VALUE OF ENDOMETRIAL NERVE FIBER DETECTION IN DIAGNOSING ENDOMETRIOSIS

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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES MEDICAL ACADEMY

Said Makari

VALUE OF ENDOMETRIAL NERVE

FIBER DETECTION IN DIAGNOSING

ENDOMETRIOSIS

Doctoral Dissertation

Biomedical Sciences, Medicine (06B)

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Dissertation was prepared at the Lithuanian University of Health Sciences Medical Academy between 2008–2012.

Supervisor

Associate professor Dr. Eimantas Švedas (Lithuanian University of Health Sciences Medical Academy, Biomedical Sciences, Medicine – 06B)

Consultant

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LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS MEDICINOS AKADEMIJA

Said Makari

NERVINIŲ SKAIDULŲ GIMDOS

GLEIVINĖJE NUSTATYMO VERTĖ

DIAGNOZUOJANT ENDOMETRIOZĘ

Daktaro disertacija

Biomedicinos mokslai, medicina(06B)

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Disertacija rengta 2008–2012 metais Lietuvos sveikatos mokslų universiteto Medicinos akademijoje.

Mokslinis vadovas

doc. dr. Eimantas Švedas (Lietuvos sveikatos mokslų universiteto Medicinos akademija, biomedicinos mokslai, medicina – 06B)

Konsultantė

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LIST OF ABBREVIATIONS

PGP 9.5 – protein gene product 9.5 NF – neurofilament

n – number of cases

DIE – deep infiltrating endometriosis CT – computerized tomography MRI – magnetic resonance imaging TVS – transvaginal ultrasound

UCH-L1 – ubiquitin carboxyl-terminal hydrolase-1 ASRM – American Society for Reproductive Medicine anti-TH – anti-tyrosine hydroxylase

anti-ACh – antiacetylcholine NGF – nerve growth factors OCPs – oral contraceptive pills TCDD – tetrachlorodibenzo-p-dioxin EPCs – endothelial progenitor cells NGF – nerve growth factor

TGFb1 – transforming growth factor beta1 VEGF – vascular endothelial growth factor CPP – chronic pelvic pain symptoms TVUS – transvaginal ultrasonography CT – computerized tomography MRI – magnetic resonance imaging TNF – tumour necrosis factor HLA – human leucocyte antigen IgG – immunoglobulin G CA125 – cancer antigen 125

HSD – hydroxysteroid dehydrogenase enzymes ER – estrogen receptors

PR – progesterone receptors

ECM – extracellular matrix molecules IGF – insulin-like growth factor HGF – hepatocyte growth factor

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1. INTRODUCTION

Endometriosis is a common gynecologic disease typically defined as the histologic presence of endometrial glands and stroma outside the uterine cavity [1]. It remains an enigmatic disease even 150 years later after it was first described by Rokitansky.

Endometriosis as a disease affects mainly reproductive aged young women regardless of their ethnicity or social status. The exact prevalence in the general population is not exactly known; however, various authors estimate this prevalence to range between 10 and 15%. Endometriosis is usually associated with a triad of symptoms including but not limited to pelvic pain, dysmenorrhea and infertility. Other presentations of the disease may include back pain, dyspareunia, dyschezia, gastrointestinal symptoms, and neuropathic pain among others.

It is one of the most variable of all gynecological conditions with conse-quent diagnosis challenges. What is more challenging is that the behavior of endometriosis is very different in the majority of sufferers. Due to its extensive pelvic involvement, diverse appearance and malignant biological behaviours including metastasis, invasiveness and recurrence, some authors go as far as to say that endometriosis is a ‘benign cancer’ [2]. Symptoms like chronic pelvic pain and infertility may suggest the presence of endomet-riosis; however, for a definitive diagnosis of endometriosis, visual inspec-tion of the pelvis at laparoscopy is the ‘gold standard’ investigainspec-tion, ideally combined with histological confirmation [3]. Yet, laparoscopy is a surgical procedure with rare but significant potential risks for the patients [4]. Fur-thermore, this procedure is time consuming for the patient, and involves significant cost to healthcare systems. Endometriosis may develop not only within the pelvis but also on other extrapelvic peritoneal surfaces. One pathologic review revealed that endometriosis has been identified on all organs except the spleen [5].

The endometriosis-associated costs to society, including delayed diag-nosis and ‘hit-and-miss' treatments, are considerable but as yet difficult to determine, so are the costs to the individual when disease symptoms inter-fere with day-to-day life at work or at home [6].

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Owing to the lack of a non- or semi-invasive diagnostic tool, principally in primary care, the delay in diagnosis between onset of pain symptoms and surgically confirmed endometriosis can be as long as 8 years in the UK and USA [8, 9]. The delay was longer in centers where women received predo-minantly state-funded health care (8.3 vs. 5.5 years) [7]. This delay is even greater in those with onset of symptoms in early adolescence, a time of life during which many doctors still believe that endometriosis does not occur. Another factor contributing to a delay in diagnosis is the failure of patients to inform their doctor of pain symptoms. The decision to complain of pain or not to doctors, are very personal decisions by individual patients. All over the world, women believe that their ‘endometriotic pains’ are a natural aspect of women’s reproductive life, and hence they do not inform their doctor. All this contributes to years of suffering and potential infertility if the disease is left untreated. Clearly, a simple non- or semi- invasive diag-nostic method may greatly help to reduce this delay, especially for minimal– mild endometriosis which cannot be diagnosed by clinical examination or ultrasound. It may be that early diagnosis and superficial surgical treatment decrease the number of patients who will develop chronic or infiltrating endometriotic disease. This may decrease the need for potent medications, referral for prolonged treatment, and deep surgical dissection.

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2. NOVELTY OF THE STUDY

In 2006 Tokushige and his team from Sydney, Australia, published the first report on the value of finding nerve fibers in the endometrial biopsies for diagnosing endometriosis. Researchers from all over the world became tempted to test the hypothesis that perhaps the demonstration of small nerve fibers and the assessment of nerve fiber density in the functional layer of endometrium of women with endometriosis could be a relatively simple surrogate marker of this condition using a semi-invasive method [12]. Shortly after, we had other researchers reporting very similar results [10, 11]. We commenced this study in 2008 only 2 years after the first reports were published. Our multidisciplinary clinical setting is ideal for performing a study of this importance, as we are a tertiary level, university hospital with expertise in laparascopic endometriosis surgery, in addition to the availability of a pathologist specialized in gynecologic pathology. We think that this topic is interesting not only in the research or laboratory setting. In the likely event that our findings will be similar to those of other researchers in this field, then our results can contribute to a ground breaking application of endometrial biospy for diagnosing endometriosis in the clinical setting as well.

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3. AIM AND OBJECTIVES OF THE STUDY

Aim:

The aim of our study was to assess the value of multiple-sensory nerve fibers present in the endometrium for diagnosing endometriosis.

Objectives:

1. To assess the significance of nerve fiber detection in endometrial

biopsy for making a diagnosis of endometriosis.

2. To compare the density of endometrial nerve fibers in patients with and without endometriosis.

3. To assess the importance of the density of endometrial nerve fibers

in pain symptoms in women with endometriosis.

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4. LITERATURE REVIEW

4.1. Burden of the problem

Endometriosis is one of the most common benign gynecological diseases. The exact prevalence of endometriosis in the general population is not known. Only a few well-conducted studies have reported data on the prevalence of endometriosis. To date, at least 100 papers have been published on the epidemiology of endometriosis [13]. Most of these reports have studied women presenting with only one of several symptoms justifying laparoscopy, for example pelvic pain, dysmenorrhea and infertility. Based on the few reliable data, the prevalence of the condition is generally considered to be around 10% [14]. The differences in the prevalence of the disease can vary by as much as 30–40 times, due to the fact that, there is very little data on the frequency of the onset of the disease in a given period (its incidence) in women without a previous diagnosis [15, 16]. Irregardless of this, temporal trends suggest an increase in the incidence of pelvic endometriosis in women of reproductive age.It has been suggested that the incidence of endometriosis tends to be higher in Asian women than in Causcasian women and the lowest in patients of African descent. However, these studies do not always take into account socioeconomic status and the availability of healthcare facilities for these women which make the results uncertain [17, 18].

Until now, the gold standard for diagnosis is the visualization of implants either at the time of laparoscopy or laparotomy, and until a simple screening test is developed, the true incidence will remain unknown. Thus far, earlier age at menarche (typically age 11 or younger) and shorter cycle lengths (27 days or less) have consistently shown an increased risk. And the highest prevalence rates are typically found in infertile couples, with ranges varying from 5 to 50% [19]. Not surprisingly, the prevalence in asymptomatic patients seeking sterilization is lower, ranging from 2% to 18%, whereas those admitted for pelvic pain is slightly higher, ranging from 5% to 21% [19]. Some report that that 20% of women with unexplained infertility were found to have endometriosis 2 years after a normal laparoscopy examination [20].

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(ovarian endometriomas) and deeply infiltrating endometriosis (DIE) [21, 22]. DIE is a specific entity histologically defined as an endometriotic lesions that extends more than 5 mm underneath the peritoneum [23, 24]. Ovarian endometriomas are the most common form of endometriosis found at the time of diagnosis, and affect approximately 55% of those with endometriosis. After surgical incision, the recurrence rate of endometrioma over 4 years has been reported to be as high as 12% [25].

We know that endometriosis has a broad, wide–ranging impact on the population. Health economic information for endometriosis is scarce, limiting our understanding of its overall economic impact. Studies reveal that approximately 51,000 women aged 15–64 years are hospitalized for endometriosis annually, and at least 5.5 million women in the United States and Canada are affected at any one time [26]. Estimates of the number of hours missed from work due to endometriosis ranged 19.2–86.4 h per patient per year [27, 28]. Another study calculated that annual costs of endometriosis reached 22 billion USD in 2002 assuming a 10% prevalence rate among women of reproductive age [29]. These costs are considerably higher than those related to Crohn's disease or to migraine. The high burden originates from the time delay between onset of symptoms and diagnosis, costly medical and surgical treatments, the chronic nature of endometriosis and the indirect costs associated with reduced quality of life and ability to work. Increasing awareness of the disease, cutting the time to diagnosis and providing centralized evidence-based specialized care are crucial steps in reducing the morbidity, health care expenditure and lost productivity associated with endometriosis.

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affected unnecessarily by such a delay [33]. There is evidence that 50% of women with endometriosis are bedridden for 17.6 days per year. In most cases, this may have a negative impact on their education and/or career [34].

Moreover, sexual dysfunction due to dyspareunia can disrupt relationships in the family, for both men and women [35]. Of utmost relevance here is the association of the disease with infertility. This condition is well-known to undermine women's quality of life [36]. This is especially relevant in countries of the developing world where infertility is socially extremely shameful and a diagnosis of endometriosis may consequently lead to social stigmatization. In fact, in many developing countries, women who are unable to conceive are often treated as outcasts, rejected by their partner, and ostracized by the society. They are often regarded as being inferior and useless [37]. The psychological wellbeing of women is not any less important than their physical wellbeing. The diagnosis of endometriosis may be psychologically distressing for women and the subsequent misuse of healthcare resources may be an important concern. Even today in the age of the internet, available information might be too scarce or on the contrary too complicated, for women to fully understand the different aspects related with endometriosis. Once women are diagnosed with the disease, they could be left with a generic diagnosis without the knowledge for discriminating between different degrees of clinical severity. More importantly, these women do not always know that there is a poor correlation between pain and the degree of endometriosis (minimal–mild–moderate–severe), as determined according to the revised staging system of American Society for Reproductive Medicine (38, 39). And they are left alone to cope with their thoughts and at many times with their disease.

A non- or semi- invasive diagnostic test for a definitive diagnosis can help resolve many of these isssues. However, the search for such a test can only be successful in the context of thorough investigation and understanding of possible pathogenic mechanisms related to the appearance of endometriotic lesions and disease progression.

4.2. Pathogenesis

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rial. Three main etiopathogenic hypothesis lines of thought have been cited for almost one century:

1) retrograde menstruation/implantation theory, postulating the implant of endometrial cells from menstrual blood reflux through the Fallopian tubes into the abdominal cavity [41], occurring under the influence of a favorable hormone environment and immunological factors which would not clear the cells out of the inappropriate site. 2) coelomic metaplasia theory, where mesothelium turns into

endo-metrial tissue [42].

3) the induction theory is a combination of the two other theories and proposes that endogenous biochemical or immunological factors induce undifferentiated cells to differentiate into endometrial tissue [43].

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disease. They may include a defective immune surveillance in the peritoneum [50], a disturbed balance between reactive oxygen species and antioxidants in the peritoneal fluid [51, 52], or the spread of endometrial epithelial progenitor cells and mesenchymal stem cells [53, 54]. Recent reports suggest that eutopic endometrium from patients with endometriosis might be more invasive and prone to peritoneal implantation as a result of altered production of various proteolytic enzymes [55].

The celomic metaplasia theory by Meyer suggests that metaplasia of the coelomic epithelium is the origin of endometriosis [42]. This theory claims that formation of endometriomas in the ovary or rectovaginal endometriosis is caused by metaplasia of the coelomic epithelium, perhaps induced by environmental factors [21, 56, 57]. This theory goes on to suggest that the parietal peritoneum is a pluripotential tissue that can undergo metaplastic transformation to tissue histologically indistinguishable from normal endometrium. Because the ovary and the progenitor of the endometrium, the müllerian ducts, are both derived from coelomic epithelium, metaplasia may explain the development of ovarian endometriosis. In addition, the theory has been extended to include the peritoneum because of the proliferative and differentiation potential of the peritoneal mesothelium. This theory is attractive in instances of endometriosis in the absence of menstruation, such as in premenarchal and postmenopausal women. However, the absence of endometriosis in other tissues derived from coelomic epithelium argues against this theory.

The induction theory proposes that some hormonal or biologic factor (s) may induce the differentiation of undifferentiated cells into endometrial tissue [58]. These substances may be exogenous or released directly from the endometrium [59]. In vitro studies have demonstrated the potential for ovarian surface epithelium, in response to estrogens, to undergo transformation to form endometriotic lesions [60].

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4.2.1. Mechanisms involved in the pathogenesis of endometriosis 4.2.1.1. Role of various types of stem/progenitor cells in the pathogenesis of endometriosis

In the last few years, there has been increased interest in the role of bone marrow derived pleuripotent undifferentiated stem cells, capable of self-renewal through numerous cycles of cell division, in the pathogenesis of endometriosis. However, of special interest were the various types of progenitor cells, which in fact arise from bone marrow derived cells but are more developmentally devoted to one line; namely endometrial stem/pro-genitor cells and endothelial prostem/pro-genitor cells.

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possibly due to excessive uterine peristaltic contractions in women with endometriosis [70]. This may cause microtrauma to the basalis layer, and increased retrograde transport of basal endometrial fragments into the peritoneal cavity. A larger proportion of menstrual fragments have basalis characteristics in women with endometriosis compared with healthy controls with respect to estrogen receptor expression. The importance of basalis-type endometrium in endometriotic lesion formation may be related to the greater numbers of stem/progenitor cells in this region [71]. In baboons which menstruate, menstrual debris induces endometriosis spontaneously or under experimental conditions [72], suggesting the presence of stem/progenitor cells in the debris. It is still not clear if there are differences in the numbers of clonogenic MSC-like cells shed in menstrual blood between women with and without endometriosis, or whether women susceptible to endometriosis may have a higher propensity to shed endometrial stem/ progenitor cells. Neither is it known whether endometrial stem/progenitor cells are shed in a retrograde manner in women with endometriosis. However, there is no doubt that long-term survival and proliferation of these lesions are crucially dependent on the formation of new blood vessels, which guarantee oxygen and essential nutrient supply [73, 74, 75]. In fact, endometriotic lesions are typically characterized by a dense vascularization [76, 77]. So far, it has been assumed that this vascularization exclusively occurs by the ingrowth of new blood vessels from the surrounding host tissue via the process of angiogenesis. However, besides angiogenesis, there also exists the possi-bility that new blood vessels develop from circulating endothelial progenitor cells (EPCs), which are recruited and incorporated into the sites of neovascularization [78]. In fact, vasculogenesis and sprouting angiogenesis most probably occur in parallel in endometriotic lesions. These observations are strongly supported by a recent study of Becker et al. [79].

4.2.1.2. Role of the peritoneal fluid

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in endometrial cells in the peritoneal fluid during menstruation or in women with endometriosis [81]. A difficulty, experienced by this and previous studies examining peritoneal fluid for refluxed endometrial cells, is that multiple markers are required to distinguish endometrial epithelial and stromal cells, mesothelial cells and leucocyte populations, technically difficult when using immunohistochemical approaches. If stem/progenitor cells are present in retrogradely shed endometrium, their concentrations in the peritoneal fluid will be lower compared with that of normal endometrial cells, making it further difficult to find them. If endometrial stem/progenitor cells are shed in retrograde menstrual debris, it is likely they will only establish ectopic lesions when transported with their niche cells [63].

Recent studies have indicated that inflammation, impaired immune response suppression and inadequate fibrinolytic mechanisms may play a role in the pathogenesis of endometriosis. Variations in genetic material might be responsible for an augmented steroid response, phenomenon of oxidative stress, and tumor suppression may furthermore contribute to the occurrence of the disease.

4.2.1.3. Altered cellular immunity

Altered cellular immunity is another proposed pathogenetic mechanism and a lack of adequate immune surveillance in the peritoneum is thought to be a cause of the disease [82]. Among the possible molecules responsible of this altered immunity, haptoglobin and monocyte chemoattractant protein 1 have been proposed [83, 84]. Several other findings support an autoimmune aetiology of endometriosis: abnormalities in function of B and T cells, high serum concentrations of IgG, IgA, and IgM autoantibodies, reduced natural-killer cell activity and familial inheritance, among others [85, 86, 87]. This

alteration in immune function, may favour the growth and progression of

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implants, inhibits NK activity around endometriosis. Moreover, with the exception of high levels of TNF-a, Th-2 cytokines dominate the peritoneal environment and Th-1 cytokines dominate the internal endometriosis environment [95, 96]. Peritoneal macrophages also produce MMP-9 and VEGF, vascular permeability and angiogenesis factors that may enhance the vascular support for ectopic endometrium [97]. B cell function and antibody production also appear to be abnormal in endometriosis. Indeed, endome-triosis has several of the characteristics of an autoimmune disease [89].

4.2.1.4. Altered inflammatory activity

Endometriosis is associated with increased inflammatory activity. Elevated serum and peritoneal fluid inflammatory markers have been observed in several studies [98]. Pelvic pain, a rather frequent symptom of endometriosis, is relieved by anti-inflammatory drugs, supporting thus the contribution of chronic inflammation to the pathogenesis of this disease [99]. Fasciani et al. [100] showed that the cells from endometrial explants can proliferate and invade a three-dimensional fibrin matrix in vitro, resulting in the formation of new glands, stroma and blood vessels consistent with early endometriosis. Several studies have shown that reduced activity of cytotoxic T cells and natural killer cells (NK) seem to play an essential role in the survival, implantation and proliferation of endometrial cells in the peritoneal cavity in women with endometriosis [101, 102]. Coexistence of autoimmune diseases with endometriosis has been reported by some researchers [103], although others have not corroborated this finding [104, 105]. Increased serum anti-endometrial antibodies suggest a link between endometriosis and otherwise unexplained infertility [106, 107].

Excess production or impaired elimination of ROS leads to increased OS which has been implicated in the development of several diseases including endometriosis [108]. Oxidative stress in the peritoneal fluid is initiated by inflammatory cells and a substrate of cellular debris while systemic OS may aggravate the established local OS in pelvic endometriosis [109].

4.2.1.5. Role of estrogen (ER) and progesterone receptors (PR)

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attenuated or dysregulated progesterone response at the molecular level is suggested in endometriosis, and interestingly, progestin-based treatment of this disorder is variably effective [111]. The effects of progesterone are mediated via intracellular progesterone receptors that are expressed from a single gene as two protein isoforms, progesterone receptor A (PR-A) and progesterone receptor B (PR-B) [112]. In normal human endometrial epithelium, both PR-A and PR-B are increased by estrogen during the proliferative phase but are reduced during the secretory phase under the influence of rising serum progesterone levels [113]. Alteration in the ratio of PR-A to PR-B was suggested as one of the possible mechanisms of progesterone resistance in endometriosis [114]. A microarray-based study in women with moderate to severe endometriosis also reported increased total PR in women with endometriosis [115]. This altered PR expression may lead to diminished PR response and decreased expression of progesterone-responsive genes in endometriosis. Multiple gene expression profiles by microarray in endometrium of women with or without endometriosis showed that several progesterone target genes were dysregulated during the window of implantation, leading to an inhospitable environment for implanting blastocyst [115, 116]. Two of the progesterone target genes that are dysregulated in endometriosis are Hox genes. HOXA expression was down-regulated in eutopic endometrium. Human studies have reported HOXA10 hypermethylation as one of the possible mechanisms by which HOXA10 levels are decreased in endometriosis [117, 118]. HOXA10 hypermethylation, a novel mechanism of HOX gene dysregulation, permanently silences HOXA10 gene expression in endometriosis. Given that HOX genes modulate some of the functions of progesterone, decreased HOXA10 expression due to hypermethylation may result in resistance to progesterone action usually seen in endometriotic tissues.

4.2.1.6. Gene polymorphism – potential culprit

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genetic risk factor for endometriosis and that they may be associated with the recurrence of endometriosis through an increase of the ERa receptor activity [124]. A case control study exploring the association of the estrogen receptor two-allele polymorphism and multiallele microsatellite polymor-phism with the occurrence of endometriosis, suggested that the variability of the estrogen receptor gene likely contributes to the pathogenesis of endometriosis [125, 126] suggest that the PVUII polymorphism of the ERa gene is associated with the risk for endometriosis. Furthermore, another study explored the association between ERa gene polymorphism and the risk of endometriosis by investigating the frequency of PvuII, XbaI and thymine-adenine (TA) repeat polymorphisms in patients with and without endometriosis in a Korean population. There was no significant difference in the allele distribution and frequency of either PvuII or XbaI polymorphisms between endometriosis patients and control subjects, but patients with stage I/II endometriosis showed a higher incidence of alleles with fewer (TA)n repeats compared with controls [127].

Polymorphisms in genes encoding cytokines or immunomodulating proteins have also been implicated in the pathogenesis of endometriosis. It has been shown that women with endometriosis diverge in their expression of several genes including heat-shock proteins, fibronectin, elastase and Toll-like receptor [128]. Toll-like receptors (TLRs) play an important role in nonacquired immunity. Data in respect to TLR signaling suggest that TLRs are closely related with the pathogenesis of autoimmune diseases [129].

4.2.1.7. Anatomic factors

Anatomic abnormalities are also considered a possible precursor of endometriosis. Vercellini et al concluded that the depth and volume of the cul-de-sac (Pouch of Douglas), differs in patients with endometriosis with or without deep lesions as compared to women with a healthy pelvis [130]. This theory would explain why most women have some degree of retro-grade menstruation but only a small percentage have endometriosis, and the presence of the disease in absence of menses.

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tions that impede emptying of menstrual blood or contribute to dysme-norrhea. Mean contraction pressure and frequency is higher and more frequent, and results in more retrograde endometrial debris in women with endometriosis than controls [133, 134]. This increase in retrograde menstrual debris may deposit more factors that could either induce nerve sprouting or contribute to neurogenic inflammation, and thus lead to continued sensitization. Also mechanical stretching can induces biochemical changes in cells, and thus it has been postulated that the mechanical stretch exerted by uterine movement stimulates the production of additional biochemical mediators in endometrial cells.

4.2.1.8. Environmental factors

Enviromental factors may also play an important role in the disease. In particular, animal models of endometriosis have provided important information on the potential influence of proton irradiation and dioxin on development of endometriosis [135, 136]. It has to be underlined, however, that there is still no epidemiological study definitively linking one class of chemicals to the risk of endometriosis, although oestrogen-like compounds in the environment have been suggested [137]. The toxins most commonly implicated are 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other diox-inlike compounds [138]. TCDD in conjunction with estrogen appears to stimulate endometriosis formation, and TCDD appears to block the progesterone-induced regression of endometriosis. In the environment, TCDD and dioxin-like compounds are waste by-products of industrial processing. Ingestion of contaminated foods or accidental contact is the most common method of exposure. Although endometriosis and TCDD were initially linked in primates, human studies also note a higher prevalence of endometriosis in women with high breast milk dioxin concentrations [139, 140]. In addition, subsequent studies have demonst-rated higher serum dioxin levels in infertile women with endometriosis compared with those in infertile controls [141].It remains to be said that the clinical manifestation of endometriosis and the presence of endometrial tissue outside the uterine cavity is probably nothing more than the end point of a combination of several aberrant biological processes.

4.3. Risk Factors for Endometriosis

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have increased exposure to endometrial material thus are at increased risk of developing endometriosis; data on other risk factors are less consistent. Endometriosis has been associated, albeit not consistently, with red hair, fair skin, freckles and light eyes. A possible co-occurrence of endometriosis and autoimmune diseases has been suggested by clinical case series and by findings derived from a cross-sectional survey conducted by the US Endometriosis Association and the US National Institutes of Health [87]. The prevalence of rheumatoid arthritis, systemic lupus erythematosus, hypo- or hyperthyroidism, and multiple sclerosis was higher in women with endometriosis than in controls in the Endometriosis Family Study. Addi-tionally, an association with non-Hodgkin lymphomas has also been suggested [142, 143]. More than 30 years ago researchers published data that women with endometriosis and an affected first-degree relative were more likely to have severe endometriosis (61%) than women without an affected first degree relative (24%) [144].The risk of endometriosis is increased up to five times in siblings [145]. In this study by Stefansson et al., the risk ratios were 5.2 for sisters and 1.56 for cousins. Other studies have shown a 7 times increased risk of endometriosis when a first degree relative has this disease, further indicating that genetic factors are important [146]. Endometriosis is inherited in a polygenic manner with a prevalence of 4–9% in first-degree relatives [147, 148]. Although monozygotic concor-dance does not reach 100%, which is expected for a Mendelian trait, higher concordance of monozygotic than dizygotic twins is reported. By the use of the linkage analysis several candidate genes with potential biological plausibility have been suggested in some studies [149, 150, 151]. Studies have also demonstrated concordance for endometriosis in monozygotic twin pairs, suggesting a familial/genetic basis [152, 153] (Table 4.1).

Table 4.1. A comparison between various risk factors of endometriosis

Risk factor/comparison OR 95% CI

Mother or sister has endometriosis/mother and sister do not

have endometriosis 7.2 2.1 to 24.3 [146]

Menstrual flow six days or more/flow less than six days 2.5 1.1 to 5.9 [154] Menstrual cycle less than 28 days/cycle of 28 to 34 days 2.1 1.5 to 2.9 [155] Consuming one or more alcoholic drinks per week/no alcohol

consumption 1.8 1.0 to 3.2 [156]

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27 OCPs = oral contraceptive pills

Endometriosis has been studied as a potential outcome of occupational exposure to dioxins and included as a secondary outcome in general studies of reduced fertility among workers in certain industries and occupations. These studies have found increased risk of endometriosis-associated infertility among workers exposed to formaldehyde [159], video display terminals, chemical dusts, or organic solvents [160]; and among workers in agricultural industries and occupations, in particular farmworkers [161]. Having ever worked as a flight attendant, service station attendant, or health worker, particularly as a nurse or health aide, was associated with increased risk of endometriosis (flight attendant: OR 9.80, 95% CI 1.08–89.02; service station attendant: OR 5.77, 95% CI 1.03–32.43; health worker: OR 1.49, 95% CI 1.03–2.15) [162]. A greater frequency of endometriosis among women of higher social class has been reported [163, 164].

On the other hand, being overweight has been associated with a lower risk of endometriosis [165]. Women with an increased body mass index have more irregular menstrual cycles and increased rates of anovulatory infertility. This might explain the association with endometriosis. Smoking is “beneficial” to female smokers by decreasing incidence of endometrial cancer and endometriosis [158, 166]. While this effect has been ascribed to diminished ovarian function and resultant estrogen diminution, there are also direct uterine effects; one cigarette constituents, benzo(a)pyrene, can inhibit endometrial cell proliferation, attachment and invasion of basement membrane possibly by down-regulating epidermal growth factor receptor and E-cadherin [167]. Additionally smoking might have a protective effect against endometriosis, because it is leads to a decrease in the circulating number of bone marrow derived stem cells; cells that have been demonstrated to be a source of endometrial progenitor cells, who in turn might play a role in the pathogenesis of endometriosis [168]. Regular physical activity might be linked with lower levels of estrogens and reduced endometriosis risk, but data on this issue are scanty [169].

4.4. Diagnosis

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simple, cost-effective and a helpful step in the process of diagnosing endometriosis. Questioning normally searches for: (I) sets of socio demo-graphic data, lifestyle characteristics and medical and reproductive history that are associated with endometriosis [9]; (II) painful symptoms that are correlated to the anatomic locations of DIE nodules [170] and contribute to diagnosing endometriosis of the bladder detrusor [171] or posterior DIE [172]; and (III) the patients' history of events occurring at adolescence that may be associated with DIE [173, 174].

Adequate primary assessment of patients with symptoms suggestive of endometriosis is of major importance and not only reduces diagnostic delay but also enables the clinician to discuss and plan appropriate treatment options. In fact, most women who suffer from endometriosis and consequant subfertility get diagnosed with the disease within the context of IVF treatment by which time the disease has already progressed to a chronic advanced-stage condition. Furthermore, the clinical manifestation of endometriosis and the presence of endometrial tissue outside the uterine cavity is probably nothing more than the end point of a combination of several aberrant biological processes.

4.4.1. Clinical Presentation

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disease, among others. The predictive value of any one symptom or set of symptoms remains uncertain as each of these symptoms can have other causes, and a significant proportion of affected women are asymptomatic. While it may be useful to diagnose endometriosis in these patients, one must not forget that these patients may suffer from multiple concurrent pain syndromes. It must be pointed out that prior to attributing pelvic pain to endometriosis other causes should be investigated. Possible bowel, bladder, psychiatric, and musculoskeletal etiologies should be ruled out. In such cases, these patients may benefit from a multi-faceted approach to chronic pain, involving an interdisciplinary team that includes pain specialists, urologists, gastroenterologists, and other non-gynecologists [177].

4.4.1.1. Definition of chronic pelvic pain symptoms (CPP) and potential bias related to it

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4.4.1.2. Symptoms of Pain in Endometriosis Sufferers

Endometriotic pelvic pain may be well recognized, but many doctors, including gynecologists, think only in terms of the common triad of dysmenorrhoea, deep dyspareunia and pain with a bowel motion. Yet, many women experience a much more complex range of pains.

The majority of women with endometriosis do in fact describe menstrual pain (dysmenorrhoea) as being intense, unbearable, cramping, gnawing, crushing or pressing. The pain is usually located in lower abdomen, lower back and referred into thighs, loin, groin, rectal area and umbilicus [184]. Most commonly, symptoms associated with the gastrointestinal system are overlooked by the gynecologist. It has been estimated that up to 80% of women with endometriosis experience colicky pains in addition to bowel type symptoms. It is essential to say that usually in irritable bowel syndrome the colic is relieved by a bowel motion, whereas in endometriosis it is not. Painful abdominal bloating is another overlooked endometriosis symptom. Two thirds of women with endometriosis will experience painful bloating every cycle, while for a quarter of women, they will only sometimes experience it. It is not uncommon, for many women with endometriosis to possess two different wardrobes to accommodate the cyclical changes in abdominal circumference. Diarrhea, constipation, and pain with a bowel motion are very common and exacerbated in the peri-menstrual stage. Bleeding from the intestines during menses is also more common than generally recognized [185].

Another type of pain usually underestimated in women suffering from endometriosis is pain from nerve entrapment. This type of pain is relatively rare, and is usually due to anatomical nerve distortion by active, fibrotic lesions, especially around the sciatic and obturator nerves [186, 187]. This pain usually radiates along the path of the trapped nerve, sometimes with associated functional disturbance, such as decrease in muscle power. Postulated nerve entrapment occurs in the complex fibrous and hypertrophic deep invasive endometriotic lesions in the rectovaginal septum in cases of deep infiltrating endometriosis, where distortion of nerve trunks appears to occur [188]. Additionally, neuropathic pain is being increasingly recognized as a significant component of persistent endometriosis pain.

4.4.1.3. Mechanisms of pain generation

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know that, pain and the perception of pain for any individual are created by activity in that individual’s central nervous system (CNS).

How endometriotic lesions engage the CNS to produce pain is still not very clear. It has generally been thought that nerve fibers already present in the pelvis somehow become stimulated by the ectopic lesions, presumably by some substance which is released from the lesions at particular times during the menstrual cycle. Leading candidates for such specific substances include the prostaglandins, bradykinin and histamine, but little work has been carried out in this area in studying pelvic pain in women. Also lesions may produce pain by compressing or infiltrating nerves near those lesions [189]. The presence of nerve growth factor (NGF) in lesions may be involved, especially in deep adenomyotic nodules where it correlates with hyperalgesia, defined as intense pain reported when pressure is exerted in the posterior fornix [189]. NGF can also act on those nerves to produce pain and facilitate nerve growth [190]. Others report an association between dysmenorrhea severity and transforming growth factor beta1 (TGFb1) in

nerve fiber bundles adjacent to endometriotic lesions. Interestingly more TGFb1 is found in lesions that are either DIE or red compared with black lesions or healthy peritoneum [191]. However, the concept of surrounding or compressing nearby nerves does not explain pain in situations where nerves are not near lesions. Indeed, pelvic pain is not correlated with nerve fibers in adhesions, peritoneum or within endometriomas (visualized by antibodies to neurofilament protein) [192].

From a pathogenetic point of view, in order for the endometriotic debris to survive after attachment it must aquire a blood supply [193]. In endometriotic lesions, there occurs a combined process of vasculogenesis and sprouting angiogenesis as reported by Becker et al. [194]. Importantly, blood vessels are innervated by sensory and sympathetic fibers [195] such that factors that act on sprouting blood vessels also act on nerve fibers (vascular endothelial growth factor (VEGF), NGF, semaphorins, netrins, slits and membrane-bound ephrins [196, 197]. Thus, when blood vessels branch to vascularize developing lesions (‘angiogenesis’), nerves inner-vating those blood vessels may also branch (‘neural sprouting’) thereby enabling nerves to invade lesions.

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signals are processed in the dorsal root ganglia and the lower spinal cord, before onward transmission of a modified signal to the thalamus, limbic system and higher centers, where pain is perceived and the emotional response is developed. In visceral organs, nociceptors tend to respond to excessive pressure, excessive stretch, ‘inflammatory’ processes and a range of injurious chemical substances. There is increasing evidence that endometriosis elicits changes in the population of uterine nociceptors. Nociceptors invade peritoneal endometriotic lesions in women [199], and are also found invading endometriotic cysts in experiments on rats [200]. The finding that lesions can be directly innervated by newly sprouted fibers gave rise to a new conceptualization of mechanisms underlying pain in endometriosis: characteristics of the ectopic growths’ newly formed nerve supply and resultant two-way communication between the growths and CNS contribute to the variable association between the growths and pain [200]. This suggests that in women with endometriosis, there is abnormal sprouting of nociceptors in the endometrium and in peritoneal endometriotic lesions. Such nerve sprouting might be caused by increased levels of nerve growth factor (NGF), since expression of NGF in endometriotic tissue is reported to be higher than in eutopic endometrium [201, 202, 203]. Complementary, this might mean that the spontaneous activity of sensory fibers is greater or they are more easily activated by stimulation [204]. Nociceptors are significantly sensitized by oestrogen, while bradykinin, histamine and interleukin-1 are probably also important sensitizers. In fact, the nociceptor is an incredibly complex organelle which can be stimulated, sensitized, inhibited or otherwise regulated by hundreds of extrinsic and intrinsic molecules, many of these arising in immune competent cells, such as mast cells, macrophages, dendritic cells, neutrophils, natural killer cells, plasma cells and probably others.

All this leads us to believe that all of the events mentioned really do have an impact on the mechanism of pain generation and pain perception in endometriosis, as it is a an estrogen dependant disease characterized by a chronic inflammation and associated with angiogenesis, neo-angiogenesis, and growth of nerve structures.

4.4.2. Physical examination

Speculum examination

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by performing a physical examination during a menstrual period [205]. One recent study found that speculum examination displayed endometriosis in 14% of patients diagnosed with deeply infiltrating endometriosis [206]. Nodularity of the cul-de-sac can be felt in patients with this form of endometriosis. Often though, no abnormalities are found on the physical examination of patients with endometriosis, and speculum exam is rarely helpful for making the diagnosis.

Bimanual examination

Bimanual examination might reveal an enlarged, tender, cystic adnexal mass that may suggest an endometrioma. In some cases, the main manifestion of endometriosis is an ovarian endometrioma. Endometriomas contain a dense, brown, chocolate-like fluid and are pseudocysts formed by the invagination of endometriosis within the ovarian cortex. These cysts are usually attached to nearby pelvic structures by adhaesions. A fixed retroverted uterus or a “frozen pelvis” can be assessed on bimanual exami-nation. On the other hand, DIE lesions are primarily found in the uterosacral ligaments or cul de sac, but may also involve the rectovaginal septum. These lesions are a nodular blend of fibromuscular tissue and adenomyosis [21, 207]. And patients with this form of endometriosis may present with deep dyspareunia and various bowel symptoms from diarrhea to dyschezia during menses, depending on the location of the deep lesions. In fact, deeply infiltrating nodules are most reliably detected when clinical examination is performed during menstruation [208] although patient acceptance might be an issue.

The bimanual examination or pelvic exam should be done as part of a standard gynecological evaluation, however whether or not it is helpful in diagnosing all cases of endometriosis is doubtful. In fact, the poor negative predictive value of the pelvic exam was demonstrated in one study of 91 patients, in which 47% of patients with surgically confirmed endometriosis and chronic pelvic pain had normal bimanual examinations [209].

4.4.3. Imaging for endometriosis

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riomas. Sensitivity and specificity of this method have been reported to be 84–100% and 90–100%, respectively [212, 213]. Endometriomas often present as cystic structures with low-level internal echoes, and occasional thick septations, thickened walls, and echogenic wall foci [214]. Color Doppler transvaginal sonography often demonstrates pericystic, but not intracystic, flow [215].

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detection of these latter forms of the disease have lead to the claims that finding new non-invasive diagnostic tests for endometriosis is a necessity.

4.4.4. Laparoscopy

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of the pathologist in identifying the characteristic features of endometriosis especially in cases of mild disease where glandular elements may not be obvious [230]. Furthermore, at laparoscopy, deeply infiltrating endometrio-sis may have the appearance of minimal disease, resulting in an under estimation of disease severity. Also, in a frozen pelvis, adhesions may completely cover endometriotic lesions. All of these arguments in fact show the limitations of laparoscopy in some cases of endometriosis. The avai-lability of a reliable noninvasive or semi-invasive test to detect the disease would thus help to avoid worthless diagnostic laparoscopies [231, 232].

4.4.5. Putative Biomarkers for endometriosis

Over the past 25 years, there has been a constant attempt to find an ideal biomarker for diagnosing endometriosis. A simple, reliable diagnostic test, in particular for minimal-mild disease, could avoid countless women having to undergo unnecessary laprascopies just for the sake of a diagnosis. Many studies have focused on identifying biomarkers in blood or urine because of the ease of sampling. Reports in the literature have described over 200 such biomarkers with various degrees of success.

4.4.5.1. Peripheral biomarkers of endometriosis

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ovarian cyst suggestive of an endometrioma. The connection between other serum markers and endometriosis has been studied in depth.

Cancer antigen 19-9 (CA 19-9), another antigenic glycoprotein, is a serum marker that has also been shown to positively correlate with the severity of endometriosis [235]. Serum placental protein 14 (PP14; glycodelin-A) was initially shown to have adequate sensitivity (59%), but this has not been confirmed by other studies [236]. Interleukin-6 (IL-6) serum levels above 2 pg/mL (90-% sensitivity and 67-% specificity) and tumor necrosis factor- (TNF-) peritoneal fluid levels above 15 pg/mL (100% sensitivity and 89% specificity) may be used to discriminate between those with or without endo-metriosis [237]. IL-8 levels are significantly elevated in women with an endometrioma compared with women with other ovarian cysts [238]. Several other serum markers have been studied, with limited diagnostic accuracy [239]. However most of these tests are rarely used outside of research settings.

4.4.5.2. Endometrial alterations and possible biomarkers

Potential biomarkers might be substances that play a role in the various mechanisms associated with the pathogenesis of endometriosis. Current medical research has examined the role of endometrial immunology, endometrial cytokines, endometrial growth factors, cell adhesion, endometrial angiogenesis, in addition to proteomics of the endometrium. Recently, more evidence is emerging that there are intrinsic differences between the eutopic endometrium of women with endometriosis and and those with an endometriosis free pelvis. It is possible to collect endometrium simply and comfortably from patients in outpatient settings without the need for

anaesthesia. The ease of endometrial biopsy means that endometrial

differences in endometriosis could be considered as potential diagnostic tools. Even markers which are more modestly altered in endometriosis have the potential to be combined into a clinically useful test.

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inconsistent on this issue. Some even reported decreased levels in affected women [241].

One study showed that the tumour necrosis factor (TNF)-a mRNA levels were elevated in women with endometriosis compared with controls during the menstrual phase [242]. On the other hand, TNF-a receptor type II levels were decreased [243].

The late secretory phase was characterized by an up-regulation of the macrophage-stimulating protein in the glandular epithelium of women with endometriosis [244]. One study has revealed an increase in levels of macrophage migration inhibitory factor in the endometrium of women with endometriosis [245]. Endometrial cytokines might be valuable in diagnosing endometriosis, however their fluctuations with regard to cycle phase diminish its value in this respect.

Endometrial immunology. In women who develop endometriosis we might have an altered immune response in the body in general and in the endometrium especially. Researchers have therefore tried to identify immunological variation within endometrial tissue, and on a background of a similar approach using serum markers.

HLA expression in the endometrium has been evaluated. An increase in the expression of HLA-DR antigens on endometrial glandular cell surfaces has been identified in women with endometriosis [246]. An earlier study however, also showed equivalent staining intensity of HLA-DR in the stroma and epithelium [247]. One group has demonstrated increased expression levels of HLA class 1 in the glands and stroma of women with endometriosis (strongest significance with stromal cells during the secretory phase [248].

From another point of view in the endometrium of women with endometriosis, levels of endometrial IgG were initially shown to be increased [249]. These authors assessed the accuracy of endometrial IgG as a diagnostic test: its sensitivity was 89%, but specificity was only 63% (levels were also increased in women with chronic pelvic inflammatory disease). This was suggested by further studies but no statistical analysis was presented [250]. However, a third study identified no glandular IgG staining in patients or controls, and equal stromal staining in both groups [251].

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Gagne et al. identified reduced numbers of CD3+, CD3+CD162 and CD3+ CD562 cells, but increased numbers of CD16+, CD16b+, CD32HLA2DR2, CD32CD45RA2 and CD562CD16+ cells in the endometrium [255]. The authors used these data in combination with clinical history (duration of menses) and serum CA125 to generate a predictive model. This showed 61% sensitivity and 95% specificity to diagnose endometriosis; these values were better than those obtained for CA125 alone. T cell subsets have been analysed by Berbic et al. [256], who found a significant increase in Foxp3+ regulatory T cells during the secretory phase of the cycle.

Recently, using immunohistochemistry and antibodies to CD68, macro-phage increases were seen during the proliferative phase [257]. Conversely, one paper has identified a significant decrease in the number of endometrial macrophages found during the early proliferative phase in women with endometriosis [258].

The issue of immune cell population levels in endometriosis remains controversial, and thus far no consistent differences between women with and without disease have been identified.

Steroids and hormones. It is well known that endometriosis is a hormone responsive disease, and that disease progression is inhibited by an anti-estrogenic environment. There has been a great deal of interest in the possibility of endometrial aromatase expression identifying women with endometriosis [259]. The original findings have been confirmed by several other groups [234, 260]. However, three papers report no detectable aromatase expression in the endometrium of patients or controls [261, 262], and one further paper found no difference in expression between infertile women with and without endometriosis [263]. We do not know how other gynecologic disease like fibroids affect the aromatase expression [264]. However, one group reported good sensitivity and specificity for aromatase expression (91 and 100%, respectively), but only for distinguishing ‘disease-free’ women from those with ‘disease’, including adenomyosis, endometriosis and fibroids [265]. The same issue was highlighted by a more recent paper, which found a relatively poor specificity of aromatase expression [266]. Wolfler et al. were able to improve the test’s diagnostic accuracy using a model incorporating aromatase expression and symptoms (the presence or absence of moderate to severe dysmenorrhoea) [267].

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was unchanged in the endometrium of affected women in one study [269], although another identified significantly increased levels in secretory phase epithelial cells [270]. Finally, one group found overall levels of oxidising (17 bestradiol estrone) and reducing (estrone 17bestradiol) 17bHSD enzymes to be equivalent in women with and without the disease [255].

Whilst no overall difference in estrogen receptors (ERs) has been shown in endometriosis, both ER forms have been investigated separately. One group found no change in ERb levels in women with endometriosis [271]. However, one study discovered that glandular expression of ERb was increased compared with controls [272] whilst a second found reduced ERb in the endothelial, stromal and perivascular compartments [273].

Cell adhesion and extracellular matrix molecules (ECM). It is possible that the endometrium of women with disease has altered expression of a variety of cell adhesion molecules, perhaps affecting how shed endometrial cells adhere. The majority of studies on this subject have assessed the expression of integrins–important proteins involved in cell to cell inte-ractions.

The b3 integrin subunit has been described as defectively expressed in women with endometriosis [274]. Using endometrial samples from women after Day 19 (the window of implantation), reduced b3 and avb3 integrin expression was found in women with endometriosis compared with healthy controls or women with other causes of infertility [275]. However, others demonstrated equivalent levels in women with infertility and endometriosis, as in fertile and infertile controls [276]. While a third group actually suggested that levels of avb3 and av integrins are increased in women with endometriosis during menses [234], highlighting the importance of knowing the cycle phase to interpret results.

Extracellular matrix molecules (ECM) have also been assessed. Protein levels of b-catenin have recently been reported as elevated in women with endometriosis during the mid-secretory phase of the cycle [277]. It seems likely that differences in cell adhesion molecules/pathways may play a role in disease pathogenesis. However, none of these proteins has yet been demonstrated to be useful as a biomarker.

Growth factors. The various growth factors might have a role to play in endometriosis, and their presence might be useful as a biomarker.

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at the TGFb superfamily member activin, important for endometrial growth and stromal decidualization. This study showed higher activin A mRNA levels in endometriosis patients throughout the cycle. Cripto expression, an antagonist of activin, was reduced during the proliferative phase only [279].

Insulin-like growth (IGF) factors are known to play an important role in diverse tissues by stimulating growth and differentiation. IGF-binding proteins (IGF-BPs) are involved in regulating transport of IGFs, but also have direct effects on cell growth [280]. Increased expression of IGF-BP3 has been found in the endometrial glands of women with endometriosis, compared with controls [281].

Hepatocyte growth factor (HGF) and its receptor, c-Met, have both been found to be expressed more highly in endometrium of women with endometriosis [282]. HGF was expressed throughout the endometrium; c-Met was found to be more highly expressed in the epithelium only.

Finally, increased levels of annexin-1 have been found in women with endometriosis, and endometrial mRNA levels of midkine and pleiotrophin appear to be increased during the secretory phase (RT–PCR) [283].

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demonstrated increased VEGF mRNA and protein levels in endometrial samples of women with endometriosis during both phases [291]. Conversely, a recent study detected increased VEGF mRNA expression in

women with endometriosis during the proliferative phase, but unchanged

VEGFR-2 expression [292]. This study also assessed levels of dopamine receptor type-2, thought to be involved in VEGF signalling regulation, and found mRNA levels to be decreased in women with endometriosis. One study was unable to detect a significant difference in VEGF mRNA levels using samples from both menstrual and secretory phases [236]. Finally, VEGF-C levels were measured by one group, and found to be significantly reduced in women with endometriosis [284]. Angiopoeitin-1 and -2 are cytokines, which regulate angiogenesis through binding to the Tie-2 (tunica interna cell kinase-2) receptor [293]. Increased endometrial Ang-1 mRNA and protein have been found in women with endometriosis, throughout the cycle [294]. Furthermore, mRNA levels of Ang-2 and Tie-2 were signifi-cantly increased in the secretory phase endometrium of women with the disease [288]. Di Carlo et al. (2009) found that secretory phase samples had increased Ang-1 and 2, both by immunostaining and RT–PCR. Several papers have assessed microvessel density (MVD) in the endometrium. An early study found no correlation between MVD or vascular surface area and presence of disease [295]; another found MVD in the endometrial stroma was increased in endometriosis. Others [276, 282] found normal endometrium to have a consistent MVD throughout the cycle, whilst women with endometriosis tended to show cyclical variation withsignificantly increased MVD in the secretory phase. This finding was replicated in another paper [283]. By immunohistochemistry, the number of endoglin positive vessels during the secretory phase was higher in women with endometriosis than in unaffected controls [296]. Platelet derived growth factor-A was decreased in the secretory endometrium of women with advanced endometriosis [297].

One study reported reduced thrombospondin-1 levels in the endometrium of women with endometriosis [280], although a more recent study found no significant change from control endometrium [298].

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Reactive oxygen and nitrogen species. One group has suggested increa-sed endothelial xanthine oxidase and catalase in women with endometriosis, although no statistical analysis was shown [300, 301], but it is not clear whether levels are altered in endometriosis when compared specifically to the healthy controls. Others reported increased levels of eNOS in women with endometriosis [269]. Whilst the data regarding reactive oxygen and nitrogen species is relatively sparse compared with other areas, there may be scope for further studies in this field.

Role of Proteomics. Proteomic analysis is becoming increasingly impor-tant in a variety of fields as evidence emerges that protein production and regulation play critical roles in disease processes. Recent studies have used new proteomic technologies to identify putative biomarkers in endometrial tissue. The first of these, although very small (three subjects, three controls) showed significantly different expression of certain peptides and proteins in women with endometriosis, using protein arrays and mass spectrometry [302]. Zhang et al. [303] also identified different protein expression using 2D electrophoresis (2DE) followed by mass spectrometry. A slightly larger study identified 223 differentially expressed protein peaks between women with and without the disease using SELDI-TOF mass spectrometry [304]. The same group assessed the mitochondrial proteome in women with endometriosis [305]. Four other studies used 2D gel electrophoresis to identify proteins that were up- or down-regulated in endometriosis [306, 307]. The first, using pooled endometrial samples, demonstrated significant changes in protein expression between women with and without disease [299]. The second study found 119 differentially regulated proteins in the endometrium of women with endometriosis; most were cell structure

proteins or involved in the immune system [300]. Some results were

confir-med by immunohistochemistry or western blotting, but the results were not all consistent. For example, peroxiredoxin 6 was up-regulated by 2DE, but down-regulated using western blotting. This study also suggested a reduc-tion in ribonuclease/angiogenin inhibitor 1, vimentin, transgelin 2 and coro-nin 1A in women with endometriosis. Still other researchers showed increa-sed expression of haptoglobin in the endometrium of women with endomet-riosis when mainly comparing ectopic and eutopic endometrium [308].

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Endometrial Biopsy and Nerve fibers. Half a centrury ago, researchers

showed that the myometrium, the endometrial-myometrial interface and the deeper portion of the basal endometrium can be innervated by nerve fibers. However, their results showed that in the normal human uterus nerve fibers are absent from the functional layer of the endometrium in healthy women [309].

In the past decade, many immunohistochemical neuronal markers like polyclonal rabbit antiprotein gene product 9.5 (PGP9.5) [310], monoclonal mouse anti-human NF [311], polyclonal rabbit anti-SP, rabbit anti-CGRP [312], polyclonal rabbit antiacetylcholine (anti-ACh) [313] and monoclonal anti-tyrosine hydroxylase (anti-TH) [314] have been employed for de-monstrating myelinated, unmyelinated, sensory A δ, sensory nerve fibers in various type of body tissue.

From the various neuronal markers, PGP9.5 is a robust pan-neuronal marker, which is becoming the neurophysiologist’s preferred immunohisto-chemical marker for defining the presence of all nerve fibers in tissue sections as it is a pan-neuronal marker. This protein, which has a molecular weight of 27kDa, was first identified by high resolution two dimensional PAGE. PGP 9.5 removes ubiquitin from other proteins and protects them from degradation by proteases [315]. This protein was originally isolated from whole brain extracts. Standard immunohistochemical techniques have demonstrated the presence of PGP9.5 in neurons and nerve fibers at all levels of the central and peripheral nervous system, in many neuroendocrine cells, in segments of the renal tubules, in spermatogonia and Leydig cells of the testis, in ova and in some cells of both the pregnant and non-pregnant corpus luteum. This antibody is suitable for staining of formalin-fixed, paraffin-embedded tissues. PGP9.5 has been shown to be a highly specific panneuronal marker for both myelinated and unmyelinated nerve fibers, including Aa, Ab, Ag, Ad, B and C fibers [303]. Most importantly this antibody has minimal cross-reaction with other tissue types. Advanced immunohistochemistry techniques have demonstrated that nerve fibers were present in human peritoneal endometriotic plaques [191] and other ectopic endometriotic lesions [188, 199] and have described the presence of certain types of nerve fibers in some of these lesions.

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to fall into two distinct categories (present or absent) which correlates with the presence of disease. This therefore has the potential to be a unique marker of endometriosis, although further work is needed to determine whether nerve fibers may also be present in other gynaecological pathology. Endometrial biopsy, with detection of nerve fibers, provided a reliability of diagnosis of endometriosis which is close to the accuracy of laparoscopic assessment by experienced gynaecological laparoscopists [10]. Although endometrial biopsy is minimally invasive, it may be regarded as unpleasant or uncomfortable. Most studies assessing patient tolerance of endometrial biopsy have shown that the procedure is associated with discomfort, but that the majority of women would be willing to undergo it again [317].

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5. MATERIAL AND METHODS

5.1 Setting

This prospective case control study was carried out in the Department of Gynecology at the Lithuanian University of Health Sciences Hospital Kauno Klinikos which is a tertiary level multidisciplinary university hospital between 2008 and 2012.

5.2 Study population

Patients admitted for laparoscopic surgery in the Department of Gynecology at the Lithuanian University of Health Sciences Hospital Kauno Klinikos were eligible to participate in the study if they conformed with the predetermined inclusion criteria of the study. Prior to enrollment eligible patients were verbally informed about the study, they were then given written information about the study. Patients were fully recruited into the study only after they signed a written informed consent. The study protocol had been approved by the Kaunas Regional Biomedical Research Ethics Committee of the Lithuanian University for Health Sciences. (Number BE-2-50).

Inclusion criteria for the study included:

1. Women in the reproductive age group undergoing laparoscopy for pelvic pain and/or infertility, or other benign disease requiring surgery (age 18-48);

2. Written informed consent.

Exclusion criteria of the study included:

1. Women currently receiving hormonal treatment for at least 3 months prior to laparoscopy;

2. Suspected or confirmed pregnancy; 3. Malignant disease;

4. Confirmed endometrial pathology (hyperplasia, polyps); 5. Unwillingness to participate in the study.

VALUE OF ENDOMETRIAL NERVE FIBER DETECTION IN DIAGNOSING ENDOMETRIOSIS

Said Makari