**1. Introduction**

132 Endometriosis - Basic Concepts and Current Research Trends

Vercellini, P., Crosignani, P., Somigliana, E., Vigano, P., Frattaruolo, M.P., & Fedele, L.

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headache in healthy subjects. *Cephalalgia*, Vol.29, No.5, (May 2009), pp. (509-519),

effect of fibroblast growth factor-9 in human endometriotic implant is regulated by aberrant production of estrogen. *Journal of Clinical Endocrinology and Metabolism*,

endometriosis? *Experimental Biology and Medicine*, Vol.235, No.6, (June 2010), pp.

the role of prostaglandins. *Expert Reviews in Molecular Medicine*, Vol.9, No.2,

Suppression of matrix metalloproteinase-9 by prostaglandin E(2) in peritoneal macrophage is associated with severity of endometriosis. *American Journal of* 

mechanisms regulate cyclooxygenase-1 and -2 in peritoneal macrophages of women with and without endometriosis. *Molecular Human Reproduction*, Vol.8,

cyclooxygenase-2 by interleukin-1beta in normal and endometriotic stromal cells. *Journal of Clinical Endocrinology and Metabolism*, Vol.90, No.1, (January 2005b), pp. Endometriosis occupies a unique position in medicine. The natural history of the disease is uncertain, its precise etiology is unknown, the clinical presentation is inconsistent, diagnosis is difficult and can be confirmed only by invasive procedures, and the treatment is poorly standardized.

It is one of the commonest benign gynaecological conditions affecting 15 – 25% of women in the reproductive age group. Yet, it displays features similar to malignancy, ranging from neo-vascularisation to local invasion and aggressive spread to distant organs (metastasis).

The most widely accepted theory to explain endometriosis is that viable endometrial cells reach the peritoneal cavity through retrograde menstruation along the fallopian tubes. Some of the cells then adhere to the peritoneal surface and proliferate in response to the ovarian hormones. However, it is well established that menstrual debris is present in the peritoneal cavity of 90% of menstruating women, suggesting that endometrial cells from only 'some women' are capable of establishing ectopic endometrial implants. Why does this happen in only 'some (10-15%) women'? There are several possible explanations for disease susceptibility, including differences in genetic predisposition (Bischoff FZ et al, 2000), increased exposure to menstrual debris, abnormal eutopic endometrium, altered peritoneal environment, reduced immune-surveillance (Sinaii N et al, 2002), and increased angiogenic capacity (Absenger Y et al, 2004).

Endometriosis, the name by itself implies an endometrial pathology but this concept has long been disputed by Thomas and Prentice in 1992. The acceptance of an endometrial origin poses the question of whether endometrium that is able to proliferate and implant at ectopic sites is in some way abnormal. The commonest site for endometriosis is the pelvis. Endometrium reaches the pelvis most commonly by retrograde menstruation which is now accepted to be an almost ubiquitous event. Endometriosis is more common in women whose normal menstrual egress is occluded by genital tract anomalies, women with an early menarche, short menstrual cycles and prolonged menses. All these factors suggest that peritoneal soiling is important but if retrograde menstruation is ubiquitous then why do not all women have endometriosis? The truth is that the presence of ectopic endometrium within the peritoneal cavity is probably universal but the association of ectopic

Genetic Polymorphisms and Molecular Pathogenesis of Endometriosis 135

clonal expansion of chromosomally abnormal cells. Cytogenetic R-banding studies on human endometriosis-derived permanent cell line (FbEM-1) showed numerous chromosomal aberrations, including monosomy X, 4q+, 5q+, trisomy 7,8,10 and tetrasomy of chromosomes 17, 18, 19 and 20 (Bouquet DJ et al, 1997). A caveat for these studies is that cultured cell-lines may be unstable, which reflects growth of selectively advantaged cells and is no longer representative of the original tissue. Comparative genomic hybridization (CGH) has also been used which revealed overrepresentation of chromosomes 1, 2, 3, 5, 6p, 7, 16, 17q, 20, 21q, and 22q, whereas chromosomes 6q, 9, 11p, 12, 13q, 18 and X were underrepresented. Subsequent FISH analysis confirmed gain at 6p and 17q and loss of 1p,

Subsequent studies were conducted using the method of *quantitative genetic analysis.* To find the genes of endometriosis from nearly 30,000 total human genes, the technique of siblingpair quantitative linkage analysis was used. Sibling-pair analysis obligatorily requires that informative DNA polymorphic markers exist every few centimorgans (10cM). The polymorphisms usually used are DNA variants, such as di-nucleotide, tri-nucleotide, or

It has been argued that findings have been inconsistent because most studies have used inappropriate controls. However, confusion is also created by underpowered studies and/or studies investigating genes without sufficient evidence to support their biological

*First,* significant association of GALT gene with endometriosis was reported with at least one allele with the N314D mutation, whereas *GALT* gene had been previously associated with Mullerian anomalies. The polymorphism causes reduced activity of the enzyme galactose-1-phosphate uridyl transferase, which is involved in galactose metabolism. Three subsequent studies involving more patients have failed to replicate the association

*Second*, genes that encode enzymes involved in detoxification, such as the glutathione Stransferase (GST) family, have been investigated based on the finding that the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) induces endometriosis in the rhesus monkey. Homozygotes for a null mutation in one of the GST family genes, *GSTM1*, were more common in endometriosis cases than in controls. Subsequently, studies in UK (Hadfield RM et al, 2001), Japanese (Morizane M et al, 2004), Chinese (Ding Y et al, 2004) and Indian (Babu KA et al, 2005) populations have failed to replicate the association. No association has been found for a mutation in a similar gene, *GSTT1*. Moreover, in a critical appraisal of all the human and nonhuman primate evidence implicating dioxin exposure as a risk factor for endometriosis, Guo SW (2004) concluded that there was insufficient evidence to support the theory, about 10 years after it was first proposed! It is possible, therefore, that research groups around the world have investigated genes involved

Although endometriosis is a benign disorder, it has also been viewed as a neoplastic process. Evidence to support this hypothesis includes molecular similarities between endometriosis and cancer; and the increased susceptibility to develop ovarian clear-cell cancer and endometrioid cancer in the presence of endometriosis. Molecular genetic

in detoxification as functional candidates based on an entirely false premise.

22q, and chromosomeX (Gogusev J et al 2000).

plausibility. *Two examples are given here.* 

tetra-nucleotide repeats.

(Stefansson H et al, 2001).

endometrium with either symptoms or anatomic distortion is not. It is these latter two groups of women that present with pathological endometriosis as the presence of ectopic endometrium alone is not pathological; this can clearly be seen in the case of infertility associated with minimal and mild endometriosis.

The endometrium shed at menstruation contains numerous cytokines and angiogenenic growth factors. These factors undoubtedly could promote proliferation of and angiogenesis around ectopic endometrial implants, allowing survival of the shed endometrium at ectopic sites. If they are of importance in the pathogenesis of endometriosis then it would be expected that differences would exist between those women with and those without endometriosis. However, no qualitative differences exist between either eutopic and ectopic endometrium nor between endometrium from women with and those without endometriosis. This observation is true for all growth factors and cytokines studied to date, and strongly suggests that endometriosis is not an endometrial disease.

If endometriosis is not primarily an endometrial disease, then it is uncertain what permits and facilitates the survival of endometrium at ectopic sites. It is becoming increasingly clear that the peritoneal fluid and its cellular constituents are important in the pathogenesis of endometriosis.

In many ways endometriosis can be considered to be a chronic inflammatory condition of the pelvis. The endometriotic implants have many different appearances but in early stages in particular, they are surrounded by new blood vessel formation which resembles an inflammatory response. The peritoneal fluid in endometriosis contains an increased size and number of macrophages that have an increased activation status. They secrete large number of cytokines and produce growth factors that support the establishment of the ectopic implants. Peritoneal macrophages are regulated by ovarian steroids and in their activated state produce increased levels of VEGF (vascular endothelial growth factor) that supports angiogenesis around the endometriotic implants. These macrophages are less susceptible to apoptosis which is the normal consequence of activation.

However, there is convincing evidence that the disease is inherited as a complex genetic trait (Kennedy S, 1997). Genetic factors accounted for 52% of the variation in liability to endometriosis in an Australian twin study (Treloar et al, 2000). The genetic relative – recurrence risk for sibs was estimated to be 2.34 in a cohort of Australian twins and their families. This risk indicates that polygenic and multifactorial etiology is far more likely to be the cause than Mendelian inheritance. Familial aggregation has been reported in humans and non-human primates (Kennedy S et al, 1995; Zondervan et al, 2004). A genome-wide Linkage study in 1,176 affected sister pair families identified a significant susceptibility locus for endometriosis on chromosome 10q26 (Treloar SA, Kennedy SH, et al, 2005).

Initially *cytogenetic studies* of chromosomal rearrangements in affected endometriotic tissue were conducted to uncover candidate chromosomal loci. Dangel et al, (1994), found no evidence of abnormalities in any of the 42 implants studied. However, with the application of chromosome-specific probes using multicolor fluoroscent in situ hybridization (FISH) technique, a significantly greater frequency of chromosome 17 aneuploidy in the endometriotic specimens was observed. It was proposed that acquired chromosome-specific alterations may be involved in endometriosis through a multistep pathway suggesting 22q, and chromosomeX (Gogusev J et al 2000).

134 Endometriosis - Basic Concepts and Current Research Trends

endometrium with either symptoms or anatomic distortion is not. It is these latter two groups of women that present with pathological endometriosis as the presence of ectopic endometrium alone is not pathological; this can clearly be seen in the case of infertility

The endometrium shed at menstruation contains numerous cytokines and angiogenenic growth factors. These factors undoubtedly could promote proliferation of and angiogenesis around ectopic endometrial implants, allowing survival of the shed endometrium at ectopic sites. If they are of importance in the pathogenesis of endometriosis then it would be expected that differences would exist between those women with and those without endometriosis. However, no qualitative differences exist between either eutopic and ectopic endometrium nor between endometrium from women with and those without endometriosis. This observation is true for all growth factors and cytokines studied to date,

If endometriosis is not primarily an endometrial disease, then it is uncertain what permits and facilitates the survival of endometrium at ectopic sites. It is becoming increasingly clear that the peritoneal fluid and its cellular constituents are important in the pathogenesis of

In many ways endometriosis can be considered to be a chronic inflammatory condition of the pelvis. The endometriotic implants have many different appearances but in early stages in particular, they are surrounded by new blood vessel formation which resembles an inflammatory response. The peritoneal fluid in endometriosis contains an increased size and number of macrophages that have an increased activation status. They secrete large number of cytokines and produce growth factors that support the establishment of the ectopic implants. Peritoneal macrophages are regulated by ovarian steroids and in their activated state produce increased levels of VEGF (vascular endothelial growth factor) that supports angiogenesis around the endometriotic implants. These macrophages are less susceptible to

However, there is convincing evidence that the disease is inherited as a complex genetic trait (Kennedy S, 1997). Genetic factors accounted for 52% of the variation in liability to endometriosis in an Australian twin study (Treloar et al, 2000). The genetic relative – recurrence risk for sibs was estimated to be 2.34 in a cohort of Australian twins and their families. This risk indicates that polygenic and multifactorial etiology is far more likely to be the cause than Mendelian inheritance. Familial aggregation has been reported in humans and non-human primates (Kennedy S et al, 1995; Zondervan et al, 2004). A genome-wide Linkage study in 1,176 affected sister pair families identified a significant susceptibility

locus for endometriosis on chromosome 10q26 (Treloar SA, Kennedy SH, et al, 2005).

Initially *cytogenetic studies* of chromosomal rearrangements in affected endometriotic tissue were conducted to uncover candidate chromosomal loci. Dangel et al, (1994), found no evidence of abnormalities in any of the 42 implants studied. However, with the application of chromosome-specific probes using multicolor fluoroscent in situ hybridization (FISH) technique, a significantly greater frequency of chromosome 17 aneuploidy in the endometriotic specimens was observed. It was proposed that acquired chromosome-specific alterations may be involved in endometriosis through a multistep pathway suggesting

and strongly suggests that endometriosis is not an endometrial disease.

apoptosis which is the normal consequence of activation.

associated with minimal and mild endometriosis.

endometriosis.

clonal expansion of chromosomally abnormal cells. Cytogenetic R-banding studies on human endometriosis-derived permanent cell line (FbEM-1) showed numerous chromosomal aberrations, including monosomy X, 4q+, 5q+, trisomy 7,8,10 and tetrasomy of chromosomes 17, 18, 19 and 20 (Bouquet DJ et al, 1997). A caveat for these studies is that cultured cell-lines may be unstable, which reflects growth of selectively advantaged cells and is no longer representative of the original tissue. Comparative genomic hybridization (CGH) has also been used which revealed overrepresentation of chromosomes 1, 2, 3, 5, 6p, 7, 16, 17q, 20, 21q, and 22q, whereas chromosomes 6q, 9, 11p, 12, 13q, 18 and X were

Subsequent studies were conducted using the method of *quantitative genetic analysis.* To find the genes of endometriosis from nearly 30,000 total human genes, the technique of siblingpair quantitative linkage analysis was used. Sibling-pair analysis obligatorily requires that informative DNA polymorphic markers exist every few centimorgans (10cM). The polymorphisms usually used are DNA variants, such as di-nucleotide, tri-nucleotide, or tetra-nucleotide repeats.

underrepresented. Subsequent FISH analysis confirmed gain at 6p and 17q and loss of 1p,

It has been argued that findings have been inconsistent because most studies have used inappropriate controls. However, confusion is also created by underpowered studies and/or studies investigating genes without sufficient evidence to support their biological plausibility. *Two examples are given here.* 

*First,* significant association of GALT gene with endometriosis was reported with at least one allele with the N314D mutation, whereas *GALT* gene had been previously associated with Mullerian anomalies. The polymorphism causes reduced activity of the enzyme galactose-1-phosphate uridyl transferase, which is involved in galactose metabolism. Three subsequent studies involving more patients have failed to replicate the association (Stefansson H et al, 2001).

*Second*, genes that encode enzymes involved in detoxification, such as the glutathione Stransferase (GST) family, have been investigated based on the finding that the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) induces endometriosis in the rhesus monkey. Homozygotes for a null mutation in one of the GST family genes, *GSTM1*, were more common in endometriosis cases than in controls. Subsequently, studies in UK (Hadfield RM et al, 2001), Japanese (Morizane M et al, 2004), Chinese (Ding Y et al, 2004) and Indian (Babu KA et al, 2005) populations have failed to replicate the association. No association has been found for a mutation in a similar gene, *GSTT1*. Moreover, in a critical appraisal of all the human and nonhuman primate evidence implicating dioxin exposure as a risk factor for endometriosis, Guo SW (2004) concluded that there was insufficient evidence to support the theory, about 10 years after it was first proposed! It is possible, therefore, that research groups around the world have investigated genes involved in detoxification as functional candidates based on an entirely false premise.

Although endometriosis is a benign disorder, it has also been viewed as a neoplastic process. Evidence to support this hypothesis includes molecular similarities between endometriosis and cancer; and the increased susceptibility to develop ovarian clear-cell cancer and endometrioid cancer in the presence of endometriosis. Molecular genetic

Genetic Polymorphisms and Molecular Pathogenesis of Endometriosis 137

Ectopic endometrium shares many morphological aspects with eutopic endometrium, but differs in its biological behaviour due to the fact that the cells are present in a different environment. Ectopic and eutopic endometrium are not synchronized in their histological changes; ectopic tissue implants present a maturation disorder with different degrees of maturation and organization in the same implant. Complete secretory modification is rarely found in endometriosis and this could be due to an impact on the production of

There are numerous suggested etiological factors in the pathogenesis of endometriotic

Epidemiological data show a familial tendency of endometriosis. Patients with an affected first-degree relative have nearly a ten-fold increased risk of developing endometriosis. Concordance of twins has also been demonstrated (Bischoff FZ et al 2000). There is evidence in both, human and non-human primates that supports the theory of a genetic basis to

Attempts to explore the role of genetic and molecular factors in the etiology of endometriosis have begun in the last decade. Genetic studies also detected an association between endometriosis and polymorphic mutations of several genes, including the Nacetyltransferase 2 gene(NAT2), the glutathione S-transferase M gene (GSTM) and Estrogen

Polymorphism literally means many forms and it is seen that on an average, every 1 in 500 base pairs of DNA varies between individuals. Variations in coding regions are rare because of the need to preserve function. Changes in non-coding regions of the genome are subject to very little selection pressure and generate allelic variations at a very high frequency. This variation can be an alteration in a single base, deletion or addition of bases and expansion or

DNA polymorphisms are the basis of all current *genetic markers*. A marker is any *polymorphic Mendelian* character that can be used to follow a chromosomal segment through a pedigree. Allelic human gene *expression variation* may be caused by changes in regulatory DNA, including sequences which regulate transcription and splicing. This type of sequence variation may often underlie the susceptibility to common diseases but quantitative methods to explore allelic variation in human gene expression have been developed only

This involves a single nucleotide which is substituted by a different nucleotide. Typically, SNPs have only two alleles. Since coding DNA accounts for only about 1.5% of the human

progesterone dependent factors like prostaglandins.

implants. However, only the genetic factors will be discussed here.

**2. Factors affecting endometriosis** 

endometriosis (Zondervan KT et al, 2001).

**2.1 Genetic factors** 

Receptor alpha gene (ER-a).

**2.1.1 DNA- polymorphism** 

contraction of repeats.

very recently (Yan H et al, 2002).

**2.1.2 Single Nucleotide Polymorphism (SNP)** 

alterations in endometrioid cancers of the ovary were analyzed (Catasus L et al, 2004). Frequent alterations are seen in beta-catenin and PTEN genes, as well as MSI in low-stage ovarian carcinomas of endometrioid type. However, allelic loss studies do not provide evidence for the 'endometriosis as tumor' theory (Prowse AH et al, 2005). Ovarian endometriosis with cytological atypia has potential for malignant transformation. Endometriotic cysts seem to be monoclonal and demonstrate aggressive growth and localized invasion of the myometrium. Malignant transformation has been documented. The proposed pathway is through loss of chromosome material by causing allelic imbalance or loss of heterozygosity (LOH).Genomic instability in any form can transform a normal cell to an abnormal or malignant cell. DNA damage can cause genetic alterations and can manifest as gross DNA damage, chromosomal instability or by a more subtle genetic change like microsatellite instability.

DNA studies examining the role of LOH in endometriotic lesions have identified candidate suppressor gene loci, including 9q, 11q and 22q (Jiang X et al 1996). Alterations in chromosome arms 5q, 6q, 11q and 22q were observed in 25-30% of women with endometriosis and associated carcinoma of the ovary (Jiang X et al 1998).

Gene expression analysis by oligo-nucleotide micro-arrays indicated inflammatory immunoreactions due to up-regulation of FCER1G and PGDS mast cell specific genes which play an important role in producing fibrosis and adhesions in endometriotic lesions (Konno R et al, 2003). Downregulated elements included the tumor suppressor Tp53, genes related to apoptosis, and the gene encoding OVGP1, a protein involved in maintenance of early pregnancy (Arimoto et al, 2003).

Eyster KM et al (2002) illustrated the use of cDNA micro-array technology by studying eutopic endometrium and endometriotic implants from three patients and reported eight genes that were over-expressed in endometriotic implants as compared to eutopic endometrium, that had roles in the cytoskeleton.

The revolutionary evolution of genetics and molecular technologies has given a new perspective to the understanding of the etiology of the perplexing disease of endometriosis. Meyer's (1919) hypothesis of coelomic metaplasia in the totipotential peritoneal cells subjected to repeated irritation by a variety of factors, as a cause of endometriosis, would explain those cases of endometriosis in primary amenorrhoea and in men on prolonged estrogen treatment. There are some unknown factors produced within the uterus which stimulate undifferentiated mesenchyme to undergo metaplastic transformation. These *unknown factors* that allow endometrial fragments to implant in the peritoneal cavity of some women and lymphatic spread to allow ectopic endometrium to develop at distant sites are presently being hypothesized to be due to genetic factors. It has been suggested that endometriosis has a genetic basis by Kennedy et al two decades back.

#### **1.1 Definition of endometriosis**

Endometriosis is defined as a disease characterized by the presence of functional endometrial cells, comprising of glands and stroma in ectopic sites outside the uterine cavity in addition to their normal presence in the innermost lining of the uterus. The ectopic endometrial tissue responds to hormones and drugs in a similar manner to eutopic endometrium.

Ectopic endometrium shares many morphological aspects with eutopic endometrium, but differs in its biological behaviour due to the fact that the cells are present in a different environment. Ectopic and eutopic endometrium are not synchronized in their histological changes; ectopic tissue implants present a maturation disorder with different degrees of maturation and organization in the same implant. Complete secretory modification is rarely found in endometriosis and this could be due to an impact on the production of progesterone dependent factors like prostaglandins.
