**7.1 PROGINS analysis**

142 Endometriosis - Basic Concepts and Current Research Trends

confirmed with the diagnosis of endometriosis and with age matched 140 normal women as controls from South India. Progesterone and Androgen Hormone Receptor *gene polymorphisms*; genomic instability at ectopic and eutopic endometrial tissues resulting in *Micro Satellite Instability* (MSI); *gene expression* in ectopic endometrial tissue by Differential Display Reverse Transcriptase Polymerase Chain Reaction (DD-RT-PCR) to identify novel genes were studied. DNA was isolated from peripheral blood and from fresh tissue samples of both ectopic and eutopic endometrium. We subsequently assessed the polymorphisms of Estrogen Receptor, TNF-alpha and TLR 4 genes. The ongoing research work involves ICAM,

Allelic imbalance in selected loci was reported by microsatellite assays in endometriosis (Goumenou et al, 2001). Three different microsatellite markers were selected based on reports from previous studies (Luokola et al, 2001; Xing et al, 1999). These three

The mononucleotide adenine (A)n repeats-( BAT-RII) and dinucleotide (CA)n repeats-

Multiplex PCR was carried out using specific primers for the microsatellite markers selected. PCR was performed to amplify the three target sequences using specific primers. Multiplex PCR was performed on blood and tissue DNA from 12 cases and a matching number of controls. Thus 60 multiplex PCR reactions were set up using specific primers for each of the

Each of the samples was analysed for all the three markers in the tissue using blood as control. MSI positivity was indicated by *mobility* difference between blood, ectopic and

MSI is defined as a difference in length due to insertion or deletion of the amplified microsatellite markers between the normal and ectopic tissue of the same individual. MSI in the ectopic tissues is assessed by the detection of alleles of novel size that are not present in the normal tissues of the same individuals. All microsatellite markers showing instability were analyzed. Samples lacking MSI were defined as Microsatellite stable

*RNA isolation and RT-PCR analysis:* Biopsy material obtained from the ectopic and eutopic endometrial tissue at the time of surgery was collected in sterile normal saline and transported to the lab for storage at minus 70°C. mRNA was isolated using mRNA direct isolation kit (Qiagen, Oligotex direct mRNA micro kit, Catalogue No. 72012) according to the manufacturer's instructions. mRNA was converted to cDNA by Reverse Transcriptase (RT) step which was followed by 45 cycles of 3-stage PCR with a specific annealing

IL-6, NOD-2, MMP-2,and MMP-9 genes polymorphism studies.

**6.1 Microsatellite instability analysis by multiplex PCR** 

**6.1.1 Single strand conformation polymorphism (SSCP)** 

eutopic tissue; presence of additional band or absence of bands.

**6.1.2 Differential display- reverse transcriptase-PCR (DD-RT-PCR)** 

microsatellite markers are as follows:

selected microsatellite markers.

(D3S1313, D9S171).

(MSS).

\*MW- Molecular Weight ladder

Fig. 1. Photograph of a 2% agarose gel stained with ethidium bromide to resolve the 306 base pair intron G insertion polymorphism of the progesterone receptor gene. The 149-base pair band represents the wild type allele (allele T1) and the 455-base pair band represents the mutant allele (allele T2). Lane 1 indicates the molecular weight markers; lanes 2 and 3 show the homozygous wild type T1T1 and the heterozygous T1T2 patterns. There is no homozygous mutant T2T2 pattern.

Genetic Polymorphisms and Molecular Pathogenesis of Endometriosis 145

Fig. 4. 2% Agarose gel showing results of multiplex PCR both from endometriotic cases and controls with the specific MSI markers and their band sizes. Lanes 1,2,3 are of a case; Lane 4

SSCP was carried out on 15% polyacrylamide gel to identify mobility shift, and/or additional bands at the three selected markers. SSCP PAGE gel showing mobility shift at BAT R II locus in ectopic tissue from a case of endometriosis. 2 cases of endometriosis showed mobility shift in the BAT R II locus associated with TGF beta R II receptor gene. One

Fig. 5. Lane 1 is the DNA ladder; Lanes 2 and 3 are blood and eutopic tissue of control;

in the ectopic tissue only and not in the eutopic tissue of the case.

**7.5 DD – RT – PCR analysis** 

Lanes 4, 5 and 6 are blood, ectopic and eutopic tissue of a case ; Additional bands are seen in all the samples of the case but not in the control, whereas mobility shift of the band is seen

Differential display RTPCR helps in identifying novel genes which are expressed or suppressed in tissues with an altered pathophysiology compared to controls using two sets

**7.4 SSCP (Single Stranded Conformation Polymorphism) analysis** 

1 2 3 4 5 6

control showed additional band in D3 locus associated with FHIT gene.

is the DNA ladder; Lanes 5,6 are of a control

BAT R II mobility shift

#### **7.2 Androgen receptor analysis**

Fig. 2. Photograph of a 2% Agarose gel stained with ethidium bromide to resolve the trinucleotide CAG repeats in AR gene encompassing Exon 1.The bands appeared between 200bp – 300bp. The Lane 4 indicates the molecular weight marker.

The small repeat changes occurring between 200bp – 300bp and heterozygotic alleles could not be identified on agarose gels. Hence, analysis was done using 12% PAGE for further analysis.

Fig. 3. Native PAGE showing heterozygous and homozygous alleles of AR gene: 200 samples processed with 12% polyacrylamide gel and silver staining showed band sizes ranging from 156 bp – 238 bp which fall within the CAG repeats range of 4-34.

#### **7.3 MSI analysis**

Multiplex PCR for 12 cases and 12 controls was carried out using blood as control. Each case had three sets of samples, viz. blood, eutopic tissue and ectopic tissue. Hence 36 multiplex PCR reactions were set up for cases. For the controls two sets of samples were set up, viz. blood and eutopic tissue. Hence 24 multiplex PCR reactions were set up for controls.

Fig. 2. Photograph of a 2% Agarose gel stained with ethidium bromide to resolve the trinucleotide CAG repeats in AR gene encompassing Exon 1.The bands appeared between

Fig. 3. Native PAGE showing heterozygous and homozygous alleles of AR gene: 200 samples processed with 12% polyacrylamide gel and silver staining showed band sizes

Multiplex PCR for 12 cases and 12 controls was carried out using blood as control. Each case had three sets of samples, viz. blood, eutopic tissue and ectopic tissue. Hence 36 multiplex PCR reactions were set up for cases. For the controls two sets of samples were set up, viz. blood and eutopic tissue. Hence 24 multiplex PCR reactions were set up for

ranging from 156 bp – 238 bp which fall within the CAG repeats range of 4-34.

The small repeat changes occurring between 200bp – 300bp and heterozygotic alleles could not be identified on agarose gels. Hence, analysis was done using 12% PAGE for further

200bp – 300bp. The Lane 4 indicates the molecular weight marker.

**7.2 Androgen receptor analysis** 

analysis.

**7.3 MSI analysis** 

controls.

Fig. 4. 2% Agarose gel showing results of multiplex PCR both from endometriotic cases and controls with the specific MSI markers and their band sizes. Lanes 1,2,3 are of a case; Lane 4 is the DNA ladder; Lanes 5,6 are of a control

### **7.4 SSCP (Single Stranded Conformation Polymorphism) analysis**

SSCP was carried out on 15% polyacrylamide gel to identify mobility shift, and/or additional bands at the three selected markers. SSCP PAGE gel showing mobility shift at BAT R II locus in ectopic tissue from a case of endometriosis. 2 cases of endometriosis showed mobility shift in the BAT R II locus associated with TGF beta R II receptor gene. One control showed additional band in D3 locus associated with FHIT gene.

Fig. 5. Lane 1 is the DNA ladder; Lanes 2 and 3 are blood and eutopic tissue of control;

Lanes 4, 5 and 6 are blood, ectopic and eutopic tissue of a case ; Additional bands are seen in all the samples of the case but not in the control, whereas mobility shift of the band is seen in the ectopic tissue only and not in the eutopic tissue of the case.

### **7.5 DD – RT – PCR analysis**

Differential display RTPCR helps in identifying novel genes which are expressed or suppressed in tissues with an altered pathophysiology compared to controls using two sets

Genetic Polymorphisms and Molecular Pathogenesis of Endometriosis 147

The eluted bands were automatically sequenced using MWG-AG, BIOTECH, Bangalore.

**I**. 350bp band showing 60/65 gene sequence showing homology with Shigella species

**II**. 250 bp showing (a) 20/20 gene sequence showing homology with Hepatocellular carcinoma- associated antigen HCA557b, (b) 24/25 gene sequence showing homology with

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

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

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

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

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.

The sequences obtained are shown below.

S. dysenteriae

**8. Discussion** 

studied.

Sequencing of differentially expressed bands:

and molecular pathogenesis of this condition.

factors in the etiology of endometriosis.

(Govindan et al. 2009)

suspicious of endometriosis throughout their reproductive life.

which included S. dysenteriae, S. boydii, S. sonnei.

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

Upper arrow: differential band of 350 bp Lower arrow: faint differential band of 250 bp

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

The eluted bands were automatically sequenced using MWG-AG, BIOTECH, Bangalore. The sequences obtained are shown below.

Sequencing of differentially expressed bands:

**I**. 350bp band showing 60/65 gene sequence showing homology with Shigella species which included S. dysenteriae, S. boydii, S. sonnei.

**II**. 250 bp showing (a) 20/20 gene sequence showing homology with Hepatocellular carcinoma- associated antigen HCA557b, (b) 24/25 gene sequence showing homology with S. dysenteriae
