| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
ORIGINAL RESEARCH |
From the 1Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Toronto, and the Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada; and 2Department of Obstetrics and Gynecology and 3Department of Quantitative Health Sciences, Cleveland Clinic Foundation, Cleveland, Ohio.
 |
ABSTRACT |
|---|
TOP
 ABSTRACT PATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
METHODS: In 118 women (75 with laparoscopically confirmed endometriosis and 43 controls), genomic DNA was extracted from blood and the PAI-1 promoter genotype was determined by polymerase chain reaction amplification of DNA using specific primers for the 4G or 5G allele followed by gel electrophoresis. A portion of the polymerase chain reaction product was purified and sequenced to confirm the gel electrophoresis results.
RESULTS: Endometriosis was more likely in patients with 4G/5G (odds ratio 38; 95% confidence interval [CI] 6–229) or 4G/4G (odds ratio 441; 95% CI 53–3,694) compared with 5G/5G PAI-1 genotype. Fifty-two of 75 women with endometriosis (69 %, 95% CI 58–79%) had the 4G/4G genotype compared with only 5 of 43 (12%; 95% CI 4–25%) controls. In contrast, the 5G/5G genotype associated with normal fibrinolysis was found in 2 of 75 (3%; 95% CI 0–9%) women with endometriosis compared with 24 of 43 (56%; 95% CI 40–71%) controls.
CONCLUSION: Hypofibrinolysis, associated with the 4G allele of the PAI-1 gene, was found significantly more often in women with endometriosis compared with controls. Persistence of fibrin matrix could support the initiation of endometriotic lesions in the peritoneal cavity, explaining why some women with retrograde menstruation develop endometriosis while others do not.
LEVEL OF EVIDENCE: II-2
Recently, we showed that cells from endometrial explants can proliferate and invade a 3-dimensional fibrin matrix in vitro resulting in the formation of new glands (immunohistochemically positive for cytokeratin), stroma (positive for vimentin), and blood vessels (positive for CD31), consistent with early endometriosis.10 In keeping with this in vitro model, it is possible that persistence of a fibrin matrix in peritoneal pockets could allow menstrually deposited endometrial fragments to initiate endometriosis in the same fashion. Factors that lead to persistence of fibrin could, therefore, play an essential role in the cause of endometriosis.
Plasminogen activator inhibitor-1 (PAI-1) is a fast-acting inhibitor of plasminogen activation, one of the major determinants of fibrin formation and degradation. Plasminogen activator inhibitor-1 is a linear glycoprotein that is composed of 379 amino acids and has a molecular weight of 48,000 kd.11 It binds rapidly to tissue plasminogen activator (t-PA) and to urinary-type plasminogen activator (u-PA) in a ratio of 1:1,12 forming a stable complex that is cleared from the circulation by hepatic cells.13 The active form of PAI-1, synthesized in platelets and endothelial cells, is unstable, with a half-life of 30 minutes.14
A common single nucleotide insertion/deletion in the PAI-1 promoter has been identified 675 bp upstream from the start codon.15 This polymorphism induces 2 alleles containing either 4 or 5 sequential guanosines (G). Individuals homozygous for the deletion allele (4G/4G) have activated gene transcription, significantly elevated plasma PAI-1 activity, and significantly diminished fibrinolytic activity (hypofibrinolysis) compared with individuals homozygous for the 5G allele (5G/5G) in which a transcriptional repressor16 results in decreased PAI-1 activity. The heterozygous 4G/5G polymorphism is also associated with decreased fibrinolytic activity.17 Of interest, the contribution of PAI-1 genotypes to the plasma PAI-1 levels seems to be greater in women than in men.18 Moreover, the hypofibrinolytic 4G/4G genotype seems to be causally associated with complications in pregnancy, including prematurity, miscarriage, stillbirth, intrauterine growth restriction (IUGR), eclampsia, and abruptio placentae, probably by producing placental insufficiency through reduced clearance of fibrin deposits.19
We hypothesized that genetic variation in the fibrinolytic system could lead to persistence of fibrin matrix in peritoneal pockets after retrograde menstruation in women who develop endometriosis. In the present study, we estimated the gene frequencies of the 4G and 5G deletion/insertion polymorphisms in the promoter of the PAI-1 gene in patients with endometriosis and in laparoscopically identified controls.
|
PATIENTS AND METHODS |
|---|
|
TOP
ABSTRACTPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Genomic DNA was prepared from peripheral blood leukocytes of the patients using a standard column-extraction technique according to the manufacturer’s instruction (Qiagen Inc, Mississauga, Ontario, Canada). The PAI-1 4G/5G-promoter genotype polymorphism was determined by 2 independent polymerase chain reaction (PCR) amplifications. The first PCR was a nonspecific reaction using a foreword primer 5'-TAA CCC CTG GTC CCG TTC A-3'; and reverse primer 5'- TTT TCC TTT GGC GAA CCA G-3' to amplify the 400 bp segment of the PAI-1 gene promoter containing the area of interest. The 400 bp product (Fig. 1A) was purified using the Qiagen DNA purification kit and was sent for automated sequencing in the DNA sequencing facility of the Samuel Lunenfeld Research Institute of Mount Sinai Hospital, Toronto, Ontario, Canada.
|
The second PCR was an allele-specific reaction where the 4G/5G-promoter polymorphism was ascertained by the following primers: 1) insertion 5G allele: 5'-GAC ACG TGG GGG AGT CAG-3' and 2) deletion 4G allele: 5'-GGA CAC GTG GGG AGT CAG-3', each in combination with 3) a common downstream primer 5'- ACC TCC ATC AAA ACG TGG AA -3' and 4) positive control upstream primer 5'-CCAGACAAGGTTGTTGACACA-3'.
The 25-µL PCR mixture contained 50 pmol allele-specific primer, 50 pmol common downstream primer, 2.5 pmol control upstream primer, 1 X PCR buffer, 2.0 mmol/L magnesium chloride, 0.2 mmol/L dNTPs, and 1.25 U Taq polymerase. All of the reagents required for PCR were purchased from Fermentas (Burlington, Ontario, Canada). The thermal cycling conditions were 94°C for 45 seconds, 62°C for 45 seconds, and 72°C for 75 seconds for 35 cycles.
This PCR reaction gave rise to 248 and 299 bp DNA fragments. Electrophoresis of the PCR products was performed on a 1.5 % agarose gel and visualized by staining with ethidium bromide, followed by ultraviolet transillumination (Fig. 1B). Patients and controls were grouped into 4G/4G, 4G/5G, or 5G/5G genotypes according to the presence of the 248 base pair PCR product generated by the 2 allele-specific primers. Automated DNA sequencing of the PAI-1 promoter region was used to confirm gel electrophoresis results in every case. Data including the demographic and clinical variables of patients and controls were collected.
We assessed the association between endometriosis and PAI-1 genotype (allele frequencies), clinical center, demographic variables (age, parity, gravidity, height, weight, and body mass index [BMI]), infertility, and pain using univariable and multivariable logistic regression. Results are reported as odds ratios and 95% confidence interval. A multivariable model was formed using backwards variable selection with a significance criterion of P < .35 to adjust the relationship of interest (endometriosis and PAI-1 genotype) for variables with even mild relationships to endometriosis. A 
2 analysis was used in subgroup analyses to assess the association between gene frequency and selected factors. The Mantel-Haenszel test for trend was used to assess an increase or decrease in the proportion with a given condition (such as pain or infertility) for increasing levels of the genotype considered as an ordinal variable (based on the level of 4G presence—none (5G/5G), half (4G/5G) or both (4G/4G).
With the given sample size of 118 patients (75 endometriosis and 43 controls) we had 80% power to detect an odds ratio of about 6 or more in either the 4G/4G or 4G/5G genotype compared the 5G/5G genotype in the odds of having endometriosis at the 0.05 significance level. The sample size proved sufficient, because the observed odds ratios are much larger. SAS 9.1 statistical software (SAS Institute, Cary, NC) was used for all analyses. A significance level of 0.05 was used.
|
RESULTS |
|---|
|
TOP
ABSTRACTPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
We found a statistically significant difference in the distribution of PAI-1 genotypes between the 2 groups on both univariable and multivariable analysis (Table 1). Patients with the 4G/5G genotype were an estimated 18 times more likely (odds ratio 18; 95% CI 4–88) to have endometriosis than those with the 5G/5G genotype, and those with the 4G/4G genotype were an estimated 125 times more likely (95% CI 23–690) than the 5G/5G genotype (P < .001). On multivariable analysis adjusting for clinical center, gravidity, and infertility, the association was even stronger (Table 1). No statistical interactions among factors in the model were found. Also, 73 of 75 patients (97%; 95% CI 91–100%) with surgically confirmed endometriosis had the 4G/4G or the 4G/5G genotype, a significantly higher percentage than the control group (19/43, 44%; 95% CI 29–60%, P < .001; Odds ratio 46; 95% CI 10–213).
Further analyses were conducted within the endometriosis and control groups to investigate the association between the PAI-1 genotype and the stage of endometriosis and the leading presenting complaint; pelvic pain or infertility (Table 2). When analyzing the genotype as a nominal variable (without ordering among the categories), no significant associations were found. However, pain increased with increasing 4G expression for endometriosis patients (P = .045) when considering genotype as an ordinal variable (increasing amount of 4G), from 5G/5G (0%) to 4G/5G (19%) to 4G/4G (40%), as did the likelihood of having stage III/IV endometriosis (P = .06, borderline significant). For control patients, the likelihood of infertility decreased with increased 4G expression (P = .02).
|
|
DISCUSSION |
|---|
|
TOP
ABSTRACTPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
We further evaluated the association between the PAI-1 genotype, the stage of endometriosis, and the leading presenting complaints, pelvic pain or infertility. On analyzing the genotype as a nominal variable, no significant associations were found with either the stage of the disease or the leading presenting symptom. However, when considering genotype as an ordinal variable, pain increased significantly with increasing 4G expression for endometriosis patients. Similarly, there was a trend toward an increased likelihood of having stage III or IV endometriosis (P = .06, borderline significant). This trend is interesting, because the more fibrinolysis was decreased, the more advanced stage of endometriosis was typically seen. Stage III or IV endometriosis is also associated with more adhesive disease.
It is known that retrograde menstruation is associated with coexistence of fibrin mesh and endometrial fragments. Fibrin formation is ultimately regulated by the proteolytic cleavage of fibrinogen to fibrin, by the activation of factors V, VIII, and XIII, and by enhancement of platelet activation.20 Immunohistochemical staining has demonstrated that tissue factor is increased in sections of decidualized stromal cells from luteal phase and gestational endometrium.21 Progestin-induced endometrial decidualization has also been associated with stimulation of PAI-1 and suppression of u-PA and t-PA, respectively, both in vivo and in vitro.22 Finally, thrombin is able to exert mitogenic and angiogenic effects20,23 and, thereby, promote regeneration of the endometrium during the late menstrual and early proliferative phases. Consequently, carriers of the 4G allele, with higher baseline levels of PAI-1, may also produce increased amounts of PAI-1 in response to retrograde menstruation, thereby leading to further inhibition of fibrinolysis. Persistence of fibrin meshes could trap endometrial fragments in peritoneal pockets for a prolonged time. We believe, therefore, that we have identified a potential causal mechanism that is likely to contribute to the initiation of endometriotic lesions.
Although endometriosis is frequently associated with extensive peritoneal adhesions,24 the relationship of these adhesions to the initiation or activity of the disease is poorly defined. Peritoneal wounds heal without adhesions in some patients, and others develop severe adhesions from similar surgical procedures. Moreover, adhesions can develop at one surgical site and not in another in the same patient.25 An experimental study has demonstrated that mice deficient in t-PA were more susceptible to adhesion formation after a surgical insult than wild-type mice.26 In addition, a recent clinical study demonstrated increased peritoneal adhesions on second-look laparoscopy in endometriosis patients with increased peritoneal fluid PAI-1 activity.27 We believe our findings of increased 4G allele of the PAI-1 gene in endometriosis patients may also clarify the potential link between adhesion formation and the pathogenesis of endometriosis.
Our data show the link between the fibrinolytic system and endometriosis. Our observation could explain why only certain women with retrograde menstruation develop endometriosis whereas others do not. Furthermore, this novel finding could form the basis of a noninvasive diagnostic test of women at risk for developing endometriosis.
|
Footnotes |
|---|
Presented in part at the 20th annual meeting of the European Society for Human Reproduction and Embryology, Copenhagen, Denmark, June 19–23, 2005.
Corresponding author: Robert F. Casper, MD, Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Toronto, and Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Room 876, Toronto, Ontario M5G 1X5, Canada; e-mail: address:rfcasper{at}aol.com.
doi:10.1097/01.AOG.0000220517.53892.0a
|
REFERENCES |
|---|
|
TOP
ABSTRACTPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
2. Sanfilippo JS, Wakim NG, Schikler KN, Yussman MA. Endometriosis in association with uterine anomaly. Am J Obstet Gynecol 1986;154:39–43.[Medline]
3. Olive DL, Henderson DY. Endometriosis and mullerian anomalies. Obstet Gynecol 1987;69:412–5.[Medline]
4. Koninckx PR, Heyns W, Verhoeven G, Van Baelen H, Lissens WD, De Moor P, et al. Biochemical characterization of peritoneal fluid in women during the menstrual cycle. J Clin Endocrinol Metab 1980;51:1239–44.[Abstract]
5. Halme J, Hammond MG, Hulka JF, Raj SG, Talbert LM. Retrograde menstruation in healthy women and in patients with endometriosis. Obstet Gynecol 1984;64:151–4.
6. Liu DT, Hitchcock A. Endometriosis: its association with retrograde menstruation, dysmenorrhoea and tubal pathology. Br J Obstet Gynaecol 1986;93:859–62.[Medline]
7. Kruitwagen RF, Poels LG, Willemsen WN, Jap PH, Thomas CM, Rolland R. Retrograde seeding of endometrial epithelial cells by uterine-tubal flushing. Fertil Steril 1991;56:414–20.[Medline]
8. Blumenkrantz MJ, Gallagher N, Bashore RA, Tenckhoff H. Retrograde menstruation in women undergoing chronic peritoneal dialysis. Obstet Gynecol 1981;57:667–70.
9. Ridley JH. The histogenesis of endometriosis: a review of facts and fancies. Obstet Gynecol Surv 1968;23:1–23.
10. Fasciani A, Bocci G, Xu J, Bielecki R, Greenblatt E, Leyland N, et al. Three-dimensional in vitro culture of endometrial explants mimics the early stages of endometriosis. Fertil Steril 2003;80:1137–43.[Medline]
11. Kruithof EK. Plasminogen activator inhibitors—a review. Enzyme 1988;40:113–21.[Medline]
12. Lindahl TL, Ohlsson PI, Wiman B. The mechanism of the reaction between human plasminogen-activator inhibitor 1 and tissue plasminogen activator. Biochem J 1990;265:109–13.[Medline]
13. Owensby DA, Morton PA, Wun TC, Schwartz AL. Binding of plasminogen activator inhibitor type-1 to extracellular matrix of Hep G2 cells: evidence that the binding protein is vitronectin. J Biol Chem 1991;266:4334–40.
14. Kooistra T, Sprengers ED, van Hinsbergh VW. Rapid inactivation of the plasminogen-activator inhibitor upon secretion from cultured human endothelial cells. Biochem J 1986;239:497–503.[Medline]
15. Dawson S, Hamsten A, Wiman B, Henney A, Humphries S. Genetic variation at the plasminogen activator inhibitor-1 locus is associated with altered levels of plasma plasminogen activator inhibitor-1 activity. Arterioscler Thromb 1991;11:183–90.
16. Dawson SJ, Wiman B, Hamsten A, Green F, Humphries S, Henney AM. The two allele sequences of a common polymorphism in the promoter of the plasminogen activator inhibitor-1 (PAI-1) gene respond differently to interleukin-1 in HepG2 cells. J Biol Chem 1993;268:10739–45.
17. Eriksson P, Kallin B, van ’t Hooft FM, Bavenholm P, Hamsten A. Allele-specific increase in basal transcription of the plasminogen-activator inhibitor 1 gene is associated with myocardial infarction. Proc Natl Acad Sci U S A 1995;92:1851–5.
18. Henry M, Tregouet DA, Alessi MC, Aillaud MF, Visvikis S, Siest G, et al. Metabolic determinants are much more important than genetic polymorphisms in determining the PAI-1 activity and antigen plasma concentrations: a family study with part of the Stanislas Cohort. Arterioscler Thromb Vasc Biol 1998;18:84–91.
19. Glueck CJ, Phillips H, Cameron D, Wang P, Fontaine RN, Moore SK, et al. The 4G/4G polymorphism of the hypofibrinolytic plasminogen activator inhibitor type 1 gene: an independent risk factor for serious pregnancy complications. Metabolism 2000;49:845–52.[Medline]
20. Dennington PM, Berndt MC. The thrombin receptor. Clin Exp Pharmacol Physiol 1994;21:349–58.[Medline]
21. Lockwood CJ, Nemerson Y, Guller S, Krikun G, Alvarez M, Hausknecht V, et al. Progestational regulation of human endometrial stromal cell tissue factor expression during decidualization. J Clin Endocrinol Metab 1993;76:231–6.[Abstract]
22. Lockwood CJ, Krikun G, Papp C, Aigner S, Schatz F. Biological mechanisms underlying the clinical effects of RU 486: modulation of cultured endometrial stromal cell plasminogen activator and plasminogen activator inhibitor expression. J Clin Endocrinol Metab 1995;80:1100–5.[Abstract]
23. Naldini A, Carney DH, Pucci A, Pasquali A, Carraro F. Thrombin regulates the expression of proangiogenic cytokines via proteolytic activation of protease-activated receptor-1. Gen Pharmacol 2000;35:255–9.[Medline]
24. Jirasek JE, Henzl MR, Uher J. Periovarian peritoneal adhesions in women with endometriosis: structural patterns. J Reprod Med 1998;43 suppl:276–80.[Medline]
25. Chegini N. Peritoneal molecular environment, adhesion formation and clinical implication. Front Biosci 2002;7:e91–115.[Medline]
26. Sulaiman H, Dawson L, Laurent GJ, Bellingan GJ, Herrick SE. Role of plasminogen activators in peritoneal adhesion formation. Biochem Soc Trans 2002;30:126–31.[Medline]
27. Hellebrekers BW, Emeis JJ, Kooistra T, Trimbos JB, Moore NR, Zwinderman KH, et al. A role for the fibrinolytic system in postsurgical adhesion formation. Fertil Steril 2005;83:122–9.[Medline]
This article has been cited by other articles:
|
|
 |
|
  C.B. Tempfer, M. Simoni, B. Destenaves, and B.C.J.M. Fauser Functional genetic polymorphisms and female reproductive disorders: Part II--endometriosis Hum. Reprod. Update, September 19, 2008; (2008) dmn040v1. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
