, i.e. the so- called 'endopelvic fascia'. These structures suspend the vagina to the pelvic sidewall between ischial spine and pubic rami. Level III is made up of connective tissue linking the distal vagina to the urethra and symphysis pubis anteriorly within the perineal mambrane, the levator ani laterally and the perineal body posteriorly (DeLancey, 2001 ), see Figure 1.1.

Increased anterior vaginal wall descent is thought to be due to disruption of levels II and III, i.e. the endopelvic fascia and its condensations, in particular at the site of attachment to the arcus tendineus fasciae pelvis, resulting in discrete fascial defects (Richardson et al., 1976, 1981, Shull and Baden, 1989). This hypothesis is as yet unproven and has been questioned (Monga, 1996). The pubourethralligaments, condensations of the most anterior and distal part of the endopelvic fascia, seem to provide an anchoring point for the proximal and central urethra and have successfully been used in incontinence surgery (Vancaillie,1995). A study using MRI imaging on a small number of subjects showed these structures to be defective in women with stress incontinence (Aronson et al., 1995).

More distal structures such as the anchoring of the urethra in the perineal membrane may also play a role (DeLancey, 1988). As there is no consensus on the exact extent (let alone functional role) of the above structures, they frequently are collectively referred to as the "paraurethral connective tissue" (Ulmsten and Falconer, 1999), the "suburethral hammock" or "urethral supports" (DeLancey, 1988, 2001). The incidence of traumatic

14 defects- as opposed to attenuated, stretched or congenitally lax structures- is unknown as none of those structures have as yet been investigated in vivo in a peripartum setting. .. It has been speculated that damage to the endopelvic fascia may not be a primary delivery- related event but occur secondary to peripartum levator trauma (DeLancey, 2001). While there is ample evidence to support the concept of neuropathic (Allen et al., 1990) or myopathic (Dimpfl et al., 1998) damage to the levator muscle in childbirth, the longterm effect of levator damage on fascial structures has not been investigated so far.

The anatomical aetiology of central and posterior compartment prolapse is also in. doubt. The main supports ofthe uterus are the cardinal and uterosacral ligaments, i.e., DeLancey's Level I; elongation and disruption of those structures is thought to result in uterine prolapse, and clinically it is evident that the uterosacral ligaments provide support to the cervix and, after hysterectomy, to the vaginal vault. Their ultimate importance in providing uterine support however has been questioned (DeLancey, 2001). As regards the posterior vaginal wall, it is thought that the posterior extension of the endopelvic fascia, also termed the rectovaginal septum or Denonvillier's fascia, provides most of the necessary structural support (Monga, 1996). It extends from the posterior cervix to the perineal body and the pubococcygeus muscle, guarding against herniation of abdominal contents (enterocele) and anterior rectal wall and rectal contents (rectocele) into tre vagina (Kohli, 2001).

A defect in Denonvillier's fascia, especially a transverse tear in the upper, less dense aspects of the rectovaginal septum, has been assumed to be the archetypal delivery­ related lesion (Kuhn and Hollyock, 1982, Chandler and Buek, 2001 ), presumably occurring with crowning of the fetal head. However, data on the incidence of rectoceles in asymptomatic nulliparous women (Shorvon et al., 1989) contradicts this theory (Karram and Porter, 2001) since they seem to be relatively common even in nulliparous women. Unfortunately, cadaver studies are oflimited use in elucidating the direct anatomical correlates of female pelvic organ prolapse and incontinence. Prolapse, after all, commonly is evident only under conditions of increased intraabdominal pressure

15 and/ or in the erect position. Cadaver tissues are "frozen" in a position they assumed when the organism was supine and in a state of rest, and biomechanical properties of tissues are irrevocably altered by fixation. The effect of dynamic manoeuvres such as coughing or Valsalva manoeuvres can only be studied in vivo which is why imaging methods such as MRI and ultrasound are indispensable to the investigation of the anatomy of pelvic organ prolapse and stress incontinence (DeLancey, 2001).

1.4 What do we know about the aetiology of the problem?

For a long time it has been suspected that the aetiology of genuine stress incontinence (GSI) and prolapse is multifactorial (Beck and Hsu, 1965, Wilson et al., 1996). Congenital, i.e., genetically determined factors (Swift and Theofrastous, 2001) appear to play a role as well as pregnancy- related, i.e., likely hormonal factors (Foldspang et al., 1999, MacLennan et al., 2000). Traumatic damage to pelvic fascial structures related to vaginal childbirth may be the third main contributor, with the relative importance of these three factors varying between patients (Swift and Theofrastous, 2001).

The link between parturition and traumatic impairment of pelvic floor structures was one of the more obvious arguments for combining Obstetrics and Gynaecology in one specialty (DeLancey, 1993). It is said that there now is compelling evidence to support the concept of permanent pelvic floor damage after childbirth (Land et al., 2001). So far, this evidence :S mainly based on neurophysiological studies implying pudendal nerve damage (Snooks et al., 1985, 1986, 1990, Jozwik and Jozwik, 2001) and some data showing a reduction in maximal urethral closure pressure and functional urethral length after vaginal delivery (van Geelen et al., 1994, Meyer et al., 1998a). However, the value of pudendal nerve neurophysiology is by no means undisputed (Vodusek, 2000), and reinnervation is the rule rather than the exception. The importance of urethral closure pressure in the causation of stress urinary incontinence is also debated (Lose, 1997).

16 The effects of pregnancy and childbirth on connective tissue integrity and biomechanics remain largely undefined. Pregnancy itself seems to change the properties of connective tissue everywhere in the body, and this seems to happen early in pregnancy as first observed in the 1930s (Abramson et al., 1934). Fascia stretches more easily and fails earlier in pregnancy than the same tissue in nonpregnant women (Landon et al., 1990), and such fascial properties have been related to the development of stress incontinence (Landon and Smith, 1998). Fascial relaxation and stretching may explain the marked changes in bladder neck support seen with radiological methods in the past (Francis, 1960).

1.4.1 Hormonal factors

Oestrogens, progestagens and relaxin have all been implicated in connective tissue alterations in pregnancy (Viidik, 1973). The latter may activate the collagenolytic system by increasing collagenase and collagen peptidase, activate fibroblasts and therefore alter the biomechanical properties of connective tissue (MacLennan, 1991, 1995). It is not clear whether such changes, if present, return to normal or near normal after childbirth, provided no traumatic damage supervenes at the time of delivery, or whether such changes are permanent. However, some permanent effect appears likely since even pregnancy ending in elective caesarean childbirth appears to be a risk factor for future symptoms of stress incontinence and surgical intervertion for pelvic floor dysfunction (MacLennan et al., 2000).

Relaxin, the most likely candidate, is an insulin- like polypeptide hormone of a molecular weight of 6000 Dalton, consisting of two chains linked by a disulfide bond (Schwabe and Bollesbach, 1990, Goldsmith et al., 1995). It seems to play a major role in reproduction, from luteal function to sperm motility, uterine activity and cervical maturation. Serum levels during pregnancy have been shown to increase until reaching a peak between 10 and 14 weeks of gestation (Petersen et al., 1994, Kristiansson et al., 1996). After this there is a slow decrease so that overall the pattern of serum levels

17 parallels those seen for HCG. Relaxin levels in maternal serum are much higher than in intrauterine fluids (Johnson et al., 1992) and seem to be determined during the first ten weeks of pregnancy (Johnson et al., 1994a,b), ie., by the events surrounding ovulation and luteinization. However, trophoblast and decidua also contribute to overall secretion (MacLennan et al., 2000). Levels are increased in pregnancies that have resulted from superovulation, even if the resulting pregnancy is a singleton (Haning et al., 1996), and multifetal reduction does not reduce relaxin levels (Johnson et al., 1994b ). In multiple pregnancy, relaxin serum levels correlate with the number of siblings. There is no diurnal variation (Petersen et al., 1994).

As shown in studies conducted on mammals and in the human, relaxin seems to induce marked changes in connective tissue, in particular at the level ofthe symphysis pubis (Samuel et al., 1996) and the cervix (Hwang et al., 1996, Eppel et al., 1999). There is inhibition of collagen biosynthesis by dermal fibroblasts in vitro (Unemori et al., 1993), and dissociation of large collagen fibrils into smaller disorganized fibrils (Blecher and Richmond, 1998). In rats treated with recombinant relaxin a reduction of total collagen content by about 2/3 was observed in symphyseal connective tissue (Samuel et al., 1996). Collagenolytic activity in the human cervix also appears to be increased by relaxin (Hwang et al., 1996).

So far, the effect of relaxin on the endopelvic fascia and its components has not been examined. The effect on joint laxity is controversial (Schauberger et al., 1996, Blecher and Richmond,1998) as is a postulated link between symptomatic pelvic girdle relaxation and back pain in pregnancy and serum relaxin levels (Kristiansson et al., 1996, 1999, Hansen et al., 1996, Petersen et al., 1994, Albert et al., 1997). Symphyseal distension does not seem to correlate with relaxin serum levels (Bjorklund et al., 2000).

The main focus in relaxin related research has been on its effect on cervix and fetal membranes. Some investigators found that serum relaxin levels predicted prematurity (Weiss et al., 1993, Petersen et al., 1992); however, these findings remain controversial.

18 Consequently, much recent work has focussed on the identification of relaxin binding sites and receptor levels (Kohsaka et al., 1998). A major stumbling block is the inaccessibility of potential target tissues.

Both progesterones and estrogens have long been suspected of having effects on connective tissue (Viidiik, 1973). Estrogens have been investigated mainly in relation to their effect on skin (Borel, 1991, Brincat, 2000) and bone (Hobson and Ralston, 2001). A lack of estrogen seems to slow collagen and elastin biosynthesis and has negative effects on the biomechanical properties of skin. There is no data as to whether such effects also occur in pelvic ligaments or fascia, although a recent study investigating the biomechanical properties of vaginal skin in women undergoing prolapse surgery showed reduced elasticity of postmenopausal tissues (Goh, 2002).

The influence of progestagen levels on connective tissue has been investigated in the uterus where progesterone inhibits collagenases, i.e., breakdown of collagen crosslinks, and therefore has a stabilizing effect (Garfield et al., 1998). Also, progesterone is a potent smooth muscle relaxant which could conceivably influence the biomechanics of pelvic support structures that contain a significant amount of smooth muscle fibres among the connective tissue making up the bulk of the structures (DeLancey, 2001).

For both progesterone and estrogen, there is no evidence that the high levels associated with pregnancy have negative effects on connective tissue biomechanics. As regards the symptom of stress incontinence, it has on the contrary been observed that an increase in oestrogen levels after the cessation ofbreastfeeding sometimes has a beneficial influence on leakage, a finding that has been attributed to effects on urethral mucosa (DeLancey, 2001).

1.4.2 Delivery- related factors

It appears highly likely that vaginal delivery can damage connective tissue structures,

19 the levator ani and/or the sensory and motor innervation of the pelvic floor, effects that may lead to prolapse as well as anal and urinary incontinence (Tetzschner et al., 1997). The link between traumatic childbirth and prolapse has been suspected for more than a thousand years (Haby Abbas, quoted in Van Dongen, 1981). A number of delivery­ related factors, such as vaginal birth, vaginal operative delivery, epidural analgesia and maternal age at time of delivery have been shown to increase the likelihood of future prolapse- or incontinence- correcting surgery or symptoms (Wilson et al., 1996, Jackson et al., 1997, Foldspang et al., 1999, Carley et al., 1999a, Chiaffarino et al., 1999, MacLennan et al., 2000). Other potential determinants of pelvic floor trauma may be the length of first and second stage of labour (Allen et al., 1990), birthweight (Allen et al., 1990) and episiotomy (Smith et al., 1989a,b, Foldspang et al., 1999).

The focus in recent years has been on anal sphincter injury (Sultan et al. 1994 , 1996) and pudendal nerve damage (Snooks et al., 1985, 1986, 1990, Jozwik and Jozwik, 2001 ). The pudendal nerve may be affected through a number of different mechanisms since the nerve is particularly vulnerable to traumatic injury in childbirth. Compression of the nerve may occur in the pudendal canal (Alcock's canal) or directly between head and pelvic sidewall. Overstretching of fibres may compromise parts of the lumbosacral plexus, the nerve itself or more distally the neuromuscular junction In any case, the end result is a functionally impaired levator ani muscle due to denervation injury (Jozwik and Jozwik, 2001). Neurogenic damage may occur on top of direct trauma to the levator ani due to haemorrhage, tearing of fibres and possibly avulsion from insertion sites (Peschers et al., 1999).

As regards damage to fascial structures, it has frequently been assumed that at least some of the distinct fascial defects seen in later life, such as paravaginal defects (Richardson et al., 1976), are due to delivery- related trauma. Translabial or transperineal ultrasound as a method was first described in the late 1980's (Kohorn et al., 1986, Grischke et al., 1986, Grischke et al., 1989, Koelbl et al., 1988, 1989b, Schaer et al., 1995). The terms trarnlabial, transperineal and perineal are synonymous and all

20 relate to ultrasound imaging performed with an external probe placed on labia maiora or pef41eum. Due to the development of this method, it has become possible and practicable to investigate the effects of vaginal delivery on the supports of the anterior vaginal wall and bladder neck.

King et al. (King, 1998) in Plymouth, UK, found no association between delivery parameters and bladder neck mobility, using an ultrasound methodology that differed in important aspects from the one employed by the author of this thesis. One hundred and three primigravid patients were examined upright and in stirrups, Valsalva force was standardized to 30 mm.Hg (approximately 40 em H20), and imaging was carried out with a full bladder. All these factors would be expected to limit mobility (Dietz et al., 1999a, 2001a), and it appears that this was indeed the case although descriptive statistics for bladder neck mobility are lacking in the publication.

Meyer et al. (Meyer et al., 1996, 1998a,b) in Lausanne, Switzerland, again used a different methodology to the one implemented in this study, imaging women with a full bladder and using the central axis of the symphysis pubis for reference. Bladder neck mobility was significantly increased after all vaginal deliveries in 149 primiparae with no differences between forceps and normal vaginal delivery. The only near- significant correlation between ultrasound indices and other delivery- related parameters was a weak positive correlation between bladder neck mobility and birthweight. Bader (Bader et al., 1995) found significant differences both between caesarean section and vaginal delivery and between normal and operative vaginal delivery as regards mobility of the bladder neck. lbwever, imaging was only undertaken postpartum so that absolute values had to be compared between patients rather than between visits. Another small study on 25 primigravid women (Peschers et al., 1996) also confirmed this finding.

As regards the other compartments, imaging studies in pregnancy so far have not presented any data relating to central and posterior compartment descent. Epidemiological studies generally do not allow distinction between compartments but

21 associations between parity or delivery lll)de and prolapse seem to apply to all three compartments (Mant et al., 1997, Anon. 2000). There are only minor methodological difficulties in extending a translabial ultrasound assessment to the central and posterior compartment (Dietz et al., 2001c). Central compartment prolapse is measured by demonstrating maximal descent on Valsalva of the leading edge of the cervix. This is easier in pregnancy since the cervix is enlarged and more sonolucent. The posterior compartment can be assessed sonographically by demonstrating maximal descent of the rectal ampulla or a rectocele. A rectocele is visible as an anterocaudal outpouching of the anorectal junction; this is as common a fmding in nulliparous women on transperineal ultrasound as it is on defecography (Shorvon et al., 1989). Overall, imaging is likely to provide better quantitative information than either the POP-Q (Prolapse assessment system of the International Continence Society) or clinical staging (Kenton et al., 1997) since the actual position of orgam (or their contents) rather than the surface topography of the vagina is assessed.

1.4.3 Other factors

Other environmental influences are obesity (Bump et al., 1992 Wilson et al., 1996), bowel dysfunction (Snooks et al., 1985, Spence- Jones et al., 1994), smoking, asthma (Rinne and Kirkinen, 1999), constipation (Wilson et al., 1999) and possibly sports such as swimming, yoga, trampolining, horseriding, parachute jumping etc.; however, the data on the latter lifestyle factors is generally inconclusive (Wilson et al., 1999). An attempt was made to assess the above factors as part of the questionnaire used in the study (see appendix), although deleterious effects are likely to be more evident in the long term, i.e., in the elderly rather than the young.

Ageing is the fmal factor in the equation. Most patients presenting with severe prolapse, i.e., procidentia or vault eversion, are elderly (Bidmead and Cardozo, 1998). The impact of ageing on pelvic floor support structures is poorly understood, although trere seems to be an increase in collagen concentration and changes in crosslinking. The result is

22 increasing loadbearing ability but decreased elasticity (Ulmsten and Falconer, 1999, Goh, 2002). In the skin, ageing reduces collagen and elastin biosynthesis and leads to a degradation of biomechanical properties (Samsioe, 1998). The effect of age on muscle has been investigated in more detail- in fact, loss of muscle mass is one of the most consistent results of ageing. This loss of muscle mass is also documented for the urethral rhabdosphincter (Perucchini et al., 1997) and the levator ani (Koelbl et al., 1989).

1.5 Is there a genetic basis for female pelvic organ prolapse (FPOP) ?

It is assumed that genetic factors contribute to or predispose a woman to tre development ofFPOP and urinary incontinence, and such factors may in fact be the single most important determinant of such pathology (Van Dongen, 1981 ). This assumption is based on a number of observations. First- degree family history of FPOP seems to increase the risk of prolapse (Van Dongen, 1981), with a relative risk of 2.4 to 3.2 (Chiaffarino et al.,l999). Furthermore, FPOP in young women is familial in 30% (Rinne and Kirkinen, 1999), and seems to occur similarly in identical twins (Van Dongen, 1981).

23 1.5.1 Genetic disorders associated with FPOP

At the most extreme end of the spectrum of normality- abnormality are congenital connective tissue defects, the most common of which are Ehlers Danlos syndrome (EDS) and Marfan Syndrome (MFS). Both are heterogeneous conditions with a wide spectrum of clinical manifestations- from no symptoms at all to death in childhood due to fatal complications. Ehlers Danlos syndrome, consisting of joint hypermobility, cutis laxa and visceral manifestations such as cardiovascular, pulmonary, ocular and gastrointestinal problems, is thought to arise from collagen or elastin gene mutations (Burrows, 1999). Marfan syndrome (arachnodactyly, joint hypermobility, vascular complications) is attributed to mutations of the :fibrillin gene (Ramirez and Pereira, 1999). It is not surprising that both MFS and EDS patients seem to have a high risk of female pelvic organ prolapse (Carley et al., 1999b, Jabs et al., 1999). Joint hyper­ mobility in its isolated form is quite common, affecting about 5% of the overall population (Kirk et al., 1967, Jessee et al., 1980) and may be more prevalent in women with both vaginal and rectal prolapse (Norton et al., 1995, Al- Rawi and Al Rawi, 1982 and Marshman et al., 1987).

The case for minimal connective tissue dysfunction due to genetic factors is supported by the remarkable clinical and genetic heterogeneity of the abovementioned syndromes. A large number of patients with connective tissue disorders are probably undiagnosed due to minimal or absent symptoms and the great clinical variability of some ofthese disorders (Burrows, 1999). It appears likely that connective tissue quality varies markedly due to abnormalities in collagen/ elastin/ :fibrillin gene structure or sequence and that such genetic variations may predispose to or contribute to the development of pelvic organ prolapse. The development of abdominal herniae (Rinne and Kirkinen, 1999) and joint hypermobility (Norton et al., 1995) are both associated with FPOP, as are striae gravidarum (Sayer et al., 1990). Mo~t recently, an association has been found between osteoporosis, arthritis and symptoms of pelvic floor dysfunction (MacLennan et al., 2000). This finding is particularly interesting in view of studies showing a link

24 between certain forms of osteoporosis and early onset osteoarthritis on the one hand and genetically determined abnormalities of collagen (Prockop, 1998) on the other hand.

While there is limited information on the genetic basis of FPOP and while proper linkage or target gene studies have yet to be performed, it appears reasonable to assume a genetic contribution to the problem. The collagen, elastin and fibrillin genes are obvious target for further study. Over the last decade, 19 collagen subtypes have been identified, their over 30 genes sequenced and numerous mutations detected. Interactions with the extracellular matrix are complex and not well understood (Burrows, 1999). Ehlers Danlos Syndrome has been linked to COLI, COL3 and COL5 mutations (Burrows, 1999), Marfan Syndrome is associated with mutations of the fibrillin gene FBNI (Kilpatrick and Phylactou, 1998, Ramirez and Pereira, 1999).

1.5.2 Ethnic origin as a determinant of FPOP

For many decades, ethnic origin has been thought to play a role in the causation of incontinence and prolapse (Knobel, 1975, Zacharin, 1977, Van Dongen, 1980, Bump, 1993, Mattox and Bhatia, 1996, Burgio et al., 1996, Marana et al., 1999). Although most early papers on this subject are of limited use due to inadequate definition of the phenotype and the presence of confounders such as socioeconomic differences in populations, there remains the oft- repeated observation that connective tissue quality and patterns of pelvic floor dysfunction seem to vary between populations (Bump, 1993, Mallett and Bump, 1994). Since commencement of this project, a recent study has revealed racial differences for one of the main parameters measured here, i.e., bladder neck mobility on Valsalva manoeuvre (Howard et al., 2000a), demonstrating higher bladder neck mobility relative to the symphysis pubis in Blacks compared to Caucasians.

25 1.5.3 Collagen biochemistry and biomechanics

Differences between the connective tissue of prolapse patients and those not suffering from pelvic relaxation have been postulated for some time (Makinen et al., 1986, Morley et al., 1996). There may be changes in collagen biosynthesis and metabolism (Falconer et al., 1998a,b). In another study, Type III Collagen was found to be reduced in affected women (Bergman et al., 1994); recently, stress incontinent women were found to demonstrate increased expression ofmetalloproteinase 1 (MMP-1)-mRNA and decreased expression of mRNA of its tissue inhibitor TIMP-1 (Chen et al., 2002). Such differences may ultimately be due to genetic variations.

Another approach is the investigation ofbiomechanical properties such as the elastic modulus (stress- strain) or load -to- failure. Kondo et al. found that the shear strength of rectus fascia was significantly reduced in incontinent patients as compared to continent women (Kondo et al., 1994). The authors concluded that "some women suffering from stress incontinence may have a hereditary disorder ofbiophysical properties of ... tissues".

26 1.6 Assessment of the genotype of Female Pelvic Organ Prolapse

From a practical point of view, analysis of connective tissue genetics is limited to genes that have already been sequenced such as the collagen genes (1-19), elastin and fibrillin. There has been considerable interest in this field over the last decade, resulting in the discovery of the genetic basis of such diverse congenital conditions as osteogenesis imperfecta, Marfan syndrome, Ehlers Danlos syndrome and epidermolysis bullosa. Mutations of genes encoding for one of the collagen monomers can also be found in certain patients with osteoporosis and osteoarthritis (Prockop, 1998).

The inheritance of these diseases is often very complex. Elastin abnormalities, some of which lead to autosomal recessive disorders such as some forms of Ehlers Danlos Syndrome (EDS), also illustrate the concept of "haploinsufficiency" which means that the heterozygous state leads to a completely different clinical picture compared to the homozygous state. As mentioned, EDS in itself is highly heterogeneous, with some forms being due to Elastin mutations, others due to COL1A1, COL1A2 or COL3A1 abnormalities (Strachan and Read, 1999). Collagen mutations are often "dominant negative", which means that a structurally abnormal protein interferes with the function of normally fOrmed protein (Strachan and Read, 1999). Marfan's Syndrome also appears heterogeneous, with an autosomal dominant form of inheritance being common.

Connective tissue makeup is of considerable complexity. There are 19 known collagen subtypes, with widely varying distributions in virtually all body tissues. Collagens are synthesized in the endoplasmic reticulum as precursor molecules (procollagens). Posttranslational modification such as hydroxylation, glycosylation, rearrangement of disulfides occurs prior to assembly of three procollagen molecules into a homo- or heterotrimer which is then secreted into the extracellular space (Baum and Brodsky, 1999). After cleavage ofC- and N- terminal ends of the procollagen molecule, these are then assembled into collagen fibrils.

27 Each collagen fibril is therefore made up of interlinked monomers which themselves consist of three polypeptide chains that fold into a triple- helical configuration (Prockop, 1998, Baum and Brodsky, 1999). It is not just the coding for any ofthe building blocks of one collagen fibril that may be deficient- the problem may lie in the rate of biosynthesis and/ or posttranslational modification. This can result in faulty trimer formation or in faulty crosslinking of those trimers to collagen fibrils (Baum and Brodsky, 1999)- many different mechanisms may result in structurally suboptimal collagen fibrils. In Osteogenesis imperfecta, probably the best- researched collagen disorder, single base substitutions result in abnormal trimer formation. The disturbance in protein structure then interferes with mineralization (Baum and Brodsky, 1999).

Collagen may be the most important constituent of connective tissue but it is by no means the only. For the purposes of this study, two other components were regarded as likely candidates: fibrillin because of its association with Marfan syndrome, and elastin as it is a commonly found component of fascial and ligamentous structures. After extensive consultation with clinicians and molecular geneticists, five target genes were selected in order of likelihood for the detection of high- risk polymorphisms for female pelvic organ prolapse: COL3A1 (coding for one of the main chains of Collagen III), COL5A1 (coding for Collagen V), ELN (Elastin), COL1A1, (coding for Collagen!), and FBN1 (Fibrillin 1). As the population of the planned study was to contain its own control group (see below for phenotype assessment), the task at hand was reduced to determining allele frequencies in subgroups of the population.

Once a decision regarding the clinical methodology, i.e., regarding sample selection and acquisition has been made, a technology for testing DNA samples has to be identified. For known sequence variations, i.e., single nucleotide polymorphisms (SNP), TaqMan

TM (see 2.6.1) allows genotyping of a large number of samples in semiautomated fashion and appeared to be the method of choice for any SNPs obtained from public or proprietary databases. Usually there is no information on the prevalence of such SNPs in the population since they often are generated by computer on comparing a number of

28 clones with the consensus sequence, i.e., the sequence determined as representative of the normal human genotype. Such SNP databases are thought to contain a significant number of common polymorphisms, making them useful starting points for investigations such as the one undertaken here (Marth et al., 2001).

A relatively novel approach to the detection of known polymorphisms is Denaturing High Performance Liquid Chromatography (DHPLC). DHPLC identifies mutations by detecting sequence variation in reannealed DNA strands (heteroduplexes), see 2.6.1.5 for a detailed description. This method efficiently detects single nucleotide and insertion/deletion variation in crude PCR products directly without DNA sequencing. DHPLC is useful for finding novel mutations and polymorphisms in genes and other genomic regions and scoring samples for known mutations, and the method can also be used to semi-quantitatively analyse merged DNA samples for allele frequencies.As a result, the method markedly reduces the cost and effort of polymorphism/ mutation detection. Most recent developments such as the proprietary MassARRAY process (Sequenom™) foreshadow fully automated screening methods that will allow analysis of single or merged (population) DNA samples for hundreds of polymorphisms at one time; however, such methods were not available in Australia at the time.

1. 7 Assessment of the phenotype of Female Pelvic Organ Prolapse

Over the course of the last 15 years the author of this study has been involved in the development of an imaging method that has the potential to replace radiological imaging in incontinence diagnostics. Transperineal or translabial ultrasound demonstrates the changes associated with female pelvic organ prolapse and Genuine Stress Incontinence and is increasingly being used as a diagnostic method in urogynaecological evaluation (Kohom et al., 1986, Grischke et al., 1986, Koelbl and Bemaschek, 1989, Grischke et al., 1989, Dietz and Wilson, 1996, Schaer, 1997). Validation of this new method against the previous 'gold standard', static lateral

29 cystourethrography and/ or fluoroscopic imaging, has generally shown good agreement (Koelbl et al., 1988, Grischke et al., 1989, Voigt et al., 1994, Dietz et al. 1998). Not just the anterior vaginal wall, but also the central compartment (i.e., the cervix and uterus) and posterior vaginal wall descent can be evaluated simply and noninvasively (Creighton et al., 1992, Dietz et al., 2001c).

Quantitation ofFPOP has until recently been limited to clinical staging (I-III or IV). The recently proposed ICS (International Continence Society) POP-Q classification system (Bump et al., 1996) does allow for quantification; however, correlation with fluoroscopy (Kenton et al., 1997) and ultrasound (Dietz, 2001c) is suboptimal, especially for the posterior vaginal wall. This is likely due to the fact that any clinical assessment relies on surface anatomy changes (e.g. laxity of the posterior vaginal wall) relative to a mobile point of reference (the hymen) while imaging methods describe actual organ descent (such as descent of the rectal ampulla) relative to the bony pelvis. Magnetic resonance imaging has also been used for prolapse quantification (Comiter et al., 1999) although for reasons of cost this modality appears impractical at the present time.

At the same time, documentation of anterior and ventral displacement of the bladder neck provides a quantitative measure of the effectiveness of a pelvic floor contraction (Dietz et al., 1998). Interobserver variability for this measure is higher than for descent ofthe anterior compartment but still acceptable (see 3.1.1 and 3.1.2). Finally, colour Doppler ultrasound imaging allows the documentation of leakage without the use of ionizing radiation (Dietz et al., 1999b) although this modality was not regularly used due to technical limitations and the logistics of the study.

As a result of these developments in imaging technology, it has now become possible to assess the phenotype, i.e., pelvic organ mobility, in a non-invasive way and without the use of ionising radiation. Quantitation is easily obtained. This methodological improvement in assessing the phenotype of female pelvic organ descent or prolapse is

30 likely to improve our chances of being able to correlate phenotype and genotype and is further described in the Methods section (chapter 2.5.5). These developments in ultrasound technology should also facilitate the identification of labour- and delivery­ related trauma to pelvic floor structures, both fascial and muscular.

1.8 Hypotheses

Prior to study commencement, a total of 15 hypotheses were defined to be tested on datasets derived from the three patient visits, laboratory testing and delivery data.

1.8.1 First Visit: Early Pregnancy assessment

Data obtained at the time of the first assessment in early pregnancy was used to test hypotheses 1- 8. Hypotheses 1, 2, 5, 6 and 7 were designed to investigate potential congenital, genetic and hormonal aetiologi.cal factors of pelvic organ hypermobility. Hypotheses 3 and 4 sought to establish the relationship between bladder neck mobility and symptoms and signs of stress incontinence in pregnancy. Hypothesis 8 was defined in order to assess the role of the levator ani in the aetiology of female pelvic organ prolapse.

Hypothesis 1: 'Pelvic organ mobility varies in relation to ethnic origin'. Hypothesis 2: 'There is an association between clinical indicators of connective tissue quality Goint hypermobility, history of hernia, epistaxis and dislocations, family history of incontinence/ prolapse, striae gravidarum, pelvic girdle pain) and pelvic organ mobility as quantified by ultrasound measurements and/ or symptoms of stress incontinence'.

31 Hypothesis 3: 'There is a correlation between frequency of symptoms of stress incontinence and anterior vaginal wall mobility in pregnancy'. Hypothesis 4: 'There is a correlation between the presence and severity of stress incontinence on paper towel test and anterior vaginal wall mobility in pregnancy.' Hypothesis 5: 'There is a positive correlation between serum relaxin and/ or progesterone levels in the first trimester and pelvic organ mobility.' Hypothesis 6: 'There are significant differences between polymorphisms of Fibrillin, Elastin and Collagen genes observed in nulliparous women with marked pelvic organ mobility and those without' Hypothesis 7: 'Above genetic factors are associated with the symptom of stress incontinence during pregnancy and postpartum' Hypothesis 8: 'There is a negative correlation between levator ani muscle function (as defined by cranioventral displacement of the bladder neck on pelvic floor muscle contraction) and pelvic organ mobility'

1.8.2 Second Visit: Third trimester assessment

Data obtained at the time of the second visit in the mid- late third trimester was used to test hypotheses 9 and 10, with the latter also requiring data on delivery outcome. Hypothesis 9 was designed to define the impact of the increasing mechanical effect of uterine growth on pelvic organ mobility. Hypothesis 10 was introduced in order to test whether delivery- related factors were independent of antepartum pelvic organ mobility.

Hypothesis 9: 'Bladder neck, uterine and rectal mobility (as measured relative to the inferoposterior margin of the symphysis pubis) increase during pregnancy, i.e., between the first and the third trimester'. Hypothesis 10: 'Delivery mode is independent of pelvic organ mobility (as measured by third trimester ultrasound)'

32 1.8.3 Third Visit: Postpartum assessment

Data obtained at the last assessment 2-5 months postpartum and data on labour and delivery outcome was used to test hypotheses 11- 15. Hypothesis 11 was designed to confirm or refute a recently published study which claimed that bladder neck descent in pregnancy is a predictor for postpartum incontinence (King et al., 1998). Hypothesis 12 sought to confirm or refute previously reported associations between delivery- related factors and postpartum stress incontinence, while hypothesis 13 was designed to test for an association between postpartum stress incontinence and ultrasonic evidence of delivery- related traumatic change in bladder neck support.

Hypotheses 14 and 15 sought to define peripartum changes in pelvic organ mobility atrl levator function as related to explanatory variables defining the events of labour and delivery. These two hypotheses therefore constitute the core component of this study. Finally, Hypothesis 16 was designed to evaluate the usefulness of 3D pelvic floor imaging in the identification of pelvic fascial trauma.

Hypothesis 11: 'Bladder neck descent at first visit is a predictor of postpartum incontinence'. Hypothesis 12: 'The symptom of postpartum stress incontinence is associated with delivery mode, birthweight and/ or length of second stage'. Hypothesis 13: 'The symptom ofpostpartum stress incontinence is related to the peripartum increase in pelvic organ mobility'. Hypothesis 14: 'Mode of delivery, birthweight and length of the second stage of labour are associated with the extent of fascial trauma as defined by a peripartum increase in pelvic organ mobility.' Hypothesis 15: 'Operative delivery, higher birthweight and increased length of the second stage oflabour are associated with a postpartal reduction in levator strength as determined by translabial ultrasound.'

33 2 Patients and Methods

2.1 Setting and study design

The work reported in this thesis was performed between January 1999 and January 2002 at the Royal Hospital for Women in Randwick, a suburb of Sydney, Australia. The Royal Hospital for Women is a tertiary hospital providing Obstetric and Gynaecological services to the population of the Eastern suburbs of Sydney. The institution has a long and distinguished tradition going back to the foundation of the Benevolent Society of New South Wales in 1819. Pioneering developments included the first antenatal clinic in New South Wales in 1912 and the first clinical ultrasound unit in Australia in 1962. In the 1960s and 1970s, some of its most distinguished medical staff played a major role in the development of diagnostic ultrasound. In 1997, the Royal Hospital for Women moved to new purpose- built premises in co- location with the Prince ofWales Hospital in Randwick. The clinical work reported here was performed in the Antenatal Clinic, the Medical Imaging Department and the Endogynaecology Clinic of the hospital.

Due to the requirements of the author's subspecialty training, supervision was shared between Professor Michael J. Bennett, Professor of Obstetrics and Gynaecology at the Royal Hospital for Women, and Associate Professor Kate H. Moore, Urogynaecologist at St. George's Hospital, Kogarah. Funding was obtained from the Research Foundation of the Royal Australian and New Zealand College of Obstetrics and Gynaecology and Mayne Nickless Health, through a RANZCOG Research Fellowship. On application, this one- year fellowship was extended to 18 months in order to allow the author to complete the clinical part of the study. The Royal Hospital for Women Foundation also contributed to funding, as did the Trust Fund of the Department of Medical Imaging at the Royal Hospital for Women. Investigations into the genetic background of incontinence and prolapse were supported by a grant originating with the University of Queensland.

35 Laboratory work was performed both on- and off- campus. Hormone analyses were undertaken by the author in the Department of Endocrinology, Prince of Wales Hospital, under supervision of Dr. Shane Brown and Lillian Tan. White cell pellet production and DNA extraction were performed by the author while supervised by Drs. Michael Buckley and Dr. Peter Taylor, George Elakis and Anita Bahar, at the Molecular Genetics Laboratory, Prince of Wales Hospital. DNA standardization, PCR and DHPLC work was undertaken at the Queensland Cancer Fund Transgenic Laboratory, Queensland Institute of Medical Research, Brisbane, Australia, under supervision of Drs. Georgia Chenevix- Trench and Mandy Spurdle, Livia Kelemen, Xiaoqing Chen, James Flanagan and Jeremy Arnold.

The study was planned as a prospective observatioml clinical study. The intention was to follow a cohort of 200 women, to be recruited over the course of one year, through their first ongoing pregnancy. Outcome parameters are both clinical (symptoms, signs of urinary incontinence and prolapse) and investigational (ultrasound quantification of pelvic organ descent and levator function, residual urine, paper towel test). For power calculations the author utilized the results of a pilot study performed under his supervision at the Department of Obstetrics ard Gynaecology, Dunedin School of Medicine, Dunedin, New Zealand, in 1997 (see 2.7.1). This pilot study showed substantial differences between primiparous women delivered by caesarean section and Forceps/ Vacuum delivery for bladder neck descent on Valsalva manoeuvre (Safari, 1997).

The main investigational outcome parameters of this study were derived from imaging data. As mentioned above, anatomical alteration of pelvic organ supports is thought to be the basis for later symptoms of stress incontinence and prolapse, just as peripartal disruption of the anal sphincter is thought to be at least partly responsible for future symptoms of faecal incontinence. In both cases, the ultimate proof of a causal relationship may require an observation period of decades due to the fact that both problems, stress urinary incontinence and faecal incontinence, may arise only after a

36 long latency. However, just as there is good evidence for a strong association between anal sphincter trauma and faecal incontinence (Sultan et al., 1996), there is now evidence of a similar association between bladder neck descent and stress incontinence (Dietz et al., 2001 b). A more in- depth discussion of the selection of outcome parameters is provided in section 2.5 .5 .1.

Urodynamic testing was considered in the planning phase of this study. However, bladder catheterisation in pregnancy for research purposes raises ethical questions. Also, the author was of the opinion that standard filling and voiding cystometry would add very little information and contribute nothing to the testing of the hypotheses in question. Finally, invasive interventions are likely to decrease patient return rates, impairing the reliability of obtained data and reducing power. The considerable additional effort and expense of cystometry appeared counterproductive to the aims of the study, an assessment that has been confirmed by recently published fmdings of urodynamic investigations in pregnancy and puerperium (Chaliha et al., 2000). The author of this study performed by the Urogynaecology Unit of StGeorge's Hospital, London, speculates that urodynamics may "not be an appropriate method to assess bladder function in pregnancy" and suggested ultrasound imaging and urethral pressure profilometry as alternative methods to be considered.

Neurophysiological investigations may also shed further light on the pathogenesis of female pelvic organ prolapse and GSI. However, even the most accepted and well­ researched of those tests, pudendal nerve terminal motor latency (PNTML), suffers from methodological problems and "says little about the function of nerve and muscle" (Vodusek, 2000). Of course, all the disadvantages of invasive testing also apply.

2.2 Selection of patient population

Lifestyle (e.g. obesity, smoking, bowel dysfunction) and the effects of pregnancy and delivery are likely to exert a strong influence on the manifestation of FPOP. It was

37 therefore planned to assess patients prior to the impact of most of these factors. Nulliparous pregnant women were selected as target population for both pragmatic and theoretical reasons. They are an easily approached group and, at this particular time in their lives, especially interested in issues related to childbirth. Blood samples are taken repeatedly as part of clinical routine. Ultrasound is a routinely used diagnostic modality in pregnancy and therefore familiar.

However, theoretical reasons also favour this group. Joint hypermobility may be associated with pelvic organ prolapse (AI Rawi and AI Rawi, 1982, Marshman et al., 1987, Norton et al., 1995) and may become more pronounced in pregnancy (Calguneri et al., 1982). If this were indeed true one could speculate that the hormonal effects of pregnancy may act as an amplifier for a preexisting connective tissue weakness, enhancing this weakness and making it more clinically apparent.

Repeated attempts were made to recruit a subgroup of women immediately after a first positive pregnancy test and after the confirmation of an intact intrauterine pregnancy; however, these attempts were largely unsuccessful due to logistical constraints. Further studies in this field should be designed to demonstrate any effect of early pregnancy on the indices of pelvic floor mobility.

2.3 Time points

In order to measure changes in pelvic orgm mobility over the course of pregnancy and the puerperium, and to test the hypotheses given in 1.8, three time points were selected:

1.) During the first trimester, i.e., shortly after booking visits in the antenatal clinic or other primary contacts with health services at our institution. In order to recruit patients in the first half of the first trimester, private obstetricians, the Imaging Department and the Infertility Unit at the Royal Hospital for Women were approached as well.

38 It is recognized that the relatively late booking of the majority of patients at our institution may impair the power of this study to assess hypothesis 1 (see 1.8.1). However, timing was found to be optimal for the assessment of serum relaxin levels which peak between 10 and 14 weeks. 2.) During the mid- to late third trimester, i.e., between 32 and 38 weeks' gestation, in order to maximize the possible impact of late pregnancy without running the risk of too many patients delivering before their scheduled visit. The mechanical effects of uterine and fetal growth have long been thought to impair pelvic organ support (Nichols, 1991). 3.) 12-16 weeks after the delivery, to document the effect oflabour and delivery as soon as possible after healing of any acute pelvic or perineal trauma sustained during childbirth. Women with symptoms of stress incontinence at three months postpartum are very likely to suffer from such symptoms in the long term (Viktrup and Lose, 2000). Recovery of neural function, i.e., reinnervation of muscular structures, should be largely complete 2 months after the delivery (Juenemann and Thuroff, 1994, Lee and Park, 2000). In one small study of postpartum levator function there were no differences between 2 months and one year postpartum (Peschers et al., 1997). Breastfeeding is expected to still influence the hormonal status at this stage. However, from a logistic point of view followup examinations may become more difficult once the baby starts to become mobile, and the above timing was deemed likely to maximize participation.

39 2.4 Patients

A consecutive series of 206 women in their first ongoing pregnancy were approached through Antenatal Clinic at the Royal Hospital for Women and recruited for the study. Figure 2.1 shows a flow chart of the study population and subsets of patients which took part in secondary projects such as the analysis of hormone levels in early pregnancy.

Recruited for 1st appointment: 206 6 excluded (twins, missed abortion, spina biflda)

First visit: 200

Molecular Genetics: 3-Dimensional Ultrasound Relaxin/ Progesterone 200 recruited 47 recruited 50 recruited 199 DNA extracted 23 seen ante- and postpartum 50 samples 188 Genotypes obtained 21 volumes analyzed 49 samples analyzed

Second visit: 173

Third visit: 169 Labour and delivery information available on 169, 2nd and 3rd visit data available on 161 women

Figure 2.1 : Flow chart of study population and subgroups.

40 The number of recruits had been determined by a power calculation, as will be described in 2.7.1. Both direct contact as well as mailouts and followup phone calls were used to acquaint women with the study. First appointments were usually arranged within one week of the booking visit and occurred between 6 and 18 weeks' gestation (see Fig. 3.1). The extremes in the distribution of gestational ages are mainly due to the patient's dates being corrected at the time of her first appointment.

2.4.1 Inclusion criteria were • First ongoing pregnancy (prev. fust trimester termination of pregnancy or miscarriage was no exclusion criterion) • no known urinary problems having required medical treatment except urinary tract infections, • ability to remain in study up to 6 months postpartum

2.4.2 Exclusion criteria were • Inability to give informed consent, • neuromuscular disorders of any kind, • other significant medical problems impairing ability to take part, • inadequate understanding of written and spoken English

One patient with spina bifida was recruited by mistake and excluded from the analysis. 3 patients were found at the time of the fust ultrasound to have a missed abortion and were excluded. 2 twin pregnancies were detected at the time of the frrst ultrasound and also excluded, leaving 200 women for analysis.

41 A subgroup of 50 patients was recruited for a study extension in order to correlate serum relaxin levels at 10- 15 weeks with pelvic ligament relaxation in a pilot project. One sample was lost, leaving 49 serum samples for analysis.

Another subgroup of 47 women were also recruited for a pilot project of the use of Volume or 3D ultrasound in the detection oftraumatic pelvic floor injury after childbirth, and 23 of those patients were seen both ante- and postpartum. The number of repeat (i.e., postpartum) visits was exclusively determined by equipment availability, as the Toshiba PowerVision 8000 system used for this purpose was provided free of charge by Toshiba Australia for a total of 6 weeks.

2.5 Clinical methods

Women were seen for their first visit at 6-18 weeks' gestation, the second visit at 32- 38 weeks' gestation and for the third and last visit at 2-5 months postpartum. Appointments were arranged by the author, at first in the Antenatal Clinic of the hospital, later in the Imaging Department (Head: Dr. Peter Warren) and the Endogynaecology Department (Head: Associate Professor Thierry Georges Vancaillie) depending on the availability of space and equipment. Repeated attempts were made to recruit patients in the early first trimester, through infertility clinics and the ultrasound department; however, these attempts proved largely :fruitless due to a lack of support from other staff and limited resources for approaching patients directly. All gestational ages were confirmed or corrected by ultrasound biometry (crown rump length or biparietal diameter) at the time of the first study appointment.

2.5.1 Interview

A standardized interview form was used for all three visits (see appendix). Ethnicity was documented for the generations of parents and grandparents. Routine antenatal data such as the estimated date of delivery, prepregnancy weight, height, medical problems

42 and drug treatment, nicotine use and sports activities were obtained from the antenatal notes and confirmed with the patient. As regards sports, a simple categorisation was attempted, with category 1 signifying occasional hobby sports, category 2 regular hobby sports, category 3 implying daily sporting activity and category 4 signifYing regular participation in competitive sporting events.

Urinary and bowel symptoms were registered in standardized fashion, using categorization to determine severity of symptoms such as stress and urge incontinence, urgency, frequency and nocturia (see datasheet, appendix 8.1). Voiding dysfunction and bowel symptoms were also queried.

As regards pelvic floor muscle use and function, women were asked whether they had had any previous teaching (and, if yes, of what nature) and whether they were in the habit of using the pelvic floor muscles at any other time, such as on intercourse. Questions regarding the motivation for use of the pelvic floor muscles on intercourse were also asked but not consistently documented. In order to detect individuals with a history suggestive of connective tissue dysfunction, women were asked whether they had in the past suffered from dislocations of peripheral joints, hernias or epistaxis. At the third and final appointment, women were asked to use a visual analog sliding scale to describe the degree of pelvic girdle (back and symphyseal) pain experienced in late pregnancy and the puerperium, excepting the delivery itself. The reading was recorded as a number between 0 and 100.

2.5.2 Family history

Patients were questioned regarding bladder problems, prolapse and operations to cure such complaints in female relatives such as sisters, mothers, aunts and grandmothers. All women were encouraged to mention the study to family members and, if possible, obtain more detailed information. All questions regarding family history were repeated at the time of the second and third visits to increase the likelihood of accurate reporting.

43 2.5.3 Joint mobility and striae gravidarum

Joint mobility varies markedly in the general population. Women mostly have more mobile joints compared to men, and mobility decreases with age (Beighton et al., 1973). Ethnic background may play a role, and joint hypermobility is associated with syndromes that are known to be due to congenital abnormalities of collagen or elastin synthesis or strt.I;ture such as Marfan and Ehlers Danlos syndromes. The method for the assessment of joint mobility was a modification of a commonly used technique (Beighton et al., 1973). The following parameters were recorded:

1.) passive dorsiflexion ofthe little fingers in the metacarpophalangeal joint, assessing flexor tendons and the palmar aponeurosis, 2.) passive apposition of the thumbs to the flexor aspect of the forearms, assessing extensor tendons and collateral ligaments, and 3.) hyperextension of the elbow joint, assessing flexor tendons and joint capsule.

Numerical values for goniometer readings were recorded. The patient was not instructed as to which hand or arm to use. However, in the presence of deformity or previous trauma the healthy limb was used.

Stretchmarks surrounding the umbilicus were recorded at the second and third appointment and counted on the right and left anterior abdomen, along an imaginary line connecting the umbilicus with the anterior superior iliac spine (see Fig. 2.2). Only stretchmarks crossing this line were counted and added for a final score.

44 I I, I

Figure 2.2: Counting of stretchmarks along a line between umbilicus and anterior superior iliac spine. In this example the count on the right lower abdomen is 7. This count was repeated on the oppo ite side and added for a final score.

2.5.4 Paper towel testing

Women were asked to not void approx. 2 hours prior to their appointment. Before bladder emptying, women were asked to perform a paper towel test as described by Miller (Miller et al., 1998). This requires the patient to place a folded paper towel between her legs after undressing, and then to cough and jump three times. Quantification is undertaken by measuring the two maximum diameters of a wet area. In women with a positive paper towel test, a 24 hr pad test (Versi et al., 1996) was given out so as to obtain more accurate data regarding severity of leakage. However, this was abandoned after approximately 3 months due to poor compliance.

45 2.5.5 Ultrasound methodology

Over the course of the last 15 years the author has been involved in research in female pelvic floor dysfunction, mainly surrounding the development and subsequent use of diagnostic imaging methodology in women with female pelvic organ prolapse and urinary incontinence. This work began in the context of an MD thesis undertaken between 1986 and 1989 at the University of Heidelberg Medical School, Germany, and continued later in several locations during the author's postgraduate medical training in Germany, New Zealand and Australia.

Originally, the purpose of the author's MD thesis at Heidelberg University was to evaluate the application of a new diagnostic method both in clinical Obstetrics and in Gynaecology (Grischke et al., 1986, Grischke et al., 1989, Dietz 1989). While this new method, i.e., translabial or transperineal ultrasound (terms which are used synony­ mously), can be used for the detection of Cervical Incompetence (Grischke et al. 1988) and Placenta Praevia (Dietz et al., 1991), its main application proved to lie in Urogynaecology (Griscke et al., 1989).

After emigration to New Zealand the author took up work in this field again when the opportunity arose on joining the unit of a renowned Urogynaecologist, Prof. Peter Donald Wilson in Dunedin, New Zealand. Several publications arose from this cooperation, most of which were concerned with further defining and evaluating the methodology of ultrasound cystourethrography (Dietz et al., 1996, 1997, 1998). This work was continued in Brisbane and Sydney, Australia. The influence of patient position and bladder filling were assessed (Dietz et al., 1999, 2001a), levator activity quantified (Dietz et al. 1998) and new imaging modalities such as Colour Doppler imaging evaluated for the detection of urine leakage (Dietz et al., 1999). The ultrasound measures used as outcome parameters in this study are the direct result of this development process.

46 2.5.5.1 Which outcome parameters should be chosen?

The main outcome parameters used in the study presented here were ultrasound indices of pelvic organ mobility on Valsalva manoeuvre. Symptoms and signs of pelvic floor dysfunction (such as incontinence and prolapse) were assessed as secondary outcome parameters. The author believes that there are considerable advantages to this approach. Analogous to the situation with faecal incontinence (Sultan et al., 1994 and 1996, Rieger et al., 1997, Damon et al., 2000), symptoms of urinary incontinence and prolapse may only appear decades after the original traumatic event. Evidently, damage to anatomical structures, whether it be nerves, muscle or fascial tissues, can be masked for decades by compensatory mechanisms, until such mechanisms fail- e.g. around the time of menopause. It therefore appears necessary to identifY and use intermediate outcome parameters such as ultrasound evidence of anal sphincter trauma (Rieger et al., 1997, Damon et al., 2000).

Mobility of the bladder neck is strongly associated with Genuine Stress Incontinence in urogynaecological patients (Dietz et al., 2001b). Figure 2.3 shows odds ratios for the diagnosis of GSI on fluoroscopy relative to bladder neck descent as measured on blinded translabial ultrasound. It was concluded from this study (Dietz, 2001 b) that increased bladder neck mobility is likely to be the single most important determinant of Genuine Stress Incontinence in the nonpregnant female. Increased mobility of the bladder neck is also more common in young parous women than incontinence itself, just as anal sphincter trauma seen on transanal ultrasound is more common than faecal incontinence. Finally, just as there is a strong association between anal sphincter defects and parity (Sultan et al., 1994, 1996), there also seems to be a strong association between parity and bladder neck mobility (Dietz et al., 2001 b). Bladder neck mobility therefore appeared to be potentially useful as an intermediate outcome measure since increased mobility seems to signifY an alteration of pelvic floor support structures associated with stress incontinence. Any factor that would affect bladder neck mobility,

47 it may be surmised, may also be expected to influence the likelihood of stress incontinence, at least in the long term.

A second argument for the substitution of intermediate outcome parameters is the nature of 'complex diseases'. Approaching this problem from the point of view of a molecular geneticist, Wright states: "One distinction between Mendelian and genetically complex disorders is that the latter often result from a combination of unfavourable intermediate phenotypes ...." and, one should add, a multitude of environmental determinants. It may therefore be preferable to "study the intermediate disease endpoints rather than the disease per se, because the disease state can be a complex and insensitive indicator of underlying pathogenic processes" (Wright et al., 1999). One of the prime models of this approach is osteoporosis, for which bone mineral density has been used as an intermediate phenotypic endpoint (Hobson and Ralston, 2001).

150,_------...,

100 ~

OR

50

0 10 20 30 40 50 60 rnm Bladder Neck Descent

Figure 2.3: Odds ratio (OR) for the diagnosis ofUrodynamic Stress Incontinence amongst 179 nonpregnant urodynamic patients relative to Bladder Neck Descent (n= 179), from Dietz 2001b.

48 As regards the other main manifestation of pelvic floor dysfunction, i.e., Pelvic Organ Prolapse, organ descent on imaging correlates well with clinical assessment of pelvic organ prolapse (Dietz et al., 2001c) and may in fact be superior to clinical assessment due to the use of fixed, bony points of reference rather than the soft tissue of the introitus or the hymenal remnant which are in themselves mobile.

In order to determine the reliability of the chosen outcome parameters, intra- and interobserver variability were investigated in preparation for the main study and reported as coefficie:rts of variation (%CV, Armitage and Berry, 1994). Results of these tests are reported in section 3.1.

2.5.5.2 Assessment methods

Translabial ultrasound was performed within 5 minutes of bladder emptying and in the supine position to maximize pelvic organ displacement on Valsalva (Martan et al., 1999, Dietz et al., 1999, 2001a). For a detailed discussion ofthe choice of methodology, see pages 143- 145. 3.5-5 MHz curved array transducers were used on a variety of commercially available ultrasound equipment (Toshiba EccoCee, Hitachi EUB 240, ATL HDI 1000, Dornier AI 5200). Since electronic calipers are standardized for reproducibility according to industry- wide standards, measurements are generally regarded as comparable between systems (Kremkau, 1998). The development of imaging methodology was supervised by Professor M.J. Bennett, Royal Hospital for Women, Randwick.

The probe was covered with a glove and ultrasound gel and placed on the perineum in a sagittal direction. This technique results in imaging ofthe symphysis pubis anteriorly, the urethra, bladder neck, vagina and cervix centrally, and the rectum and anal canal posteriorly (see Fig. 2.4 to Fig. 2.6 for illustration of some measured parameters). A cough may result in these structures becoming more clearly visible. If labia and/ or pubic hair impairs image quality, the labia may be parted, resulting in a more direct

49 contact between the perineum/ anterior vaginal wall and the ultrasound probe. Once the probe is placed correctly, the urethra is identified by moving the transducer from sagittal to parasagittal positions until the urethra is visible in its entirety. If imaging of the cervix, rectum or anal canal is desired, the probe may have to be angled slightly more laterally. However, all data acquired for this study was obtained from sagittal and parasagittal planes. Table 2.11ists all assessed ultrasound parameters of pelvic organ mobility.

RVA-R Retrovesical angle at rest RVA-S Retrovesical angle at stress/ on Valsalva ROT Rotation ofproximal urethra on Valsalva BSD-RV Vertical distance between bladder neck and inferoposterior border of symphysis pubis at rest BSD-SV Vertical distance between bladder neck and inferoposterior border of symphysis pubis on Valsalva BND Bladder neck descent (vertical) on Valsalva BND-H Bladder neck displacement (horizontal) on Valsalva Oblique displacement Bladder neck descent (oblique or total) on Valsalva Cystocele Maximal caudal displacement of the posterior bladder wall on Valsalva, relative to inferior border of symphysis pubis. Cervix Maximal caudal displacement of the leading edge of the cervix uteri on Valsalva, relative to inferior border of symphysis pubis. Rectum Maximal caudal displacement of the rectal ampulla on Valsalva, relative to inferior border of symphysis pubis.

Table 2.1: Parameters used to describe pelvic organ position and descent on transperineal ultrasound.

50 urethra \

cranial

Fig. 2.4: Field of view for translabial ultrasound (midsagittal orientation).

at rest

Fig. 2.5: Parameters meastred for assessment of the anterior vaginal wall: BND is descent of the bladder neck on Valsalva. RVA-R is the retrovesical angle (between proximal urethra and trigone) at rest, RVA-S is the retrovesical angle on straining.

51 Bladder contour on valsalva at rest

cranial

Fig. 2.6: Parameters meastred for assessment ofthe anterior vaginal wall: "Rotation" is rotation of the proximal urethra on Valsalva, "Cystocele" is maximal bladder descent on straining, relative to the inferior border of the symphysis pubis.

2.5.5.3 Valsalva manoeuvre

The patient was asked to cough and strain. Since straining results in more marked displacement of the bladder neck than a cough (Peschers et al., 2001), the latter was used to document mobility. Verbal feedback was given in order to optimize the Valsalva manoeuvre. To reduce the effect of varying Valsalva pressure, the results of at least three Valsalva manoeuvres were measured on- screen and documented by printout. The best (i.e., the most effective in achieving descent) manoeuvre was used for analysis.

The position of the bladder neck, leading edge of a cystocele or posterior bladder base, the cervix and rectal ampulla were determined relative to the inferoposterior margin of the symphysis pubis (see Fig. 2.4 for schematic drawing, Figs. 2.7- 2.10 for examples of findings and measurements). Numerical fmdings for descent of the anterior and posterior vaginal wall as well as for the cervix were recorded relative to the inferopo-

52 sterior margin of the symphysis pubis which is easily identified in the midsagittal view (see Figs. 2.7- 2.10). Rotation of the proximal urethra on Valsalva was measured by comparing urethral inclination to the vertical (incident beam) at rest and on Valsalva. In practical terms, the angle between proximal urethra and the incident beam was measured both at rest and on Valsalva and the first measurement (e.g. 20 degrees) deducted from the second (e.g. 70 degrees), resulting in a value for rotation of 50 degrees.

Total bladder neck descent in a dorsocaudal direction was calculated with the help of the formula

Oblique descent (Valsalva)= Square root ((BND vertical/+(BND horizontazj),

treating oblique descent as the hypotenuse of a right- angled triangle. In the presence of reflex levator activation on Valsalva manoeuvre, which was particularly noticeable and common in late pregnancy, attempts were made to make the patient aware of the effect ofboth manoeuvres. Great care was taken to obtain an unimpeded Valsalva manoeuvre. The confounding effect of levator activation, in particular in nulliparous women, has been noted by other investigators (Peschers et al., 2001) and is a phenomenon that midwives and obstetricians are familiar with. It was at times necessary to attempt tiring the levator by asking the patient to perform a prolon­ ged levator contraction. The prompt "try to push the transducer away from you" also can help overcome involuntary levator activation and help the patient relax.

In very few instances (<1%) no Valsalva manoeuvre without levator activation could be obtained despite best efforts. In such cases, multiple coughs were used with a cine loop function (storage of at least 32 consecutive frames which can be examined after freezing the on- screen image) to obtain an approximation of the effect of a Valsalva manoeuvre. Generally however, the displacement obtained with coughing is less than the displacement resulting from a correct Valsalva manoeuvre (Howard et al., 2000).

53 At both followup visits patients were asked at the outset to not divulge any information relating to previous visits or, in the case of the postpartum appointment, any information relating to labour and delivery mode. As the patient covered her abdomen with a drawsheet prior to the ultrasound examination, the examiner remained blinded to delivery mode until after completion of the final ultrasound assessment.

As regards the choice of reference point for measurements of pelvic organ displacement, both the central axis of the symphysis pubis (Schaer et al., 1995, Meyer et al., 1996) as well as the inferoposterior margin or anterior inferior border of the symphysis pubis (Grischke et al., 1989, Creighton et al., 1992, King et al., 1998, Wijma et al.,2001) have been described. The author has chosen the latter method for reasons of

practicability. The main disadvanta~ of a technique utilizing the inferoposterior margin is that, in principle, an angle error may be introduced by rotating the transducer around the horizontal axis during Valsalva, which is most likely in women with marked descent. Such unwarranted transdooer movement is however easily controlled for and avoided as a change in the main axis of the symphysis pubis is readily apparent on printouts. While it is possible to avoid any potential angle error by using the central axis of the symphysis as reference (Schaer et al., 1995), the author has found this approach impractical due to the fact that the whole symphysis pubis has to be imaged, losing view of the posterior compartment. The Schaer method requires resident software that allows angle measurements, ani the central axis of the symphysis pubis may be difficult to obtain with any accuracy in elderly women due to calcification of the interpubic disc. For a discussion of this issue, see also page 143.

The central and posterior compartment can be assessed without any modification of the methodology presented above. Figure 2.8 demonstrates descent of the rectal ampulla and shows the typical appearances of a rectocele distended by stool. As regards the cervix, it is more easily visualized in pregnancy due to increased size and the increased prominence of cervical glands. The more descent of the cervix, the more easily visua­ lized it is (see Fig. 2.9). A high cervix and a small rectocele is demonstrated in Fig. 2.10

54 Fig. 2.7: Translabial view ofValsalva manoeuvre: images at rest (1) and on Valsalva (r). Arrow indicates bladder neck. BND is 22.4 - (- 3.lmm) = 25.5 mm.

Fig. 2.8: Measurement of bladder neck position at rest and on Valsalva, relative to the inferoposterior margin of the symphysis pubis (white arrow). BND = 34 mm- 28.3 mm =5.7 mm. There is a rectocele to 12 mm below the symphysis pubis (black arrow).

55 Fig. 2.9: Measurement of descent of the uterine cervix relative to the inferoposterior margin of the symphysis pubis. Cervix descends to 4.3 mm below ths symphysis.

Fig. 2.10: First degree rectocele to - 10 with high cervix at +44 and bladder neck at+8. The horizontal line represents the level of the inferoposterior margin of the symphysis pubis.

56 In this study, cervix and rectal ampulla could not be reliably imaged in a small number of patients (4% of cases for the cervix, 6% for the rectal ampulla). Failure to image the cervix may be due to a very high, posterior cervix or a large rectocele which can completely obscure the cervix. The rectal ampulla may be difficult to identify in the absence of rectal filling and in women with excellent posterior compartment support.

For a more extensive description of the assessment of central and posterior compartment descent as well as a validation of the method by comparison with clinical prolapse assessment, see a previous publication of the author (Dietz eta., 2001c).

2.5.5.4 Pelvic floor muscle (=levator ani) activation

The role of the levator ani complex in the preservation of urinary continence in the female, i.e., its role in functional anatomy, is not well understood. Clinicians looking after women with incontinence and/ or prolapse often do not even assess levator function although pelvic floor muscle exercise training is the mainstay of conservative management ofboth stress and urge incontinence (Wilson and Herbison, 1995, Wilson et al., 1999, Mantle, 2001). The role of pelvic floor muscle exercises in the management of female pelvic organ prolapse is less well defmed although it is assumed that levator muscle training may result in a narrowing of the urogenital hiatus, therefore reducing symptoms of prolapse and possibly slowing progression..

The translabial ultrasound parameter used to define levator function in this study, cranioventral displacement of the bladder neck on maximal levator activation, has been shown to correlate well with palpation and perineometric assessment of levator strength (Dietz et al., 2002a). Its main advantages are the fact that it is completely noninvasive (particularly appreciated in pregnancy), and the ease with which biofeedback teaching can be undertaken.

57 For the purpose of assessment oflevator function, patients were asked to perform a pelvic floor muscle contraction. If a woman was unable to do so, standard help was given, asking her to "attempt to stop the flow of urine or stop wind from escaping, squeeze and pull up". Three effective contractions were documented on videoprinter, with the displacement of the internal meatus relative to the inferoposterior margin of the symphysis pubis measured in two dimensions. Oblique displacement was calculated according to the same principle as oblique displacement of the bladder neck on Valsalva (see 2.5.5.4), treating oblique displacement as the hypotenuse of a right- angled triangle. The most effective levator contraction (i.e., the one resulting in the highest numerical displacement of the bladder neck) was used for analysis. Figure 2.11 (see page 67) shows a typical pelvic floor contraction as documented by translabial ultrasound.

The presence of a reflex levator activation at least once on at least three episodes of coughing was also noted. As a postural muscle, the levator complex would be expected to automatically (reflexly) compensate against sudden increases in intraabdominal pressure. Despite the logic behind such an assumption it has been claimed that reflex levator activation on coughing is a learned response ('the knack') and not a reflex or involuntary response (Staskin, 2001). Another authority has claimed that automatic levator activation with coughing is rare (Schuessler 1994).

At the first visit, patients were asked whether they had had any previous instruction and whether they consciously used their pelvic floor, e.g., on intercourse. Intercourse may be a hitherto underestimated everyday trigger of pelvic floor activity. Anecdotally, the author had previously observed unexpectedly strong levator activity in older women who reported that they commonly contracted the levator muscle on intercourse.

There is no data in the literature on the prevalence of such practices although it could potentially explain part of the marked variation observed between individuals. Most young women in Australia first become acquainted with pelvic floor function through the efforts of popular magazines emphasizing the use of the levator muscle group on

58 intercourse, promising them and their partners new dimensions of sexual fulfillment. Regardless of whether this is in fact true, it appeared prudent to further investigate this issue. Answers were recorded as 'yes' or 'no'.

The pelvic floor assessment as outlined above was performed in identical fashion at all three appointments. Subsequent to the first pelvic floor assessment a transabdominal early pregnancy ultrasound was carried out, confirming vitality and dates and excluding twin pregnancy. At the time of the second assessment, only fetal vitality was confirmed. No additional abdominal scanning was undertaken at the time of the third appointment.

2.5.5.5 Intra- and interobserver variability of ultrasound measurements

At the outset of this project, intraobserver variability and two forms of interobserver variability were determined for three randomly selected groups of20, 20 and 47 patients (see 2.7.3 for statistical aspects). This activity was seen as crucial since ultrasound measurements, regardless of the context, are recognized as highly operator- dependent. The colleague with whom the author undertook both interobserver series, Dr. Anneke Steensma, an Obstetrician and Gynaecologist and Fellow at the Endogynaecology Department, Royal Hospital for Women, Sydney, had previously undergone more than 6 months' training in the use oftranslabial ultrasound. During this time she had assessed over 100 patients under supervision of the author. This teaching resulted in a similar examination and evaluation technique being used by both examiners.

Common pitfalls that could be expected to increase interobserver variability between different examiners are the instructions that are given for Valsalva manoeuvre and pelvic floor contraction. It also is crucial to pay attention to such confounders as bladder filling, the presence of a full rectum, and unwanted levator activity. Scanning should be undertaken in the supine position and after bladder (and, if necessary, bowel) emptying, a technique which maximizes descent of pelvic organs with a Valsalva manoeuvre (Dietz et al., 1999b, Martan et al., 1999, Dietz et al., 2001a) . Any teaching in the

59 technique oftranslabialpelvic floor assessment has to assess these factors if :findings are to be reproducible between examiners.

Intraobserver variability was determined by the author obtaining two different Valsalva manoeuvres and levator contractions (first series, n=20). Interobserver variability was tested in two different ways: in the second series (n=20), the same stills for Valsalva and levator contraction were used for on-screen measurements by both the author and Dr. Steensma, in a blinded fashion. In a third series (IF 47), both the author and Dr. Steensma independently and in a blinded fashion asked the patient to perform Valsalva manoeuvres and a levator contraction, both selecting the best of at least three manoeuvres and performing on- screen measurements. This last series, giving true interobserver variability, describes the variability of individual examination technique in addition to the variability ofValsalva manoeuvres and the variability of evaluation of stills. Results are given in detail in 3.1.1- 3.1.2; Figure 2.12 demonstrates a dotplot of BND measurements obtained in this interobserver series.

50 • 40 • N,__ • • CJ.) • • • • 2: 30 • ••• CJ.) • (/) ..0 ••• • 0 • •••• • • • • 0 • • • • z • • • • co ••• • 10 • • • • • • • t 'IIi II' Ill' I 1 Ill 1 IIi I ill 1: Ill!' I IIi I i 111111! IIi i I' 1 '10 20 30 40 5() BND Observer 1

Fig. 2.12: Dotplot of repeat measurements ofbladder neck descent obtained by two blinded examiners within the same hour (n= 47). Scales in mm. Coefficient of variation 0.21, Intraclass correlation coefficient 0.79, implying excellent agreement.

60 2.5.5.6 Standardization of Valsalva manoeuvre

Valsalva manoeuvres were standardized by giving patients a set of standard instructions (women were asked to 'do their best', i.e., perform a maximal push) and by using the most effective of at least three Valsalva manoeuvres for numerical evaluation as explained in 2.5.5.3. The author is not aware of any other method, published or otherwise, that would reduce the influence of the two most important confounders, i.e., Valsalva force and levator coactivation, to a similar or superior degree.

The option of numerical standardization of Valsalva force by measurement of Valsalva pressure was deliberated extensively prior to study commencement. True intraabdominal pressures can only be obtained invasively, i.e., with pressure sensors placed in the stomach by swallowing or in the vagina or rectum by manual insertion (Howard et al., 2000). The use of such methods would have affected recruitment considerably and was therefore decided against, in particular as there is no published data available to demonstrate better reproducibility or accuracy of an assessment method using pressure sensors.

In order to avoid invasive techniques, attempts have been made to standardize Valsalva force with the help of spirometric devices (King et al., 1998). The pressures used to standardize Valsalva manoeuvres in this study were very low in comparison to a normal cough or spontaneous straining (30 mm Hg vs. 60- 120 mmHg as shown by Enhorning, 1976, Howard et al., 2000, and Peschers et al., 2001) and probably insufficiently provocative. It has recently become clear that Valsalva manoeuvres have to reach a strength of about 60 em H20 to provide for close to maximal displacement of the bladder neck (Martan et al., 2001). The vast majority of women will attain Valsalva pressures within the plateau part of the pressure- displacement curve, i.e., well above 60 em H20, which means that formal standardization ofValsalva pressures may be quite unnecessary (Martan et al., 2001).

61 Finally, it may be argued that a spirometric technique will at best measure intrathoracic pressures, with diaphragmatic, abdominal wall and pelvic floor contractions being obvious confounders. Such a technique may in fact encourage reflex levator activation which is a major confounder of pelvic organ mobility.

For an in- depth discussion of the methodology employed in this study and by others, see Chapter 4. The validity of a new method is shown by comparison with previously used assessment systems as has been undertaken by the author both for pelvic organ descent (Dietz eta., 2001) and for levator function (Dietz et al., 2002). Reproducibility or repeatability is tested by test- retest and/ or interobserver series which are presented in this thesis, with good to excellent repeatability of measurements shown for bladder neck descent, the main outcome parameter of the study. The author is not aware of any other ultrasound method used in tre assessment of the female pelvic floor for which such data is available.

Finally, the validity of a new method is further proven by the power it provides for studies of a certain outcome parameter. Most hypotheses tested in this study could be refuted or confirmed unequivocally, justifying and supporting the choice of the methodology described above. No other imaging method described to date has provided similar power to answer the research questions posed in this study.

62 2.5.5.7 3D Ultrasound Imaging

As a pilot project within the main study, the author attempted to further define the extent and nature of potential traumatic damage to pelvic floor structures by using 3D ultrasound. No such data is currently available in the literature. Several leading manufacturers of ultrasound equipment were approached and asked for support of such a project. Toshiba Australia agreed to supply both an ultrasound system (Toshiba Powervision 8000 v 3.0) as well as the necessary hard- and software for analysis of the resulting volumes (see below). The ultrasound system was made available on three separate occasions and for approx. two weeks on each occasion. It therefore became possible to conduct a pilot study into the use of 3D ultrasound to document pelvic floor trauma after vaginal childbirth.

47 women already enrolled in the main project were recruited for this pilot study. 23 of them attended at a time when 3D equipment was available. After routine imaging as described above several 3D datasets were acquired both in the anteroposterior and in the transverse direction. This involved the storage of a varying number of frames (> 100) which are then converted into a volume dataset similar to the raw data obtained on CT or MRI. Imaging was repeated during a moderate Valsalva manoeuvre. In total between 3 and 16 volumes were acquired per patient. The lack of a position sensor required repeat attempts in a large number of acquisitions due to asymmetrical movement of the transducer or faulty positioning. Both symmetry of the sweep and lateral reach were confirmed immediately after data acquisition and the process repeated as often as necessary to ensure inclusion of the paravaginal areas and both levatores down to the anal canal. The same procedure was followed at the time of treir return approximately 3 months postpartum.

Analysis of the volume datasets was performed with the author blinded against delivery details and all parameters of anterior vaginal wall descent, on a desktop computer (micron clientpro) supplied by Toshiba Australia, using proprietary software

63 (Powerview ™), several months after the data acquisition. Analysis was limited to a qualitative assessment of transverse or tilted transverse sections. As the software imposed limitations regarding acquisition angle and volume configuration, anteroposterior sweeps were not systematically evaluated. Quantitative evaluation of volumes requires a position sensor or automated volume acquisition, techniques that were not available to the author at the time.

Identification of structures was easiest in the transverse plane and in "opacity mode", a proprietary method of image data representation which renders pixels semitransparent. In order to orient 2D transverse images along the plane of the levator ani muscle, a pivot of about 20- 30 degrees was used.

Of the most technically satisfactory datasets, printouts were obtained at three levels, demonstrating the levator ani and the paravaginal structures in the lower, middle and upper vagina. Medilink Australia provided a Codonics NP 1660 M Medical Printer and the media required to produce medical quality printouts of approx. 850 transverse slices.

"Tenting" of the vagina (see Fig. 2.13), i.e., continuity of the vagina and paravaginal tissues with the pelvic sidewall, was described on those three levels and scored as 'present', 'indeterminate' and 'absent' for both right and left. Identification of sides was made possible by consistent direction of acquisition: sweep direction always was from the patient's right to the patient's left since pulling on the transducer cord provided for a smoother sweep. This results in the right side of any resulting slice representing the patient's left. The view obtained is therefore equivalent to a view of the urogenital diaphragm from below. The right side of any image shown signifies the patient's left and vice versa.

64 Fig. 2.11: Translabial ultrasound at rest (1) and on levator activation (r). Vertical (cra­ niocaudal) displacement is 4.5 mm, horizontal (dorsoventral) displacement 16.1 mrn.

Pelvic Sidewall

Levator Levator ani ani

Fig. 2.13: 'Tenting' of the vaginal fornices (arrows): vaginal and paravaginal tissues are in continuity with the pelvic sidewall.

65 Fig. 2.14: 3D imaging ante- and postpartum, transverse sections at Level II. Unilateral loss of paravaginal tenting on left.

Fig. 2.15: 3D imaging ante- and postpartum, transverse sections at Level II, with marked bilateral loss of tenting.

66 In order to determine the relevance of the parameter 'tenting' for overall support of the anterior vaginal wall, antepartum and postpartum tenting for the above- mentioned three levels was compared. Women with reduced tenting in any levels were compared to those without change, with bladder neck descent and urethral rotation being used as parameters of anterior vaginal wall support. Reduced tenting was also tested against delivery mode.

In a second attempt to define peripartum change in paravaginal support, ante- and postpartum images of all three levels were directly compared (see Figures 2.14 and 2.15), rated unchanged, better, worse or indeterminate, and the above analysis repeated.

Valsalva manoeuvres and pelvic floor contractions did not yield volume da1:asets that could be consistently evaluated. The marked displacement of structures on Valsalva in many women made identification of landmarks in transverse sections difficult; often the only structure to be detected easily was the widened urogenital hiatus between a laterally displaced levator. Results are presented in section 3.6.

2.5.6 Other tests considered prior to study commencement

Invasive testing, such as urethral pressure profilometry and filling cystometry, had been considered in the planning of the study. The former is controversial as a diagnostic tool in incontinent women (Lose, 1997), and the latter has been criticised for its poor correlation with patient symptoms (Salvatore et al., 1999, Chaliha et al., 2000). In view of these shortcomings, the additional benefit to be derived from these methods did not seem to outweigh the increased cost and effort, let alone the probable reduction in patient compliance. This decision is borne out by a recently published study showing virtually no useful results from the performance of over 400 filling cystometries in pregnancy and puerperium. This study, performed at St. George's Medical School, ~ . London, also suffered from a poor return rate of 56% (Chaliha et al., 2000). Poor retUrn

67 rates of 60% or lower are commonly reported for similar studies employing invasive testing (Nel et al., 2001).

Pudendal nerve terminal motor latencies (PNTML) were also considered for inclusion. Pudendal nerve damage as a result of vaginal childbirth has been lmown for some time (Snooks et al., 1985, 1986, 1990, Allen et al., 1990). However, the correlation between neurophysiological findings and function is notoriously poor (Vodusek, 2000), to the degree that a recent editorial demanded more basic research into the validity of the method before advocating its use, even in a research setting (Vodusek, 2000). Also, the invasiveness of the test was regarded as very likely to reduce compliance with followup arrangements.

As a result of these deliberations, a completely non-invasive study protocol was developed, with the aim of maximising return rates while obtaining all information necessary to investigate the hypotheses defmed under 1.8.

2.5. 7 Collection of delivery data

In order to optimise the collection of delivery data, a data collection form was developed, explained to labour ward staff (medical and midwives) and stapled to patients' antenatal record (see appendix). A colour- coded collection box was installed in Labour ward and checked several times a week during the study period. Forms were collected and the information entered in the database after the final visit. A raffle for an A$ 50 book voucher was announced for all completed and signed forms to improve return rates.

Since only Ill forms were returned, the medical records of all patients were reviewed after their last visit for both antenatal and delivery details. When the information in the patient's records disagreed with data entered on the labour ward assessment form, all

68 available information was reviewed. Typical errors on the data collection forms were erroneous adding up of passive and active second stage, and these were corrected.

Total duration of the second stage oflabour was defined as the interval between confirmation of full cervical dilatation and delivery of the infant. Active second stage was taken as the time between commencement of active pushing and delivery. Passive second stage was defined as the difference between total and active second stage. In cases of vaginal operative delivery, an attempt was made to define station (0-3 em below the ischial spines), difficulty (easy, moderate, firm, very firm) and record the number ofpulls (1-5); see 8.1. Information on presentation was frequently missing from Theatre notes on Caesarean sections.

In at least four instances, patients delivered at other hospitals. Attempts were made to obtain information on the delivery details of these women, and three forms were received from within Australia and overseas.

2.6 Laboratory methods

2.6.1 Genetics

To reiterate some of the issues raised in the introduction, the author would like to propose the hypothesis that abnormal mobility of the pelvic organs, i.e., a wealmess of the supporting fascial structures of urethra, bladder, cervix and rectum, constitutes a complex "disease" with a genetic background or contribution, similar to the vast majority of problems physicians and surgeons deal with every day. Most human disease states are not unifactorial as in Mendelian diseases but multifactorial. Combinations of favourable and unfavourable genetic and environmental factors result in an infinite variety of clinical outcomes. The further the phenotypic result is from original gene action, the less likely is it to show a simple Mendelian pattern (Strachan and Read,

69 1999). For example, an abnormality in a defmed protein is more likely to be Mendelian than a birth defect such as cleft palate, and schizophrenia is extremely likely to be caused by a complex array of genetic and environmental factors. For an in-depth discussion of this topic see Strachan and Read, 1999, esp. Chapter 3.

For those involved in molecular genetic research, complex diseases such as endometriosis or osteoporosis, coronary artery disease, schizophrenia and pelvic organ prolapse pose a major challenge. Until recently, tackling such complex diseases was impossible due to the absence of genetic markers. The most common of such markers (thought to make up 85% of all genomic variation in the human) are single nucleotide polymorphisms (SNPs). Due mainly to the human genome project, a large number(> 1.400.000 at the time of analysis) of polymorphic markers or Single Nucleotide Polymorphisms are now known. The number of SNPs available on public and proprietary databases has been increasing at a near exponential rate over the last 3-4 years. To give an example for one of the main target genes used for this study, the number of SNPs documented for the gene of Collagen 3Al has increased from about 5 to over 90 within the timeframe of this thesis.

As discussed in the introductory section of this thesis, it was decided to limit genetic investigations to a target gene approach, concentrating on the collagen, elastin and :fibrillin genes mentioned in the Introduction of this thesis (COLlAl coding for the one of the main chains of Collagen I), COL3Al coding for Collagen III), COL5Al (Collagen V) ELN (Elastin) and FBNl (Fibrillin 1). The prioritization of target gene candidates is an essential step in the process (Strachan and Read, 1999), and advice was obtained from a number of clinical and non-clinical sources. As a result, it was decided to initially investigate COL3Al as the most likely candidate in this group. This was done on the understanding that such would constitute a pilot study and was unlikely to yield defmitive results as only approx. 5-10% ofknown SNPs ofthe above target genes would be covered.

70 All techniques described in this section, with the exception of DNA sequencing, were performed by the author under supervision of staff at molecular genetic facilities in Sydney and Brisbane.

2.6.1.1 Preparation of blood sample and DNA extraction

10 ml ofEDTA blood were taken from 199 women enrolled in the study. All had consented to this in writing (see 8.2 for consent forms). One blood sample could not be obtained due to the patient having left Australia shortly after her first visit. The EDTA blood was stored in a refrigerator at +4 deg until processing later the same week (max. 5 days later). At the Cytogenetics Laboratory, South Eastern Area Laboratory Services, Prince of Wales Hospital, Sydney (Director: Dr. Michael Buckley), the author then prepared white cell pellets in the following manner:

The sample was lysed in 40 mls NH4Cllysis buffer and left for about 30 minutes until clearing could be observed. After spinning the sample in a laboratory centrifuge (Labofuge G2, Heraeus, Germany) for 10 minutes at 3000- 4000 U/min, the supernatant was poured off and the process repeated with 49 mls ofNH4Cllysis buffer to lyse all red cells and remove haemoglobin and cell wall fragments. Following this, the supernatant was again poured off. Then 1 ml Tris EDTA buffer was added as storage medium to stabilize the DNA. The white cell pellet was resuspended and transferred to an Eppendorf container for repeat spinning in a microcentrifuge. After pipetting off the supernatant, the Eppendorf container with tre white cell pellet was stored at temperatures at or below -15 degrees centigrade.

Once all samples had been obtained, DNA extraction was performed, also in the Cytogenetics Unit at Prince ofWales Hospital, with the help of two scientific officers, Anita Bahar and George Elakis, who supervised the author for this procedure. White cell pellets were thawed in a heating block and resuspended in Tris EDTA buffer. After homogenisation the cell membranes were lysed by overnight digestion in Proteinase K

71 (15 microliters per pellet) with a detergent, SDS (approx. 50 microliters), in a waterbath at 50 degrees centigrade. The next morning the DNA extraction was performed according to the method described by Miller (Miller et al., 1988). The clear supernatant resulting from the digestion process was pipetted off and added to 2 ml of 80% Ethanol. This precipitates the DNA (helped by shaking) which becomes visible, looking like a ball of cotton wool. The DNA is then removed from the alcohol with the help of a plastic hook, washed in buffer and then resuspended. The resuspended DNA is then refrozen and kept at -10 de g. Centigrade.

After transfer of the samples to the Queensland Cancer Fund Transgenic Laboratory, Queensland Institute of Medical Research (Head: Georgia Chenevix- Trench), standardization of DNA concentration was undertaken by the author. Spectrophoto­ metric analysis at 260 nm of an aliquot diluted 1: 100 and subsequent dilution of samples to a stock solution of either 50 or 500 ng/microliter followed. From the stock solution a working aliquot of 10 ng/ microliter was produced which was the DNA concentration used in all subsequent work.

As a first step and in order to validate the process up to this point, Polymerase Chain Reaction (PCR) was performed by the author for ER5 ', a ubiquitous cell housekeeping gene commonly used for DNA quality control. A random selection of 20 DNA samples obtained over the whole study period were utilized, and good amplification (PCR product) of the chosen sequence was obtained for all 20 samples, confirming them to be of sufficient quality for further work.

2.6.1.2 Identification of Polymorphisms

Searches by the author in the public domain (dbSNP, HGMD Cardiff, University of Leicester Collagen Genetics Database, NCBI OMIM, biochip.org and others) as well as proprietary databases (Celera Discovery System) yielded approximately 90 single nucleotide polymorphisms for the first candidate gene, COL3Al.

72 COL3A1 codes for Procollagen III, a fibrillar protein that is found commonly in extensible connective tissues, often in association with Collagen I. The gene spans approx. 40.000 base pairs on the short arm of chromosome 2 (location 2p31). It consists of 50 exons of varying length (see Fig. 2.16 for a graphic representation), with the genomic DNA expressed as mRNA of about 5.5 kB size

Ill I II I II I II llllllllllllll II Ill II !IIIII I II IIIII II.

Fig. 2.16: Schematic representation ofCol3A1: Vertical bars represent exons. Intervening spaces are introns. Total length 39020 base pairs.

Not all SNPs or variations of the genomic DNA are likely to be phenotypically relevant: many are found in noncoding regions of the gene, i.e., in introns. Others are situated in exons, ie.e, the coding region, but do not alter the actual nucleotide coded for by a codon of three base pairs. Silent (intronic or noncoding) SNPs were therefore excluded, leaving approx. 50 coding SNPs in COL3A1 to be investigated. Population frequency data was not available on any of those databases at the time of repeated searches (Jan. 2001- Oct. 2001); however, a recent study looking at the potential usefulness ofpublic domain SNPs has shown that, in three different ethnic populations, of>1200 SNPs tested randomly, about 50% were common, i.e., the minor allele being found in 20% or more of the population (Marth et al., 2001). This implies that a considerable proportion of public domain SNPs should be detectable in a population of 199 samples as represented in this study.

2.6.1.3 Preparation of template DNA and primers

Initially, it was planned to use TaqMan™, (Applied Biosystems, Foster City, Ca., USA) for analyzing patient DNA for the presence of a certain SNP at a time, a proprietary

73 automated system that employs fluorophore probes to detect the wild type or mutant allele. However, the technical requirements of the system, as well as the physical structure of the sequence in question, limit opportunities for primer and probe design. The author found it impossible to design primer- probe combinations for all SNPs identified on database searches, due to the clustering of a large number of SNPs in certain exons of Col 3A1. In consultation with Dr. Chenevix- Trench, it was therefore decided to use the technique of Denaturing High Performance Liquid Chromatography (DHPLC, see below) to screen entire exons. Table 2.2lists the exons selected as potentially containing the largest number of SNPs, and the SNPs listed for those exons. Amplicon length refers to the base pair length of the resulting PCR product.

In order to test the quality of the DNA obtained and the population frequency ofthe selected po1ymorphisms, a first primer pair was ordered and used for PCR amplification. Primer titration was also performed at this stage. The result was separated with agarose gel electrophoresis, documenting good amplification of the PCR product.

As a next step, all available patients samples were plated out onto three masterplates containing 12x8= 96 wells (96 well microtest tissue culture plates, Becton Dickinson, Franklin Lakes, NJ, USA) with 30 microliters of H20 and 30 microliters of 10 ng/J..Ll DNA solution, resulting in a dilution of 5 ng/ J..Ll of patient DNA. These masterplates were then used for the manufacture of 3x6= 18 PCR plates (Thermo- Fast R 96 well, AbGene, Epsom/ Surrey, UK) for further analysis, using a robotic plating machine (Beckman Biomek 1000 Automated Laboratory workstation, Beclanan Coulter, Fullerton, Ca. USA). These plates were then dried and frozen at -20 deg., awaiting the arrival of suitable primers.

74 SNPs Amplicon (mRNA base number) Length

Exon 23 1735, 1763, 1772 Exon 24 1817, 1861 445

Exon 31 2293,2329,2330 Exon 32 2348,2361,2384 440

Exon 39 2816,2825,2843,2870,2887,2923 Exon 40 2986,2996,3014,3032 400

Exon 48 3644,3652,3662,3671,3679,3680 230

Table 2.2: Exons and SNPs covered by the selected amplicons, amplicon length.

2.6.1.4 PCR methodology

Polymerase chain reaction (PCR) is a technique that allows the almost unlimited amplification(= multiplication) of any desired fragment or target sequence of DNA (the 'amplicon'). It vvas developed in the mid-80s and is now one of the fundamental technologies of molecular genetics (Strachan and Read, 1999). Primers, i.e., short (15- 30 base pair) oligonucleotide sequences are designed to be specific for the target sequence, i.e., to hybr:idise there and nowhere else in the genome. After adding these primers to the template DNA, together with DNA precursors (the four nucleoside triphosphates dATP, dCTP, dGTP and dTTP) and a heat- stable DNA polymerase (in this case a bacterial enzyme, TAQ, obtained from Thermus aquaticus), they start up the synthesis of new complementary DNA strands.

75 This process is a chain reaction as the new strands will act as fresh template against which further synthesis then occurs. After 25 or more cycles of successive heating and cooling (denaturation at 93-95 degrees, reannealing at the specific Tm of this particular product, and DNA synthesis at about 70-75 degrees), the resulting PCR product consists almost exclusively of the target sequence or "amplicon". The machines used for this process are called 'thermal cyclers' which are computer- controlled to provide for optimal amplification conditions. Samples are usually contained in a well plate which fits slots in a metal block that is successively heated and cooled.

Four PCR primer pairs were designed by the author to produce amplicons covering exons 23/24, 31/32, 39/40 and 48, containing a total of 27 coding SNPs, more than half of those described for COL3Al, and ordered from a commercial supplier of oligonucleotides (GeneWorks, Adelaide, SA 5000, Australia,). DHPLC methodology works best for fragments of up to 600 bp, and therefore DNA product size was kept below this limit. Once these primer pairs were obtained, they were tested on a small number of samples already proven to contain good quality amplifiable DNA (for PCR mix see Table 2.3).

H20 4.9 f.!l 1Ox PCR buffer (Applied Biosystems, 2 mM) 1.0 f.!l MgCt(25 mM) 0.6 f.!l 1Ox NTPs (Mix of 4 nucleoside- trophosphates, 2mM each) 0.4 f.!l Primer (Forward, 20 f.!M) 0.4 f.!l Primer (Reverse, 20 f.!M) 0.4 f.!l

Amplitaq Gold TM (Applied Biosystems) 0.2 f.!l

Table 2.3: PCR Mix for all subsequent primary PCR reactions (number given for 10 f.!l reaction with 1.5 f.!l of DNA (10 ng/f.!l) added.

76 Hold 10 Cycles 25 Cycles Hold (Touchdown 68-59° 1 cycle each)

95.0 95.0 95.0 10.00 0.20 0.20 72.0 68.0 72.0 72.0 0.20 0.20 0.20 10.00

Table 2.4: Touchdown PCR protocol used with ABI Gene Amp TM PCR System 9700. Numbers above line indicate temperature, numbers below line time in minutes and seconds for this particular temperature. For 10 cycles the lowest temperature in the cycle (annealing step) is lowered by one degree per cycle until 58 degrees is reached. 25 cycles at 58 degrees follow.

Exons 23/24 Exons 29/30 Exons 39/40 Exon 48

Fig. 2.17: Agarose gel electrophoresis for all four tested amplicons (exon 23/24, exon 31/32, exon 39/40 and exon 48 ofCol3Al). Amplicon 1 containing Exons 23/24 did not amplify despite repeated attempts.

The primer pair for exons 23/24 did not produce any PCR product despite repeated attempts; further tests were limited to the other three primer pairs. These three tested

77 well (see Fig. 2.17) and were used to amplify 20 samples oflmown quality. The PCR

mix and touc~down protocol used for all subsequently tested amplicons are given in Tables 2.3 and 2.4. All PCR reactions were checked on agarose gel. Failed PCRs were repeated until all DNA samples had been successfully amplified (205 samples for 196 patients).

2.6.1.5 DHPLC methodology

PCR Primers for use with DHPLC were designed using ABI Prism Primer Express TM (Applied Biosystems, Foster City, Ca., USA), producing amplicons (DNA strands defined by the forward and the reverse primer) containing between 3 and 71mown coding polymorphisms (see Table 2.2). These were tested for DHPLC suitability using the programme DHPLC Melt (public domain, Stanford Genome Technology Centre, http://insertion.stanford.edu/meltl.html), prior to PCR. DHPLC of a minimum of61 samples for each amplicon was used by the author to screen for the presence of SNPs in order to give 95% power to detect SNPs of a population frequency of 0.05 or above.

A Varian HELIX DHPLC system (Varian Australia, Mulgrave Vic 3171) was employed for this purpose. After testing at the two temperatures predicted by DHPLC Melt (see above) and two degrees above the higher of the two temperatures, one common polymorphism was detected in the Exon 31/32 amplicon, with a number of less common polymorphisms detected only once each. All patterns detected by DHPLC in this test run were then sequenced (see below). Only the one amplicon yielding a common polymorphism (Exon 31/32) was tested on DHPLC for all 196 samples.

DHPLC is a high performance liquid chromatography method, with the D standing for "denaturing". It is most suitable for amplicons of up to 600 bp. The stationary phase is a column of a polystyrene-divinylbenzene copolymer. The mobile phase mediates bin­ ding of DNA to the stationary phase. Acetonitrile is used to wash out DNA between samples. Fragments come off the column depending on size and/or presence ofhetero­ duplexes. As the DNA is eluted from the column, it passes through an UV detector.

78 Mutations and polymorphisms, i.e., small variations in base sequence, can be detected due to the formation of so- called "heteroduplexes" between mismatched nucleotides (i.e., incompletely annealed strands of wild type and variant allele) in double stranded DNA amplified by PCR. Sequence variation results in a mix of heteroduplexes and homoduplexes during reannealling of wild type and mutant DNA (Figure 2.18).

Hom odu pl exes Hap loid Systr m Wt + Mt II II II II HEAT ~~ n ) trn ~~~~ Jl Jl Allele A Allele B Dip lon s ysteJI\ \t \( H eta-o duplexes

Fig. 2.18: Heteroduplex formation (Source: http://www.arl.arizona.edu/lmse/dhplcinfo.htm)

When this mixed population is analyzed by HPLC, the heteroduplexes elute from the column slightly earlier than the homoduplexes because of their reduced melting temperature. This alters the resulting chromatographic profile (see Fig. 2.19 for a public domain illustration available from http://www.arl.arizona.edu/lmse/dhplcinfo.htm.)

The fact that DHPLC of a given PCR product detects only heterozygotes (i.e., patients who carry one normal ('wild type') allele and one mutant allele) and not those who are homozygous for the mutant allele, can be overcome by DNA mixing. This requires the merging of known wild type DNA with each sample to generate false 'heterozygotes' in samples that are homozygous for the mutant allele (Strachan and Read, 1999). Since no common coding SNPs were detected in this pilot study, this technique was not used.

79 Homozygote., CT '' T G C "' --···----- ·---. l"" f. i ' ! . l__/ . t- \. . - ' . . ~.'t .., 't J.Qn. t~ (N r(l

Het~oz~{~go~te~------, c ,. .A. T T T G C + f\ .fY'/Y vy·v~~

Fig. 2.19: Identifying heterozygosity with DHPLC (Source: http://www.arl.arizona.edu/lmse/dhplcinfo.htm)

2.6.1.6 equencing methodology

Sequencing of samples that had produced aberrant DHPLC patterns was performed by the DNA Peptide Unit at QIMR (Head: Macky Edmundson). The author prepared PCR product for sequencing by cleaning the product of a second touchdown PCR which had been tested on an agarose gel to confirm presence of sufficient product.

A QIA-QUICK TM P R purification kit (QIAGEN Pty Ltd, Clifton Hill, Vic.) was u ed which i designed to purify DNA fragments from PCR. This allows for binding of DNA to a stationary membrane in a pin column after dissolving in a proprietary buffer. The DNA is eluted with the help of 10 mM Tris-Cl buffer at pH 8.5. Part of this cleaned product was then subjected to a special PCR programme which allows incorporation of BigDye ™ (Applied Bio ystems, Foster City, Ca., USA) marker. Only one primer pecific for the particular amp Iicon tested is used, producing a mix of arq:>licon fragment of varying lengths, all terminating in a marker that is specific for the Ia t ba e

0 in this particular fragment. The succession of markers for successive lengths of product then allows determination of the nucleotide succession in the amplicon tested.

Prior to sequencing the product has to be cleaned again. This is performed by adding 70% isopropanol, mixing and leaving it at room temperature for approx. 20 minutes. After spinning for 20 minutes in a microcentrifuge at 13.000/ min the DNA precipitates while primers, excess oligonucleotides and other PCR constituents remain in the supernatant and can be aspirated off. The resulting pellet is rinsed with 70% isopropanol again, re- spun, and the supernatant pipetted off again. Samples are dried under vacuum.

2.6.1. 7 Association analysis

Once all DHPLC patterns were attributed to a certain SNP, the author was able to perform an analysis for associations between such patterns and clinical parameters. However, the only SNP found to be sufficiently common to allow such analysis, was identified as a "silent", ie.e., noncoding SNP. Association testing was still carried out on the remote chance oflinkage to another (undetected) abnormality, comparing continuous phenotype variables between homozygote and heterozygote patterns with the help of t- Test statistics. This was also repeated for homozygote versus heterozygote patterns for all 3 amplicons.

81 2.6.2 Relaxin serum levels

For a pilot group of 50 women, second blood samples (1 0 ml) were taken at the time of the first appointment, i.e., between 9 and 16 weeks. The sample size was determined arbitrarily as there was no data available to perform power calculations. All gestational ages were confirmed or corrected at the time of the fust study appointment. The blood was spun down, the serum separated and frozen. An enzyme- linked immunosorbent assay (hRLX Elisa, Immundiagnostik Bensheim, Germany) was carried out by the author at the Department of Endocrinology, South Eastern Area Laboratory Services, Prince of Wales Hospital, Sydney (Director: Dr. S. Brown), under the supervision of Senior Scientist Lillian Tan. One sample was lost, leaving 49 serum samples.

Reagents were prepared as follows: The supplied ELISA wash buffer concentrate was diluted 1: 10 with deionized water. The supplied biotinylated second antibody was diluted 1: 1000 in Elisa wash buffer, as was the horseradish peroxidase- labelled streptavidin. For the calibration curve, a Relaxin standard was diluted successively to obtain concentrations between 4 and 250 pg/ml with the dilution buffer serving as the 0 pg/ml control.

Serum samples were retrieved and thawed, then diluted 1 :5 in sample dilution buffer. 100 microliter of diluted sample was the added to a precoated microtiter plate after washing of the latter with 5x 250 f.Ll of ELISA wash buffer. The plates were then incubated overnight at a temperature of 2-8 degrees centigrade.

The next day, the contents of the plate were decanted and washed five times with ELISA wash buffer. Subsequently, 100 f.Ll ofbiotinylated anti-hRelaxin antibody was added and the plates incubated for 2 hours at 2-8 degrees. After this and decanting/ washing of the microtiter plates as above, 100 microliter of PO- labelled streptavidin were added and the plates again incubated for 1 hr at 2-8 degrees. Again plate content was decanted and washed. The final step was adding 100 f.Ll of substrate. The reaction

82 was stopped after 30 minutes at room temperature, using the supplied stop solution and mixing to provide for homogenisation. Absorption at 450 nm was measured with an automated ELISA microtiter plate reader, against a reference standard of 620 nm.

The resulting readouts were then converted to serum levels using the calibration curve and a spline algorithm since the calibration curve for this test is nonlinear. (A spline algorithm is used to estimate points on a curve using more than two points when plotting results for standard concentrations). Due to the originall :5 dilution of serum samples, the resulting figures were then multiplied x5 to arrive at actual serum concentrations. All samples tested fell well within the calibmtion curve as a result of this dilution although actual serum levels in the first and early second trimester of pregnancy are often above the maximum calibration concentration for this test. The %CV (coefficient ofvariation, =standard deviation(SD) divided by the mean expressed as a percentage) for the hRLX Elisa was determined at between 5.9 and 13.6 for the working range of the test which constitutes acceptable precision for a manual assay.

2.6.3 Progesterone levels

Progesterone levels were determined by D. Garrett BSc (Biomed) at the Endocrine Laboratory, School of Obstetrics and Gynaecology, University of New South Wales, Randwick, Australia (Head: Prof. M.J. Bennett), employing a Vitros ECI automated analysis system (Johnson& Johnson Immunodiagnostics). The Vitros progesterone assay uses a competitive immunoassay technique, i.e., results depend on competition between progesterone present in the sample and a horseradish- peroxidase labelled progesterone for limited binding sites on antiprogesterone antibodies in the liquid phase. Effects of binding proteins are eliminated by the use of a blocking agent. The antiprogesterone antibody is captured in streptavidin- coated wells, and the bound conjugate is measured by utilizing a luminescent reaction that is picked up by the automated system and converted to progesterone levels using the calibration curve. This

83 calibration curve is nonlinear, i.e., a modified 4 parameter logistic curve fit function has to be used by the computer controlling the automated system to allow conversion.

25 microliter serum samples were all tested on the same day and with the same lot of reagents. Samples were stored at -20°C and defrosted to 21 °C before testing. All samples tested fell within the test- specific calibration curw (0- 178 nmol/L). The manufacturer quotes a precision of between 4.4 and 8.2 % for CV within the range of levels tested (Within-day, within-calibration and within- laboratory).

2. 7 Statistical Analysis

2. 7.1 Power calculations, sample size

A pilot stilly performed in 1996/ 97 under supervision of the author at the Department of Obstetrics and Gynaecology, Dunedin School ofMedicine, University of Otago, Dunedin, New Zealand was used for power calculations (Safari, 1997). Forty- three of 287 eligible primiparous women (15%) consented to a clinical and ultrasound assessment on average 2.8 years after childbirth. There were significant differences between women delivered by Forceps/ Ventouse and those delivered normally or by Caesarean as regards bladder reck descent even in that small group.

It was estimated that antepartal recruitment and repeated visits as well as an appointment only 3 months postpartum should result in a follow-up rate of75%, singifigantly higher than those documented in the literature (Chaliha et al., 2000) for studies using invasive techniques. Based on the above- mentioned pilot data and this follow-up rate, recruitment of200 women, i.e., a final sample size of 150 women, was estimated to provide over 90 % power to detect a statistically significant difference between normal delivery and Forceps/ Ventouse as regards bladder neck descent, and over 95 % power to detect a statistically significant difference between vaginal operative delivery and Caesarean Section. These numbers were calculated assuming

84 21% Caesarean section, 15% vaginal operative deliveries and 64% normal vaginal deliveries (data for Royal Hospital for Women, 1998).

In the event, a minimum of 153 datasets were available for all main outcome parameters, guaranteeing adequate power. Lower numbers were tested in associated pilot studies such as hormonal analysis and 3D ultrasound, for which no power calculations had been performed due to lack of data.

2.7.2 Descriptive statistics

Mean and Standard Deviation are given for normally distributed data. No quantitative data was skewed enough to require a different format. Whenever the data was not sufficiently represented by mean and standard deviation, the author used histograms or dotplots.

2. 7.3 Analytical statistics

In order to define test- retest and interrater reliability of ultrasound measures ofbladder neck descent and levator contraction, the coefficient of variation (CV =standard deviation divided by the mean, expressed as percentage) was used (Armitage and Berry, 1994) as well as ther lntraclass Correlation Coefficient (ICC 1,1 or ICC 2,1) (Rosner, 1995). The CV was employed to define repeatability of the Relaxin ELISA. The Kolmogorov- Smimov test was used to confirm normal distribution of data for all tested parameters. Only striae gravidarum and passive second stage were clearly not normally distributed. The 2-sample t- test was used for comparison of two normally distributed sets of quantitative continuous data, e.g. to compare ultrasound data between ethnic groups. In the absence of normal distribution, the Mann Whitney U test for independent samples was employed.

85 Three or more sets of continuous quantitative data were tested using analysis of variance, with one- way ANOVA being performed after stratifying data for such parameters as delivery mode, followed by Tukey's pairwise comparison (Armitage and Berry, 1994). Quantitative data was analyzed for changes over time using a 2- sample t­ test, e.g. for changes in ultrasound from one time point to the next. Categorical parameters were tested cross- sectionally by Chi square test. Pearson's correlation coefficient or Spearman's correlation coefficient were used to test for association between two sets of quantitative continuous variables, the former for normally distributed data and the latter for skewed distributions. Logistic regression analysis was used to analyze the relative contribution of first stage, passive and active second stage to changes in pelvic organ mobility and levator function, and the relative contribution of delivery mode and second stage length was determined by stepwise omission of factors from the regression model. To analyze pelvic organ mobility versus operative delivery, a binary logistic regression model was used, yielding predicted probabilities for the outcome ofNormal Vaginal Delivery.

86 3 Results

3.1 Validation of ultrasound imaging methodology

In order to validate the main ultrasound parameters, i.e., bladder neck descent on Valsalva manoeuvre and oblique lift on pelvic floor muscle contraction, consecutive blinded assessments were con:iucted by the author and Dr. A. Steensma. Methodology was kept identical. Patients were selected randomly, i.e., joint assessments were conducted whenever staffing/ equipment availability made this possible. In a series for intraobserver variability, two consecutive assessments were performed on the same patient by the author. In a second series, the same still photographs were used by two assessors for blinded on- screen measurements, and in a third series each investigator obtained his/ her own still photographs for true interobserver variability.

3.1.1 Intraobserver variability

For this parameter the author used data from 2 separate printouts per patient that had been obtained by him (n= 20). Printouts had been produced in a nonblinded fashion but had not originally been obtained for the purpose of comparison, as only the most effective of three manoeuvres (Valsalva or levator contraction) were used for the purposes of the main study. In this series, intraobserver variability was found to be 12% (coefficient of variation CV= 0.12) for bladder neck descent, with a mean difference of3.4 mm (range 0.1- 11.2 mm, SD 2.8) . The corresponding values for levator strength were a coefficient of variation of 0.20 and 1.7 mm (range 0- 6.21 mm, SD 1.8 mm) for the mean difference between measurements.

3.1.2 Interobserver variability

One series of interobserver variability measurements (n= 20), with two assessors measuring the same still photographs, yielded the following results: The coefficient of variation (CV)

87 for bladder neck descent on Valsalva was 0.08, with an average difference in measurements of 1.98 mm (0- 4.5 mm, SD 1.27). The Intraclass correlation coefficient ICC (2.1) was 0.98. For levator strength as measured by oblique bladder neck displacement, CV was 0.19 and the average discrepancy between measurements was 1.92 mm (0.04- 4.72 mm, SD 1.5).

For the third series of variability assessment (separately obtained printouts and blinded analysis, n= 47) which constituted a true interobserver series, the CV vvas 0.21 for BND with an average difference between measurements of 5.8 mm (0- 14.5 mm, SD 4.2 mm); the Intraclass Correlation coefficient ICC (2,1) for this series was 0.79 which by definition implies "excellent" agreement. For levator strength as expressed by oblique lift of the bladder neck relative to the symphysis pubis, the corresponding data were CV= 0.26 and 2.56 mm (0- 10.3 mm, SD 2.15 mm) for the difference between measurements.

3.2 First visit: late first/ early second trimester assessment

Two hundred women were enrolled, with first assessments undertaken between 6 and 18 weeks' gestation (median 12 weeks, range 6-18 weeks). Average age at enrolment was 29 (17-42) years. Average prepregnancy weight was 61.8 (43- 105) kg, height was 165 (148- 189) em.

Figure 3.1 gives a histogram of gestation at the time ofthe fust assessment. The distribution is due to the fact that booking visits at the Royal Hospital for Women in Randwick are usually arranged by General Practitioners and between 10 and 14 weeks' gestation. A few earlier enrolments were mainly due to women presenting directly to Antenatal clinic. Recruitment was not attempted if women had passed 14+0; however, in a number of cases gestational age was corrected at the time of the assessment ani women remained in the study provided the new gestational age was below 18+ 1.

88 50 r-

i- 40

r-- (') 30 c Q) 1-- ::::lo- 20 ~ r- LL - 10

0 I f------1 5 7 9 11 13 15 17 19 Gestation

F ig. 3.1: Gestational age (completed weeks) at presentation for first visit, n= 200.

Twenty women smoked which was weakly associated with stress incontinence (p= 0.044) but not with any of the ultrasound parameters measured. One hundred and seven women mentioned sporting activities which were categorized according to frequency/ intensity (see 2.5.1.).

3.2.1 Ethnic background

Ethnicity of all four grandparents was recorded for all 200 women at the time of the first visit. There was considerable ethnic diversity but, due to the ethnic structure of the Eastern Suburbs of Sydney, most of this variation was within the Caucasian group. The only other ethnic group of significance was Asian patients, most of them Chinese, Chinese­ Indonesian or Indonesian. In 23 women at least two major ethnic groups were found amongst the four grandparents; they were omitted from further analysis.

89 On testing the 16 women of Asian extraction against 161 women of Caucasian background, the incidence ofsymptoms of stress and urge incontinence did not differ significantly.

Of the clinical indices of joint hypermobility, only elbow hyperflexion showed a significant difference, with the 16 Asian women having significantly higher values recorded (185.4 vs. 189.6 degrees, p= 0.008 on unpaired t- test).

As regards ultrasound indices of pelvic organ mobility, significant differences between Asian and Caucasian women were detected for virtually all tested parameters, with all trends and associations showing reduced pelvic organ mobility in Asian women. These results are shown in more detail in Table 3.1. Levator strength as determined by ultrasound, on the other hand, was not significantly different.

Oblique Parameter RVA-S Rotation BSD-SV BND descent Cystocele Cervix Rectum

(degrees) (degrees) (mm) (mm) (mm) (mm) (mm) (mm)

Asians 132.5 25 17.8 13.2 16.9 17.5 43.9 20.9 (n=16) SD 25.4 SD 16.7 SD8.9 SD6.7 SD7.6 SD9.5 SD 12 SD 13.9

Caucasians 145 39.5 11.8 19.7 24.9 11.7 39.2 12 (n=161)

SD26.4 SD 25.3 SD 11 SD 10.6 SD 11.0 SD 11.4 SD 18.1 SD 18.5

P= 0.081 0.005 0.023 0.002 0.001 0.035 0.17 0.04

Table 3.1: Translabial ultrasound findings in Asian and Caucasian women. All trends and significances indicate reduced pelvic organ mobility in Asian women (t- test statistics).

90 3.2.2 History of physical activity

An attempt was made to stratify for physical activity prior to pregnancy as explained in 2.5.1. Groups 3 (daily sports) and 4 (regular competitive sports) were merged as only one patient fell in the latter category. Physical activity prior to pregnancy did not correlate with stress incontinence at the time of the interview, nor were there any significant correlations between physical activity and ultrasound indices of pelvic organ mobility or levator strength.

3.2.3 Symptoms

At the time of the first appointment 24 (12%) women suffered from stress incontinence (seven of those noticed at least daily leakage), 15 (7.5%) from urge incontinence (none leaking more than once a week), 44 (22%) complained of frequency (voiding at least 9 times a day on average), and 86 (43%) of nocturia (at least 2 episodes per night).

Symptoms did not correlate significantly with ultrasound measures of mobility (see Table 3.2 for an example), although there was a weak trend towards increased bladder neck mobility in stress incontinent women.

SI N Mean SDev SE Mean

no 176 19.1 10.6 0.80 yes 24 21.7 9.2 1. 9 p= 0. 2

Table 3.2: Relationship between BND and the symptom of stress incontinence in early pregnancy (n= 200).

3.2.4 Paper towel testing

Originally, it had been intended to perform 24 hour pad testing in all women showing a positive paper towel test. However, return rates proved to be only between 30 and 40% in total. 24 hour pad testing was therefore abandoned after approx. three months.

91 Paper towel testing was positive in 23 out of 200 women. The correlation between reported symptoms of incontinence and paper towel test was poor: of the 23 women with positive tests, only 5 had in fact reported symptoms, and of the 24 women who had complained of stress incontinence, only 5 showed a positive paper towel test (not significant on X2 test). The result of the paper towel test was not significantly correlated with bladder neck mobility.

3.2.5 Joint mobility, personal and family history

Table 3.3 gives joint mobility data for the first assessment. In one woman wrist

hyperflexion and 5th finger hyperextension could not be assessed due to bilateral contractures as a result ofprevious trauma. All joint mobility measurements seemed to

decline with age; however, this was only significant for 5th finger hyperextension (r= - 0.155, p= 0.028).

Wrist Fifth finger Elbow Hyperflexion hyperextension hyperextension

Average 169 73 186 Max. 210 150 210 Min. 90 35 165 SDev. 17 16 6.5

Table 3.3: Joint mobility data (n= 200) in degrees

Joint mobility indices did not significantly differ between continent and stress incontinent patients, and there was no consistent correlation between bladder neck mobility and joint mobility. Only thumb approximation to forearm (wrist hyperflexion) correlated wealdy with descent ofthe cervix (r= 0.17, p= 0.017), the rectal ampulla (r= 0.168, p= 0.019) and rotation of the proximal urethra (r=-0.144, p= 0.043).

92 A personal history of hernias, dislocations or clinically relevant epistaxis was very uncommon (dislocations and epistaxis in two patients each) which precluded further analysis. A family history of incontinence (n=49), prolapse (n= 31) and surgery for either (n= 26) in first- degree relatives was much more common. A family history of incontinence (n= 49) correlated weakly with pelvic organ mobility. BND (18.5 vs. 21.9 mm, p= 0.034 on unpaired t- test) and maximal cystocele descent (12.8 vs. 9.2 mm, p= 0.047) were both higher in women with a positive history. This did not hold true for a history of prolapse (n= 31) or operations for such conditions (n= 26).

3.2.6 Ultrasound assessment

In all 200 women recruited for the study, a detailed assessment of pelvic organ mobility and levator function was carried out by translabial ultrasound at the time of their first visit between 6 and 18 weeks' gestation (see Methods). All except one dataset are complete; in this one case it was impossible to visualize the cervix uteri. Table 3.5 recapitulates the information given in Table 2.1 in Methods and describes the parameters used.

The large number of parameters given here may seem confusing at first. However, different authors have in the past used a large variety of measures for pelvic organ descent. Retrovesical angle, rotation of the urethra, position of the bladder neck on Valsalva and overall displacement of the bladder neck have all been utilized to define anterior vaginal wall mobility. The fact that all those parameters are given here is intended to facilitate comparison with past and future studies in this field.

93 RVA-R Retrovesical angle at rest RVA-S Retrovesical angle at stress/ on Valsalva ROT Rotation of proximal urethra on Valsalva BSD-RV Vertical distance between bladder neck and inferoposterior border of symphysis pubis at rest BSD-SV Vertical distance between bladder neck and inferoposterior border of symphysis pubis on Valsalva BND Bladder neck descent (vertical) on Valsalva BND-H Bladder neck displacement (horizontal) on Valsalva Oblique displacement Bladder neck descent (oblique or total) on Valsalva Cystocele Maximal caudal displacement of the posterior bladder wall on Valsalva, relative to inferior border of symphysis. Cervix Maximal caudal displacement of the leading edge of the cervix uteri on Valsalva, relative to inferior border of symphysis. Rectum Maximal caudal displacement of the rectal ampulla on Valsalva, relative to inferior border of symphysis pubis.

Table 3.5: Translabial ultrasound parameters used to describe pelvic organ position and descent at rest and on Valsalva.

3.2.6.1 Pelvic organ mobility

Table 3.6 shows descriptive statistics for ultrasound parameters of pelvic organ position and descent. Figure 3.2 gives a histogram ofbladder position ('Cystocele') on Valsalva at the first visit. It is apparent that measurements that would normally be regarded as abnormal were common: 47 women (24%) demonstrated Cystocele values ofO or lower, implying bladder descent to or beyond the inferior margin of the symphysis pubis. 29% of all women showed BND of 25 mm or higher on Valsalva, and most of those (49 of 58)

94 were stress continent. Similar variations were observed for all three compartments: Figures 3.3 and 3.4 show histograms for cervical and rectal descent.

Oblique RVA-R RVA-S Rot BSD-RV BSD-SV BND BND-H descent Cystocele Cervix Rectum

Mean 108.6 145 39.3 31.3 11.9 19.4 13.4 24.5 11.8 39.1 12.4

Max. 140 180 100 48 34.4 46.4 39 55.4 34.4 87 58

Min. 90 90 0 23 -11.8 -20.4 3.6 -16 -21 -30

SDev. 12.1 26.8 25.2 3.6 11 10.5 6.98 10.8 11.4 18.1 18.6

Table 3.6: Descriptive statistics for ultrasound parameters of pelvic organ descent in the first/ early second trimester (n= 200). Negative values for 'BSD-SV', 'Cystocele', 'Cervix' and 'Rectum' infer descent below the symphysis pubis on Valsalva. For explanation of parameters see Tab. 3.5.

Bladder position on Valsalva

30

20 - ~ c (]) :::l 0" ~ LL 10

0

-20 -10 10 20 30 40 Cystocele

Fig. 3.2: Histogram of bladder position on Valsalva ('Cystocele') in early pregnancy. Positive values are above, negative are below the symphys1is pubis (measurements in mm, n=200).

95 Cervical position on Valsalva

25 - -r- - 1- - (;' 15 c Q) ::J 0"' r- ~ 10 LL 1- ,.- 1-- 5 rll.rr-1 0 Hl-,

0 50 100 Cervix

Fig. 3.3: Cervical position above the symphysis pubis (on maximal Valsalva). Positive values are above, negative below the symphysis pubis (measurements in mm, n= 199).

Position of the rectal ampulla on Valsalva

r- ;-

;--

;- - ;-- - (;' c ;- - Q) ;-- i- 6- 10 - ~ LL

i-

0 rl ll_o

-40 -30 -20 -10 0 10 20 30 40 50 60 rectum

Fig. 3.4: Position of the rectal ampulla on maximal Valsalva. Positive values are above, negative are below the symphysis pubis (measurements in mm, n= 200). 3.2.6.1.1 The influence of previous pregnancies

96 About one third of women recruited to the study had been pregnant prior to the index pregnancy, with prior gestations ending in first trimester terminations and/ or miscarriages. Only previous pregnancies that had progressed beyond the first trimester were considered an exclusion criterion. The histogram in Fig. 3.5 demonstrates the frequency of previous pregnancies. For 114 women the index pregnancy was the first, for 59 the second and for 13 the third pregnancy. Eight, two, two and one respectively were pregnant for the fourth, fifth, sixth or seventh time. In one case this information was not available.

100 -

fY c Q) ::l. C'" 50 - u..~

I 0 - I I I I I I I I 2 3 4 5 6 7 gravida

Fig. 3.5: Gravidity of enrolled patients (n= 199).

As early pregnancy may theoretically produce an irreversible (likely hormonal) effect on pelvic connective tissue, women with/ without previous first trimester pregnancies were compared for indices of pelvic organ mobility (see Table 3.7). There were no significant differences between the two groups. In addition, ANOVA analysis was performed to test for a cumulative effect of previous pregnancies. Again, no significant differences could be detected for ultrasound indices of pelvic organ mobility with respect to gravidity.

97 no previous pregnancy previous (n= 114) pregnancy (n= 85) p mean (SD) mean (SD) RVA-S (deg) 143.7 (SD 27.5) 146.2 (SD 25.) 0.5 ROT (deg) 39.0 (SD 24.7) 39.1 (SD 26.0) 0.9 BND (mm) 18.3 (SD 10.2) 20.7 (SD 10.7) 0.1 Cystocele (mm) 12.6 (SD 11.2) 10.9 (SD 11.5) 0.3 Cervix (mm) 39.6 {SD 18.2) 38.6 (SD 18.1) 0.7 Rectum (mm) 13.4 (SD 18.9) 11.4(SD 18.2) 0.5

Table 3.7: Previous first trimester pregnancy had no significant influence on parameters of pelvic organ mobility (n= 199).

3.2.6.2 Levator function

Of the 200 women recruited, all except 4 managed to contract the levator muscle either spontaneously on command or after short instruction, with an oblique bladder neck lift of 0 - 19.9 (mean 8.5, SD 3.9) mm. Only 41 (21 %) had ever been professionally taught (verbal teaching only). A spontaneous contraction on simple instruction was obtained in 167 women (82%). Advice as regards the performance of a levator contraction was necessary in 92 women (45%) in order to obtain an optimal contraction, e.g., to avoid activation of rectus abdominis, adductores or glutei. A reflex levator activation on coughing was seen in 113 women (58%). This was not associated with presence or absence of stress incontinence: of 171 stress- dry women, 99 showed a cough reflex as did 14 of24 stress incontinent women (58% of both groups). Also, there was no correlation between levator strength as measured on ultrasound and symptoms of stress incontinence: continent women demonstrated an oblique lift of mean 8.5 (SD 3.9) mm versus mean 8.7 (SD 4.4) mm in stress incontinent women.

98 On testing for correlations between ultrasound parameters of pelvic organ descent and levator function, significant correlations were detected for most tested parameters (see Table 3.8). Increased pelvic organ mobility was consistently associated with more marked cranioventral displacement of the bladder neck on levator activation.

Parameter RVA-S Rot BSD-SV BND Obi. descent Cystocele Cervix Rectum

Pearson's r 0.282 0.319 -0.295 0.262 0.249 -0.313 -0.155 -0.191 p= <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.029 0.007

Table 3.8: Correlations between levator strength as determined by translabial ultrasound and indices of pelvic organ mobility (n= 200). All correlations (positive or negative) signify more marked elevation of the bladder neck in women with more mobile pelvic organs.

The best predictor of a strong levator contraction was use of the levator on intercourse (see Table 3.9), while previous verbal teaching did not appear to influence measurements. ANOVA analysis was used to test for any association between levator strength and the level of sporting activity prior to pregnancy; however, there was no significant relationship detected (p= 0.9 for oblique lift on ultrasound versus level of sporting activity).

Motivational factors for the use of tre levator on intercourse mentioned were boy- friends, mothers, other female relatives and, most commonly, articles in popular maga-zines, e.g. "Cosmopolitan" and "Cleo"; however, this was not consistently documented.

PFM use on intercourse N Mean {mm) SD SE Mean

no 109 6.88 3.38 0.32 yes 85 10.53 3.46 0.38

Table 3.9: The relationship between regular conscious use ofPFM on intercourse and levator activity as determined by ultrasound (n= 194), p< 0.001.

99 3.2.7 Hormones in early pregnancy and pelvic organ mobility

3.2.7.1 Relaxin

Relaxin serum levels were determined by the author, under supervision of Lillian Tan, at the Endocrinology Laboratory, Prince ofWales Hospital, Randwick. An enzyme-linked immunosorbent assay (hRLX Elisa, Immundiagnostik Bensheim, Germany) was used on 49 serum samples obtained between 9 and 16 weeks' gestation. Tests were performed on two occasions a fortnight apart since one of the well plates used for tre first run yielded no results. This required re-freezing of samples which, according to information provided by the manufacturer Immundiagnostik Bensheim, should not affect serum levels unless performed more than three times. The second run was successful. Relaxin levels were determined at a mean of 184.79 (range 45.28- 394.55) pg/ mi.

X X 360 +

Relaxin X X X X X X 240 + X X X XX X X X - X 23 2 X X X X X X X XX 120 +x X XX X X X X XX X XXX X

X

+------+------+------+------+------+------gestation 9.6 10.8 12.0 13.2 14.4 15.6 p=0.9

Fig. 3.6: Relaxin serum levels plotted against gestational age (n= 49).

100 X X 360+

Relaxin- X XXX XX 240+ X X X X X 2x - X XX X XX X X X X X XX X XX X 120+ X X X xxxx X XX X X XX X

X

+------+------+------+------+------+------BND 0.0 8.0 16.0 24.0 32.0 40.0 p= 0.65

Fig. 3.7: Relaxin serum levels plotted against bladder neck descent on Valsalva (n= 49).

There was no correlation with any of the clinical indices of joint hypermobility or ultrasound parameters of pelvic organ mobility, neither at the first or the second visit nor with changes observed between first and second visit. Relaxin serum levels also did not correlate with gestational age as shown in Fig. 3.6. Fig. 3.7 shows a scattergram of serum levels against bladder neck descent.

3.2.7.2 Progesterone

Progesterone serum levels (n= 49) were determined on an automated analysis system (Vitros ECI, Johnson& Johnson Immunodiagnostics), utilizing a competitive immunoassay technique .. The only significant correlation detected was between gestational age (9-15 weeks) and serum progesterone (r= 0.488, p< 0.001), see Fig. 3.8. There were no significant or near significant correlations between serum progesterone and joint hypermobility or ultrasound indices of pelvic organ mobility (bladder neck descent, proximal urethral rotation, descent of cystocele, retrovesical angle on Valsalva). Fig. 3.9

101 shows a scattergram of progesterone serum levels against bladder neck descent (r= 0.148, p= 0.3), the paraJlleter with the closest correlation.

Progesterone-

0 150+

0 0 0 0 00 0 0 0 00 0 100+ 2 0 0 00 0 0 0 0 00 0 0 0 0 00 0 2o 00 0 - 00 0 0 0 0 o2 0 50+ 0

+------+------+------+------+------+ gestation 9.6 10.8 12.0 13.2 14.4 15.6 p< 0.001

Fig. 3.8: Progesterone serum levels plotted against gestational age (n= 49).

Progesterone-

0 150+

0 0 00 0 0 00 0 0 0 0 100+ 00 00 0 0 0 0 - 0 o2 0 0 00 0 2oo 0 000 - 0 0 0 0 0 0 00 0 0 50+ 0

+------+------+------+------+------+-BND 0.0 8.0 16.0 24.0 32.0 40.0 p=0.3

Fig. 3.9: Progesterone levels versus bladder neck descent on Valsalva (n= 49).

102 3.3 Second visit: Third trimester assessment

One hundred and seventy three of the 200 women originally enrolled (86.5%) were seen during the 3rd trimester, i.e., between 31 and 38 completed weeks. Table 3.10 summa-rizes reasons for non-attendance. Only 5 patients were unwilling to continue when contacted. 11 were unable to do so because of miscarriage/ termination of pregnancy or premature delivery, in one case two hours before her planned appointment. Eight women had moved away from Sydney. Three were lost to followup (1.5%).

Miscarriage/ stillbirth <28weeks 4 Termination of pregnancy 2 Premature Delivery 5 moved out of Sydney 8 unwilling to continue 5 lost to followup 3

Table 3.10: Reasons for non-attendance at 2nd visit (n= 27).

60

50

t)>- 40 c Q) :::1 30 0" Q) 1- u.. 20

10

0 I -r-· I -T-·-r- ~-- I I - ----r-1 30 31 32 33 34 35 36 37 38 Gestation in completed weeks

Fig. 3.10: Histogram for gestation at 2nd visit

103 Figure 3.10 shows a histogram of gestation at the time of the second visit. The wide spread results from the fact that at least three attemps were made to see everybody enrolled in the study in order to reach as many women as possible. This necessitated accommodating them as far as possible regarding the timing of this appointment. In some instances this meant moving dates forward or backward by up to one month. The mean for the timing of the second appointment was 35.5 weeks (range 31.6- 38.3, SD 1.2 weeks).

3.3.1 Symptoms

At the time of the second assessment, 68 women complained of stress incontinence (39% ), 21 ofurge incontinence (12%), 58 of frequency (34%) and 111 ofnocturia (64%). This is a significant increase over symptoms at the time of the first visit for stress incontinence (p= 0.01), frequency (p= 0.013) and nocturia (p< 0.001) but not for urge incontinence (p= 0.13). The development or worsening of symptoms during the course of pregnancy did not correlate with changes in bladder neck mobility nor with absolute values of parameters of anterior vaginal wall mobility at 36 weeks' gestation.

As a recent paper has linked higher relaxin serum levels in early pregnancy with a relatively lower prevalence of stress incontinence in late pregnancy (Kristiansson et al., 2001), we tested relaxin serum levels obtained in early pregnancy against stress incontinence reported at the second visit. Women who were suffering from stress incontinence at this point in time in fact were shown to have nonsignificantly higher relaxin levels on 2 sample t test than those who were continent (continent mean 166, SD 60.2, vs. incontinent mean 209, SD 84.5; p= 0.063).

3.3.2 Paper towel testing

Paper towel data was available for 172 women, 66 of which complained of stress incontinence at the time of the third trimester appointment. Overall, 23 women leaked on paper towel testing. This was no significant increase over first trimester paper towel testing,

104 and the average severity of the leak as defined by measurement of the wet patch on the paper towel had increased nonsignificantly. Sixteen women with a positive paper towel test had a history of stress leakage, and 50 others with complaints of stress leakage produced a negative paper towel test. This implies that the paper towel test was positive in only 16 out of 66 women who complained of stress incontinence (24%).

Nevertheless, as opposed to first visit data, women with symptoms of stress incontinence were much more likely to have a positive paper towel test (p= 0.001 on X2 test). When severity of symptoms was tested against the result of the paper towel test, a highly significant correlation was observed (p=0.001 on ANOVA). There was no correlation between paper towel test result and bladder neck mobility.

3.3.3 Joint hypermobility and striae gravidarum in the third trimester

Correlations for joint hypermobility angle measurements between first and second appointments varied markedly. While Pearson's correlation coefficients were 0.581 for wrist hyperflexion and 0.535 for fifth finger hyperextension, only 0.215 was reached for elbow hyperextension. All correlations are significant (p<0.001 for wrist and fifth finger, p<0.005 for elbow).

Between the first and second visit the only significant change observed was a reduction in wrist hyperflexion (169.4 deg SD 17.5 vs. 162.4 deg SD 17.4, p

105 3.3.4 Third trimester ultrasound assessment

Measurements were obtained for all 173 women who attended for their second visit. Exceptions were cystocele descent in one and cervical descent in 2 cases; the rectal ampulla was not visible in 10 women due to suboptimal imaging conditions or poor documentation. In four cases reflex levator activation could not be overcome despite all efforts at teaching (see 2.5.5.3).

3.3.4.1 Pelvic organ mobility

Table 3.11 shows descriptive statistics for pelvic organ mobility parameters as ascertained by ultrasound.

Oblique RVA-R RVA-S ROT BSD-RV BSD-SV BND descent Cystocele Cervix Rectum

(degrees) (degrees (degrees) (mm) (mm) (mm) (mm) (mm) (mm) (mm)

Mean 109.5 157 44 29.2 7 22.2 27.1 7 36.8 16.5

Max. 140 180 100 45.6 43 43.8 48.6 43 72 60

Min. 90 90 10 18 -22.5 7 -22.5 -10.7 -35.6

SDev. 12.8 23.7 25.6 4.3 11.5 10.3 10.7 11.5 14.5 20.9

Table 3.11: Descriptive statistics for ultrasound parameters used to define pelvic organ mobility (n= 173 for all parameters except Cystocele, n= 172, Cervix, n= 171, and Rectum, n = 163). Negative values for 'BSD-SV', 'Cystocele', 'Cervix' and 'Rectum' infer descent below the symphysis pubis on Valsalva.

None of the indices of pelvic organ relaxation except descent of the cervix correlated with gestational age. For the latter Pearson's correlation coefficient was -.258 (p= 0.001), suggesting a significant descent of the cervix with advancing gestation. On comparing the means (39.1 vs, 36.8) however it appears that this effect is minor.

106 A plot showing correlations between first and second visit measurements is given in Fig. 3.11. Pearson's correlation coefficient is 0.495 (p< 0.001). The change in bladder neck descent between first and second visit was on average 2.8 mm (range - 32 to +40.8 mm, SD 10.4 mm). Fig. 3.12 shows a histogram of the observed differences, illustrating that a few aberrant measurements are balanced by a large majority between +10 and - 7 mrn .

• 40- • • • • ••• • :t:::: • ••• • • -~ .. • • > • .. .. • ~- • ,, .. (/) • I • • I -L.. • • • t;::: • • 0 2>- • . • t • • z • • a • • ••• •• • •• • m ...... ·,: .. .' ~ • • • • • 10 - . ··r~- . . • ,., . • • • •• • ...• I ··-'· .. . . • • 0 - • • I I I I I I I I I I 0 5 10 15 20 25 30 35 40 45 BND second visit

Fig. 3.11: Measurements for bladder neck descent at the first and second visits plotted against each other. Pearson's correlation coefficient is 0.493 (n= 173).

The observed change in measurements between the first and second visits was not dependent on weight gain (r= 0.07, p= 0.3) or any other tested parameters, in particular not on gestational age or the difference in gestational age between the first and second visits.

107 40

30 ~ c (!) :::::1 20 0"' ....(!) LL 10

0

-40 -30 -20 -10 0 10 20 30 40 50 Change in BND from first to second visit

Fig. 3.12: Differences in BND between 1st and Illrd trimester visits (n= 173).

The difference between first and second visit measurements reached significance for BSD­ SV (11.9 vs. 7.0 mm, p< 0.001), BND (19.4 vs. 22.2 mm, p= 0.01), oblique descent (24.5 vs. 27.1 mm, p= 0.018) and maximal cystocele descent (11.8 vs. 7 mm, p< 0.001). Retrovesical angle at rest, rotation of the urethra, cervical and rectal descent were not significantly different, see Table 3.12.

Oblique RVA-R RVA-S Rotation BSD-RV BSD-SV BND descent Cystocele Cervix Rectum (degrees) (degrees (degrees) (mm) (mm) (mm) (mm) (mm) (mm) (mm) 151 appointment mean 108.6 145 39.3 31.3 11.9 19.4 24.5 11.8 39.1 12.4 2nd appointment mean 109.5 157 44 29.2 7 22.2 27.1 7 36.8 16.5

p= n.s. <0.001 n.s. <0.001 <0.001 0.01 0.018 <0.001 n.s. n.s.

Table 3.12: Changes in parameters of pelvic organ mobility between the first and second antepartal appointment (t- test statistics).

108 3.3.4.2 Levator function

Levator function was assessed by translabial ultrasound in all 173 women returning for their second visit. Of the four women who had not been able to manage a visible contraction at the time of the first visit, three returned, and two of those were now able to contract the levator on command, although additional instruction was required in one. As a result, all but one of 173 women were able to contract the levator at the time of the second assessment. The strength of levator activity as observed by translabial ultrasound was increased over the previous visit: 11.2 (SD 4.5)mm vs. 8.5 (SD 3.9) mm, p< 0.001. Fig. 3.13 shows a histogram of the observed changes in oblique lift of the internal urethral meatus on levator contraction.

20

~ c Q) ::J g 10 L.. LL

0

-10 -5 0 5 10 15 Change in levator strength

Fig. 3.13: Histogram of changes in oblique lift (displacement of the internal meatus on levator contraction) between first and second visits; n= 173, p< 0.001 for the difference.

Fig. 3.14 shows the correlation between first and second visit measurements oflevator strength by ultrasound (r= 0.592, p< 0.001).

109 20- • • • • • ;<: • • • ·;;;r.n • . " • • • • • • •••• • • ...... r.n . . .. ·~. - ~ ~ I ':· • a; 10 - . ' ·'" . _,., : . ¢::: .' .• • • I ... " • • ' • • ... .. ~- . Q) ...... _.,.. . :::l . . ,. .2" ...' ' ' "., ...... '' • r.. •\. .0 , " ' . 0 ""• •• • .. 0 - • • • I I I 0 10 20 Oblique lift at second visit

Fig. 3.14: Corrrelation between first and second visit measurements of levator strength by translabial ultrasound (n= 173, Pearson's r= 0.592, p< 0.001).

3.4 Delivery data

Information on labour and delivery was obtained for all 173 women who had attended for their third trimester appointment and for 10 others, giving data on 183 deliveries. Only ten patients were lost to follow- up (5%); 7 others were known to have had a miscarriage, termination or stillbirth before the completed 26th week of gestation.

Primarily, this information was obtained by issuing each patient with a labour and delivery assessment form which was filled in by the attending midwife/ doctor at the time of the delivery. 111/200 forms were returned (55.5%), three from other hospitals as far away as Wales. One woman was known to have delivered at another institution in Australia; attempts to obtain delivery information were unsuccessful in this instance. The hospital notes of all women were subsequently reviewed, the information on the returned forms checked and missing data entered. In cases where conflicting information was contained in notes and delivery data sheet, the more plausible alternative was selected after review of

110 the notes entered by midwife and doctor at the time of the delivery. Also, patients were asked to provide information at the time of their postpartum appointment, after completion of the assessment to preserve blinding. In two instances other hospitals were contacted with a request to provide missing data. Table 3.13 explains the reasons for incomplete data on labour and delivery

Originally enrolled 200

Termination ofpregnancy 2 Miscarriage (first trimester) 3 Miscarriage/ Stillbirth (second trimester) 2 Lost to follow- up 10

Delivery data available 183

Table 3.13: Reasons for incomplete delivery data

Apart from information on vaginal operative deliveries (mentioned in detail below) all labour and delivery data is complete and verified by hospital records. Table 3.14 to 3.17 record descriptive statistics.

Mean or *Median Range SD or **IQR

Gestational age at delivery 278 198- 301 14 days Length of first stage 492 0- 1263 284 mm Length of passive second stage 0* 0-270 0-55 min** Length of active second stage 52.6 0-255 49 mm Birthweight 3471 930- 5160 583 g

Table 3.14: Descriptive statistics for quantitative parameters (n= 183). Median and interquartile range for length of passive second stage.

111 The onset of labour was spontaneous in 123 women. In 60 cases the timing of delivery was determined by medical intervention (induction of labour or elective Caesarean Section). 58% of all women in this study opted for epidural analgesia (n= 107), and 59% had an indwelling catheter inserted (n= 109). Episiotomies were performed in 34 women (25% of vaginal deliveries), of which none extended to a third or fourth degree tear. Table 3.15 shows the frequency of perineal trauma overall.

n % Episiotomy 34 25% Tears I 20 15% II 50 37% Ill 5 4% IV 1 1%

any trauma 110 81%

Table 3.15: Perineal trauma amongst women who delivered vaginally (n= 135)

At the time of delivery, the presentation was Occipitoanterior in 144 women, Occipitoposterior in 18 women, occipitolateral in 3 women (all delivered by Caesarean Section) and Breech in 5 women. In 13 cases (all Caesarean deliveries) the presentation had not been documented. Table 3.16 shows delivery mode data, with Tab. 3.17 summarizing the 29 vaginal operative deliveries. No vaginal operative delivery included rotation of the fetal head by more than 45 degrees.

n %

Normal Vaginal Delivery 106 58% Elective Caesarean Section 12 7% Emergency Caesarean Section 36 20% Vaginal Operative Delivery 29 16% Vacuum 26 14% Forceps 10 5%

Table 3.16: Delivery mode (n= 183). 7 Vacuum deliveries were completed by Forceps.

112 Station n Pulls N Difficulty n

0 2 1 3 1 15

1 13 2 11 2 8 2 11 3 5 3 1 3 1 4 4 4 1 n.a. 2 5 3 n.a. 4

n.a. 3

Tab. 3.17: Vaginal operative deliveries(n= 29).

3.4.1 Pelvic organ mobility as predictor ofNormal Vaginal Delivery

With delivery data collection complete, it became possible to test hypothesis 10, i.e, to test whether pelvic organ mobility is associated with delivery mode. The analysis was performed on those data sets that contained both a 36 week visit and delivery information

Patients who were delivered by elective Caesarean Section (n=9) were excluded since the state of pelvic connective tissue structure would not be expected to have any influence on delivery indications such as Breech presentation (n=5), Placenta Praevia (n=1), intrauterine growth retardation (n=l), severe back pain (n=1) and abnormal cardiotocogram (n=1). This left 162 datasets. Table 3.18 shows differences in pelvic organ mobility between those who went on to deliver by normal vaginal delivery (n= 99) and tlnse who did not (n= 63). Numbers are lower than those given in Table 3.16 (n= 162 instead ofn=183) due to prelabour caesarean births and deliveries which occurred in women who had not attended their second appointment. Weak but signify-cant correlations were also found between most measures of pelvic organ descent and length of second stage (rotation r=-0.258, p=0.002, BSD-SV r= 0.224, p= 0.009, BND r=-0.183, p= 0.034, Cystocele r= 0.221, p= 0.01, Cervix r=0.221, p=0.01, Rectum r= 0.211, p= 0.017), with passive 2nd stage being more affected than active 2nd stage.

113 Oblique Rotation BSD-SV BND descent Cystocele Cervix Rectum

Normal vaginal delivery (n= 99) 49.1 4.3 24.2 28.6 4.4 35.2 13.8 Operative delivery (n= 63) 37.5 10.6 19.4 23.5 10.5 39 20.9

P= 0.004 0.001 0.004 0.02 0.001 0.1 0.037

Table 3.18: Relationship between antepartum indices of pelvic organ mobility and subsequent delivery mode (n= 162); t- test statistics. For all tested parameters, normal vaginal delivery was associated with higher pelvic organ mobility although this did not reach significance for the central compartment.

Parameters of pelvic organ descent proved to be predictors of normal vaginal delivery (NVD): Figure 3.15 shows a regression analysis of maximum cystocele descent at the third trimester appointment against the predicted probability of a normal vaginal delivery. This regression model was significant at p= 0.002.

114 1.0

0 .9

.8

.7

.6

~ .5 :.aro ..0 ....0 .4 c. "0 Q) t5 .3 '6 0 ~ a.. .2 -30 -20 -10 0 10 20 30 40 50

CYSTOCEL

Figure 3.15: Regression plot for likelihood ofNVD against maximal cystocele descent (Cystocel) at 32-38 weeks (p=0.002).

115 3.5 Third visit: Postpartum assessment

Fig. 3.16 shows a histogram of the interval between delivery and postpartum assessment. 169 women were seen on average 92.7 days (range 20- 217, SD 18.8) after delivery oftheir child. In three cases the followup appointment had to be arranged less than 60 days postpartum to allow attendance. Four women were seen more than 120 days postpartum. Of those 169 women, 162 had been seen at 36 weeks which means that complete datasets (36 week visit- labour/ delivery datal 3months postpartum visit) for the assessment of delivery­ related changes were available in 162 cases.

50

40 :>. 0 ffi 30 ::J 0" ~ 20 LJ..

10

0

0 100 200 Days after Childbirth

Fig. 3.16: Timing ofthe 3rd (postpartum) visit (n= 169).

Miscarriage/ SB <28weeks: 4 Termination of pregnancy 2 Moved within Australia 6 Moved Overseas 6 unwilling to continue 10 lost to followup 3

Table 3.19: Reasons for non-attendance at 3rd visit (n= 31 ).

116 Table 3.19 gives the reasons for norrattendance: 12 women were unable to attend due to their having left Sydney, three were lost to followup and probably also had moved away. Ten women either expressed an unwillingness to return for their followup appointment or did not attend three consecutive appointments. A postpartum followup appointment was not arranged in women with miscarriages, terminations or stillbirths before 28 weeks gestation (n=6).

3.5.1 Bladder symptoms at the postpartum visit

Approximately three months postpartum, 34 women (20%) complained of stress incontinence. One experienced stress leakage several times a day, seven noticed it about once a day. Incontinence occurred about once a week in 10 women and about once a month in 16 women. Compared to antepartal symptoms, this is a marked reduction from 68/ 173 (39%), p< 0.001 on X' test.

In order to test Hypothesis 11: 'Bladder neck descent at first visit is a predictor of postpartum incontinence', this ultrasound parameter was tested for predictive value for symptoms of stress incontinence postpartum. No significant or near- significant associations or correlations were observed. In particular, there were no differences in bladder neck descent or proximal urethral rotation between women who were stress incontinent (mean BND 20.0, SD 12.9 mm, mean urethral rotation 42.6, SD 28.2 degrees) and stress continent postpartum (mean BND 19.3, SD 9.9 mm, mean urethral rotation 38.2, SD 24.9 degrees).

To test hypothesis 12: 'The symptom of postpartum stress incontinence is associated with delivery mode, birthweight and/ or length of second stage', stress incontinence at the third visit was correlated with these delivery- related factors. Neither birthweight nor epidural analgesia, active or total second stage differed between stress continent and stress incontinent women.

117 Delivery mode however did correlate with postpartum incontinence; on chi square testing, caesarean section was clearly protective against stress incontinence. Only 2/44 women were stress incontinent after caesarean section (5%), whereas this was the case in 32 of 125 women after vaginal delivery (26%, p= 0.003 on X2 test). Operative vaginal delivery did not seem to increase this risk further: 4 out of25 women (16%) after Vacuum or Forceps delivery complained of stress incontinence postpartum. For testing of postpartum symptoms against increases in pelvic organ descent, see section 3.5 .4.1.5.

Other urinary tract symptoms were reported less commonly: Urge incontinence occurred about once a month in five women, about once a week in another five; two women reported urge leakagl approx. once daily. Compared to antepartum, this implies a nonsignificant decrease in urge incontinence: a reduction from 21/ 173 to 12/ 169 (p= 0.1 on X2 test).

16 women (10%) suffered from mild frequency (9-12 voids a day), a marked decrease over antepartal symptoms (p< 0.001 on X2 test). Nocturia was reported by 26 women (15%), of whom 23 averaged 2 voids per night and three experienced 3-4 voids per night. This also constitutes a significant reduction from 58% antepartum (p< 0.001 on X: test).

Symptoms of voiding dysfunction such as hesitancy, poor stream, stop- start voiding, straining to void, the feeling of incomplete emptying and the need to revoid were reported by 34 women. The collected data would allow a more detailed analysis which, however, was thought to be outside the scope of this work.

3.5.2 Postpartum paper towel testing

At the postpartum visit, 11 out of 169 women had a positive paper towel test; and again not all of those ( 6 out of 11) had reported the symptom of stress leakage. The frequency of a positive paper towel test was non-significantly lower than at both other visits. Those women with a positive test were more likely to complain of symptoms of stress incontinence (X2 test, p= 0.003). They also showed higher values in the peripartal change

118 in bladder neck descent (mean 13.6 [SD 12.3] mm versus mean 5.9 [SD 10.4] mm) than those with a negative test although this trend did not reach significance (p= 0.084 on 2- sample t- test).

3.5.3 Postpartum joint hypermobility, striae gravidarum and pelvic girdle pain

Table 3.20 shows joint hypermobility indices at the postpartum visit.

Wrist V Digit Elbow hyperflexion hyperextension hyperextension Average 164.9 70.8 185.9 Max. 195.0 100.0 210.0 Min. 120.0 30.0 160.0 SDev. 16.9 14.2 7.3

Table 3.20: Joint hypermobility at the postpartum visit (n= 169). All values indicate goniometer readings in degrees.

There were no significant differences between anteparta1 and postpartal measurements for wrist hyperflexion (mean 162.4 vs. 164.9 deg.), fifth digit hyperextension (mean 70.8 vs. 70.8 deg.) and elbow hyperextension (mean 184.7 vs. mean 185.9 deg.) Abdominal striae gravidarum (stretchmarks) were ascertained in 166/ 169 women and found in 55 (33%). In 111 women no striae were found in the area assessed (anterior abdominal wall between umbilicus and the anterior superior spine of the iliac crest, see 2.5.3.). The maximum number of striae counted was 40, the minimum two. There was no correlation between indices of pelvic organ mobility or stress incontinence on the one hand and joint hypermobility or striae gravidarum on the other hand when this aspect of hypothesis 2 was tested.

In order to ascertain pain caused by pelvic girdle relaxation, women were asked to use a visual anlog scale to rate their experience of lower back and symphyseal pain in late pregnancy and the puerperium, excepting the delivery itself (see 2.5.1). Readings were

119 taken in 164 women. The average reading was 27.1 (range 0-100, SD 28.1). Again, there

were no signific~nt or near significant correlations between any of the main indices of pelvic organ descent on the one hand and the degree of reported pelvic girdle pain on the other hand (part of hypothesis 2). However, when the visual analog scale reading was analyzed versus symptoms of stress incontinence, the latter was associated with higher VAS readings for back pain experienced in late pregnancy and the puerperium (mean 37.3, SD 29.5 vs. mean 24.5, SD 27.2, p= 0.029 on 2-sample t-test).

3.5.4 Ultrasound assessment at the postpartum visit

An ultrasound assessment of pelvic organ mobility and levator strength was performed in all 169 women presenting for their postpartum visit. Cervix and I or rectum were not reliably imaged in six cases each.

3.5.4.1 Pelvic organ mobility

3.5.4.1.1 Descriptive data

Table 3.21 shows descriptive statistics for pelvic organ mobility. In comparison to measurements at the second visit, many of those parameters had undergone significant change (see Tab. 3.22). Excepting the retrovesical angle on Valsalva, all indicators of pelvic organ mobility are significantly increased compared to the antepartum measurements, and in all cases this change is towards increasing mobility.

A plot showing correlations between second and third visit BND measurements is given in Fig. 3.17. Pearson's correlation coefficient is 0.49 (p< 0.001) with third trimester measurements and 0.51 with first visit measurements (p< 0.001), n= 162.

120 Oblique RVA-R RVA-S Rot BSD-RV BSD-SV BND descent Cystocele Cervix Rectum (degrees) (degrees (degrees) (mm) (mm) (mm) (nun) (mm) (mm) (mm)

Mean 109.3 153.4 61.3 29 0.5 28.5 32.1 0.1 20.8 2.9

Max. 150 180 120 38.1 29 50 51.5 29 51 43

Min. 90 80 0 19.2 -23.1 5.8 8.4 -23.1 -15 -33.1

SDev. 11.3 28 34.4 3.5 11.5 10.6 10.3 12.3 15.9 19 Table 3.21: Descriptive statistics for ultrasound parameters used to defme pelvic organ mobility at approx. 3 months postpartum (n= 169). Negative values infer descent below the symphysis pubis on Valsalva.

BSD- BSD- Oblique RVA-R RVA-S Rot RV sv BND descent Cystocele Cervix Rectum (degrees) (degrees (degrees) (mm) (mm) (nun) (nun) (nun) (mm) (mm) n= 161 161 159 161 161 161 161 161 157 153

AP mean 109.5 157 44 29.2 7 21.3 27.1 7 36.8 16.5

PP mean 109.3 153.4 61.3 29 0.5 28.5 32.1 0.1 20.8 2.9

p= n.s. n.s. <0.001 n.s. <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

Table 3.22: Comparison of ultrasound data ante- and postpartum. In all instances significant associations show increased pelvic organ descent postpartum.

Changes in the above parameters of pelvic organ mobility (ROT, BSD-SV, BND, oblique descent and descent of cystocele, cervix and rectal ampulla) were presumed to be indicative of birth trauma to fascial support structures. These parameters were therefore correlated with all labour/ delivery related factors that could be assumed to contribute to such trauma.

Fig. 3.18 shows a histogram ofperipartal changes in BND measurements. It is evident that changes towards hypermobility were observed in a large number of women.

121 ~ __J------, • . ..•.• 40- • .•• • 35- • • • • •••.. f ..: ••• Xl- ..... f •• ..-- 25 - . . -- ~,. o.' ...... , -....t.·l, . z a>­ • • • • • • • • a:l • ...... ·~ .. 15 - , .. . . . 10 - .. ' ...... • .. _.. ' ... . .' • 5- • .. ... ' . 0- • I I I I I I 0 10 20 30 40 50 BND_2

Fig. 3.17: BND at third trimester appointment and postpartum visit (n= 161), r= 0.49.

40

30

~ c Q) :::J 20 0" ~ u. 10

0

-20 -10 0 10 20 30 40 BND change antepartum- postpartum

Fig. 3.18: Histogram ofBND changes from second to third visit (n= 161).

122 3.5.4.1.2 Gestational length and induction oflabour

There were no significant correlations between length of gestation in women who delivered vaginally and indices of pelvic organ mobility or the increase in the values of those indices peripartum. No significant differences were detected beween spontaneous and nonspontaneous onset of labour.

3.5.4.1.3 First and second stage of labour

As regards length of the first stage of labour, there were no significant correlations observed with change in the indices of pelvic organ mobility, neither for the whole dataset nor for a subset containing vaginal deliveries only. However, all tested parameters of pelvic organ mobility change, i.e., all parameters of presumptive traumatic fascial damage, correlated positively with the length of the second stage of labour, i.e., from full cervical dilatation to delivery. Table 3.23 gives the significance of correlations between total length of second stage and passive/ active second stage on the one hand, and above- defmed parameters of presumptive fascial trauma on the other hand. As the data for passive second stage was skewed (D=0.284 on Kolmogorov- Smirnov Test), Spearman's correlation coefficient was used.

As described in 2.7.3, a linear regression model was constructed using length of :first stage, passive and active second stage as predictors for the response peripartum BND change, in order to obtain a numerical measure of the relative importance of first and second stage of labour. This yielded the equation

BND change= 2.79 + 0.00305 1st stage+ 0.0112 2nd pass+ 0.0342 2nd act, demonstrating that the relative weighing of first stage, passive and active second stage is approximately as 1: 3. 6 : 11.2, with active second stage being by far the strongest contributor. For active second stage this analysis reaches significance (p= 0.03).

123 Change in Urethral Bladder neck Cystocele Cervical Rectal Parameters rotation descent descent descent descent

Total 2nd stage r= 0.192 r=0.171 r=-0.232 r=-0.211 r=-0.288 p=0.016 p=0.031 p=0.003 p=0.009 p=<0.001 Passive 2nd stage r=0.131 r=0.121 r=-0.18 r=-0.144 r=-0.19 p=0.10 p=0.128 p=0.024 p=0.077 p=0.019 Active 2nd stage r=0.180 r=0.170 r=-0.231 r=-0.176 r=-0.223 p=0.024 p=0.032 p=0.003 p=0.029 p=0.005

Table 3.23: Pearson's r and p values for correlations between parameters of fascial trauma and total/ active second stage (see ANOVA graphs for n). Spearman's correlation used for passive second stage. All relationships are positive in the sense that increased length of second stage was associated with increasing pelvic organ mobility/ descent on Valsalva.

Numerically, this model implies that every half. hour in active second stage results in an increase in BND of one mm on average (1 I 0.0342 = 29.2). Regression analysis for other parameters of anterior vaginal wall descent had similar results, with change in Cystocele descent being the most sensitive of them all (p= 0.016 for the regression model):

Cystoc change 2-3 = -1.73 - 0.00348 1st stage -0.0203 2nd pass -0.0593 2nd act

The relative weighing of first, passive and active second stage is as 1: 5.8: 17.This implies that one mm increase in cystocele descent is to be expected on average after 17 minutes in active second stage (1 I 0.05.93= 16.8).

Change in mobility of the cervix did not yield significant results on regression analysis using the above predictors (p= 0.1), but change in the position of the ampulla recti on Valsalva reached the highest level of significance (p= 0.001 for the regression model), albeit showing a different pattern of weighing:

Rectum change = - 10.3 + 0.0111 1st stage - 0.110 2nd pass - 0.100 2nd act The relative weighing is 1: 10: 9, showing passive second stage being at least as strong a predictor as active second stage. The implication is that for every 10 minutes in active or

124 passive second stage, the change in position of the rectal ampulla will increase by more than one nun. This weighing differs from the one documented for the anterior compartment in that passive second stage seemed to have an effect on the posterior compartment that was over and above its effect on anterior compartment indices (see Figure 3.19).

However, when regression modeling was used to test whether this effect of second stage on anterior and central compartments was independent of the delivery mode, virtually the whole effect on the outcome parameter was explained by delivery mode, with active and passive second stage both becoming nonsignificant (see 3.5.4.1.5). The opposite was true for the posterior compartment: merging delivery modes and length of second stage into a model showed that delivery mode had virtually no independent effect.

18.------~

16 -t------i

14 -t------i

12 +------l 1st stage 10 -t------1 o • pass. 2nd stage 8 -t------1 o act. 2nd stage 6 -t------:===::::j

4+----

2 -t------

2 Anterior compartment Posterior compartment

Fig. 3.19: Relative weighing of first stage, passive and active second stage of labour for its effect on mobility of the anterior and posterior compartment. The posterior compartment seems to be affected equally by passive and active second stage; this does not seem to be the case for the anterior compartment.

125 3.5.4.1.4 Birthweiglt

The correlation between birthweight and indicators of fascial trauma was tested in a subset ofvaginal deliveries only (n= 121) with otherwise complete datasets. None ofthe parameters correlated significantly with birthweight although there were consistent trends towards increased pelvic organ descent with increasing birthweight (see Table 3.24).

Oblique Correlation with Birthweight ROT BSD-SV BND descent Cystocele Cervix Rectum

Pearson's correlation coefficient 0.15 -0.16 0.15 0.12 -0.14 -0.04 0.02

P= 0.089 0.08 0.09 0.19 0.13 0.65 0.84

Table 3.24: Correlation between parameters of fascial trauma and birthweight (vaginal deliveries only, n= 121).

3.5.4.1.5 Delivery mode

All ultrasound imaging parameters defined as indicative of fascial trauma were analyzed against delivery mode. Those women who had undergone a failed vacuum delivery with subsequent forceps were categorized with primary forceps deliveries. A distinction between primary and secondary forceps is made impractical by the low numbers.

ANOVA allowed ranking of delivery mode for potential to cause trauma to anterior vaginal wall support structures. From least to most traumatic this appears to be: Prelabour

Caesarean Section (CIS)- CIS in 1st stage- CIS in 2nd stage- Normal vaginal delivery­ Ventouse delivery- Forceps. In order to give relative values, ANOVA analysis was carried out after categorizing patients into these six groups (see Figures 3.20-26).

126 N Mean 8Dev ------+------+------+------

Prelabour C/8 11 -2.73 25.33 (------*------) C/8 in stage I 23 6.52 24.97 (----*----) C/8 in stage II 6 8.33 26.39 (------*------) NVD 94 19.57 30.01 (- -*-) Vacuum Del. 16 18.75 39.31 (-----*----) Forceps Del. 9 52.22 24.89 (------*------) ------+------+------+------0 25 50

Fig. 3.20: Change in proximal urethral rotation on Valsalva relative to delivery mode (n= 159). Increased rotation is signified by positive numbers (p= 0.001 for ANOVA).

Delivery mode N Mean 8Dev ------+------+------+------Prelabour C/8 11 1.84 8.41 (------*-----) C/8 in stage I 23 -2.47 10.35 (----*---) C/8 in stage II 7 -4.41 6.14 (------*------) NVD 95 -7.30 10.88 (-*-) Vacuum Del. 16 -9.64 14.64 (----*-----) Forceps Del. 9 -19.07 8.52 (------*------) ------+------+------+------20 -10 0

Fig. 3.21: Change in bladder neck position on Valsalva after different modes of delivery (n= 161). Increased descent is signified by negative numbers (p= 0.001 for ANOVA).

Delivery mode N Mean 8Dev -+------+------+----- Prelabour C/8 11 -2.27 8.75 (------*------) C/8 in stage I 23 2.63 9.50 (----*-----) C/8 in stage II 7 4.00 4.58 (------*------) NVD 95 7.24 10.17 (--*--) VD 16 9.22 14.72 (------*-----) FD 9 14.49 6.53 (------*------) -+------+------+------+------8.0 0.0 8.0 16.0

Fig. 3.22: Change in bladder neck descent (vertical) on Valsalva after different modes of delivery (n= 161 ). Increased descent is signified by positive numbers (p= 0.002 for ANOVA).

127 As regards the main outcome parameter used to defme anterior vaginal wall support, i.e., bladder neck descent on Valsalva manoeuvre, delivery mode proved to be a strong determinant of peripartum change. Prelabour Cesarean Section led to an average reduction in bladder neck descent of 2.27 mm, almost canceling out the average gain in BND observed during pregnancy (2.8 mm). Bladder neck descent increased postpartum for every other delivery mode, in the order of Caesarean Section during first stage, C/S in second stage, normal vaginal delivery, Ventouse and Forceps deliveries. The same pattern was observed for all other parameters of anterior vaginal wall descent (Figures 3.20- 3.24).

Tukey's pairwise comparisons were carried out with the ANOVA; examples for the most important outcome parameters are given below. For urethral rotation, pairwise comparisons remained significant between Forceps and prelabour Caesarean Section, Caesarean Section in first stage and Normal Vaginal Delivery. For bladder neck descent, prelabour C/S remained significant against normal vaginal delivery and Forceps; the same held true for C/S in first stage versus Forceps. For cystocele position, pairwise comparisons remained significant for Forceps versus all other delivery groups except Ventouse.

The effect of delivery mode on central and posterior compartment descent was less obvious (see Fig. 3.25 and 3.26).

128 Delivery mode N Mean SDev ---+------+------+------+--- Prelabour C/S 11 -6.06 8.63 (-----*-----) C/S in Stage I 22 0.80 8.67 (---*---) C/S in stage II 7 2.93 6.27 (------*------) NVD 93 5.57 9.97 ( -*-) Vacuum Del. 16 9.03 14.43 (----*----) Forceps Del. 10 14.83 7.29 (-----*-----) ---+------+------+------+--- -10 0 10 20

Fig. 3.23: Change in total (oblique) bladder neck descent after different delivery modes (n= 161). For significances between groups see Table 3.25. Increased descent is signified by positive numbers (p< 0.001 for ANOVA).

Delivery mode N Mean SDev -----+------+------+------+-

Prelabour C/S 11 1. 58 8.63 (-----*------) C/S in stage I 23 -1.31 13.37 (---*---) C/S in stage II 7 -2.31 11.43 (------*------) NVD 95 -8.06 12.65 (-*-) Vacuum Del. 16 -9.92 14.44 (----*----) Forceps Del. 9 -20.87 8.86 (------*-----) -----+------+------+------+- -24 -12 0 12

Fig. 3.24: Change in cystocele position on Valsalva relative to delivery mode (n= 161). For significances between groups see Table 3.25. Increased descent is signified by negative numbers (p= 0.001 for ANOVA).

Delivery mode N Mean SDev --+------+------+------+---- Prelabour C/S 11 -11.95 13.61 (------*------) C/S in stage I 22 -4.30 18.42 (-----*------) C/S in stage II 7 -19.59 14.58 (------*------) NVD 90 -18.65 17.80 (--*---) Vacuum Del. 15 -19.44 17.93 (------*------) Forceps Del. 8 -22.45 21.62 (------*------) --+------+------+------+---- -30 -20 -10 0

Fig. 3.25: Change in position of the cervix on Valsalva relative to delivery mode (n= 157). Increased descent is signified by negative numbers (p= 0.019 for ANOVA).

129 Delivery mode N Mean 8Dev -----+------+------+------+-

Prelabour C/8 10 -3.68 22.13 (------*------) C/8 in stage I 21 -0.15 20.56 (----*----) C/8 in stage II 7 -32.14 13.95 (------*------) NVD 90 -12.67 24.25 (--*--) Vacuum Del. 16 -20.61 23.35 (------*------) Forceps Del. 9 -32.96 23.22 (------*------) -----+------+------+------+- -40 -20 0 20

Fig. 3.26: Change in position of the rectal ampulla on Valsalva relative to delivery mode (n= 153). Increased descent is signified by negative numbers (p= 0.002 for ANOVA).

Interestingly, a Caesarean in 2nd stage seemed to have no protective effect on descent of the ampulla recti which parallels findings for levator ani strength (see below).

The impact of delivery mode was confirmed with regression analysis, using birthweight, first and second stage and delivery mode:

Predictor Coef 8E Coef T p Constant -2.93 32.96 -0.09 0.929 NVD -6.932 2.701 -2.57 0.011 VD2 -8.893 3.934 -2.26 0.025 FD -11.206 4.642 -2.41 0.017 gest age 0.0491 0.1286 0.38 0.703 1st stag -0.003222 0.003883 -0.83 0.408 2nd pass -0.02668 0.02336 -1.14 0.255 2nd act -0.00909 0.02596 -0.35 0.727 BW -0.002565 0.002381 -1.08 0.283

NVD, Ventouse and Forceps delivery were the strongest factors in the model, accounting for virtually all the difference attributable to second stage. No other factors reached significance on their own as long as these three delivery modes were still present. Conversely, the length of second stage (or any of its constituents) did not seem to confer an independent effect on any of the parameters of anterior vaginal wall descent. Using

130 Cystocele change as an example, the strongest model resulted from omission of all factors except these three, making it highly significant:

Cystoc change 2-3 = - 0.87 - 7.09 NVD - 9.35 VD 2 - 11.5 FD (P<0.0001).

Predictor Coef SE Coef T p Constant -0.870 1.920 -0.45 0.651 NVD -7.092 2.309 -3.07 0.003 VD2 -9.353 3.433 -2.72 0.007 FD -11.454 4.490 -2.55 0.012

To test the magnitude to which childbirth had contributed to the degree of pelvic organ mobility observed at the postpartum visit, the increase in bladder neck descent from the third trimester to the postpartum appointment was represented as a fraction of the descent observed at the postpartum visit. This allowed direct estimation of the relative importance of childbirth for pelvic organ mobility observed in a given patient or group. Fig. 3.27 shows ANOVA testing of BND measurements after stratification for delivery mode. The differences between delivery groups were significant on ANOVA (p<0.003). On t- Test, women delivered by Caesarean showed a markedly lesser contribution of childbirth to bladder neck mobility (mean 4% only) than those delivered vaginally (mean 33%). This result was also significant (p= 0.003).

Delivery Mode N Mean SDev ------+------+------+------Prelabour.C/S 14 0.0486 0.3738 (------*------) C/S in Ist St. 22 0.0132 0.6744 (-----*------) C/S in IInd St. 8 0.0800 0.4304 (------*------) NVD 99 0.3472 0.3055 (--*--) Vacuum Del. 16 0.1794 0. 8683 (------*------) Forceps Del. 10 0.5310 0.2076 (------*------+------+------+------0.00 0.30 0.60

Fig.3.27: Relative contribution of childbirth to bladder neck mobility observed at the postpartum visit. The mean could be interpreted as giving the relative contribution of childbirth to mobility in this group. To give an example: amongst patients delivered by Forceps, bladder neck descent postpartum was to 53% due to childbirth (p= 0.003 for ANOVA).

131 This derived parameter was also tested against the symptom of stress incontinence as a potential measure of the degree of traumatic injury to the suburethral hammock. Women with stress incontinence more than once a week and women with worse stress incontinence than at their first appointment were shown to demonstrate a higher contribution of childbirth to bladder neck mobility although these relationships were not highly significant (see Tables 3.25 and 3.26).

81>=1/wk 81<1/wk p= Delivery contribution to BND 47% (8D 28) 32% (8D 25) 0.048

Table 3.25: The contribution ofbirth trauma to bladder neck descent vs. stress incontinence postpartum (n= 169).

81 worse 8 I same or better p= Delivery contribution to BND 44% (8D 30) 32% (8D 25) 0.044

Table 3.26: The contribution of birth trauma to bladder neck descent vs. stress incontinence postpartum II (n= 169)

Finally, the data was stratified for the severity of postpartum stress incontinence and tested against the delivery contribution to bladder neck mobility. Figure 3.28 shows an ANOVA analysis of this data. Despite the visually obvious trend the analysis was not significant due to wide confidence intervals.

132 Stres N Mean SDev -----+------+------+-- Incontinence

less than 1/m 135 0.3227 0.2504 (-*) 1/m to < 1/wk 16 0.3363 0.3029 (---*---) 1/wk to < 1/d 10 0.4350 0.3186 (-----*----) approx 1/d 7 0.4929 0.2498 (-----*------) more often 1 0.6700 0.0000 (------*------) -----+------+------+------+ 0.30 0.60 0.90 1.20

Fig. 3.28: ANOVA testing of the contribution of childbirth to bladder neck mobility versus stress incontinence postpartum (n= 169, p= 0.2)

3.5.4.1.6 Presentation, epidural analgesia and perineal trauma

Occipita- posterior presentation was associated with caesarean section as delivery mode (p< 0.001 on t-test), but not with indices of presumptive pelvic floor trauma for any of the three compartments when tested in a subset containing only vaginal deliveries. In the same group, epidural anaesthesia was not associated changes in ultrasound measurements. Finally, no significant differences between groups with different degrees of perineal trauma were detected on ANOVA testing regarding any of the ultrasound indices of pelvic organ descent.

3.5.4.2 Levator function

Cranioventral displacement of the bladder neck on levator contraction was significantly reduced postpartum (8.82 (SD 4.21) mm vs. 11.21 (SD 4.5) mm, p< 0.001) compared to the third trimester visit, although there was no difference between early pregnancy and postpartum measurements. This reduction in the effect of a levator contraction was taken as indirect evidence of impairment of function, and the same delivery- related factors as in 3.5.4.1 were tested for associations with observed change.

133 Gestational age at delivery showed no significant effect nor did the length of the first stage of labour, in contrast to second stage duration. The active component of second stage duration correlated somewhat more strongly than the passive component (r=-0.196, p= 0.013 vs. r= -0.147, p= 0.066 on Spearman's correlation), but when the reduction in levator strength was tested against total second stage of labour, the correlation became more significant (-0.225, p= 0.004) although it remained weak.

A regression analysis similar to the one given in 3.5.4.1.3 was also performed. Although this modeling did not reach significance for change in levator strength (p= 0.07), the same pattern as in rectal descent (see 3.5.4.1.3) was observed for the levator ani:

Oblift change 2-3 = -1.71 + 0.00072 1st stage -0.0139 2nd pass- 0.0129 2nd act,

The relative weighing rated passive second stage about as strongly as active second stage: 1: 19: 17.9. The numerical effect appears smaller not the least due to the numerically lower values obtained on measuring the effect of a levator contraction as opposed to the effect of a Valsalva manoeuvre. A reduction of oblique lift on ultrasound of one mm resulted on average after 72 minutes in passive second stage (1/ 0.0139) and after 78 minutes (1/0.0129) in active second stage.

Birthweight did not correlate with levator strength but delivery mode did: Caesarean section appeared to be weakly protective (means-1.17 mm vs. -2.87 mm, p= 0.031) when tested against vaginal delivery. Operative vaginal delivery resulted in a more marked reduction (-3.83 mm vs. -1.17 mm for caesarean section, p= 0.022). Other differences between delivery groups showed nonsignificant differences, resulting in a ranking for traumatic potential that was different from the ranking established for the anterior and central compartment (see Fig. 3.29), although mt significant on ANOVA (p= 0.08). The effect of a prelabour caesarean section does not seem to differ from a procedure in first stage, and a C/S before full dilatation was markedly different from a procedure done after this point in time (-0.61 mm vs. -3.9 mm, p= 0.009). A Caesarean in second stage appears

134 to exert no discernible protective effect on longterm levator function relative to any form of vaginal delivery, including forceps.

Level N Mean 8Dev ------+------+------+------

Prelab. C/8 11 -1.327 5.709 (------*------) C/8 Ist st. 23 -0.270 3.363 (------*------) C/8 IInd st 7 -3.900 2.217 (------*------) NVD 95 -2.553 4.757 (---*--) Vacuum del. 16 -4.175 5.470 (------*------) Forceps del 9 -3.956 2.544 (------*------) ------+------+------+------5.0 -2.5 0.0

Fig. 3.29: Reduction in levator strength as determined by translabial ultrasound after different forms of delivery (n= 161, ANOVA p= 0.08).

Moreover, when length of second stage and delivery mode were analyzed jointly in a regression model, it was apparent that delivery mode had no significant relationship with the outcome parameter independent of delivery mode, similar to the pattern observed for posterior compartment descent but very different from the pattern seen for anterior and central compartment data.

Fetal presentation and epidural anaesthesia had no effect on reduction of levator strength on univariate analysis. Also, there was no association between different degrees of perineal trauma and a peripartum change in levator strength.

In conclusion, the obvious reduction in levator strength observed at the postpartum visit was generally less strongly associated with factors thought to produce pelvic floor trauma than most indices of pelvic organ descent.

3.6 3D Ultrasound imaging

23 women were included in a pilot study assessing the use of volume or 3D ultrasound in the detection of delivery- related pelvic floor trauma. They were seen on average at 35.6 weeks' gestation (range 33.1- 36.7 weeks) and 3 months postpartum (range 2.3-4.1 mo.).

135 16 delivered vaginally (14 normal vaginal deliveries, 2 ventouse deliveries), 7 underwent Caesarean Section (one before the onset of labour, 5 in first stage and one in second stage).

As quantitative analysis is precluded by the acquisition mode (see 2.5.5.7), this pilot study was limited to a qualitative assessment of anterior compartment supports (i.e., endopelvic/ paravaginal fascia, arcus tencineus fasciae pelvis, ATFP, and arcus tendineus levator ani, ATLA), see also Figures 2.13- 2.15. The integrity of paravaginal supports is supposedly shown by ,tenting" of the lateral vaginal fornices towards the arcus tendineus fasciae pelvis- i.e., the state of the suburethral hammock (DeLancey, 2001). An attempt was made to evaluate this ,tenting" on the three levels of central urethra (plane 1), bladder neck (plane II) and upper vagina (plane III). Tenting was scored as 'present', 'indeterminate' and 'absent' for both right and left paravaginal areas.

At their antepartum visit, only three patients out of 23 exhibited absence of tenting at one of three planes, with no significant differences in anterior vaginal wall mobility between those three and the remaining women (BND 21.2, SD 10.9 in 20 women with intact tenting, BND 27.8, SD 14.1 in 3 without, p= 0.5). Intact tenting was seen with a wide range of anterior vaginal wall mobility (BND range 5.4 mm to 41.6 mm).

Two postpartum volumes were found to be of inferior quality, leaving 21 sets of antepartum/ postpartum volumes to be evaluated. The subsequent analysis is limited to those 21 volume pairs. Definite changes were observed in five women. These changes occurred on all three planes selected. Table 3.27 gives data on antepartum and postpartum tenting. Of the five women with reduced tenting, four had had a vaginal delivery, one had been delivered by Casearean Section after 10 hours in first stage., with the head palpable at the spines.

136 Peripartal change in paravaginal support Plane1 Plane 2 Plane 3 overall

Same 17 16 17 16 Better 0 0 0 0 Worse 2 4 3 5 Indeterminate 2 1 1 0

Table 3.27: Peripartal changed in paravaginal support as imaged in the transverse plane.

On the assumption that loss of paravaginal support should be evident as a peripartum increase in urethral rotation and bladder neck descent, an analysis was carried out comparing these parameters in women with and without loss of tenting. Results are shown in Table 3.28. While there is an apparent difference between the two groups, the standard deviations are so large that a sample of approx. 270 observations would be required to provide 80% power to prove this difference in either bladder neck descent or urethral rotation as statistically significant.

Any tenting All tenting lost (n=5) intact (n=16) P=

BNO change (SO) -16.4 (21.2) -9.36 (11.3) 0.5 Rotation change (SO) 38 (41.5) 23.1 (36.6) 0.5

Table 3.28: Peripartum change in ,tenting" on transverse imaging and parameters of anterior vaginal wall descent.

Direct visual comparison of ante- and postpartum films derived from 3D volumes was possible in 21 women (see Figs. 2.14 and 2.15). Table 3.29 gives results for changes in Bladder neck descent and urethral rotation in women who appeared unchanged and those

137 that appeared to have suffered a deterioration in paravaginal support. In 12 cases there seemed to be no .change, in 6 there was apparent deterioration, and in two cases postpartum images seemed to show better supports.

Paravaginal support postpartum same {n=12) worse (n=6) p=

BND change (SD) -9.14 {10.3) -9.8 (17.5) n.s. Rotation change (SD) 20.8 (34.5) 25 (37.2) n.s.

Table 3.29: Peripartum changes in apparent paravaginal support and parameters of anterior vaginal wall mobility.

3. 7 Molecular Genetics

Blood samples were obtained from 199 patients, an:l in ten cases two samples were available. Four samples (all of different patients) yielded no amplifiable DNA, leaving samples of 195 patients for analysis (see Table 3.30). Due to time constraints, analysis was limited to seven exons (23, 24, 31, 32, 39, 40, 48) of the gene for Collagen 3A1, the most promising of the selected target genes. After DNA purification and standardisation, primers were designed for four PCR products encompassing these seven exons. In total, 27 known polymorphisms detected in database searches (see 2.6.1) were expected to be covered by DHPLC of those products.

138 Patients recruited 200 Blood taken 199 DNA extraction successful 195 PCR successful 195 DHPLC successful 193 Genotype available 188

Table 3.30: Number trail for molecular genetic analysis

Despite repeated attempts, exon 23/24 could not be amplified by the primers obtained. Exons 31/32, 39/40 and 48 amplified well, so that PCR products of 195 patients were successfully obtained for all three amplicons. DHPLC was performed for all PCR products ofamplicon 1 (exons 31/32) and 61 samples each ofamplicons 2 (exons 39/40) and amplicon 3 (exon 48). All DHPLC was performed both at all predicted temperatures and two degrees above (T+2).

Only amplicon 1 yielded a common aberrant DHPLC pattern suggestive ofheteroduplex formation (see Fig. 3.30) as well as several less common patterns. No aberrant patterns were found for amplicon 2 whereas amplicon 3 did produce one heteroduplex patternalbeit uncommon (1 out of 61 products), and only on analysis at 2 degrees above the predicted temperature (see Fig. 3.31).

139 3.50

1.41niJ1A<

Fig. 3.30: DHPLC profiles for a PCR product (amplicon 1) containing Exon 31 and 32 of Col3A 1 in a series of 41 samples. Homo- and heterodup lex profiles are both shown, the homoduplex dark blue and the common heteroduplex red (arrow). Similar appearances were obtained for the other 154 samples of amp Iicon 1.

7

i S

;:s

()

-t O f 2.2S 250

Fig. 3.31 : Heteroduplex pattern identified in amplicon 3 at T+2 (arrow).

Sequencing of samples was conducted to identify all detected heteroduplex patterns. The samples used for amplicon 3 were not successfully sequenced despite repeated attempts within the timeframe available. Samples for amplicon 1 however were sequenced

140 successfully, identifying the common heteroduplex pattern seen in Fig. 3.30 as a silent polymorphism (GGT<>GGC) at position 2346 ofCol3A1, SNP ID CV1729157 (Celera SNP database, 2001). In 36 cases DHPLC analysis was performed at least twice due to partial failure of a plate, and in all cases identical classifications were obtained.

Of the mentioned 195 PCR products only two DNA samples did not yield clean DHPLC patterns despite at least 2 attempts; DHPLC was successful in 193 patients. In 10 women 2 samples were available; DHPLC classifications agreed in all ten cases. However, when deidentified samples were compared to the previously laid down code, it was found that in five cases unambiguous identification was impossible due to clerical errors, i.e., duplicate or very similar patient names. In total, only 188 out of 200 women could therefore be atttributed a genotype (see Table 3.30).

The common polymorphism mentioned above (GGT<>GGC) was observed by DHPLC unequivocally in 72 out of 188 women (38%) of whom unequivocally identified samples were obtained. In ten women other aberrant patterns were observed which in most instances were close to but not identical to either the normal homozygote or the heterozygote pattern. On sequencing, six ofthose ten patients seemed to be homozygotes while the remaining four could not be identified despite three attempts at sequencing. 106 of 188 patients (56%) showed an obviously normal homozygous pattern.

Fig. 3.32 shows sequencing of the common polymorphism identified on DHPLC. As sequencing proved that this heterozygote pattern was due to a silent polymorphism, i.e., a mutation which does not result in alteration of the gene product, association analysis was not expected to yield any results. It was performed nevertheless because of the slight possibility oflinkage disequilibrium between this silent mutation and other, phenotypically manifest mutations. Table 3.31 shows a comparison of parameters of anterior, central and posterior compartment descent for women showing the identified silent polymorphism (heterozygote pattern) and those showing a normal homozygote DHPLC pattern. No significant differences were detected.

141 Fig. 3.32: Sequencing of the common polymorphism (GGT<>GGC) identified on DHPLC (right) compared to wild- type sequencing for the same locus on the left. Arrow identifies position 2346 of COL3Al.

Heterozygote pattern Homozygote pattern Parameter (silent polymorphism) (normal) p= n= 72 n= 106

Urethral rotation (deg) 40.7(SD26.1) 35.7 (SD 23.7) 0.2

Bladder neck descent (mm) 19.9 (SD 10) 18.3 (SD 10.5) 0.3

Cystocele descent (mm) 11.5(SD10.4) 13.1 (SD 11.5) 0.3

Descent of Cervix (mm) 37.9 (SD 17.6) 40.9 (SD 16.4) 0.2

Rectal descent (mm) 12.8 (SD 16.8) 13.5 (SD 18.9) 0.8

Table 3.31: Comparison of parameters of anterior, central and posterior compartment descent for women showing the identified silent polymorphism and those showing a normal homozygote DHPLC pattern (t- test statistics).

142 4 Discussion

As postulated in the introduction to this thesis, modem obstetrics is faced with a dilemma. Both clinical obstetrics and a large part of the community see vaginal delivery as a desirable outcome of pregnancy while there is an ever- growing body of evidence linking vaginal childbirth with significant future maternal morbidity, and while more and more women opt for elective Caesarean Section to avoid such presumptive morbidity.

The main purpose of the study presented here was to define and measure peripartal changes in pelvic organ support that are indicative of trauma to pelvic fascia. Once such changes are defmed, risk factors can be sought. It is clear that individual risk assessment will be essential for accurate and meaningful counseling of pregnant women (Brubaker, 2001), and for future intervention trials. The author hopes that this thesis will make a significant contribution towards this goal.

The main outcome parameters of this study are ultrasound measures of pelvic organ descent, measured against the inferoposterior margin of the symphysis, supine and after voiding. The methodology used for this purpose has, to a large degree, been developed by the author; consequently there is a lack of outside validation to date. An alternative methodology, developed for measuring bladder neck descent by ultrasound (Schaer et al., 1995) has utilized the central axis of the symphysis as a point ofreference. The author decided against the use of this (not externally validated) method since it requires orthopaedic measurement software, and not all ultrasound systems used by the author provided such software. Another disadvantage is that this alternative method requires imaging of the whole symphysis pubis. Due to the limited footprint of modem curved array transducers this makes simultaneous imaging of the posterior compartment almost impossible.

As regards patient position and bladder filling, the two main variables in terms of imaging technique, two previous studies of the author have addressed these issues. In a study on 132 women undergoing urodynamic assessment (Dietz et al., 2001 a), bladder

143 neck descent on Valsalva was significantly higher in the supine position (24.1 (SD 11. 7) rmn vs. 18.7 (SD 8.3) rmn, p< 0.001), mainly due to a higher location of the bladder neck at rest in the supine position. This fmding contradicts a smaller study (Schaer et al., 1996), a discrepancy that is probably explained by the fact that Schaer et al. performed ultrasound with catheters in situ and 300 mls ofbladder filling.

Bladder filling is the other main variable in assuring reproducibility, with both the author's own work (Dietz et al., 199b) and data in the literature (Martan et al., 1999) agreeing that an empty bladder shows increased mobility when compared with the full organ. There was a small but significant difference (bladder neck descent on 19 rmn, SD 12 rmn vs. 17 rmn, SD 10 rmn, between up to 50 m1 of bladder filling and maximum capacity (mean 355 ml) in 109 women seen for urodynamic assessment (Dietz et al, 1999b); a similar difference of 1.5 rmn between measurements taken at 300 m1 and after emptying was seen in a small study of 20 patients, although it is not clear whether this difference reached significance (Martan et al., 1999). Again this is in apparent contrast with work by Schaer et al. (Schaer et al., 1996) who concluded that there were no significant differences between 100 m1 and 500 m1 bladder filling in a study with 30 women undergoing urodynamic assessment. This discrepancy may be due to the presence of a catheter in this last- quoted study or the fact that no testing was performed on an empty bladder.

In conclusion, examinations for this thesis were performed supine and after bladder emptying which, in the opinion of the author, maximizes numerical findings for bladder neck descent on Valsalva manoeuvre.

The mainstay of any assessment of methodology is an evaluation of validity and reliability/ repeatability (Deyo et al., 1991). The first, i.e., evaluation ofvalidity, is generally undertaken by comparing a new method against a previous 'gold standard'. Such testing has been undertaken by the author against fluoroscopic cysturethrography (Dietz and Wilson, 1996), clinical prolapse assessment methods such as a modified Baden Walker classification and the res prolapse assessment system res POP-Q (Dietz

144 et al., 2001 c) and the evaluation of levator strength by palpation and perineometry (Dietz et al., 2002a). In all instances good agreement was demonstrated with established methods.

As regards reliability or repeatability of the main outcome parameter used in this study, bladder neck descent on Valsalva manoeuvre, several test- retest series have been undertaken either by the author or by staff trained by him. Excellent repeatability of bladder neck descent (Coefficient of variation, CV= 0.21, Intraclass correlation coefficient 0.79) was documented (see 3.1.2) as part of this thesis. This has since been confirmed in another, unrelated series of test- retest variability in which a CV = 0.219 and Intraclass correlation coefficient of 0. 7 5 was reached after an average interval of 46 days (n= 48, unpublished own data, see appendix 8.4). In this instance, the test- retest series was performed independently by two research nurses trained by the author, providing for a degree of outside validation. There are currently no other studies available in the literature giving robust measures of repeatability for the methodology used by the author or for alternative methodologies.

The findings of repeatability stu:lies conducted as part of this thesis demonstrate excellent repeatability of the method used here to document bladder neck descent. However, repeatability of other parameters of pelvic organ descent such as urethral rotation, cervical or rectal descent has not yet been examined, The author acknowledges this shortcoming of his thesis. Ongoing work by the author and others will hopefully provide such data in the near future.

The variability of measurements demonstrated in the quoted interobserver series is likely to be due mainly to the variation in Valsalva strength and concomitant levator activation (CV = 12% in intraobserver series, see 3.1.1). Other confounders are bladder and rectal filling and the accuracy of the detection of landmarks such as symphysis pubis and bladder neck. In late pregnancy, concomitant activation of the levator ani seems to increase in prevalence, even in the patients of this study who had had instructions in the performance of a Valsalva manoeuvre at their first visit. In an unrelated series, the author

145 has recently found that it is much more difficult to obtain an effective Valsalva manoeuvre in third trimester women who have not previously been instructed (unpublished own data). This is not an academic issue but likely to impact on any study designed along similar lines to the one presented here. The observation may explain some of the discrepancies between this study and published literature (see 4.4.2.1).

It is possible that the presence of the fetal head and/or the growing size of uterus and pregnancy could increase the likelihood of involuntary levator activity and/or augment the basal tone of this muscle. This is an intriguing concept in view of the fact that both maximal urethral closure pressure and functional urethral length have been found to increase during pregnancy (Iosif et al., 1980). Whatever the reason, midwives and obstetricians are familiar with the phenomenon that nulliparous women often require significant teaching to optimize the expulsive effort.

146 4.1 Early Pregnancy assessment

4.1.1 Ultrasound data

Before discussing early pregnancy results in relation to the hypotheses listed in 1.8, the author would like to comment on descriptive statistical data obtained for the main outcome parameters describing pelvic organ mobility in nulliparous women in early pregnancy. It may come as a surprise that a significant number of those young and largely asymptomatic women displayed pelvic organ hypermobility. Of 200 women seen between 6 and 18 weeks' gestation, 29% showed bladder neck mobility of25 mm or higher, and incidentally most of them (49/ 58) were stress continent. Similar figures were obtained for mobility of the central and the posterior compartment.

There is some supporting evidence in the literature for the finding that many nulliparous continent women show signs of bladder neck hypermobility. In a study using magnetic resonance imaging, bladder neck descent on Valsalva in 30 nulliparous women varied widely, with the lowermost position of the bladder neck ranging :from 22 mm above to 23 mm below a pubococcygeal reference plane (Law et al., 2001). This range of 45 mm is remarkably similar to the one reported here (range 34.4 mm above to 11.8 mm below the symphysis pubis(= 46.2 mm) for position of the bladder neck on Valsalva).

The author of a recently published ultrasound study (Peschers et al., 2001), using similar methodology to this thesis, showed that bladder neck mobility amongst 39 young asymptomatic nulliparous volunteers varied between 4 and 31 nun, with the mean being 15 mm. This value is lower than the 19.5 mm bladder neck descent documented previously by the author in 52 nulliparous women undergoing urogynaecological . assessment (Dietz, 2002c) and the 19.4 mm measured in this study amongst 200 nulliparous women in early pregnancy, but very close to an average of 17.4 mm observed by the author in a series of 116 nulliparous women between 18 and 23 years of age (own unpublished data). It is clear :from all this data that there is a significant minority of nulliparous women demonstrating a degree of bladder neck mobility that would be

147 regarded as abnormal by many investigators and classified as a first or even second degree cysto- or cysto-urethrocele clinically.

A potential criticism of the study presented here may be derived from the fact that the first assessment of pelvic organ mobility occurred in early pregnancy, introducing the potential confounder of hormonal effects associated with gestation itself. The logistical problems of following a nonpregnalt cohort of women through pregnancy precluded direct observation of the effect of early pregnancy.

148 4.1.1.1 Pelvic organ descent

4.1.1.1.1 Ethnic background

The potential role of ethnicity was covered by

Hypothesis 1: 'Pelvic organ mobility varies in relation to ethnic origin'

Racial background has long been discussed as one of the determinants of female pelvic organ prolapse and incontinence (see 1.5.2). Cadaver dissections have demonstrated marked differences in pelvic support structures between Caucasian and Chinese women (Zacharin, 1977). The pubourethralligament, endopelvic fascia and its attachment to the obturator fascia, i.e., the fascial white line, were found to be markedly thicker in Asian women, and their attachments to be intact in a larger percentage of women. However, this apparent superiority of Asian pelvic supports has recently been disputed (Brieger et al., 1996), with the author speculating that progressive Westernization of lifestyle might explain the increasing incidence of female pelvic organ prolapse and stress incontinence in Hong Kong Chinese women.

Recently, ultrasound methodology has been developed that allows noninvasive quantitation of pelvic organ descent in all three vaginal compartments (Dietz et al., 2001c). For the anterior compartment, translabial ultrasound has already been employed to evaluate racial differences between black and Caucasian Americans (Howard et al., 2000). While results were not completely conclusive, probably due to a lack of power, evidence points towards greater bladder neck mobility in black women as compared to Caucasians.

In the study presented here, ethnicity was defmed by ascertaining the ethnic background of all four grandparents. In order to avoid the confounding effect of mixed ethnicity, only women with grandparents of homogeneous ethnicity were considered although intermarriage within ethnic groupings was not an exclusion criterion for this analysis. For

149 example, a woman with Italian- Nigerian parents was excluded from analysis whereas a woman with Chinese- Vietnamese parentage was included as of Asian ethnicity. There are obvious limitations to this approach, one of them being the purely subjective and historical nature of the racial classification used in this study. However, imaging data showed significantly less pelvic organ mobility in Asian women compared to Caucasians (see Table 3.1). This was true for the anterior compartment (Asians [n= 16], BND 13.2 mm vs. Caucasians [n= 161], BND 19.7 mm, p= 0.002) and posterior compartments (Asians [n= 16], 20.9 mm vs. Caucasians [n= 161], 12 mm, p= 0.04 for rectal descent) while this difference did not reach significance for the cervix (p= 0.17).

It is conceivable that bias might have influenced findings, as the author was aware of ethnicity at the time of the assessment. It would be rather difficult to blind the ultrasound operator against the ethnicity of the patient. However, compared to clinical assessment, Ultrasound prolapse quantification may reduce the potential impact of such bias dre to the standardized procedure of obtaining measurements. Another wealmess of the data presented here is the fact that no information was obtained on socioeconomic factors which would influence lifestyle and nutrition.

To the Imowledge of the author, this is the first study to support in vivo the previously mentioned finding on cadaver dissection that women of Asian ethnic background show stronger pelvic fascial support structures (Zacharin, 1977). The above- defined hypothesis is supported by the presented data. Consequently, the case for a congenital contribution to the phenotype of female pelvic organ mobility appears strengthened. This congenital contribution is most likely due to a heritable factor or trait, further justifying a molecular genetic approach to the investigation of pelvic floor disorders.

4.1.1.1.2 Family history and joint hypermobility

The value ofjoint hypermobility as a predictor of stress incontinence has been controversial, with both positive (Tincello et al., 2000) and negative findings (Chaliha et al., 1999) having been reported. The later of the two studies used a similar methodology

150 to the one reported here, with joint mobility scores tested against both ante-and postpartum symptoms of stress incontinence. The authors concluded that joint mobility was not a useful predictor of either ante- or postnatal incontinence. Claims regarding an association between prolapse and joint mobility appear to be better supported (Norton et al., 1995).

The author is not aware of any previous attempt at correlating joint mobility with imaging data on pelvic organ mobility. In principle, more accurate prolapse assessment should increase the likelihood of detecting a potential association. We therefore tested

Hypothesis 2: 'There is an association between clinical indicators of connective tissue quality (joint hypermobility, history of hernia, epistaxis and dislocations, family history of incontinence/ prolapse, striae gravidarum, pelvic girdle pain) and pelvic organ mobility as quantified by ultrasound measurements and/ or symptoms of stress incontinence'.

In the study presented here, thumb approximation to forearm correlated with descent of the cervix (p= 0.019), rectum (p= 0.017) and rotation of the proximal urethra (p= 0.043) but not with any of the other indices such as bladder neck descent and cystocele descent (see chapter 3.2.5). There were no significant associations between continence status and joint mobility, confirming the fmdings reported by others (Chaliha et al., 1999). It appears that, even if an association between joint mobility and pelvic organ mobility may argue for the existence of generalized minimal connective tissue dysfunction in women with pelvic organ hypermobility, such an association may be too weak for it to be clinically useful as part of a potential risk scoring system.

A history of incontinence in first degree relatives correlated weakly with BND (18.5 vs. 21.9 mm, p= 0.034) and maximal cystocele descent (12.8 vs. 9.2 mm, p= 0.047), see chapter 3.2.5. However, this was not demonstrated for a history of prolapse or operations for such conditions. Since a personal history of dislocations, hernias or epistaxis was very uncommon, this part of the hypothesis can not be tested.

151 The postpartum components of this hypothesis, i.e., striae gravidarum and pelvic girdle pain, were tested as presented in chapter 3.5.3, with no significant relationships with pelvic organ mobility recorded. However, a significant relationship was found between higher Visual Analog Scale readings for back pain and stress incontinence at the postpartum visit (p= 0.029 on 2-sample t-test).

Hypothesis 2 was therefore not substantiated regarding striae gravidarum but confirmed in several other aspects: firstly as regards an association between back pain in late pregnancy and stress incontinence postpartum, and secondly as regards the observation that joint hypermobility and a family history of incontinence seem to be associated with increased anterior compartment mobility. However, the clinical significance of such weakly significant relationships remains doubtful, in particular considering the multiple analyses performed.

4.1.1.1.3 Symptoms of lower urinary tract dysfunction

The prevalence of both the symptom of stress incontinence and the finding of a positive paper towel test in the study presented here fall well within reported prevalence rates as discussed in the Introduction. At the time of the first visit, 12% of all women reported stress incontinence at least once a month, and this figure had risen to 3 9% by the third trimester appointment. Objective leakage as confirmed by paper towel test had risen non­ significantly from 11.5 to 13% which is in keeping with a study reporting urodynamically diagnosed Genuine Stress Incontinence in 9% of primigravid women at 34 weeks' gestation (Chaliha et al., 2000).

As Genuine Stress Incontinence is strongly associated with bladder neck mobility in nonpregnant women (Bader et al., 1996, Dietz et al., 200lb), it appeared important to test for the existence of such a relationship in pregnancy. First visit ultrasound imaging data was therefore used to test

152 Hypothesis 3: 'There is a correlation between frequency of symptoms of stress incontinence and anterior vaginal wall mobility in pregnancy'.

and

Hypothesis 4: 'There is a correlation between signs of stress incontinence on paper towel test and anterior vaginal wall mobility in pregnancy.'

There are a number of reports correlating anatomical findings in pregnancy with symptoms. The earliest such paper describes appearances on X-ray cystourethrography in pregnancy and puerperium (Francis, 1960). In a series of 83 women, a qualitative assessment of the bladder neck was performed. The increase in retrovesical angle (which usually is associated with hypermobility) ''was a striking feature in patients with stress incontinence" (Francis, 1960). However, while it is stated that 90% of all pregnant women with symptoms of stress incontinence showed signs ofhypermobility (in this case, an increased retrovesical angle), no statistical testing was performed.

Two observational trials using ultrasound imaging in nulliparous women did not demonstrate any association between stress incontinence and bladder neck mobility (Meyer et al., 1998a,b, Wijma et al., 2001). It was concluded that stress incontinence in pregnancy must be explained by other factors (Wijma et al., 2001). The findings of the study reported here agree with this in that there was only a weak trend towards increased bladder neck mobility in stress incontinent women (p= 0.2, see Table 3.2). Hypothesis 3 is therefore not supported by the results ofthis study.

Testing of hypothesis 4 confirmed these findings in that no significant correlation was found between presence or severity of leakage on paper towel test on the one hand and mobility of the bladder neck on the other hand (see Chapter 3.2.4 and Table 3.2). While a different pad test regime was used by Wijma et al., results regarding pad testing were again similar in that no correlation was detected between measures of bladder neck hypermobility and objective leakage.

153 This fmding may seem confusing in view of the clear associations between bladder neck descent and GSI on video- urodynamics observed in older, symptomatic patients (Dietz et al., 2001b). However, one should not overlook the fact that increased mobility in young nulliparous women is likely to be atraumatic, signifying increased elasticity but not disruption of the suburethral hammock. In a small subgroup of the study population, 3D ultrasound was used to demonstrate vaginal 'tenting', a fmding on transverse plane ultrasound or MRI which is thought to signify intact paraurethral and paravaginal support structures (see chapter 3.6). Tenting was found to be absent in only three patients out of 23, and this never at more than one of three levels. Hypermobility may not in fact matter much for continence as long as the endopelvic fascia and pubourethralligaments are intact. This issue is discussed in more detail in section 4.7.1.3.

The finding of varying correlations between reported symptoms and results of the paper towel test (absent at the first visit and highly significant at the second visit) agrees with published literature. Generally speaking, only a minority of women reporting stress incontineme in pregnancy will be shown to be stress incontinent on objective testing (Chaliha et al., 2000). Correlations between reported stress incontinence and pad weight have been weak (Wijma et al., 2001), and at any rate the standard 1-hour ICS pad test has been shown to be a poor objective measure of incontinence due to poor repeatability (Simons et al., 2001). Poor correlations between objective loss as measured by paper towel test and self: reported stress incontinence have also been demonstrated in nonpregnant women (Miller et al., 1999).

The merits and/ or disadvantages of the paper towel test are beyond the scope of this study. It may suffice to state that, on the one hand, its convenience and simplicity make it an attractive objective measure of stress incontinence, on the other hand it seems to suffer from a relatively low sensitivity. Reproducibility seems high (Miller et al., 1998); however, further work seems needed to define the validity of the test in relation to pad test regimes.

154 4.1.1.1.4 Relaxin and Progesterine serum levels

It has been argued that pregnancy itself and not the act of childbirth is the dominant factor in the pathogenesis of stress incontinence and pelvic organ prolapse (MacLennan et al., 2000). Such arguments seem supported by the fact that the incidence of stress incontinence is highest in late pregnancy and decreases markedly postpartum (Nel et al., 2001, Mason et al., 1999). Effects of pregnancy may be hormonal or related to the mechanical effect of a growing intraabdominal mass. We therefore tested

Hypothesis 5: 'There is a positive correlation between serum relaxin and/ or progesterone levels in the first trimester and pelvic organ mobility.'

Relaxin and progesterone have both been implicated in the pathogenesis of female pelvic organ prolapse (see chapter 1.4.1) although the role of relaxin in human pregnancy is controversial. Progesterone, a strong smooth muscle relaxant, could conceivably influence the biomechanics of pelvic 'ligaments' which contain a significant amount of smooth muscle fibres (DeLancey, 2001). The above hypothesis was tested by ELISA analysis of 49 consecutive blood samples obtained at the first appointment. After failure of the first kitset samples had to be re- frozen which, according to information provided by the the manufacturer (lmmundiagnostik Bensheim, Germany), should not affect detected serum levels unless repeated more than three times. No significant or near­ significant correlations between relaxin or progesterone levels in the first trimester and ultrasound indices of pelvic ligament relaxation could be detected (see chapter 3.2.7, figures 3.7 and 3.9). This hypothesis therefore has to be rejected.

After commencement of the experimental work for this thesis, a study was published linking higher relaxin serum levels in early pregnancy with a relatively lower prevalence of stress incontinence in late pregnancy (Kristiansson et al., 2001). A secondary data analysis was performed to investigate this issue. Women who were suffering from stress incontinence at the third trimester visit were in fact shown to have had higher early pregnancy relaxin levels than those who were continent (continent mean 166, StD 60.2,

155 vs. incontinent mean 209, SD 84.5), although this fmding did not quite reach significance (p= 0.063). In retrospect, sample size may not have been adequate. However, fmdings seem to be in direct contradiction to the results presented by Kristiansson. The role of relaxin in gestational stress incontinence deserves further study.

In summary, this study has not yielded any evidence for a major role of hormonal factors in the pathogenesis of pelvic organ hypermobility; however, it may well be possible that relaxin has an effect on other components of the urinary continence mechanism.

4.1.1.1.5 Mutation detection

As discussed in chapter 1.5, there is some evidence for a congenital and/ or genetic contribution to the aetiology of female pelvic organ prolapse and stress urinary incontinence. This would imply an association between the phenotype ofpelvic organ mobility and known or unknown genetic variations and necessitate a search for differences in the genotype of women with varying phenotypes. The commonest variation in the human genome is due to single nucleotide polymorphisms (SNPs, see chapter 1.6), and any work in this field would likely have to focus on the detection of such genetic variants.

Due to the enormity of this task, an approach was selected that would allow the establishment of a methodology for future studies in this field. After extensive consultation with clinicians and molecular geneticists, five target genes were selected in order of likelihood for the detection of high- risk polymorphisms for female pelvic organ prolapse: COL3A1 (Collagen 3), COL5Al (Collagen 5), ELN (Elastin), COL1A1(Collagen 1) and FBN1 (Fibrillin 1). Only the first of those was tested, and of this only seven exons in total, representing more than half of all SNPs listed in public and proprietary databases at the time of the experimental work (Oct. 2001).

156 Hypothesis 6: 'There are significant differences between polymorphisms of Fibrillin, Elastin and Collagen genes observed in nulliparous women with marked pelvic organ mobility and those without'

and Hypothesis 7: 'Above genetic factors are risk factors for the symptom of stress incontinence during pregnancy and postpartum'

were assessed by attempting detection of single nucleotide polymorphisms (SNPs) by denaturing high pressure liquid chromatography (DHPLC, see 2.6.1) after selection of suitable exons and PCR amplification of such. Over the course of four months of full­ time laboratory work at the Queensland Institute of Medical Research, the author was able to develop a methodology that would allow a comprehensive testing of the above hypotheses. In 195 out of200 women, PCR and DHPLC were successful, resulting in the production of homo- and heteroduplex patterns which allow identification of single nucleotide polymorphisms.

The developed methodology has been shown to be eminently practicable and did in fact lead to the detection of one common silent (noncoding) SNP (see Figures 3.30 and 3.32). However, it is also evident that in order to completely cover the selected target genes, at least one year of full- time laboratory work and funds in the order of A$ 200.000- 500.000 would be required. Only less than half of the known polymorphisms (as of early 2002) for one out of five target genes (COL3A1), i.e., approximately one- tenth of polymorphisms currently known for the five chosen target genes, could be assessed. Furthermore, it is by no means certain that structural protein genes are responsible for the wide variation observed in pelvic organ mobility- this variation may theoretically be due to genes determining connective tissue metabolism or genes involved in other, currently unknown ways.

157 Consequently, the above hypotheses can not be tested on the basis of available data. For directions for future research, in particular other and possibly more rewarding molecular genetic approaches, see chapter 5.3.

4.1.1.2 Levator strength

It has been suggested that levator damage resulting from pudendal nerve trauma (Allen et al., 1990) or from direct injury is the primary insult leading to connective tissue failure. In this hypothesis, functional or structural impairment of the pubococcygeus and pubovisceral muscles is seen as the primary insult, resulting in progressive stretching of unsupported fascial structures (De Lancey, 1994, 1996, 2001) and eventually in pelvic organ prolapse and incontinence. Formal testing of this hypothesis would entail a longitudinal study extending over many years, possibly decades. Not surprisingly, there is currently no experimental data supporting this concept.

However, the fact that a significant percentage of nulliparous women in this study (29%) showed a bladder neck descent of 25 mm or more in early pregnancy (i.e., long before potential traumatization of the levator) does cast doubt upon the validity ofthis concept. Evidently, many women show anterior vaginal wall relaxation without levator trauma. To further investigate this issue the author tested

Hypothesis 8: 'There is a negative correlation between levator ani muscle function (as defined by cranioventral displacement of the bladder neck on pelvic floor muscle contraction) and pelvic organ mobility'

At the first trimester visit, significant correlations were detected for all ultrasound indices of pelvic organ descent and levator strength as defined by translabial ultrasound (see Table 3.8). However, all correlations (positive in the case ofRVA-S, Rotation, BND and oblique descent and negative in the case of BSD-SV, Cystocele, cervical and rectal descent) indicate increased lift of the bladder neck in women with more marked pelvic organ descent. The most likely explanation for this finding is that displacement of the

158 bladder neck on levator contraction depends not just on the force exerted by the contraction, but also on tissue compliance: the higher the compliance, the higher the observed lift.

The above hypotresis could not be substantiated on the basis of available data. On the contrary, the observed correlations seem to strongly argue against hypothesis 8 as tested in nulliparous women.

4.2. Third trimester assessment

4.2.1 Ultrasound data: Pelvic organ descent

If pregnancy rather than childbirth were the dominant factor in the pathogenesis of pelvic organ prolapse and stress incontinence, then such an effect would be expected to be evident either in early pregnancy if due to hormonal factors, or in late pregnancy if due to the effect of the growing intraabdominal mass of uterus and pregnancy. As mentioned, symptoms of stress incontinence are well known to increase during pregnancy (Mason et al., 1999), and this effect was also observed in this study. On the other hand, it has been stated that the position of bladder neck and urethra within the pelvic cavity are not changed by pregnancy per se (van Geelen, 1994). We therefore tested the hypothesis

Hypothesis 9: 'Bladder neck, uterine and rectal mobility (as measured relative to the inferoposterior margin of the symphysis pubis) increase during pregnancy, i.e., between the first and the third trimester'.

The hypothesis can be regarded as substantiated for the anterior vaginal compartment, i.e., the bladder neck and anterior vaginal wall. The difference between first and second visit measurements reached significance for BSD-SV (11.9 vs. 7.0 mm, p< 0.001), BND (19.4 vs. 22.2 mm, p= 0.01) and maximal cystocele descent (11.8 vs. 7 mm, p< 0.001), see Table 3.13. Rotation of the urethra, cervical and rectal descent were not significantly

159 different. Overall, the observed change between first and second visit data was small compared to the change after childbirth itself (e.g. BND 2.8 mm vs. 7.2 mm, see below).

As regards bladder neck mobility, these results confirm those of another imaging study published since inception of the work presented here. Wijma et al. used a different parameter to define anterior vaginal wall mobility, i.e., the 'angle of the urethrovesical junction' (Wijma et al., 2001). In this study, the angle used to describe bladder neck mobility on Valsalva increased from 76.6 to 84.9 degrees (p<0.01), with nonpregnant controls measured at 73.5. These relative changes are remarkably similar to the ores observed by the author, with bladder neck descent in nulliparous controls at means of 19.5 mm (Dietz et al., 2002c) and 17.4 mm (unpublished own data), early pregnancy mean at 19.4 mm and the late pregnancy mean at 22.2 mm (see Tables 3.6 and 3.12).

Meyer (Meyer et al., 1998) saw no increased mobility ofthe bladder neck in pregnancy compared to the nonpregnant state, and no increase in this parameter during pregnancy. The methodology of this study was similar to the one reported by the author; however, findings such as an average bladder neck descent of only 7 mm with Valsalva in pregnant nulliparae, raise serious doubts as regards potential confounders such as levator activation and strength of Valsalva manoeuvre. Findings did not appear logical as remarked in the Editorial comments to this study. Other imaging studies (Peschers et al., 1996, Bader et al., 1995, Toosz- Hobson et al., 1997, King et al., 1998, Lange et al., 2000) did either not assess changes during pregnancy or focused on other parameters such as width of the urogenital hiatus.

4.3 Labour and delivery outcome

4.3.1 Predictors of labour and delivery

Obstetric intervention in labour such as Vacuum or Forceps delivery or emergency Caesarean Section is associated with a significant increase in maternal and neonatal morbidity. Urinary incontinence (Farrell et al., 2001), faecal incontinence (MacArthur et

160 al., 2001) and defaecation disorders (Karasick and Spettell, 1997) as well as pelvic organ prolapse (Swift and Theo:frastous, 200 1) are all increased in some or all of those interventions. Beyond the immediate area of interest of this thesis, there are a number of other important associations between such intervention and general health problems (Brown and Lumley, 1998), postnatal depression (Rowe- Murray and Fisher, 2001) or even posttraumatic stress disorder (Reynolds, 1997) and neonatal trauma with its diverse potential sequelae (Puza et al., 1998). Identification of antenatal risk factors for such intervention undoubtedly would hold major promise in terms of reducing somatic and psychological trauma as well as the cost of service delivery, especially in primiparous women, and would be of importance well beyond the prevention of pelvic floor morbidity.

In the context of this study the question was raised whether pelvic organ descent as measured antepartum and delivery outcome were independent of each other. The author therefore determined to analyze antepartum ultrasound data for its predictive value for the length of second stage and delivery mode. There is no evidence in the literature of any prior attempts to use ultrasound imaging parameters or other measures of pelvic organ mobility for the prediction of intervention in labour. The parameters to be discussed here are those describing pelvic organ mobility, testing

Hypothesis 10: 'Delivery mode is independent of pelvic organ mobility as measured by ultrasound in the third trimester of pregnancy'

For all tested parameters, normal vaginal delivery was associated with higher pelvic organ mobility, and this proved highly significant for most tested parameters excepting central compartment descent (see Table 3.19). A regression model using cystocele descent was significant at p= 0.002 (see Fig. 3.15). This association was also present for the length of second stage, especially passive second stage, suggesting that the biomechanical properties of pelvic organ support structures, i.e., 'pelvic compliance', do indeed influence progress in labour and the likelihood of obstetric intervention. Consequently, the above hypothesis is rejected.

161 Whether this correlation between greater pelvic organ mobility and enhanced progress in labour is of sufficient magnitude to be clinically useful, is a question not answered by this study and will require further study (see chapter 5.3.2 re. suggestions for future work). At the time of proofreading (9.03), independent confirmation of this finding has just become available (Balmforth et al., 2003).

4.4 Postpartum assessment

4.4.1 Symptoms

4.4.1.1 Prediction of postpartal Stress Incontinence

It has been claimed that increased mobility during pregnancy correlates with postpartum incontinen::e, i.e., constitutes a risk factor (King et al., 1998). If this were the case then a translabial ultrasound in conjunction with routine antenatal scanning could be used to define a high- risk group which could then benefit from intervention such as intensive pelvic flor muscle exercises (King et al., 1998). The author therefore tested

Hypothesis 11: 'Bladder neck descent at first visit is a predictor of postpartum incontinence'.

As described in 2.5.5.6, the methodology used in the quoted study from Plymouth, UK, differs from the one used by the author of this thesis. While the author did not standardize for Valsalva manoeuvre and performed measurements with the patient supine and after bladder emptying, King et al. used standardized low pressure Valsalva manoeuvres (30 mm Hg or approx. 40 em of H20) and conducted their assessment with the patient sitting upright with their feet in stirrups, and at varying bladder volumes.

In the study presented here there were no significant or near significant correlations between BND or proximal urethral rotation at first visit and postpartum stress incontinence (see chapter 3.5.1). Hypothesis 11 was therefore rejected. Even in view of

162 the methodological differences discussed above, this finding is surprising and needs further elucidation, in particular as the Plymouth study has already been used to justify clinical intervention (Reilly et al., 2002). It appears possible that the methodology of the Plymouth study served to identify women with particular characteristics- e.g. absent reflex activation of the levator muscle- that may in themselves predispose to postpartum incontinence. At the very least one may state that further investigation of this issue is indicated before clinical interventions should be advocated on the basis of this parameter.

4.4.1.2 Postpartal stress incontinence and delivery mode

As discussed in the introduction, the symptom of stress incontinence is strongly associated with parity in epidemiological studies. A number of delivery- related factors, such as vaginal birth, vaginal operative delivery, epidural analgesia and maternal age at time of delivery have been shown to increase the likelihood of future prolapse- or incontinence- correcting surgery or symptoms (Carley et al., 1999a, Chiaffarino et al., 1999, MacLennan et al., 2000, Foldspang et al., 1999, Wilson et al., 1996, Farrell et al., 2001 ). Other potential determinants of pelvic floor trauma may be the length of first and second stage oflabour (Allen et al., 1990), birthweight (Allen et al., 1990) and episiotomy (Smith et al., 1989a, Foldspang et al., 1999). The author tested

Hypothesis 12: 'The symptom of postpartum stress incontinence is associated with delivery mode, birthweight and/ or length of second stage'.

in order to determine the extent to which these associations applied to the study reported here.

Caesarean section was clearly protective of the symptom of postpartum stress incontinence (p= 0.003) but no other significant correlations were observed. In particular, neither birthweight nor length of second stage (active or passive) differed between stress continent and stress incontinent women.

163 Hypothesis 12 can be considered substantiated as regards delivery mode. The concept of a partly protective role for Caesarean Section is supported by the data obtained in this study which agrees with epidemiological studies in the literature assessing this factor (Jackson et al., 1997, Carley et al., 1999, Mason et al., 1999, Persson et al., 2000, Farell et al., 2001, Arya et al., 2002). Elective Caesarean Section may be even more clearly protective than unplanned procedures (Digesu et al., 2001) although a lack of power precludes analysis of this issue here.

As regards other parameters such as birthweight, no significant associations with stress incontinence were demonstrated. Studies describing correlations between these factors and stress incontinence or the need for its surgical correction generally benefit from larger sample sizes. This is the case for associations described with epidural analgesia (Carley et al., 1999, Persson et al., 2000), birthweight (Persson et al. 2000, Swift et al., 2001) and episiotomy (Alling Moller et al., 2000). In some instances, discrepancies of findings may be due to a lack of power in the study presented here. Power calculations for the effect of epidural analgesia on stress incontinence (OR 1.27 in primiparous women) based on the study of Persson (Persson et al., 2000) indicate that a total sample of over 600 patients may be required to reach 80% power to detect such a small difference at the p= 0.05 level.

4.4.1.3 Postpartal stress incontinence and fascial trauma

As discussed above, a peripartal increase in pelvic organ mobility was regarded as evidence of fascial impairment. While 29% of the study population showed signs of bladder neck mobility of 25 mm or above in early pregnancy, it has to be assumed that this hypermobility was nontraumatic in origin. This implies that the suburethral hammock in those. women was very likely intact despite their anterior vaginal wall laxity. An intact suburethral hammock may protect against stress incontinence even if it is highly elastic.

It is postulated that traumatic hypermobility due to fascial trauma sustained in childbirth may be more likely to be associated with stress incontinence. The author therefore tested

164 Hypothesis 13: 'The symptom of postpartum stress incontinence is related to the peripartum increase in pelvic organ mobility'.

There were no significant correlations between either the ultrasound parameters of anterior vaginal wall descent or peripartum changes in such parameters and the postpartal symptom of stress incontinence. However, when the relative magnitude of delivery­ induced changes in bladder neck mobility was considered, then those women with relatively more marked changes were more likely to experience stress incontinence more than once per week and stress incontinence that was worse than at their first appointment. These analyses reached significance (p= 0.048 and p= 0.044), see Tables 3.26 and 3.27. The hypothesis appears substantiated by available data.

It appears likely however that a number of confounders influence the relationship between alteration of fascial support and the symptom of postpartal stress incontinence, the most obvious being impairment (whether by direct trauma or neuropathy) oflevator and urethral rhabdosphincter function.

4.4.2 Ultrasound data

4.4.2.1 Pelvic organ descent

Descent of pelvic organs on Valsalva maneouvre measured by translabial ultrasound may be used as a surrogate measure of fascial trauma leading to stress incontinence and prolapse. The author therefore tested

Hypothesis 14: 'Mode of delivery, birthweight and length of the second stage of labour are associated with the extent of fascial trauma as defined by a peripartum increase in pelvic organ mobility.'

165 Of the three factors connective tissue trauma, neuropathy and myopathy, the first is most likely to contribute to hypermobility on Valsalva manoeuvre or a lower position of the bladder neck at rest, cystocele, rectocele and uterine prolapse, at least in the short term.

Delivery mode showed a strong and highly significant relationship with changed pelvic organ mobility. This was true for all tested ultrasound parameters, including cervical and rectal mobility. The order of traumatic potential seems to be prelabour Caesarean Section, Caesarean Section during the first stage oflabour, Caesarean Section in second stage, Normal Vaginal Delivery, Vacuum delivery and Forceps delivery. On ranking the above delivery modes for ANOVA testing the results were returned as significant for urethral rotation (p= 0.001), BSD-SV (p= 0.001), BND (p= 0.002), Cystocele descent (p= 0.001), Cervical descent (p= 0.02) and descent of the ampulla recti (p= 0.002), as slx>wn in Figures 3.20- 3.26, chapter 3.5.4.1.5. Low numbers for some ofthe above groups clearly restrict the power of this study to show significance between individual delivery modes. Nevertheless, this hypothesis appears substantiated.

As discussed in detail in the introduction, comparison with previous work in this field is problematic due to differences in methodology. Two prospective studies involving a smaller number of nulliparous women did not fmd any association between parameters of anterior vaginal wall mobility and delivery data (King et al, 1998, Meyer et al., 1998) while two other, even smaller studies (Bader et al., 1995, Peschers et al., 1996) did show associations between delivery mode and bladder neck mobility. Generally, means and ranges for bladder neck descent were lower in these studies which, as discussed above, is likely due to confounders such as bladder filling and levator activation.

Birthweight did not correlate significantly with any of the indices of pelvic organ mobility altlx>ugh there were trends towards increased evidence of fascial trauma with higher birthweights (see Table 3.25). Studies showing a correlation between birthweight and surgically treated stress incontinence (Persson et al., 2000) or severe prolapse (Swift and Theophrastous, 2001) used larger sample sizes; it therefore appears probable that the

166 lack of a statistically significant association between birthweight and parameters of fascial trauma is due to a lack of power.

Length of second stage was found to correlate significantly with peripartal changes in pelvic organ mobility, and this was true for all tested parameters of pelvic organ descent and for all three compartments, with increased descent of the posterior compartment showing the strongest correlation with length of second stage (see chapter 3.5.4.1.3, Table 3.24).

All parameters of pelvic organ descent except rectal descent showed the same pattern, i.e., the numerical impact of active second stage was about three times the impact of passive second stage. Rectal descent, on the other hand, demonstrated a very similar pattern to the one observed for a peripartum change in levator function, i.e., equal impact of passive and active second stage (see Fig. 3.19). This was confirmed on testing the effect of delivery mode since a caesarean in second stage, while protective for the other compartments, had no apparent protective effect for rectal descent, and this was similar to the patterns observed for levator function (see 4.4.2.2.).

It may be speculated that trauma to the posterior compartment may be related less to actual fascial damage arising from crowning of the fetal head and more to denervation occurring due to prolonged presence of the fetal head within the true pelvis, regardless of actual delivery mode. This is supported by discrepancies in fmdings between studies using neurophysiological outcome parameters (Allen et al., 1990, Sultan et al., 1994) or faecal incontinence (MacArthur et al., 1997) and those focusing on postpartum stress incontinence (Viktrup and Lose, 1993, Wilson et al., 1996, Farrell et al., 2001, Arya et al., 2002). Denervation injury to the pudendal nerve may follow a similar pattern as increased posterior compartment descent in that second stage caesarean section is not protective, whereas anterior compartment trauma follows the pattern observed in studies examining the relationship between delivery mode and postpartum stress incontinence.

167 None of the above- quoted published imaging studies appear to have had the power to detect these associations. In epidemiological studies the length of second stage is only infrequently assessed as this parameter is difficult to obtain retrospectively. Where it was attempted, some authors have shown a link between symptomatic pelvic floor dysfunction and the length of second stage (Baird and Caputo, 2001) while others failed to detect such a relationship (Farrell et al., 2001).

4.4.2.2 Levator function

It was thought that levator strength may be a more sensitive indicator for the changes caused by a traumatic delivery since there is ample evidence for such trauma in the literature, both from neurophysiological (Allen et al., 1990) and, more recently, imaging studies (Tunn et al., 1999a,b, DeLancey et al.,1999, Peschers et al., 1999) using magnetic resonance. Despite this recent work, it is still disputed whether the resulting damage is neurogenic (Smith et al., 1989, Allen et al., 1990) or myogenic (Dimpfl et al., 1998) in origin. Regardless of the aetiology of impairment, its existence has been clearly shown, and several authors have postulated that it constitutes the primary insult in the development of incontinence and prolapse (Smith et al., 1989, DeLancey 1994, 1996, 2001).

The parameter used in this study was displacement of the bladder neck on maximal levator contraction as measured by translabial ultrasound, a technique first described by Peschers et al. (Peschers et al., 1997) and used by the author in several studies leading up to this thesis (Dietz et al., 1998, Dietz et al., 2002a). While intra- and interobserver variability are somewhat higher for levator strength compared to bladder neck descent on Valsalva (see 3.1), the impact of outliers (likely mainly due to faulty Valsalva manoeuvres in BND measurements) may be less as suggested by better correlations between first and second visit measurements (r= 0.49 for BND, r= 0.59 for levator strength). Ultrasound measures of levator function seem to correlate well with the more traditional assessment methods (Dietz et al., 2002a).The author therefore tested

168 Hypothesis 15: 'Mode of delivery, birthweight and length of the second stage of labour are associated with a postpartal reduction in levator strength on voluntary contraction as measured by displacement of the internal urethral meatus relative to the inferoposterior margin of the symphysis pubis.'

To the author's knowledge, only two papers have discussed the impact of vaginal delivery on levator strength using ultrasound imaging. One (Peschers et al., 1997) had very low numbers (8 primiparous women seen after 6-15 months) and was inconclusive. The other (Meyer et al., 1998), presenting data on 149 primiparae, showed no significant differences in pelvic floor function between delivery modes. It was planned to replicate these results since they appeared counterintuitive in view of the evidence for pudendal nerve and levator damage in labour. Due to the limitations of the quoted imaging studies, no power calculations could be performed for the hypothesis; this part of the thesis therefore has to be regarded as a pilot study.

Any differences in pelvic floor muscle function before and after childbirth may of course be time- dependent and influenced by posttraumatic reinnervation and training effects. All study participants underwent routine postnatal verbal instruction in pelvic floor muscle exercises. Re-innervation of muscle affected by neurapraxia should be virtually complete (Juenemann and Thuroff, 1994) by three months postpartum. However, it is acknowledged that other factors such as breastfeeding and general physiological factors associated with the puerperium may still influence levator function at that time. This may limit the degree to which assessments three months after childbirth are representative of the permanent post- partum state.

Overall, the effect of a levator contraction, as quantified by cranioventral displacement of the bladder neck, was reduced by about 22%, from an average of 11.2 to an average of 8.8 mm (p< 0.001), see chapter 3.5.4.2. This is comparable to perineometric data showing that squeeze pressures were reduced by 1/3 at about 2 months postpartum (Allen et al., 1990), and assessment of levator activity by palpation has shown a very similar numerical effect of childbirth (Sampselle, 1990). In one of the imaging stu::lies mentioned above,

169 intravaginal squeeze pressure on perineometry was significantly reduced in primiparous women- again by about 1/3. Ultrasound data was obtained in a similar fashion to the study reported here, but no significant reduction in bladder neck displacement on levator contraction was demonstrated in a group of 25 primiparae after normal vaginal delivery.

Delivery mode: The correlation between delivery parameters and levator strength observed in the study discussed here was generally weaker tl:an comparable correlations with parameters of anterior compartment fascial supports (see chapter 3.5.4.2). This finding may be due to methodological limitations; after all, intra- and interobserver variability of this parameter were found to be consistently higher than for bladder neck descent on Valsalva (see 3.1).

Regardless of the strength of associations however, it was found that the pattern of progressive trauma was somewhat different from the pattern observed for fascial damage in the anterior and central compartments (see 4.7.2.1.). A Caesarean in second stage did not seem to confer any protective effect at all. Women who underwent Caesarean Section after attaining full dilatation were shown to have a similar reduction in levator strength as women who were delivered by Ventouse or Forceps, and this reduction in strength was significantly lower in patients delivered by Caesarean Section during first stage (p=0.009).

It appears that damage sustained by the levator plate is dependent less on the actual passage of the fetal head through the pelvic outlet and more on its presence within the pelvic cavity at full dilatation. This is consistent with the concept of neurapraxia due to stretching of the pudendal nerve in second stage (Snooks et al., 1985, 1986). It appears clear that a Caesarean Section after prolonged labour may result in neuropathy even if the woman never enters active second stage (Sultan et al., 1994), just as it may result in faecal incontinence (MacArthur et al., 1997).

On ANOVA testing on the delivery categories mentioned above, results did not reach significance for the ultrasound parameter used to define levator strength (p= 0.08), see

170 Figure 3.29. However, some comparisons between categories showed significant results although the pattern was different from the one observed for fascial trauma. On comparing women who had undergone vaginal versus caesarean delivery, the latter seemed to be protective of the levator (loss of strength -1.17 vs. -2.87 mm for NVD, p= 0.031. Comparing Caesarean Section against operative vaginal delivery, this relationship was somewhat stronger (-1.17 for CIS vs. -3.83 mm for operative vaginal delivery, p= 0.022. There was no difference between NVD and operative vaginal delivery on univariate analysis. A relatively clear difference emerged between Caesareans performed prior to second stage and in second stage (-0.61 vs. -3.9mm, p= 0.009).

Total length of second stage correlated with a reduction in levator strength (p= 0.004),

with active 2nd stage showing a stronger association than passive second stage (see chapter 3.5.4.2). In a regression model however, passive and active second stage were of about equal weight, paralleling the effect seen on descent of the posterior compartment. In a second regression model using delivery mode and length of second stage, the former added virtually nothing to the strength of the model, suggesting that changes in levator function are determined by events preceding the actual delivery. Controlling for length of second stage in essence caused the effect of delivery mode to disappear.

It seems that mechanisms of injury differ between anterior and central compartment fascial supports on the one hand and posterior compartment supports/ levator function on the other hand, as discussed in 4.4.2.1. One explanation for this observation would be that support of the posterior compartment relies relatively more on muscular (i.e., levator complex) support than on fascial structures. This appears very likely from review of the anatomical literature which provides prima facie evidence for this concept. The puborectalis muscle inserts into the rectum and anal canal and forms a loop around the anorectal junction (DeLancey, 2001), likely providing significant support to those structures. Of all pelvic organ landmarks observable on translabial ultrasound, the effect of a levator contraction is most evident at the level of the rectal ampulla.

171 After childbirth there is permanent divarication of the levator ani muscles (Haadem, 1994), whether due to neuropathy, direct trauma or myopathy. Impairment of the levator complex would be expected to be associated with posterior compartment problems leading to a "hernia en glissade of the posterior wall" (Haadem, 1994). A long second stage (even in the absence of a vaginal delivery) seems to result both in increased descent of the rectal ampulla and in reduced levator fimction as observed on translabial ultrasound.

The anterior and central compartments, on the other hand, are secured by more defined fascial structures; muscular support through the levator complex is much less direct due to the presence of the urogenital hiatus. Not surprisingly, the pattern of injury appears different, with the actual passage of the fetal head resulting in the most marked degree of trauma, and with Caesarean delivery conferring some protection even if performed after full dilatation. As mentioned above, this is consistent with the contrast between studies examining pudendal nerve injury (Allen et al., 1990, Sultan et al., 1994) or faecal incontinence (MacArthur et al., 1997) and those focusing on postpartal stress incontinence as an outcome parameter (Viktrup and Lose, 1993, Wilson et al., 1996, Farrell et al., 2001).

The two imaging papers available on the subject of levator function before and after childbirth both lack the power to assess any link between length of second stage and levator fimction. However, there is good evidence for a link between a longer second stage and increased pudendal neuropathy (Allen et al., 1990, Sultan et al. 1994) which seems to support the results obtained here. As regards perineometry, data on postpartal intravaginal pressure measurements was provided in one of the above- mentioned imaging studies (Peschers et al. 1997), but again numbers were insufficient to assess any correlation with length of second stage. This is even more true for recent attempts to define levator trauma using magnetic resonance imaging (Tunn et al., 1999a,b).

Birthweight: In the data presented here birthweigt did not correlate with a change in levator fimction (r= 0.123, p= 0.12). There is little data in the literature regarding a

172 potential link; correlations, if any, were weak (Allen et al., 1990) or nonsignificant (Meyer et al., 1998). Since birthweight is only one ofmmy factors determining the degree of trauma suffered during childbirth and since there is a strong relationship between birthweight and maternal body habitus, this does not appear surprising.

4.5 Other results not covered by hypotheses

4.5.1 Levator function

There is very little information on what constitutes normal pelvic floor muscle activity. In fact, nobody !mows how much spontaneous activity of the pubococcygeus and puborectalis muscle is experienced by the 'normal' woman although it is assumed that this muscle exhibits constant low- level activity similar to other postural muscles (DeLancey, 2001). Two potential forms of 'spontaneous' activity seemed worthy of further investigation using first visit imaging data: one being the use of the levator muscle complex on intercourse, the second being the incidence of reflex levator activation on coughing and its supposedly protective function as regards stress incontinence.

There was a highly significant relationship between regular use of the levator on sexual intercourse and levator strength as determined by ultrasound (p< 0.001, see Table 3.9). It is not clear however whether a congenitally strong levator prompts its use on intercourse or whether regular use of the pelvic floor muscles on intercourse strengthens them to such· a marked degree.

It is nevertheless hypothesized that information on sexual practices involving the pelvic floor musculature in the popular media may have a beneficial effect. Sexual practices may be amongst the lifestyle factors that influence pelvic floor morbidity, an issue that deserves further study.

As discussed in chapter 2.5.5.4, it has been claimed that reflex levator activation is rare or a learned response (Schuessler, 1994, Staskin, 2001). The data reported in chapter 3.2.6.2

173 contradicts this contention since a large proportion (57%) of young nulliparous women, of whom none had had formal physiotherapy teaching, visibly contracted the levator muscle on coughing. The true rate should be higher still since weak reflex contractions would likely only be detectable by neurophysiological means. Such a reflex contraction should protect pelvic organs, especially the bladder neck, from downwards displacement (Miller et al., 1996). It has been shown to augment urethral closure pressure and therefore is likely to be an important component of the female continence mechanism (Van Loenen and Vierhout, 1997). However, there was no correlation between the presence of a visible reflex levator contraction on coughing and stress incontinence (see 3.2.6.2).

4.5.2 3D Ultrasound for the identification of pelvic fascial trauma

Due to limited availability of the necessary equipment, only a small subgroup of the original study population was assessed by 3D ultrasound. As a result, the power of this pilot project to demonstrate significant peripartum changes is limited. Another limiting factor is the lack of quantitation due to the absence of a position sensor (see 2.5.5.7.). Clinical examinations were not performed to increase patient compliance although it is acknowledged that such assessments would have been desirable. Their absence is one of the many shortcomings of this pilot study. Nevertheless, the author believes that important insights into the aetiology of pelvic floor disorders may be g;:tined from the data presented here.

The main advantage of 3D or "volume" ultrasound is the fact that it allows access to the transverse plane, i.e., the plane in which both levator hiatus and paravaginal attachments to the arcus tendineus can be observed. Magnetic resonance imaging in wome~ with pelvic floor disorders has demonstrated that paraurethral and paravaginal supports can be identified in the transverse plane (Klutke et al., 1990), being evident as a H- shaped vagina with anterolateral tenting to-wards the pelvic sidewall. In 1999 the first description of ultrasound .imaging of paravaginal supports was published; the authors used a transrectal approach (Wisser et al., 1999).

174 Of the 23 women investigated by translabial 3D ultrasound in this pilot study, the paraurethral/ paravaginal support structures suspending the urethra and vagina to the pelvic sidewall- or at least their effect on the vagina- were clearly visible in all but three women (see 3.6). Even in those three women, this anterolateral fixation of the vagina (see Fig. 11) was evident in two out of three planes assessed. This observation was defined as 'tenting' of the vagina towards the pelvic sidewall. The only study to assess this parameter in nulliparous women was published in 2001 (Ochsenbein et al., 2001), showing tenting to be visible in 14 out of 14 patients examined. Ochsenbein et al. used transrectal ultrasound to obtain images of the paravaginal supports; no attempt was made to assess those supports at different levels.

The presen:;e of tenting has been understood to signify intact paravaginal fascial supports of urethra and vagina to the arcus tendineus fasciae pelvis (DeLancey, 2001). If this is indeed true, then the wide range of values obtained for anterior vaginal wall mobility in nulliparous women with intact tenting would suggest that bladder neck hypermobility can occur with intact paravaginal support structures. This finding contradicts the belief that the presence of tenting signifies good bladder neck support (Klutke et al., 1990). Put another way, hypermobility may not just be due to paravaginal defects but may also be caused by stretched or highly compliant but otherwise intact paravaginal fascia.

Despite the limitations of the method used in this study, it was possible to document marked changes to paravaginal supports in some women, while in others no alterations were seen. Avulsion of the paravaginal fascial attachments from the pelvic sidewall was suspected in five patients, four after normal vaginal delivery and one after a Caesarean Section performed late in the first stage of labour, with the fetal head palpable at the level of the ischial spines.

Somewhat contrary to expectations however, such qualitative changes did not correlate significantly with an increase in anterior compartment mobility in those patients. While the women with loss of tenting did show increased bladder neck descent and urethral

175 rotation, the wide confidence intervals made this difference nonsignificant (see Table 3.29).

There are two potential explanations for this finding. Either the technology and methodology lacked accuracy, resulting in insufficient power, or increased bladder neck mobility postpartum may not in fact equate to avulsion of fascial structures. It is conceivable that such peripartum change may be due to stretching or attenuation of support structures rather than outright tearing as suggested in the past (DeLancey, 1993).

The author would then like to hypothesize that hypermobility as observed in parous women may be due to either of those two mechanisms or, as observed above, to preexisting congenital laxity or increased compliance of structures which nevertheless remain intact. Any combination of these three mechanisms appears possible at one given site in one particular patient- or at different sites in this one patient, explaining the infinite variation in the presentation of female pelvic organ prolapse- and possibly also the fact that hypermobility alone, while a strong predictor (Dietz, 2001 b), does not equate to Genuine Stress Incontinence. An intact suburethral hammock may keep the patient dry, whether it is rigid or elastic, in contrast to an avulsed or tom structure.

As far as the author is aware, the pilot study presented here is the first attempt at evaluating peripartum changes in paravaginal fascial attachments. Further studies in this field are urgently needed. A whole surgical paradigm - the site- specific defect or defect­ specific approach (Richardson et al., 1976, Miklos et al., 1998) which currently enjoys widespread popularity, in particular among laparoscopic surgeons- rests on the existence of defects that may well be absent in a significant proportion of patients with Genuine Stress Incontinence and I or pelvic organ prolapse.

176 5 Summary of main findings

A prospective observational study was conducted at a tertiary obstetric unit in order to define the effect of pregnancy and childbirth on the lower urinary tract and pelvic floor. 200 women were recruited and assessed at 6-18 weeks. The assessment consisted of an interview (covering lower urinary tract symptoms, family history and ethnicity) and an examination for joint mobility in the upper limb, as well as translabial ultrasound imaging. The latter technique was used to determine the mobility of pelvic organs on maximal Valsalva manoeuvre and on pelvic floor muscle contraction, with the strongest of three manoeuvres used for numerical evaluation. Blood was taken for extraction of DNA and, in a subgroup of 50 women, for determination of relaxin and progesterone serum levels.

The above assessment (excluding the phlebotomy) was repeated at 32- 38 weeks (attended by 173 women) when an assessment for abdominal striae was added. Women were invited to return for a postpartal appointment, and 169 (84.5%) attended. This last assessment also contained the documentation of pelvic girdle pain in pregnancy and puerperium, documented with the help of a sliding visual analog scale. At the third appointment the examiner was blinded against delivery information which was only collected after all women had completed the study. Delivery data was obtained via especially designed data collection sheets and the institutional database and was available for all 173 women who attended the third trimester appointment as well as for 10 others who had delivered prematurely..

Primary outcome parameters were symptoms of stress urinary incontinence, paper towel testing and ultrasound measures of pelvic organ descent and levator function. The main explanatory parameters used were the mode of delivery, length of first and second stage of labour, and birthweight. A number of secondary outcome parameters and explanatory parameters were used to test a total of 15 hypotheses as listed in 2. 7.

177 5.1 Conclusions

The main conclusions to be drawn from this study are as follows:

5.1.1. Hypothesis 1: 'Pelvic organ mobility varies in relation to ethnic origin'

It has been claimed in the past that women of Asian extraction may benefit from stronger pelvic support structures compared to Caucasian women. In support of this concept it was found that Asian women showed significantly less pelvic organ mobility than Caucasian women (p= 0.002 for bladder neck descent). No such data has been available in the literature to date.

As an unexpected finding not covered by any specific hypothesis, this study has found that pelvic organ mobility in young nulliparous women varies to a degree hitherto unknown. More than Y4 of the population tested here exhibited abnormal bladder neck mobility of 25 mm or more; measurements ranged from 1 to 46.4 mm. This constitutes substantial new evidence for a congenital contribution to female pelvic organ mobility.

5.1.2. Hypothesis 2: 'There is an association between clinical indicators of connective tissue quality (joint hypermobility, history of hernia, epistaxis and dislocations, family history of incontinence/ prolapse, striae gravidarum, pelvic girdle pain) and pelvic organ mobility as quantified by ultrasound measurements and/ or symptoms of stress incontinence'.

Joint hypermobility (p= 0.017 for wrist hyperflexion) and a family history of incontinence (p= 0.034) seem to be associated with increased anterior compartment mobility. This finding again strengthens the concept of a congenital contribution to the problem of stress urinary incontinence and female pelvic organ prolapse. So far, no such data has been reported in the literature. No other significant relationships were observed; in particular, striae gravidarum and pelvic girdle pain did not correlate with

178 pelvic organ mobility. There was a weakly significant relationship between pelvic girdle pain and postpartum stress incontinence (p= 0.029); the clinical relevance of this and the above findings is doubtful in view of the multiple analyses performed.

5.1.3. Hypothesis 3: 'There is a correlation between frequency of symptoms of stress incontinence and anterior vaginal wall mobility in pregnancy'; and Hypothesis 4: 'There is a correlation between the presence and severity of stress incontinence on paper towel test and anterior vaginal wall mobility in pregnancy.'

No significant correlations between bladder neck mobility and symptoms and signs of stress incontinence in pregnancy were observed, in accordance with published literature. It is trerefore speculated that other (hitherto undefmed) factors are responsible for stress incontinence in pregnancy, at least in nulliparous women. In contrast to data obtained in nonpregnant older women, bladder neck descent does not seem to confer an increased risk of stress incontinence in the group assessed here.

5.1.4 Hypothesis 5: 'There is a positive correlation between serum relaxin and/ or progesterone levels in the first trimester and pelvic organ mobility.'

Relaxin and Progesterone serum levels were not associated with pelvic organ mobility in early pregnancy in this first study to assess the effect of both hormones on pelvic fascial supports. Furthermore, mobility of the anterior vaginal wall in early pregnancy was similar to the means and ranges observed for this parameter in nonpregnant nulliparous women. Hence there is no evidence for a significant hormonal contribution to alterations in pelvic organ support in pregnancy and childbirth, an issue that has been debated at length.

5.1.5 Hypothesis 6: 'There are significant differences between polymorphisms of Fibrillin, Elastin and Collagen genes observed in nulliparous women with marked pelvic organ mobility and those without' and

179 Hypothesis 7: 'Above genetic factors are risk factors for the symptom of stress incontinence during pregnancy and postpartum'

The above hypotheses could not be tested on the basis of available data. However, the author believes that the considerable effort expended in establishing a methodology for the identification of single nucleotide polymorphisms (SNPs) has not been wasted. Denaturing high- pressure liquid chromatography seems to be an appropriate technique for screening the DNA of patients whose phenotype has been defined by translabial ultrasound. To the lmowledge of the author, the present study constitutes the first use of molecular genetic techniques in the investigation of urinary incontinence and prolapse. It appears highly likely that, given enough time and resources, clinically relevant findings will result from such work. For directions for future research, see chapter 5.3.

5.1.6 Hypothesis 8: 'There is a negative correlation between levator ani muscle function (as defined by cranioventral displacement of the bladder neck on pelvic floor muscle contraction) and pelvic organ mobility'

Recent claims that levator wealmess or damage is the primary event in the causation of pelvic organ descent could not be substantiated in this study; there were consistent weakly positive correlations between bladder neck hypermobility and displacement of the bladder neck on levator contraction (r= 0.26, p

However, several other relevant findings were recorded outside the testing of formal hypotheses. Levator function in young nulliparous women seems to vary widely, with an oblique bladder neck lift ofO -19.9 mm documented in early pregnancy. Only very few women were unable to contract the levator at all. Verbal instruction alone was insufficient in about half of all women assessed. A reflex levator contraction on coughing was present in more than half of all women despite them

180 never having been taught the 'lrn.ack', implying that a true reflex was observed. This latter finding contradicts claims in the literature that consider reflex levator activation a learned response.

Furthermore, use of tre levator muscle on intercourse was significantly associated with more marked bladder neck displacement on levator contraction (p< 0.001), pointing to the impact of lifestyle factors on pelvic floor muscle function. This novel observation may have consequences for pelvic floor muscle exercise teaching and prophylactic public health intervention.

5.1.7 Hypothesis 9: 'Bladder neck, uterine and rectal mobility (as measured relative to the inferoposterior margin of the symphysis pubis) increase during pregnancy, i.e., between the first and the third trimester'.

Most parameters of pelvic organ descent showed significant increases between early and late pregnancy appointments (p< 0.001 for bladder neck descent). The cause of the observed changes remains unclear but may be due to hormonal influences or the mass effect of pregnancy. So far, there is very little (and conflicting) evidence on this issue in the published literature.

5.1.8 Hypothesis 10: 'Delivery mode is independent of pelvic organ mobility as measured by ultrasound in the third trimester of pregnancy'

This hypothesis was refuted very clearly as there seems to be a significant relationship between the mobility ofpelvic organs and labour outcome. Less mobility was associated with longer second sta~ oflabour and operative delivery (p= 0.002 for bladder descent versus delivery mode). Pelvic organ mobility may be a measure of pelvic compliance and a potential predictor of progress in labour. This fmding is potentially of major significance to Obstetrics, in particular in view of the rising tide of elective Caesarean Section on maternal request. Together with other predictors of outcome such as station of the fetal head, cervical length, maternal age,

181 body mass index and weight gain in pregnancy, this parameter may allow the construction of a predictive model for operative intervention in labour. A prospective study is now in progress to further investigate whether such a model can attain sufficient accuracy for use in clinical intervention trials (see 5.3.2)

5.1.9 Hypothesis 11: 'Bladder neck descent at first visit is a predictor of postpartum incontinence'.

This hypothesis could not be substantiated in the current study which contradicts previous work in this field. Discrepancies may be due to methodological differences (see 4.4.1.1).

5.1.10 Hypothesis 12: 'The symptom of postpartum stress incontinence is associated with delivery mode, birthweight and/ or length of second stage'.

In this study Caesarean section was partly protective of postpartum stress incontinence (p= 0.003) which agrees with published epidemiological evidence, but neither birthweight nor length of second stage (active or passive) differed between stress continent and stress incontinent women.

5.1.11 Hypothesis 13: 'The symptom of postpartum stress incontinence is related to the peripartum increase in pelvic organ mobility'.

This hypothesis was substantiated although significance levels were low. The degree of change in anterior vaginal wall mobility seems to be associated with postpartum worsening of symptoms of stress incontinence (p= 0.044). This suggests that it is not hypermobility as such which adversely affects continence, but possibly the traumatic alteration of support structures. However, further work will be needed befOre such conclusions can be drawn with any degree of certainty.

182 5.1.12 Hypothesis 14: 'Mode of delivery, birthweight and length of the second stage of labour are associated with the extent of fascial trauma as defined by a peripartum increase in pelvic organ mobility.'

Vaginal childbirth, and in particular vaginal operative delivery, seems to increase pelvic organ mobility. This effect was highly significant and observed in all three vaginal compartments (all p< 0.001 ). The duration of second stage correlated weakly but significantly with increased urethral (r- 0.19, p= 0.016), bladder (r--0.23, p= 0.003), uterine (r- 0.21, p= 0.009) and rectal descent (r- -0.29, p= <0.001) postpartum. The pattern of change varied between compartments. Increased anterior and central compartment descent was associated with the length of active second stage and with any form of vaginal delivery. The integrity of the posterior compartment appears to be equally affected by passive and active second stage, similar to the effect of parturition on levator function (see below), with the actual delivery being of lesser importance. The author is not aware of any study in the published literature that has demonstrated the presumed traumatic effect of vaginal childbirth on pelvic organ support structures in such a comprehensive way. The data presented here may well go a long way towards settling the debate as to whether such damage is done by pregnancy or delivery.

All forms of Caesarean Section, but especially prelabour Caesarean, were associated with relatively less anterior compartment descent, with postpartum measurements in women after prelabour Caesarean Section practically returning to early pregnancy values. This implies that elective Caesarean Section is likely to protect women from the increase in pelvic organ mobility associated with late pregnancy and vaginal childbirth. These findings may partly explain the protective effect of elective Caesarean delivery for future symptoms of pelvic floor disorders observed in epidemiological studies and, in the work presented here, its protective effect on stress incontinence three months postpartum. Such fmdings are of major importance in informing the debate surrounding the issue of elective Caesarean Section.

183 Birthweight did not correlate with parameters of pelvic organ descent which appears to be in agreement with most epidemiological and clinical data published to date.

An interesting finding not covered by a hypopthesis defined prior to study commencement was the association betweenantepartal pelvic organ mobility and the extent ofperipartal change in these parameters. Women with little congenital pelvic organ mobility seem to be those that show the most marked increases in organ mobility from vaginal childbirth. It appears that stiff pelvic support structures are more likely to suffer traumatic disruption than those which are more elastic (p

As a pilot study, 3D volume ultrasound was used in a small subgroup of women to assess paravaginal fascial attachments before and after childbirth, the first such study in the published literature to date. Findings consistent with avulsion of paravaginal supports from the pelvic sidewall were demonstrated in 5 out of 21 tested women, four of them delivered vaginally. There was no clear association with increased bladder neck mobility. It appears that anterior vaginal wall hypem1obility may in fact be due to stretching or attenuation of fascial structures rather than actual disruption or avulsion; however, further work using 3D ultrasound or MRJ imaging seems needed to substantiate these findings.

184 5.1.13 Hypothesis 15: 'Mode of delivery, birthweight and length of the second stage of labour are associated with a postpartal reduction in levator strength on voluntary contraction as measured by displacement of the internal urethral meatus relative to the inferoposterior margin of the symphysis pubis.'

Childbirth reduces bladder neck displacement on levator contraction (p< 0.001), a finding that agrees with published literature on levator strength on palpation. This effect is still evident 3 months postpartum, at a time when reinnervation should be complete. The main factor affecting levator trauma seems to be the length of second stage, both active and passive (Spearman's r =-0.225, p= 0.004 for total second stage). A Caesarean Section in the second stage of labour appears to exert no discernible protective effect on longterm levator function relative to any form of vaginal delivery, including forceps. Consequently it appears that it is not crowning but full engagement of the fetal head and its duration that determines the peripartal deterioration in levator function. This observation adds support to the concept that delivery- related damage to the levator muscle is primarily neurogenic, a claim that hitherto was mainly based on neurophysiological evidence. Again, there was no correlation between levator function and birthweight.

5.2 Implications for clinical practice

What are the potential implications of the presented work for obstetric practice? It is evident that vaginal childbirth significantly alters both fascial supports of pelvic organs and levator muscle function, whether this is due to stretching and attenuation of fascial structures or actual disruption. Such alterations are in addition to the known traumatic effect of vaginal childbirth on the anal sphincter and the pudendal nerve. It may therefore be argued that the data obtained in this work further strengthens the argument for elective caesarean section in primigravid women.

However, it is also evident from the presented data that some women appear to be more at risk of traumatic pelvic floor damage than others. There are those who

185 already exhibit a marked degree of pelvic organ mobility antepartum- these women seem less likely to sustain pelvic floor trauma, and they seem in fact more likely to have an uncomplicated, normal vaginal delivery. It would be a waste of scarce healthcare resources to offer an elective caesarean section to these women; and it would deprive them of an experience central to their lives, and of the sense of achievement associated with successful vaginal childbirth.

On the other hand it may be possible to characterize a subgroup of women who are less likely to deliver without operative intervention and/ or who are more likely to suffer pelvic floor trauma if they in fact do achieve a vaginal delivery. Such women may benefit from elective caesarean section in a number of ways. The intervention could potentially confer benefits regarding present and future health of mother and child and may well be cost- effective, in the short term due to a reduction in emergency obstetric intervention, and in the long term due to decreased pelvic floor morbidity.

5.3 Directions for future research

5.3.1 Defining the genetic basis for female pelvic organ prolapse and incontinence

Two promising directions for future molecular genetic research have opened up as a result of the work described in this thesis. On the one hand, further target gene analysis oftre five above- mentioned target genes in the DNA of women enrolled in this and other studies may yield results defining the relationship between genotype and phenotype. On the other hand, a pilot study is in progress under the direction of the author, at the Queensland Institute of Medical Research (QIMR) in Brisbane, Australia, using a twin study approach to define heritability of the phenotype "pelvic organ mobility". A comparison of mono- and dizygotic twins allows a relatively straightforward approach to the investigation of heritability which is why much effort worldwide has gone into the establishment of twin registries and databases.

186 Since 1992, an adolescent cohort has been recruited from schools in Brisbane and surrounding areas of southeastern Queensland. As these women are still mostly nulliparous, they constitute an excellent sample in which to assess the heritability of pelvic organ mobility. At the time of writing (9/03), about 230 clinical assessments have been carried out as part of this project. A first attempt at genetic modelling has demonstrated that bladder mobility in nulliparous women is indeed a heritable trait (Dietz et al., 2003)

After demonstration and quantification of heritability, linkage studies will be required to determine candidate genes. A genomic screen (ABI Prism Linkage Mapping Set Version 2 (PE Applied Biosystems) with 400 markers at "'10 eM coverage) has been performed on these twins and is available for future linkage analysis at QIMR, although it is likely that much higher numbers of clinical assessments will be needed for this purpose.

In the long term, it is to be hoped that prophylactic measures will reduce the considerable morbidity from pelvic floor disorders. In order to develop and introduce prophylactic measures, high risk groups will have to be identified. After definition of target genes by linkage analysis one would attempt to isolate genetic risk factors, i.e. certain polymorphisms associated with pelvic floor dysfunction The definition of high- risk patients and risk factors associated with childbirth has the potential to change obstetric practice and will allow us to tar~t intervention such as physiotherapy and elective caesarean section in a much more rational and effective way. Ultimately, the goal is a major reduction in morbidity from female pelvic organ prolapse and urinary incontinence.

5.3.2 Prediction of intervention in labour

The strength of the association between pelvic organ mobility and delivery mode seems to indicate that this issue is also worthy of further investigation. Other factors such as maternal age, body mass index, weight gain in pregnancy, cervical length,

187 position and mobility of the fetal head could be combined with pelvic organ mobility to develop a model that could act as a clinically useful predictor. This has been attempted by the author (Dietz, 2002b) in a pilot project but is outside the scope of this thesis as its focus was the effect of pregnancy and delivery on the pelvic floor. Prior to the findings demonstrated in this study, only maternal age (Ecker et al., 2001, Bell et al., 2001), cervical length (Ware and Raynor, 2000) and engagement of the fetal head on palpation (Roshanfekr et al., 1999) have been shown to be (albeit weak) predictors of either operative delivery or Caesarean Section.

The accurate identification of nulliparous women at high risk for operative delivery has the potential to significantly reduce short- and longterm maternal and fetal morbidity, decrease the likelihood of mentally or physically traumatic childbirth, the utilization of after- hours resources and the associated increased complication rates. On the other hand, a large proportion of women may receive reassurance regarding their potential course of labour, allowing the choice of a low- intervention, low­ technology environment with more confidence. Future work will have to focus on defining appropriate predictive models, with definitions of 'low- risk' vs. 'high- risk' and cutoffs to be determined by intervention trials.

Advice given to women on the basis of an accurate risk assessment may prove to be eminently cost- effective since emer~ncy obstetric intervention is not just stressful and traumatic to the patient but also veryresource- intensive. The aim of further research in this field would be to confirm the role of potential predictors in a prospective study, after which a randomized controlled trial could be undertaken to test the effect of an offer of elective Caesarean Section in nulliparous women at high risk of operative delivery. On the other hand, women at low risk of operative delivery may be offered birthing in a less technical environment such as a Birth Centre. Outcomes may well be improved at both ends of the spectrum, resulting in safe low- technology birthing for an increased number of nulliparous women and equally safe elective Caesarean delivery for the minority of women with a very low likelihood of successful normal vaginal delivery. At the time of writing (9/03), a

188 prospective study is 60% completed at Royal Prince Alfred Hospital, Sydney, which will allow a more accurate definition of the predictive value of the parameters mentioned above.

5.3.3 Prediction and prevention of pelvic floor trauma

Further work, parallel to that suggested in 5.3.2., may serve to confirm and/ or more accurately define the usefulness of antepartum pelvic organ mobility as a predictor of fascial trauma. Advances in realtime 3D ultrasound now facilitate investigations such as the 3D pilot study conducted as part of this thesis. The author has recently undertaken a pilot study determining pelvic organ support in 53 young nulligravid women with the help ofsuch equipment (Kretz Voluson 730, GE Ultrasound). It is to be hoped that this work will help define 'normality' and allow us to detect traumatic changes in symptomatic women later in life. At the time of writing (1/03) the author is involved in a study using 3D ultrasound imaging to assess paravaginal support tissues in urogynaecological patients and correlate fmdings with a blinded clinical assessment. With the help of volume ultrasound imaging, future investigators in this field will hopefully also be able to more accurately pinpoint peripartum changes in fascial integrity and compliance.

If confirmed by other investigators, antepartum pelvic organ mobility may serve to select those women most at risk of suffering impairment of pelvic organ supports. Fortunately, those women seem to be precisely the ones most likely to require emergency intervention in labour. This implies that intervention trials as proposed in 5.3.2. should include clinical or imaging parameters of pelvic organ mobility in order to determine whether antepartum risk assessment for intervention in labour has the potential for reducing pelvic floor morbidity. Testing of such a hypothesis may well require a major collaborative effort and is likely to involve long-term follow-up. In any event, either of the above developments has the potiential to significantly change clinical practice, not just in Gynaecology, but in Obstetrics as well.

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225. Thorp, J.M.,Jr., Norton, P.A., Wall, L.L., Kuller, J.A., Eucker, B., and Wells, E. Urinary incontinence in pregnancy and the puerperium: a prospective study. Am.J.Obstet.Gynecol. 181 (2):266-273, 1999.

204 226. Tincello, D.G., Adams, E.J., Booth, K., Cording, V., and Richmond, D.H. Antenatal prediction of postnatal urinary incontinence in nulliparous women. lnt.Urogyneco/.J 11(S1):S35, 2000.

227. Tunn, R., Delancey, J.O., Howard, D., Ashton Miller, J.A., Quint, L.E., and Goedicke, J. Evaluation of the levator ani muscle structure using different MR imaging techniques. lnt.Urogynecoi.J 1O:S56, 1999a. (Abstract)

228. Tunn, R., Delancey, J.O., Howard, D., Thorp, J.M., Ashton-Miller, J.A., and Quint, L.E. MR imaging of levator ani muscle recovery following vaginal delivery. lnt.Urogynecoi.J. 10(5):300-307, 1999b.

229. Ulmsten, U. and Falconer, C. Connective tissue in female urinary incontinence. [Review] [34 refs]. Curr. Opin. Obstet. Gynecol. 11 (5):509-515, 1999.

230. Unemori, E.N., Beck, L.S., Lee, W.P., Xu, Y., Siegel, M., Keller, G., Liggitt, H.D., Bauer, E.A., and Amenta, E.P. Human relaxin decreases collagen accumulation in vivo in two rodent models of fibrosis. J.lnvest.Dermatol. 101 (3):280-285, 1993.

231. Vancaillie, T.G. Plication of the pubourethralligament: a new method for the treatment of genuine stress urinary incontinence. Gyn. Endoscopy 4(S1):30-3, 1995.

232. van Dong en, L. The anatomy of genital prolapse. S.Afr.Med.J. 60(9):357 -359, 1981.

233. van Geelen, J.M., Lemmens, W.A., Eskes, T.K., Martin, C.B., Jr. The urethral pressure profile in pregnancy and after delivery in healthy nulliparous women. Am.J.Obstet.Gynecol. 144:636-49, 1982.

234. van Geelen, J.M. Delivery and Urethral sphincter incontinence. In: Pelvic Floor Reeducation- Principles and Practice, edited by Schuessler, B., Laycock, J., Norton, P., and Stanton, S.L.London:Springer, 1994,p. 111-118.

235. van Loenen, N.T. and Vierhout, M.E. Augmentation of urethral pressure profile by voluntary pelvic floor contraction. lnt.Urogynecoi.J. 8(5):284-287, 1997.

236. Versi, E., Orrego, G., Hardy, E., Seddon, G., Smith, P., Anand, D. Evaluation of the home pad test in the investigation of female urinary incontinence. Br.J.Obstet.Gynaecol. 103:162-67, 1996 .

237. Versi, E., Harvey, M.A., Cardozo, L., Brincat, M., and studd, J.W. Urogenital prolapse and atrophy at menopause: a prevalence study. Int. Urogynecoi.J. 12(2):1 07-110, 2001.

238. Viidik, A. Functional properties of collagenous tissues. lnt.Rev.Connect. Tissue Res. 6:127-215, 1973.

239. Viktrup, L. and Lose, G. Epidural anesthesia during labor and stress incontinence after delivery. Obstet. Gynecol. 82(6):984-986, 1993.

240. Viktrup, L. and Lose, G. Lower urinary tract symptoms 5 years after the first delivery. lnt.Urogynecoi.J 11 (6):336-340, 2000.

241. Vodusek, D.B. Clinical neurophysiological tests in urogynecology. lnt.Urogynecoi.J 11(6):333-335, 2000.

205 242. Voigt, R., Halaska, M., Michels, W., Martan, A., Starker, K., and Voigt, P. Examination of the urethrovesical junction using perineal sonography compared to urethrocystography using a bead chain. lnt.Urogynecoi.J 5:212-214, 1994.

243. Walters, M.D. and Weber, A.M. Anterior vaginal wall prolapse with and without genuine stress incontinence. In: Textbook of Female Urology and Urogynaecology, edited by Cardozo, L. and Staskin, D.London:lsis Medical Media, 2001 ,p. 588-597.

244. Ware, V. and Raynor, B.D. Transvaginal ultrasonographic cervical measurement as a predictor of successful labor induction. Am. J. Obstet. Gynecol. 182(5): 1030-1032, 2000.

245. Weiss, G., Goldsmith, L.T., Sachdev, R., Von Hagen, S., and Lederer, K. Elevated first- trimester serum relaxin concentrations in pregnant women following ovarian stimulation predict prematurity risk and preterm delivery. Obstet. Gyneco/. 82(5):821-828, 1993.

246. Wijma, J., Weis Potters, A.E., de Wolf, B.T., Tinga, D.J., and Aarnoudse, J.G. Anatomical and functional changes in the lower urinary tract during pregnancy. Br.J. Obstet. Gynaecol1 08(7):726-732, 2001.

247. Wilson, D. and Herbison, P. Conservative management of incontinence. Curr. Opin. Obstet. Gynecol. 7( 5):386-392, 1995.

248. Wilson, P.D., Herbison, R.M., and Herbison, G.P. Obstetric practice and the prevalence of urinary incontinence three months after delivery. Br.J. Obstet. Gynaecol. 103(2):154-161, 1996.

249. Wilson, P.D., Bo, K., Bourcier, A.P., Hay Smith, E.J., Staskin, D.R., Nygaard, I.E., Wyman, J.F., and Shepherd, A. Conservative management in women. In: Incontinence: First International Consultation on Incontinence, edited by Abrams, P., Khoury, S., and Wein, A. 1999

250. Wisser, J., Sch? r, G., Kurmanavicius, J., Huch, R., and Huch, A. Use of 3D ultrasound as a new approach to assess obstetrical trauma to the pelvic floor. Ultraschai/.Med. 20(1):15-18, 1999.

251. Wright, A.F., Carothers, A.D., and Pirastu, M. Population choice in mapping genes for complex diseases. Nature Genetics 23(4):397-404, 1999.

252. Zacharin, R. A Chinese Anatomy- the pelvic supporting tissues of the Chinese and Occidental female compared and contrasted. Aust.N.Z.J.Obstet.Gynaecol. 17:1-11, 1977.

206 7 Own peer- reviewed publications and awards based on this Thesis

7.1 Abstracts

1. Dietz, H.P., Tan, L., Brown, S., Garrett, D., Vancaillie, T. Do relaxin and progesterone serum levels affect pelvic organ mobility? Abstract, ICS 2001 Seoul

2. Dietz, H.P., Steensma, A., Vancaillie, T. Levator activity in nulliparous women. Abstract, IUGA, 2001 Melbourne

3. Dietz, H.P., Steensma, A.B., Moore, K.H. The effect of childbirth on pelvic organ mobility. Neurourol. Urodyn. 21:361-62, 2002

4. Dietz, H.P.,: The case for a congenital contribution to Female pelvic organ prolapse and stress incontinence Aust NZ J Obstet Gyneco/42, S12; 2002.

5. Dietz, H.P., T.G. Vancaillie: Do Asian women have superior pelvic fascia? Abstract, ICS 2002 Heidelberg

6. Dietz, H.P: Defining a congenital contribution to Female pelvic organ prolapse and Genuine Stress Incontinence Int. Urogyneco/. J. 13: S58, 2002

7. Dietz, H.P., Moore, K.H.: Who suffers most from pelvic floor trauma during childbirth? Abstract, RANZCOG/ RCOG 2002 Sydney

8. Dietz, H.P., T.G. Vancaillie: Pelvic Organ hypermobility: how much is congenital, how much is due to childbirth? Abstract, RANZCOG/ RCOG 2002 Sydney

9. Dietz, H.P., Bennett, M.J.: Can we predict the course of labour? Aust. NZ. J. Obstet. Gynaeco/. 42: S 16; 2002.

10. Dietz, H.P.: Ultrasound determination of fetal head engagement predicts delivery mode. ASUM 2002 Gold Coast

11. Dietz, H.P.: Translabial ultrasound can define pelvic compliance and predict progress in labour. ASUM 2002 Gold Coast

12. Dietz, H.P.: Translabial 3D Ultrasound of the Pelvic Floor: The effect of parturition on paravaginal fascia. ASUM 2002 Gold Coast

13. Dietz, H.P., Steensma, A.B., Moore, K.H. The effect of childbirth on levator function. AGES 2003 Melbourne

14. Dietz, H.P., Moore, K.H. Voiding function in pregnancy and puerperium. Submitted to IUGA 2003, Buenos Aires

15. Dietz, H.P., Steensma, A.B., Eldridge, A., Grace, M., Clarke, B. Test- retest reliability of the ultrasound assessment of bladder neck mobility. Submitted to IUGA 2003, Buenos Aires

16. Dietz, H.P., Eldridge, A., Grace, M., Clarke, B. Does pregnancy affect pelvic organ mobility? Submitted to IUGA 2003, Buenos Aires

17. Dietz, H.P. and Moore, K.H.: Does good levator function protect against delivery­ related pelvic floor trauma? RANZCOG 2003 Auckland

207 7.2 Original articles

1. Dietz, H.P., Steensma, A., Vancaillie, T.G.: Levator strength in nulliparous women Int. Urogynecol. J. 2003; 14: 24-26

2. Dietz, H.P., Steensma, A.B. Which women are most affected by peripartal pelvic floor trauma? In print, Eur J Obstet Gyneco/ Reprod Bioi

3. Dietz, H.P. Do Asians have superior pelvic organ support structures? In print, Int. Urogynecol. J.

4. Dietz, H.P., Moore, K.H., Steensma, A.B. Pelvic organ mobility is associated with delivery mode. Aust NZ J Obstet Gynecol. 43: 70-74; 2003.

5. Dietz, H.P., Hastings, R.: Three- dimensional ultrasound imaging of the pelvic floor: the effect of parturition on paravaginal fascia Ultrasound Obstet Gynecol2003; 21: 589- 595

6. Dietz, H.P., Bennett, M.J. The effect of childbirth on pelvic organ mobility. Obstet Gynecol. 2003; 102: 223-228

7. Dietz, H.P., Bennett, M.J. Predicting Operative Delivery. ASUM Bulletin 5 (4):4-8; 2002.

8. Dietz, H.P.: Translabial 3D Ultrasound of the Pelvic Floor: The effect of parturition on paravaginal fascia. ASUM Bulletin 6 (2003); 16-17.

9. Dietz HP. Levator function before and after childbirth. In print, Aust NZ J Obstet Gynecol

10. Dietz, H.P., Bennett, M.J. Predicting Operative Intervention in Labour Submitted to Acta Obstet Gynaecol Scand.

7.3 Awards

2002 Segelov Traveling Fellowship, Royal Prince Alfred Hospital, Sydney

2002 Toshiba ASUM Scholarship

2002 Best Research Presentation, ASUM 2002

2003 Christopher Kohlenberg Medal for Best Original Research Annual NSW/ QLD Joint Meeting ofRANZCOG

208 8 Appendix

8.1 Datasheets

....._j.,L ~...... ~ D ~~...... RHW Name/ Address/DOB Date I I I

Ethnicity: ......

Pregnancy: EDD: ...... Complic: ...... Meds: ...... Comments: ......

Lifestyle: .. Smoking ...... Sports ...... Wt pre preg: ...... Ht: .... . Wt current: ...... S~m~toms:

Sl: 1/mO 1/w 0 1/d 0 more 0 Urgency: defer <10' D <5' D <1' D Ul: 1/mO 1/w 0 1/d 0 more 0

Frequency: 9-120 13-16 0 17+0 just in case? Nocturia: 20 3-40 5+0 Voiding: hesitancy, poor stream, straining, dribble, incomplete empty, need to revoid, hover, deviated stream, dysuria, haematuria, UTI Bowel symptoms: Straining, constipation, manual evacuation, haemorrhoids, faecal urgency, incontinence, soiling, flatus incontinence.

Famil~ History:

Bladder: ...... Prolapse: : ...... Operations: : ...... Comments:

209 Medical Assessment:

Stretch marks (number): Joint hypermobility: 1): thumb opposition to forearm: 2.):Vth digit passive hyperext.>55° 3.) elbow hyperextension >190°

Paper towel test:

Flowmetry: Voided vol...... max. flow ...... avg. flow ...... Voiding pattern: ...... max accel......

Translabial US:

Residual urine: ...... ml

RVA-R ~--1-----liRVA-5 Rot. Funneling D

Rest Valsalva Rest2 Lev. contr. BND (vertical) BND (horizontal) EJEJEJEJ Spontaneous? On intercourse? Ever taught? 1----1 Corrected? Cough reflex?

Cystocele

Uterine descent

Rectocele

Comments: ......

210 Name/ Address/ DOB D

Date of delivery DOD

Labour: (please mark)

First stage (hrs) I I Second stage (mins) not actively pushing

active pushing

Epidural yes no

Indwelling catheter no to 12h 13-24h 24h+

Delivery: (please mark if applicable) Birthweight: ......

Position OA OP other: ......

Operative delivery Ventouse Forceps Station: 0 +1 +2 +3

Pull: to 2 3 4 5+ Easy Mod. Firm Veryfrrm

Rotation

Episiotomy yes no

Tears I II Ill IV Comments:

211 8.2 Consent forms

Royal Hospital for Women, Randwick RHW

SUBJECT INFORMATION STATEMENT AND CONSENT FORM "The lower urinary tract and pelvic floor in pregnancy and puerperium"

You are invited to participate in a study of the effect of pregnancy and childbirth on bladder function. We hope to learn how much pregnancy and the birth of a baby influence bladder function and the function of the pelvic floor. You were selected as a possible participant in this study because you are pregnant with your first child.

A large number of women develop bladder problems during and after pregnancy, and not a lot is known about the causes. Until recently we were unable to properly investigate bladder function because tests would have involved the use ofXrays. Now we use ultrasound which is thought to be completely harmless.

If you were to agree to take part in this study we would ask you to have: 1.) an interview before you have reached 18 weeks in your pregnancy 2.) four tests, at 5-18 and 36 weeks and again 6 weeks and 6 months following the birth of your child. The assessment takes about 15 minutes. You will be asked to bring in a pad worn at home for one hour while following written instructions asking you to cough, jump on the spot etc., pass urine on a special toilet and then have an ultrasound test. The ultrasound imaging is similar to the ultrasound done to check on baby's growth, for instance, with the difference that the scanner has to be placed between the legs instead of on the abdomen. This is necessary because the pelvic organs cannot be seen well from above. We'll ask you to cough and bear down, and also to squeeze (contract the pelvic floor).

We also would like to analyze some of your blood which would be taken at the same time as other pregnancy routine blood tests (no extra needleprick!). We are trying to find out whether changes in the genes which determine the strength of your connective tissue may be responsible for incontinence and prolapse in later life.

We cannot guarantee that you personally will receive any benefits from this study, however we hope that results may help us to improve care for women in the future. Any information about you that is obtained in connection with this study will remain confidential and will be disclosed only with your written permission. However, the results of the study may be published or disclosed to other people in a way that will not identify you. The data from this study will be stored on computer for 10 years and then disposed ofby erasure.

Whether you take part in this study or not, it will not make any difference to the medical care you will receive from the Royal Hospital for Women. If you decide to take part in the study, you can still withdraw at any time and this will not make any difference to your medical care either.

Limited funds are available to assist with parking fees. If you have any questions at any time Dr Peter Dietz (Telephone Number: 93 82 6111) will be happy to answer them. You will be given a copy of this form to keep.

212 Royal Hospital for Women, Randwick

SUBJECT INFORMATION STATEMENT AND CONSENT FORM "The lower urinary tract and pelvic floor in pregnancy and puerperium"

(continued)

I have read the above and would like to participate in the study. I understand that having or not having these tests will in no way influence the care I am receiving and that I can withdraw from this study at any time.

I have been given the opportunity of asking any questions relating to any possible physical and mental harm I might suffer as a result of my participation and I have received satisfactory answers.

I also understand that any proprietary rights derived from the analysis of genetic material from my blood will rest with the investigators.

Signature of subject Signature of witness

Please PRINT name Please PRINT name

Date Nature of Witness

Signature(s) ofinvestigator(s)

Please PRINT Name

213 Royal Hospital for Women, Randwick RH\tV

CONSENT FOR COLLECTION, STORAGE AND TESTING OF BLOOD FOR RESEARCH

I understand and consent to the following: (please initial)

The blood sample will only be used in the study ""The lower

I.______J_ I urinary tract and pelvic floor in pregnancy and puerperium"

The study has been approved by the Ethics Committee of the Royal I.______._ I Hospital for Women, Randwick

._l _____,l I have consented separately to my participation in the study

I IMy.blood sample may be stored for 10 years after completion of tile '------__. proJect

._l ______.l I will be able to obtain a report on tile outcome of tile project

I or my attending doctor will be advised if the project produces information which could be of value to me or my family I.______, I Testing of parents' and childrens' blood may reveal non- paternity or I.______. I non- maternity of a presumed natural parent. If blood or DNA is stored, it may not remain in a suitable state for I.______._ I testing ...... has explained to me and I understand the consequences and procedures involved in storage and testing of blood samples or DNA derived from such samples and I have had the opportunity to ask questions. I am satisfied with the explanation and the answers to my questions.

Signature of Research Subject Date

214 8.3 Declaration

The work in this thesis was carried out at the Royal Hospital for Women and Prince of Wales Hospital, Sydney, Australia, and at the Queensland Institute of Medical Research, Brisbane, Australia, between January 1999 and January 2002. I personally planned the studies that comprise this thesis and undertook more than 95% of the clinical and ultrasound assessments myself. Apart from the determination of progesterone levels and fascial biomechanical testing, all laboratory work reported here was also performed by myself.

A large number of staff at the above institutions supported me with help and supervision and are named in Acknowledgements. The main study and its extensions were approved by the South Eastern Sydney Area Health Board under Protocol number 99/ 184. Informed Consent was obtained from all participants.

I hereby declare that this submission is my own work and to the best of my knowledge it contains no material prevoiusly published or written by another person, nor material which to a substantial extent has been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis.

I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.

Hans Peter Dietz

215 8.4 TEST- RETEST AND INTERRATER RELIABILITY OF THE ULTRASOUND ASSESSMENT OF BLADDER NECK MOBILITY H P Dietz, A.B. Steensma, Ann Eldridge*, Marlene Grace*, Barton Clarke+ Royal Prince Alfred Hospital, Sydney, Queensland Institute of Medical Research, Brisbane, *Royal Women's Hospital, Brisbane, Australia

Objective: Bladder neck mobility is a strong predictor of Genuine Stress Incontinence in women with pelvic floor dysfunction (Dietz et al., 2002). It is usually assessed by fluoroscopic or ultrasound imaging, although only the latter allows for quick and convenient quantification. The objective of this study was to internally validate translabial ultrasound, a method increasingly used in urogynaecology (Koelbl and Hanzal, 1995, Schaer, 1997), for the determination of bladder neck descent by providing comprehensive test- retest data. Both patient- specific and operator- dependent factors are thought to influence the accuracy of ultrasound determination of bladder neck descent. Methods: In four series of229 repeat assessments in 181 nulliparous young women recruited for two separate studies of pelvic floor function, four different forms of test­ retest reliability protocols were followed. In all instances, patients were examined by translabial ultrasound, supine and after bladder emptying, in order to maximize bladder neck descent on Valsalva manoeuvre (Dietz et al., 1999, 2001 ). The best of a series of at least three effective Valsalva manoeuvres were used for evaluation. The position of the bladder neck was determined against the inferoposterior margin of the symphysis pubis. All measurements were taken by the first author or by personnel trained by him (ABS, AE, MG) for a minimum of 100 patient assessments. All repeat measurements were performed by operators blinded to previous assessments. Documentation was obtained by videoprinter printouts and on videotape. The first series (n= 114) determined the variabilityofValsalva manoeuvres by comparing three effective manoeuvres assessed by the same operator during the same session, within a timeframe of 10 minutes. The second series (n=20) documented the interobserver variability of two observers measuring on the same still printout of one given Valsalva manoeuvre. The third series (n= 47) represented interobserver variability during the same session, with a maximum of 10 minutes between assessments. The fourth series (n= 48) represented a test- retest comparison after an interval of 46 (range 32- 122) days. Ethics committee approval had been obtained for both studies of pelvic floor function, and all subjects gave informed consent. Results: The first series demonstrated a variability ofbladder neck descent (BND) of 16% for three effective Valsalva manoeuvres (%CV= 0.16). The Intraclass Correlation (ICC (1,1) was 0.92. Three women were excluded from analysis since they had been unable to produce an effective Valsalva manoeuvre due to persistent levator activation. In the second series,% CV for BND on maximal Valsalva was 0.08, with an average difference in measurements of 1.98 mm (0- 4.5 mm, StD 1.27). The ICC (2,1) was calculated at 0.98. For the third series, the %CV was 0.21 for BND with an average difference between measurements of5.8 mm (0- 14.5 mm, StD 4.2 mm), ICC (2,1) = 0.79. The fourth series yielded a %CV of0.219, with an average difference between measurements of 4.87 mm), and ICC (1,1) = 0.75. All ICC values indicate excellent agreement.

216 Conclusions: Bladder neck mobility or the magnitude of bladder neck descent seems to be a determinant of continence in women presenting with symptoms of pelvic floor dysfunction. Until now, measurements of this parameter have not been validated. The authors have attempted to provide such internal validation in four series of measurements assessing different factors contributing to the variability of a translabial ultrasound assessment of this parameter. The strength ofValsalva manoeuvres varies considerably (as implied by a %CV of0.16 in the first series), contributing the major share of test- retest variability. Standardization of Valsalva pressure has in fact been attempted (King and Freeman, 1998) although this is unlikely to be successful without intraabdominal pressure sensors. In many settings the placement of such sensors appears impractical, and at any rate even standardization of Valsalva pressure would not control for concomitant levator activation, likely to be a significant confounder. Operator recognition of landmarks such as symphysis pubis and bladder neck is an important operator- dependent factor, contributing a %CV of 0.08 (second series). Teaching would likely be the main determinant of the magnitude of this error. Interobserver variability on the same day yielded a% CV of0.21 which would be expected to include all sources of variation discussed above. The last series of retesting by the same observer after an average of 46 days (%CV of 0.219) may include other confounders such as teaching effects and variations in bladder and rectal filling. All the above factors need to be considered when comparing data before and after intervention as well as between units. It appears that the assessment of bladder neck descent by translabial ultrasound can be perfonred with excellent test- retest and interrater reliability, provided operators are adequately trained and examination conditions are standardized.

References

Dietz HP, Clarke B, Herbison P. Bladder neck mobility and urethral closure pressure as predictors of Genuine Stress Incontinence Int. Urogyn. J. 13 (2002); 289-293 King JK, Freeman RM. Is antenatal bladder neck mobility a risk factor for postpartum stress incontinence Br.J.Obstet. Gynaecol. 1998;105:1300-07. Koelbl H, Hanzal E. Imaging of the lower urinary tract. Curr.Opin.Obstet.Gynecol. 1995;7:382-85. Schaer ON. Ultrasonography of the lower urinary tract. Curr. Opin. Obstet. Gyneco I. 1997 ;9: 313-16. Dietz HP, Wilson PD. The influence ofbladder volume on the position and mobility ofthe urethrovesical junction. Int.Urogynecol.J. 1999; 10:3-6. Dietz HP, Clarke B. The influence ofposture on perineal ultrasound imaging parameters. Int. Urogyn. J. 2001;12:104-06.

217