Genetic Variants in Fibrinolytic System-Related Genes in Infertile Women With and Without Endometriosis

Abstract

Aims: The aim of this study was to evaluate urokinase-type plasminogen activator gene (uPA) and thrombin-activatable fibrinolysis inhibitor gene (TAFI) genotypes in a group of infertile women with and/or without endometriosis and controls. Methods: A case-control study comprising 180 infertile women with endometriosis, 68 women with idiopathic infertility, and 152 fertile women as controls was carried out. Detection of uPA (C422T/rs2227564) and TAFI (G438A/rs2146881) polymorphisms was performed by TaqMan polymerase chain reaction. The results were statistically analyzed and a p-value of <0.05 was considered significant. Results: We found no association among both uPA or TAFI polymorphisms and endometriosis-related infertility (p=0.920 and p=0.356, respectively) or idiopathic infertility (p=0.502 and p=0.392, respectively) comparing to controls, even considering minimal/mild and moderate/severe endometriosis separately. Both uPA and TAFI polymorphisms were in Hardy-Weinberg equilibrium for all studied groups. The combinatory analysis of both uPA and TAFI polymorphisms to endometriosis-related infertility, idiopathic infertility, and control group showed no statistical difference to any combination. Conclusion: The data suggest that, in the Brazilian population, genetic variations in both uPA and TAFI were not relevant to endometriosis and/or infertility.

Introduction

Endometriosis is a common gynecological disease, defined as the growth of endometrial tissue outside the uterine cavity that often results in a vast array of gynecological problems including dyspareunia, dysmenorrhea, pelvic pain, and infertility (Giudice and Kao, 2004; Barbosa et al., 2009, 2010). It affects 3%-10% of women in their reproductive years and 20%-50% of women with infertility. Immune system disorders, genetic predisposition, altered peritoneal environment, and endometrial alterations are believed to increase the susceptibility to disease (Ramón et al., 2005; Bianco et al., 2009; Gomes et al., 2010; Vilarino et al., 2011).

The plasminogen activator system may be involved in the endometriosis physiopathology, wherein local extracellular proteolysis plays a crucial role. There is evidence of a modified fibrinolytic activity in the eutopic endometrium of women with endometriosis that could result in endometrial fragments with a high potential to adhesion onto the peritoneal lining, degradation of the extracellular matrix components, invasive growth by pericellular proteolysis, and cell migration into the surrounding tissue (Gilabert-Estelles et al., 2006).

Some evidence suggests that the eutopic endometrium from women with endometriosis shows an abnormal expression of several components of the plasminogen activator system, such as urokinase-type plasminogen activator gene (uPA) (Gilabert-Estellés et al., 2003; Ramón et al., 2005). uPA is a serine protease that is mainly implicated in cellular proteolysis. Besides its fibrinolytic activity, uPA also regulates the cell migration process under physiologic and pathological conditions, such as angiogenesis, embryo implantation, inflammation, and tumor metastasis (Irigoyen et al., 1999; Binder et al., 2007).

Moreover, it has also been reported that thrombin-activatable fibrinolysis inhibitor gene (TAFI) may play a role as a modulator of the cellular functions of the plasminogen activator system (Swaisgood et al., 2002; Guimarães et al., 2007; Zorio et al., 2008), as it can tune angiogenesis and matrix proteolysis by controlling plasminogen binding to plasma clot matrix and to cell surface, on one hand, and by regulating cell migration, on the other hand (Guimarães et al., 2007; Zorio et al., 2008).

The plasminogen activator inhibitor-1 (PAI-1) gene, a member of the serine protease inhibitor family, is a main regulator of the endogenous fibrinolytic system (Zorio et al., 2008). Our group previously evaluated PAI-1 (4G/5G) genotypes in a group of infertile women with or without endometriosis. The genotype frequencies were statistically different between infertile women with endometriosis and controls (p=0.003), suggesting that PAI-1 4G/5G polymorphism may be associated with a risk of endometriosis-associated infertility (Gonçalves-Filho et al., 2011).

Based on this observation, we have hypothesized a possible relationship between endometriosis and/or infertility and polymorphisms (uPA and TAFI) of fibrinolytic system-related genes.

Material and Methods

Patients

One hundred eight infertile women with endometriosis (mean age: 35.4+2.1 years) from the Endometriosis Outpatient Clinic of the Human Reproduction and Genetics Center of Faculdade de Medicina do ABC were studied. Women with endometriosis diagnosed by laparoscopy were selected and classified according to the American Society for Reproductive Medicine (ASRM, 1997), with histological confirmation of the disease. In the endometriosis group, the stage of the disease was found to be minimal/mild (stage I and II) in 76 cases (42.2%) and moderate/severe (stage III and IV) in 104 cases (57.8%). Sixty-eight women with idiopathic infertility (mean age: 34.1+6.0 years) were also selected at the Human Reproduction Center of Faculdade de Medicina do ABC. For the control group, 152 fertile women (mean age: 39.5+3.9 years) were recruited especially for this study among a group of patients having tubal ligation at the Family Planning Outpatient Clinic of Faculdade de Medicina do ABC. In all of them, absence of endometriosis was confirmed.

The cause of infertility was investigated according to the minimum propedeutic procedure for infertile couples: hormonal and biochemical profile, testing for sexually transmitted diseases, imaging examinations, investigation of genetic and/or immunological abnormalities, semen analysis of the partner, hysterosalpingography, hysteroscopy, and laparoscopy (performed in all women up to 36 years old and also in patients over 36 whenever there were symptoms or abnormalities on imaging examinations). If none of these examinations revealed an abnormality, infertility was considered idiopathic. Women with endometriosis who did not achieve pregnancy after at least six natural or induced cycles after laparoscopy were considered infertile. All women whose partner had any factor associated with infertility were excluded.

Clinical data and peripheral blood samples were collected only after explaining the objectives of the study, and all participants signed an informed consent form, as approved by the local research ethics committee (No. 033/2011).

Genotyping

Peripheral blood was collected from each patient and control into an ethylenediaminetetraacetic acid-containing test tube. Genomic DNA was extracted from peripheral blood lymphocytes according to Lahiri and Nurnberg (1991).

Detection of the C422T (rs2227564) polymorphism in the exon 5 region of uPA gene (10q24, MIM 191840, GenBank ID 5328) and G438A (rs2146881) polymorphism in the promoter region of TAFI gene (13q14.11, MIM 603101, GenBank ID 1361) was achieved using the TaqMan polymerase chain reaction (PCR) system with commercially available TaqMan primers and probes, provided by Applied Biosystems^® (Foster City, CA). The assays were performed using TaqMan Universal Master Mix, with ∼50 ng of DNA per reaction. The PCR conditions were as recommended by the manufacturer: 40 denaturation cycles of 15 s at 95°C and 1 min annealing/extension at 60°C.

Statistical analysis

Statistical analyses were carried out using SPSS for Windows 11.0 (SPSS, Inc., Chicago, IL). The chi-squared test was used to detect differences in allele and genotype frequencies between patients and controls and to evaluate the Hardy-Weinberg equilibrium. Genetic Power Calculator (Purcell et al., 2003) was used to estimate the statistical power of the results concerning individual polymorphism data, which showed 83% of power to detect the genetic effects regarding the association with endometriosis-related infertility and <50% of power considering idiopathic infertility for the allele frequencies and sample size in the present study. The odds ratio (OR) was used to measure the strength of the association between the frequencies of studied polymorphisms and endometriosis and/or infertility. Association analysis was performed to evaluate the interaction between the polymorphisms. All p-values were two-tailed, and 95% confidence intervals (CIs) were calculated. A p-value of <0.05 was considered significant.

Results

Genotypes and allelic frequencies of the uPA and TAFI polymorphisms in the infertile women with and without endometriosis and controls are shown in Table 1.

Table 1.

Genotypes and Allele Frequencies of the Urokinase-Type Plasminogen Activator Gene and Thrombin-Activatable Fibrinolysis Inhibitor Gene Polymorphism in Infertile Women with Endometriosis or Idiopathic Infertility and a Control Group

				Genotypes			Alleles
				n (%)	n (%)	n (%)	n (%)	n (%)
Gene	SNP	Population studied	n	CC	CT	TT	C	T	p^a	OR (95% CI)
		Endometriosis-related infertility	180	129 (71.7)	46 (25.5)	5 (2.8)	304 (84.4)	56 (15.6)	0.920	1.01 (0.66-1.54)
uPA	C422T	Minimal/mild endometriosis	76	58 (76.3)	17 (22.4)	1 (1.3)	133 (87.5)	19 (12.5)	0.479	0.78 (0.44-1.38)
	rs2227564	Moderate/severe endometriosis	104	71 (68.3)	29 (27.9)	4 (3.8)	171 (82.2)	37 (17.8)	0.565	1.18 (0.74-1.90)
		Idiopathic infertility	68	52 (76.4)	15 (22.1)	1 (1.5)	119 (87.5)	17 (12.5)	0.502	0.78 (0.43-1.42)
		Controls	152	108 (71.0)	41 (27.0)	3 (2.0)	257 (84.5)	47 (15.5)

				GG	GA	AA	G	A
		Endometriosis-related infertility	180	107 (59.4)	68 (37.8)	4 (2.8)	282 (78.3)	78 (21.7)	0.356	0.83 (0.58-1.19)
TAFI	G438A	Minimal/mild endometriosis	76	44 (57.9)	31 (40.8)	1 (1.3)	119 (78.3)	33 (21.7)	0.507	0.83 (0.52-1.32)
	rs2146881	Moderate/severe Endometriosis	104	63 (60.6)	37 (35.6)	4 (3.8)	163 (78.4)	45 (21.6)	0.438	0.83 (0.54-1.26)
		Idiopathic infertility	68	32 (47.1)	32 (47.1)	4 (5.8)	96 (70.6)	40 (29.4)	0.392	1.25 (0.80-1.96)
		Controls	152	89 (58.5)	50 (32.9)	13 (8.6)	228 (75.0)	76 (25.0)

Versus control group.

OR, odds ratio; CI, confidence interval; SNP, single-nucleotide polymorphism; uPA, urokinase-type plasminogen activator gene; TAFI, thrombin-activatable fibrinolysis inhibitor gene.

Single-marker analysis revealed no association between endometriosis-related infertility and control group considering C422T uPA polymorphism (p=0.920, OR=1.01, 95% CI=0.66-1.54), not even when the women with endometriosis were separated according to disease stage in minimal/mild (p=0.479, OR=0.78, 95% CI=0.44-1.38) and moderate/severe endometriosis (p=0.565, OR=1.18, 95% CI=0.74-1.90). No statistical difference was found between idiopathic infertility and control group (p=0.502, OR=0.78, 95% CI=0.43-1.42).

Considering G438A TAFI polymorphism, similar results were found. No difference was observed between endometriosis-related infertility (p=0.356, OR=0.83, 95% CI=0.58-1.19) and idiopathic infertility (p=0.392, OR=1.25, 95% CI=0.80-1.96) comparing to controls. When we studied the patients with minimal/mild endometriosis and moderate/severe endometriosis separately, no difference was found (p=0.507, OR=0.83, 95% CI=0.52-1.32 and p=0.438, OR=0.83, 95% CI=0.54-1.26, respectively).

All studied groups were in Hardy-Weinberg equilibrium for both uPA and TAFI polymorphisms.

The combinatory analysis of both uPA and TAFI polymorphisms to endometriosis-related infertility, idiopathic infertility, and controls showed no statistical difference to any combination.

Discussion

In this study, we hypothesized that fibrinolytic system-related gene polymorphisms might be involved in the pathogenesis of endometriosis and/or infertility in Brazilian population. We concomitantly examined the C422T polymorphism in uPA gene and G438A polymorphism in the TAFI gene in patients with endometriosis-related infertility or idiopathic infertility and control group and assessed the association of genotype and allele frequencies with the risk of endometriosis or infertility. To our knowledge, this is the first study to evaluate a possible association between these polymorphisms and endometriosis and/or infertility.

We found no association between both uPA or TAFI polymorphism and endometriosis-related infertility or idiopathic infertility comparing to controls, even considering minimal/mild and moderate/severe endometriosis separately. Combinatory analysis also did not show a haplotype associated either to endometriosis or to idiopathic infertility. Our findings suggest that, in the Brazilian population, genetic variations in both uPA and TAFI were not relevant to endometriosis and/or infertility risk.

There were some limitations for the present study. Only one single-nucleotide polymorphism was genotyped for each gene, and these markers may not provide full coverage for genetic test. Besides, serum levels of uPA and TAFI were not measured to correlate them with genetic variation in such genes. Further, our study had a relative low statistical power to detect association between the studied polymorphisms and idiopathic infertility. However, the statistical power is strong enough to detect association considering the endometriosis-related infertility group.

Some authors have hypothesized an association of fibrinolytic system-related gene polymorphisms and endometriosis, such as PAI-1 4G/5G polymorphism. Bedaiwy et al. (2006) found the genotype 4G/4G in 69% of the women with endometriosis and in only 12% of the control group and 5G/5G was present in 3% of the women with endometriosis and in 56% of the controls. In contrast, Ramón et al. (2008) showed a similar PAI-1 genotype distribution in patients and controls. Similar findings were found by Gentilini et al. (2009). Our group (Gonçalves-Filho et al., 2011) previously observed a significant difference between genotypic frequencies of the PAI-1 4G/5G polymorphism in infertile women with endometriosis compared with controls, especially in women with advanced disease. Regarding the infertile women without endometriosis, no significant differences from controls were detected.

Ethnic and geographic variation could influence the distribution of the polymorphisms, leading to a genetic heterogeneity in different populations possibly because of divergent evolutionary lineages resulting in separate clusters of distinct geography (Vogel et al., 2001). So, the same allele may have different patterns of association with markers and haplotypes in different populations (Neale and Sham, 2004).

Genetic studies provide one important approach to define causal pathways influencing endometriosis. Finding genetic variants contributing to complex diseases such as endometriosis is far more difficult, because the contribution of individual genes is small, many genes contribute to an individual's risk of developing the disease, and disease risk is often modified by environment. Many studies are required for both the discovery and replication steps with sufficient power to detect the small effects of any individual variants (Montgomery et al., 2008). Thus, it would be of great interest to characterize the actual relation between fibrinolytic system-related gene polymorphisms and endometriosis and/or infertility in a large number of cases in a different population.

In conclusion, the present study showed that, in Brazilian women, genetic variations in both uPA (C422T) and TAFI (G438A) were not relevant to endometriosis and/or infertility risk.

Footnotes

Acknowledgments

The authors thank FAPESP for granting a Scientific Initiation scholarship (No. 2010/13206-8) to student Ariel Brandes and also thank Tatiana G. Lerner, Juliana S. Teles, and Raqueila Gonçalves for their help in laboratory procedures.

Disclosure Statement

The authors disclose that no conflicts of interest exist.

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