**8. Discussion**

146 Endometriosis - Basic Concepts and Current Research Trends

of five and seven arbitrary primers. A number of common bands were seen between eutopic and ectopic samples from the same patient. However, in three cases studied differential bands of 45bp in the first set, and 350bp region in the second set were observed in the ectopic tissue of patient suffering from severe endometriosis, stage IV. DD-RTPCR using

first set of 5 primers. Fig. 6.

Fig. 6. DD-RT-PCR using second set of seven primers

1 2 3 1 2 3

(before eluting), and in lane 3 on the right side (after eluting).

Fig. 7. Lane 1: 100 bp ladder; Lane 2: Eutopic tissue; Lane 3: Ectopic tissue.

Differentially expressed bands are seen in ectopic tissue in lane 3, as shown by the arrows

Upper arrow: differential band of 350 bp Lower arrow: faint differential band of 250 bp One hundred and six (106) cases of endometriosis, diagnosed at laparoscopy and/or laparotomy were staged according to the Revised American Fertility Society (rAFS) classification and were enrolled in the study. One hundred and forty (140) controls were also included in the study who comprised of women who either had no symptoms, or no endometriosis at the time of laparoscopy/laparotomy which was performed for other indications. Mild endometriosis (stages I & II) was diagnosed in 72% of cases and advanced endometriosis (stages III & IV) was diagnosed in 28% of cases in the cohort studied.

The aim of the present study was to identify individuals clinically suffering from endometriosis and establish genetic and molecular markers for understanding the cellular and molecular pathogenesis of this condition.

Few well-designed epidemiologic studies of risk factors for endometriosis exist. Eskanazi B et al, (1997) conducted a review of more than 100 published studies and found that only 6 ( 1 cohort and 5 case-control studies) included a surgically case confirmed group, provided clear criteria for control selection, and considered potential confounding factors in the analysis. Hence, the importance of the present study is that all the cases of endometriosis included were confirmed by laparoscopy/laparotomy and the controls were age matched women who were surgically proven to have no endometriosis or had no symptoms suspicious of endometriosis throughout their reproductive life.

Endometriosis plaques have been shown to have estrogen, progesterone and androgen receptors, and they grow in the presence of estrogen but atrophy when exposed to androgens. Since endometriosis is a hormone-sensitive disease, in the present study the role of hormonal genes polymorphism was analyzed in women with surgically confirmed diagnosis of endometriosis. This is the first Indian study to evaluate the hormonal genetic factors in the etiology of endometriosis.

The PROGINS polymorphism has been shown to be associated with endometriosis in Caucasian women (Weiser et al, 2002) including Italian women (Lattuada D et al, 2004). *Our study established 5% prevalence of PROGINS polymorphism in Indian population and showed no susceptibility to endometriosis.(Govindan et al 2006).* Estrogen receptor-α gene (T/C) Pvu II polymorphism in Endometriosis and Uterine Fibroids has been studued by our group. It was observed to be significantly associated with endometriosis in Asian Indian population. (Govindan et al. 2009)

Genetic Polymorphisms and Molecular Pathogenesis of Endometriosis 149

sequences in ectopic tissue of endometriosis which was hither-to not associated with

A world-wide literature search through Medline, PubMed and Google, showed no reported work associating either the Shigella species nor the HCAA gene with endometriosis. These

The HCAA gene is a likely candidate for causing endometriosis, which needs to be further

*Shigella* are gram-negative, non-motile, aerobic and facultatively anaerobic bacilli from the family Enterobacteriaciae.1 *S. dysenteriae, flexneri, boydii,* and *sonnei* are highly infectious strains that can cause dysentery in humans with an ID**50** of only 100-200 bacteria. Bacterial diarrhoea in humans caused by Shigella species due to the shiga toxin is called shigellosis. It causes inflammation in the small and large bowel, which may extend to the pelvic peritoneum. Although this organism has not been earlier associated with endometriosis, it can be hypothesized that a bacteria related to shigella which induces pelvic peritoneal inflammation, may be playing a role in the etiology of endometriosis. This may not be farfetched after the Nobel Prize winning discovery of Warren and Marshall (Julie Parsonnet 2005), who found that inflammation caused by Helicobacter pylori is responsible for

Studies on the pathogenesis of *Shigella* have revealed unique methods of mucosal invasion that result in the lesions seen with infection. Because most lesions are often centered on gutassociated lymphoid tissue (GALT) and spread outward, it is suspected that the bacteria make their initial entry into the body through the normally *phagocytic macrophage cells* overlying the lymphoid tissue (Salyers AA et al.1994) Additional studies have revealed that through a complex process involving multiple genes found on both a large plasmid and on the *Shigella*  chromosome, attachment of the bacteria to mucosal epithelial cells stimulates a structural alteration of the normally nonphagocytic epithelial cell cytoskeleton and actin filaments to cause uptake of the organism in a manner similar to phagocytosis. Once within the intracellular vacuole of the invaded cell, a hemolysin produced by *Sh*i*gella* causes release of the organism into the cytoplasm. The *Shigella* then rapidly multiply and migrate along polymerized actin filaments to reach the plasma membrane so that adjacent cells can be invaded (Keusch GT et al, 1993). Early in the course of disease, low numbers of *Shigella*  organisms can be found by electron microscopy within mucosal epithelial cell vacuoles. As the disease progresses, fibrinous exudate replaces the dead epithelial cells (Brady AG et al, 1998). Death of epithelial cells and sloughing of mucosa creates the ulceration, pseudomembrane

formation, hemorrhage, and inflammatory response that typifies shigellosis.

our group to explain the molecular pathogenesis of endometriosis.

An additional aspect of virulence involves the production of an exotoxin, shiga toxin, by *S. dysenteriae*. Shiga toxin also enhances the lipopolysaccharide-mediated release of cytokines, such as interleukin-1 and tumor necrosis factor-alpha (Kodati V et.al, 2009), which likely contributes to the vascular damage leading to renal failure seen in a complication of shigellosis, hemolytic uremic syndrome. Subsequent to the identification by sequencing of shigella bacterial association with peritoneal inflammation, a hypothesis was proposed by

results open up new possibilities for the etiology of endometriosis.

esophageal/gastric cancer (Forbes et al, 1994).

endometriosis.

investigated.

The highly polymorphic trinucleotide repeat (CAG) in AR varies in length and methylation pattern which affects both AR expression and function.The AR gene CAG polymorphism has been associated with a number of benign and malignant conditions, eg. polycystic ovarian syndrome in women, and male infertility and prostate cancer in men. Its association with endometriosis in Italian women did not constitute an important factor of genetic predisposition (Lattuada D et al, 2004). The range of CAG repeats varies from 9-31 in Japanese population (Hsieh Y et al, 2001) and 14-32 in Italian population. However, there are no studies reported for the AR polymorphism in Indian women with endometriosis.

*Our study proposes that the 19 CAG repeats may be associated with increased risk of endometriosis in our population.* AR gene CAG repeat polymorphism may become a useful marker to predict the future development of endometriosis and to permit early therapeutic intervention in women at high risk of developing endometriosis (Shaik et al.2009)

Since endometriosis clinically mimics cancer with proliferation, angiogenesis and metastasis, three markers associated with carcinogenesis were selected for MSI analysis. TGF-beta II receptor gene is a putative tumor suppressor. It has been found that the TGFBRII gene was inactivated in a subset of colon cancer cell lines exhibiting MSI. Once generated, the proliferative advantage of cells with inactivated type II receptor would drive colon tumor progression (**Markowitz et al 1995)**. This pathway may also be operative in other human pathologies like endometriosis. Human FHIT (fragile histidine triad) protein is encoded by the FHIT putative tumor suppressor gene (Barnes et al 1996). Aberrant transcripts of the FHIT locus were found in approximately 50% of esophageal, stomach, and colon carcinomas (Ohta et al 1996). The results from several studies showed aberrant regulation of several cell cycle proteins, including CDKN (Kim et al 2005).

In endometriotic lesions the differentiation of glands and stroma is absent as the lining is attenuated, lost or replaced with granulation tissue and dense fibrous tissue. Hence it is difficult to get suitable ectopic endometrial tissue from cases for DNA isolation. Therefore MSI could be assessed in 12 cases only and tissue samples were obtained from 12 controls. Out of the 12 cases 2 (16.66%) showed microsatellite instability in the TGFbetaRII gene. *This is the first study reporting an association of TGF-beta receptor II gene with endometriosis in Indian women.* None of the samples studied showed any instability with regards to the other two markers. This suggests that D3S1313 and D9S171 may not be important markers for endometriosis.

In the present study mRNA was isolated from ectopic and eutopic tissues and DD-RT-PCR was carried out twice using 7 different sets of arbitrary primers. **Ectopic endometriotic tissue of one case showed unique band of 45bp after DD-RT-PCR using the first set, while a band in the region of 350bp was obtained using the second set of primers.** Both the 45bp and 350bp bands were cut out, eluted and automatically sequenced. The 45bp band gave multiple errors during sequencing, whereas the 350bp band could be sequenced successfully. Sequencing was carried out twice from the latter band. Both the sequences were analyzed using the BLAST search (National Centre for Biotechnology Information-NCBI; Google Search). It revealed a 60/65bp, 96% homology with Shigella dysenteriae, boydii and sonnei species. This sequence also matched a smaller 20/20bp stretch of 100% homology with human Hepatocellular carcinoma associated antigen (HCAA). Hence, a simple cost-effective technique like DD-RT-PCR has enabled the identification of novel gene

The highly polymorphic trinucleotide repeat (CAG) in AR varies in length and methylation pattern which affects both AR expression and function.The AR gene CAG polymorphism has been associated with a number of benign and malignant conditions, eg. polycystic ovarian syndrome in women, and male infertility and prostate cancer in men. Its association with endometriosis in Italian women did not constitute an important factor of genetic predisposition (Lattuada D et al, 2004). The range of CAG repeats varies from 9-31 in Japanese population (Hsieh Y et al, 2001) and 14-32 in Italian population. However, there are no studies reported for the AR polymorphism in Indian women with endometriosis.

*Our study proposes that the 19 CAG repeats may be associated with increased risk of endometriosis in our population.* AR gene CAG repeat polymorphism may become a useful marker to predict the future development of endometriosis and to permit early therapeutic

Since endometriosis clinically mimics cancer with proliferation, angiogenesis and metastasis, three markers associated with carcinogenesis were selected for MSI analysis. TGF-beta II receptor gene is a putative tumor suppressor. It has been found that the TGFBRII gene was inactivated in a subset of colon cancer cell lines exhibiting MSI. Once generated, the proliferative advantage of cells with inactivated type II receptor would drive colon tumor progression (**Markowitz et al 1995)**. This pathway may also be operative in other human pathologies like endometriosis. Human FHIT (fragile histidine triad) protein is encoded by the FHIT putative tumor suppressor gene (Barnes et al 1996). Aberrant transcripts of the FHIT locus were found in approximately 50% of esophageal, stomach, and colon carcinomas (Ohta et al 1996). The results from several studies showed aberrant

In endometriotic lesions the differentiation of glands and stroma is absent as the lining is attenuated, lost or replaced with granulation tissue and dense fibrous tissue. Hence it is difficult to get suitable ectopic endometrial tissue from cases for DNA isolation. Therefore MSI could be assessed in 12 cases only and tissue samples were obtained from 12 controls. Out of the 12 cases 2 (16.66%) showed microsatellite instability in the TGFbetaRII gene. *This is the first study reporting an association of TGF-beta receptor II gene with endometriosis in Indian women.* None of the samples studied showed any instability with regards to the other two markers. This

In the present study mRNA was isolated from ectopic and eutopic tissues and DD-RT-PCR was carried out twice using 7 different sets of arbitrary primers. **Ectopic endometriotic tissue of one case showed unique band of 45bp after DD-RT-PCR using the first set, while a band in the region of 350bp was obtained using the second set of primers.** Both the 45bp and 350bp bands were cut out, eluted and automatically sequenced. The 45bp band gave multiple errors during sequencing, whereas the 350bp band could be sequenced successfully. Sequencing was carried out twice from the latter band. Both the sequences were analyzed using the BLAST search (National Centre for Biotechnology Information-NCBI; Google Search). It revealed a 60/65bp, 96% homology with Shigella dysenteriae, boydii and sonnei species. This sequence also matched a smaller 20/20bp stretch of 100% homology with human Hepatocellular carcinoma associated antigen (HCAA). Hence, a simple cost-effective technique like DD-RT-PCR has enabled the identification of novel gene

suggests that D3S1313 and D9S171 may not be important markers for endometriosis.

intervention in women at high risk of developing endometriosis (Shaik et al.2009)

regulation of several cell cycle proteins, including CDKN (Kim et al 2005).

sequences in ectopic tissue of endometriosis which was hither-to not associated with endometriosis.

A world-wide literature search through Medline, PubMed and Google, showed no reported work associating either the Shigella species nor the HCAA gene with endometriosis. These results open up new possibilities for the etiology of endometriosis.

The HCAA gene is a likely candidate for causing endometriosis, which needs to be further investigated.

*Shigella* are gram-negative, non-motile, aerobic and facultatively anaerobic bacilli from the family Enterobacteriaciae.1 *S. dysenteriae, flexneri, boydii,* and *sonnei* are highly infectious strains that can cause dysentery in humans with an ID**50** of only 100-200 bacteria. Bacterial diarrhoea in humans caused by Shigella species due to the shiga toxin is called shigellosis.

It causes inflammation in the small and large bowel, which may extend to the pelvic peritoneum. Although this organism has not been earlier associated with endometriosis, it can be hypothesized that a bacteria related to shigella which induces pelvic peritoneal inflammation, may be playing a role in the etiology of endometriosis. This may not be farfetched after the Nobel Prize winning discovery of Warren and Marshall (Julie Parsonnet 2005), who found that inflammation caused by Helicobacter pylori is responsible for esophageal/gastric cancer (Forbes et al, 1994).

Studies on the pathogenesis of *Shigella* have revealed unique methods of mucosal invasion that result in the lesions seen with infection. Because most lesions are often centered on gutassociated lymphoid tissue (GALT) and spread outward, it is suspected that the bacteria make their initial entry into the body through the normally *phagocytic macrophage cells* overlying the lymphoid tissue (Salyers AA et al.1994) Additional studies have revealed that through a complex process involving multiple genes found on both a large plasmid and on the *Shigella*  chromosome, attachment of the bacteria to mucosal epithelial cells stimulates a structural alteration of the normally nonphagocytic epithelial cell cytoskeleton and actin filaments to cause uptake of the organism in a manner similar to phagocytosis. Once within the intracellular vacuole of the invaded cell, a hemolysin produced by *Sh*i*gella* causes release of the organism into the cytoplasm. The *Shigella* then rapidly multiply and migrate along polymerized actin filaments to reach the plasma membrane so that adjacent cells can be invaded (Keusch GT et al, 1993). Early in the course of disease, low numbers of *Shigella*  organisms can be found by electron microscopy within mucosal epithelial cell vacuoles. As the disease progresses, fibrinous exudate replaces the dead epithelial cells (Brady AG et al, 1998). Death of epithelial cells and sloughing of mucosa creates the ulceration, pseudomembrane formation, hemorrhage, and inflammatory response that typifies shigellosis.

An additional aspect of virulence involves the production of an exotoxin, shiga toxin, by *S. dysenteriae*. Shiga toxin also enhances the lipopolysaccharide-mediated release of cytokines, such as interleukin-1 and tumor necrosis factor-alpha (Kodati V et.al, 2009), which likely contributes to the vascular damage leading to renal failure seen in a complication of shigellosis, hemolytic uremic syndrome. Subsequent to the identification by sequencing of shigella bacterial association with peritoneal inflammation, a hypothesis was proposed by our group to explain the molecular pathogenesis of endometriosis.

Genetic Polymorphisms and Molecular Pathogenesis of Endometriosis 151

Thereby the bacteria reach the lamina propria of the colonic mucosa (Fig. 8). It is hypothesized that by the same mechanism the bacteria can enter the blood stream and/or travel across the colon wall to reach the outer peritoneal surface of the colon which is in close proximity to the posterior uterine surface, the site which incidentally happens to be the commonest site of early endometriosis (Cul-de-sac or Pouch of Douglas) as shown in Fig. 8. We propose that the peritoneal reaction to this bacterial invasion may be similar to any antigenic response by the host immune system resulting in the activation of macrophages and production of cytokines characteristic of acute inflammatory response. The endometrial cells that are shed during the retrograde menstruation into the cul-de-sac adhere to the inflamed peritoneal and ovarian surfaces and come under the influence of circulating ovarian hormones. The thus implanted endometrial cells in the peritoneum progress to endometriosis through angiogenesis. Our postulated bacterial hypothesis proposes that shigella or shigella-like organisms may be the trigger for the immunological changes in the pelvic peritoneum which initiate the etiopathogenesis of endometriosis. The inflammatory hypothesis is further reinforced by our subsequent research on TNF-alpha -C850T polymorphism, which showed significant association with endometriosis. (Lakshmi KV et al 2009) The bacterial hypothesis is supported by our recent research work on TLR-4 (A896G) polymorphism. Toll- Like Receptor 4 is specific for recognition of the molecular pattern of gram-negative bacteria. TLR-4 is present on the surface of endometrial cells. TLR-4 A896G is a functional polymorphism resulting in hypo-responsiveness of the receptor, causing peritoneal inflammation in the female pelvis. The molecular micro-environment of the cul-

de-sac becomes favourable for initiation of endometriosis.( Latha M et al 2011)

Endometriosis remains a difficult clinical problem today and warrants more extensive research to understand the disease pathology. The future is to confirm early diagnosis by non-invasive test using a panel of potential genetic and molecular bio- markers. A long term goal is to be able to identify genetic determinants that contribute to the expression of the

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**10. Conclusion** 

**11. References** 

different phenotypes seen in endometriosis.

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