Clinica Chimica Acta 431 (2014) 118–124

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Invited critical review

New markers in pelvic inflammatory disease Shun-Fa Yang a,b, Tzu-Fan Wu a,1, Hsiu-Ting Tsai c,d, Long-Yau Lin a,e,f, Po-Hui Wang a,e,f,⁎ a

Institute of Medicine, Chung Shan Medical University, 110, Section 1, Chien-Kuo North Road, Taichung 40201, Taiwan Department of Medical Research, Chung Shan Medical University Hospital, 110, Section 1, Chien-Kuo North Road, Taichung 40201, Taiwan c School of Nursing, Chung Shan Medical University, Taiwan d Department of Nursing, Chung Shan Medical University Hospital, Taiwan e School of Medicine, Chung Shan Medical University, Taiwan f Department of Obstetrics and Gynecology, Chung Shan Medical University Hospital, 110, Section 1, Chien-Kuo North Road, Taichung 40201, Taiwan b

a r t i c l e

i n f o

Article history: Received 12 December 2013 Received in revised form 29 January 2014 Accepted 5 February 2014 Available online 11 February 2014 Keywords: Pelvic inflammatory disease Biomarkers Prediction Clinical course Single nucleotide polymorphisms

a b s t r a c t Pelvic inflammatory disease (PID) is a common infection in women of reproductive age. However, diagnosis of PID can be difficult due to the wide variation in the symptoms and signs, ranging from subtle or mild symptoms to severe pain in the lower abdomen. Clinical diagnosis alone has only 87% sensitivity and 50% specificity. Therefore, identifying biological factors that are useful for early diagnosis and correlating their expression with the severity of PID could provide significant benefits to women suffering from PID. Pentraxin 3 (PTX3), E-cadherin, myeloperoxidase, stromal cell-derived factor 1 (SDF-1) and the matrix metalloproteinase-9 (MMP-9)/MMP-2 ratio are potential candidates for detecting PID reliably. As PID is often subtle, highly sensitive PID detection methods are needed to promote the prevention of severe sequelae. Growth arrest-specific 6 (Gas6), in combination with its soluble tyrosine kinase receptor, sAxl, could elevate the sensitivity to 92%, which was higher than all other markers tested. Moreover, PTX3, D-dimer and YKL-40 concentrations can predict the clinical course of PID. Although single nucleotide polymorphisms of biomarker genes are not associated with the development of PID, myeloperoxidase SNP −463 G/A and SDF-1 SNP 801 G/A may affect the aggravated expression of their biomarkers in PID. © 2014 Published by Elsevier B.V.

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Introduction to pelvic inflammatory disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Clinical features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biomarkers in pelvic inflammatory disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Pentraxin 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Matrix metalloproteinase-9 level and its ratio to matrix metalloproteinase-2 . . . . . . . . . . . . . . . 2.3. Neutrophil gelatinase associated lipocalin and its complex with matrix metalloproteinase-9 . . . . . . . . 2.4. Growth arrest-specific 6 and its soluble tyrosine kinase receptor sAxl . . . . . . . . . . . . . . . . . . 2.5. Other biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biomarkers and clinical course of pelvic inflammatory disease . . . . . . . . . . . . . . . . . . . . . . . . . Single nucleotide polymorphisms and biomarker expression and pelvic inflammatory disease . . . . . . . . . . . 4.1. Myeloperoxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Stromal cell-derived factor 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. E-cadherin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Urokinase-type plasminogen activator (uPA) and soluble urokinase-type plasminogen activator receptor (suPAR) 4.5. Other biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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⁎ Corresponding author at: Institute of Medicine, Chung Shan Medical University, Department of Obstetrics and Gynecology, Chung Shan Medical University Hospital, 110, Section 1, Chien-Kuo North Road, Taichung 40201, Taiwan. Tel.: +886 4 24739595x21721; fax: +886 4 24738493. E-mail address: [email protected] (P.-H. Wang). 1 Equal contribution as first authors.

http://dx.doi.org/10.1016/j.cca.2014.02.004 0009-8981/© 2014 Published by Elsevier B.V.

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5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction to pelvic inflammatory disease

2. Biomarkers in pelvic inflammatory disease

Pelvic inflammatory disease (PID) is a common infection in women of reproductive age [1]. It is an ascending polymicrobial infection that leads to inflammation of the upper genital tract that results from microorganisms that colonize the endocervix and ascend to the endometrium and fallopian tubes and may also affect the neighboring pelvic organs [2,3]. Subsequently, neutrophils are recruited in abundance in the infected lesions and, thereafter, cause many disease manifestations, including endometritis, pelvic peritonitis, tubal abscess, and salpingitis [3–5]. An infectious blended mass of ovary and fallopian tube, termed as a tuboovarian abscess (TOA), often arises as a consequence of PID, which may subsequently result in severe sequelae such as tubal factor infertility, ectopic pregnancy, and chronic pelvic pain.

C-reactive protein (CRP) was first discovered in 1930 by Tillet and Frances, who identified a substance in the sera of patients with acute pneumococcal pneumonia [12]. It forms a precipitate when combined with polysaccharide C of Streptococcus pneumoniae. CRP is synthesized by hepatocytes, and its production is stimulated by cytokines, particularly IL-6, IL-1 and tumor necrosis factor, in response to infection or tissue inflammation [13]. CRP is an acute phase protein widely used as an indicator of infectious or inflammatory conditions. It has been studied in the diagnosis and prediction of severity of pelvic inflammatory disease [14]. Lehtinen et al. revealed that the overall sensitivity and specificity of CRP in the diagnosis of PID were 74% and 67%, respectively, using a cutoff level of 2.0 mg/dl [15]. Though sensitive, its nonspecific nature limits its clinical use [16].

1.1. Clinical features 2.1. Pentraxin 3 Low abdominal pain is the main presenting symptom in women with PID. Because of the potential for significant consequences if treatment is delayed, treatment should be initiated based on clinical judgment without waiting for confirmation from laboratory or imaging tests. Even if these symptoms are mild, PID is included into differential diagnosis in reproductive women who present with lower abdominal or pelvic pain and cervical motion or pelvic tenderness. Microorganisms that cause PID may be divided into two categories: sexually transmitted pathogens and lower genital tract flora. Sexually transmitted infections include Neisseria gonorrhoeae, Chlamydia trachomatis, and Mycoplasma genitalium [6,7]. Endogenous, bacterial vaginosis-associated lower genital tract organisms include Prevotella spp., Peptostreptococci sp., Gardnerella vaginalis, Escherichia coli, Haemophilus influenza, and aerobic streptococci [8,9].

1.2. Diagnosis Based on the Centers for Disease Control and Prevention (CDC) criteria, the diagnosis of PID is made if the patients have lower abdominal pain or pelvic pain of no other origin accompanied by one of the following criteria: uterine tenderness, adnexal tenderness, or cervical motion tenderness. In addition to these criteria, the patient should have at least one of the following minor criteria: oral temperature more than 38.3 °C, abnormal cervical or vaginal mucopurulent discharge, an abundance of white blood cells (WBCs) on microscopic inspection of vaginal secretions, elevated erythrocyte sedimentations, elevated C-reactive protein (CRP), or laboratory documentation of N. gonorrhoeae or C. trachomatis [2]. However, diagnosis of PID is sometimes difficult because of the wide variation in the symptoms and signs, ranging from subtle or mild symptoms to severe pain in the lower abdominal region. Delays in diagnosis and treatment probably contribute to inflammatory sequelae in the upper reproductive tract and, subsequently, lead to severe sequelae. Prevention of these sequelae depends on the clinician's alertness to make an early diagnosis and initiate aggressive treatment. There is no single symptom, physical finding, or laboratory test that is sensitive or specific enough to definitively diagnose PID [10]. Clinical diagnosis alone is 87% sensitive and 50% specific [11]. It is therefore of value to identify biological factors that could be used for early diagnosis and correlate their expression levels with the severity of PID.

Pentraxins are a superfamily of multifunctional conserved proteins characterized by a cyclic multimeric structure and are regarded as acute phase proteins due to their elevated production in response to inflammatory conditions [17]. Pentraxin 3 (PTX3) was the first member of this family to be discovered [18]. Its plasma level is low in normal conditions but increases rapidly during sepsis, endotoxic shock and other inflammatory and infectious conditions [19–23]. Elevated plasma PTX3 was also observed in a restricted set of autoimmune disorders [24]. In small vessel vasculitis, PTX3 levels are correlated with the clinical activity of the disease and represent a candidate marker for monitoring the disease [25]. Chang et al. found that the plasma PTX3 level is significantly elevated in patients with PID compared to controls [26]. In their study, women who were breast feeding; pregnant; taking antibiotics due to other infection or inflammatory conditions, such as sepsis or autoimmune disorders; suspected of having tumors originating from any organ, such as cervix or ovary; or had undergone a gynecologic operation within 3 weeks before admission were excluded. If the cutoff level of the plasma PTX3 concentration was determined at 2.87 ng/ml, the adjusted odds ratio with its 95% confidence interval of plasma PTX3 concentration for PID risk was 12.5 and 3.87–41.37 after adjusting the plasma PTX3 (positive test if ≥cutoff level 2.87 ng/ml) and CRP levels (positive Table 1 The sensitivity, specificity and likelihood ratio for a positive result (LR+) of biomarkers for the prediction of pelvic inflammatory disease.a Biomarkers

Sensitivity

Specificity

LR+b

CRP [15] Pentraxin 3 [26] MMP-9 [30] MMP-9/MMP-2 ratio [30] NGAL [40] NGAL/MMP-9 complex [40] Gas6 [46] sAxl [46] Gas6 and sAxl [46] Osteopontin [54]

74.0% 84.4% 76.6% 76.6% 76.6% 78.1% 76.6% 75.0% 92.2% 82.3%

67.0% 81.4% 65.0% 68.8% 57.1% 58.6% 65.7% 60.0% 42.9% 60.0%

2.24 4.54 2.17 2.46 1.79 1.89 2.23 1.88 1.61 2.06

CRP, C-reactive protein; MMP-9, matrix metalloproteinase-9; NGAL, neutrophil gelatinase associated lipocalin; Gas6 and sAxl, plasma growth arrest-specific 6 and its soluble tyrosine kinase receptor, sAxl. a The data were collected or calculated from the cited references. b LR+ = sensitivity/1-specificity.

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test if ≥cutoff level 0.7 mg/dl), as well as WBC (positive test if ≥cutoff level 8000/mm3) and neutrophil counts (positive test if ≥cutoff level 5500/mm3), using a logistic regression model. Moreover, PTX3 exhibited higher sensitivity (84.38%; Table 1) and a lower false-negative rate (15.63%) than CRP (79.69% and 20.31%, respectively) for detecting PID [26]. In addition, plasma PTX3 had a significant correlation with CRP, WBC counts and neutrophil counts in PID patients. 2.2. Matrix metalloproteinase-9 level and its ratio to matrix metalloproteinase-2 Matrix metalloproteinases (MMPs) are key proteinases involved in extracellular matrix (ECM) degradation in physiological and pathological conditions [27]. To date, there are more than 20 MMPs recognized to affect ECM proteolysis [28,29]. Wang et al. found that MMP-2 alone is not predictive of PID and first utilized the plasma MMP-9 and MMP-9/ MMP-2 ratio to predict PID [30]. In patients with PID, 80% have a plasma MMP-9 level higher than 115 ng/ml or a MMP-9/MMP-2 ratio higher than 2.15. The sensitivities of the MMP-9 level and the MMP-9/MMP-2 ratio for the prediction of PID were both 76.6%, with specificities of 65.0% and 68.8%, respectively. Although there were significant correlations between MMP-9 and WBC or neutrophil counts, as well as significant correlations between the MMP-9/MMP-2 ratio and WBC or neutrophil counts, there were no significant correlations between the MMP-9 and CRP levels or between the MMP-9/MMP-2 ratio and CRP. The plasma CRP level was also significantly decreased after treatment for PID. However, they did not compare the sensitivity and specificity between the MMP-9 and CRP levels or between the MMP-9/MMP-2 ratio and the CRP level [30]. 2.3. Neutrophil gelatinase associated lipocalin and its complex with matrix metalloproteinase-9 Neutrophil gelatinase-associated lipocalin (NGAL), also referred to as lipocalin 2, is a 25-kDa secretory glycoprotein that was originally identified as a glycoprotein in complex with MMP-9 in human neutrophils [31–33]. It binds with MMP-9 covalently to form a Stable 135-kDa disulfide-linked heterodimer [34]. NGAL is regarded as a “stress protein”, which is induced and produced by numerous types of cells after exposure to various stress conditions [35]. It is also an acute phase protein that is up-regulated in human epithelial cells under different inflammatory conditions [36,37]. The NGAL/MMP-9 complex has been demonstrated to prevent MMP-9 from degradation and increase MMP-9 enzyme activity in breast and gastric cancers [38,39]. Tsai et al. first demonstrated that the levels of plasma NGAL or NGAL/MMP-9 complex are increased in patients with PID compared with those in normal controls and decrease significantly after treatment [40]. The level of the plasma NGAL/MMP-9 complex has a significant correlation with the level of NGAL. Tsai et al. also found that plasma NGAL and the NGAL/MMP-9 complex may act as diagnostic adjuvant biomarkers for PID. In patients with PID, approximately 80% have plasma levels of NGAL or NGAL/MMP-9 complex level N10.04 ng/ml or 2.33 ng/ml, respectively. 2.4. Growth arrest-specific 6 and its soluble tyrosine kinase receptor sAxl Growth arrest-specific protein 6 (Gas6) was initially identified as a protein produced by growth-arrested fibroblasts. Gas6 is expressed in many tissues, including vessel endothelial cells, bone marrow cells, lungs and ovaries [41], and has been demonstrated to be important for the activation of endothelium and the invasion of inflammatory cells into the vessel walls [42]. Tyro3, Axl and Mer are members of the TAM family of receptor tyrosine kinases, and Gas6 has growth factorlike properties through its interaction with receptor tyrosine kinases of the TAM family [43,44]. Soluble Axl (sAxl) has a higher affinity than Tyro3 and Mer for Gas6 [45]. This family of receptors has been recognized to act as regulators of

inflammation [42]. Wang et al. found that the concentrations of plasma Gas6 and sAxl are significantly increased in the patients with PID compared to the healthy controls [46]. Using 7.5 and 15.2 ng/ml as the cutoff concentrations of plasma Gas6 and sAxl to detect PID, the adjusted odds ratios were 4.17 and 4.36, respectively. The correlation between Gas6 and sAxl in the plasma of the patients with PID and the healthy women was significant. The novel application of Gas6 in combination with sAxl as a biomarker has high sensitivity and is superior to CRP in detection of PID, and thus could facilitate earlier diagnosis of PID. Gas6 in combination with sAxl also has better sensitivity than PTX3; thus, the false negative rate for PID detection from these biomarkers is decreased. However, Gas6/sAxl is less specific for PID than PTX3; thus, the false positive rate of PID diagnosis for healthy women is increased using Gas6/sAxl. In addition, the likelihood ratio for a positive result using PTX3 is higher than for Gas6/sAxl. Therefore, if the prevention of early sequelae from subtle PID is not considered, PTX3 is better than Gas6/sAxl when considering only its use as a diagnostic tool. 2.5. Other biomarkers Tsai et al. revealed that the serum level of cathepsin B increases and the level of cystatin C decreases significantly in patients with PID compared to healthy controls [47]. The ratio of cathepsin B to cystatin C increased significantly in patients with PID before they received treatment compared to controls and then decreased significantly after treatment. Osteopontin is a phosphorylated acidic glycoprotein that comprises functional domains for calcium binding, phosphorylation, glycosylation, and extracellular matrix adhesion [48]. It is produced by osteoblasts, osteoclasts, vascular smooth muscle cells, and endothelial cells in addition to activated immune cells such as T cells and macrophages [49–51]. Osteopontin has been found to act as a proinflammatory cytokine [52,53]. Wang et al. showed that the level of plasma osteopontin is elevated in patients with PID compared to that of healthy women and decreases significantly after treatment [54]. When the cutoff level of the plasma osteopontin concentration was set to be 58.53 ng/ml, the adjusted odds ratio of plasma osteopontin for PID risk was 3.87 (95% confident interval: 1.30–11.51). In the studies cited above, the cutoff levels of the tested biomarkers were selected to differentiate women with PID from healthy women. Based on these findings, women with plasma levels of the tested biomarkers ≥ cutoff levels would have increased risk (odds ratio) to suffer from PID. However, the sensitivities, specificities and likelihood ratios for a positive result were used to compare these biomarkers for clinical application (Table 1). We found that the plasma PTX3 levels and the MMP-9/MMP-2 ratio have better sensitivity, specificity and likelihood ratio for a positive result for detecting PID. However, PID is often subtle. As such, high sensitivity PID detection methods are needed to prevent severe sequelae. Measurement of Gas6 and sAxl in combination may elevate the sensitivity to 92%, which is highest among all of the markers discussed herein. To date, no study correlates the plasma levels of these biomarkers with urinary tract infection (UTI), a very common infectious disease; thus their potential clinical utility is not compromised by the presence of UTIs. 3. Biomarkers and clinical course of pelvic inflammatory disease In addition to the prediction of PID, some biomarkers are correlated with the clinical course of PID. Terao et al. found that high CRP levels are independently associated with a poor clinical course of PID, with a cutoff value for CRP at 4.4 mg/dl [55]. Moreover, Demirtas et al. defined CRP N11.5 mg/l to be the best predictor of TOA [56]. They found that a high level of CRP is associated with a longer duration of hospitalization and a higher disease severity, and the level is statistically significantly associated with TOA size of ≥5 cm. Chang et al. found that PTX3 concentration not only predicts the presence of PID with lower false-negative

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rate than CRP but is also affiliated with the presence of TOA [26]. Patients with PID who received surgery tended to have higher plasma PTX3 concentrations than those who did not receive surgery. Additionally, PTX3 exhibited a significant correlation with the length of the hospital stay. Franchi et al. demonstrated that in patients who underwent destructive surgery, pre-surgery D-dimer was more than double that of nonsurgically treated patients before they received medical therapy [57]. Thus, D-dimer levels appear to be correlated to the need for destructive surgery and, consequently, to the severity of the disease. While comparing D-dimer values with CRP, it is evident that D-dimer sensitivity in terms of the identification of severe forms of PID is higher. Although Gas6 and sAxl were predictive of PID, neither Gas6 nor sAxl was associated with the incidence of TOA, surgery, or length of hospital stay [46]. The chitinase-3-like1 (CHI3L1) protein is encoded by the chitinase 3-like 1 (CHI3L1) gene located on human chromosome 1q32.1 [58]. This glycoprotein is often referred to as YKL-40 and is known as a pro-inflammatory cytokine of chitinase family [59,60]. YKL-40 is a 40 kD secreted protein identified by its signature N-terminal sequence of tyrosine (abbreviated as Y), lysine (K) and leucine (L) [61]. Lee showed that the level of plasma YKL-40 is significantly elevated in patients with PID compared to that in controls; however, after age stratification, the significance is restricted to women aged 30 years or older [62]. YKL-40 levels declined significantly after PID patients received treatment. Although both plasma YKL-40 and CRP were elevated in patients with TOA, PID patients with surgery exhibited higher YKL-40 concentrations than those without surgery and only the plasma YKL-40 concentration was significantly associated with the length of the hospital stay.

4. Single nucleotide polymorphisms and biomarker expression and pelvic inflammatory disease Single nucleotide polymorphisms (SNPs) are highly abundant, stable, and distributed throughout the genome; however, these genetic variations are associated with diversity in the population, individuality, susceptibility to diseases, and individual response to medicine [63]. SNPs occur when a single nucleotide in the shared sequence of a gene differs between the members of a species or paired chromosomes in an individual. Single nucleotide polymorphisms are associated with the occurrence and development of certain diseases and may affect susceptibility to PID [63].

4.1. Myeloperoxidase Myeloperoxidase is a lysosomal enzyme that is stored in the azurophil granules of polymorphonuclear leukocytes and is released into the phagosome [64–67]. It may serve as a catalase to catalyze a reaction between hydrogen peroxide and chloride to generate hypochlorous acid (HOCl) for bacteria killing by neutrophils [64,66]. If individuals have a myeloperoxidase deficiency, they are susceptible to severe infection [68]. A base mutation from G to A (G → A) is found for a bi-allelic gene polymorphism at the promoter region position −463 of myeloperoxidase gene on chromosome 17q23 [69,70]. The high-expression bi-allelic G/G genotype has been associated with an increased risk of diseases with inflammatory components, such as Alzheimer's disease [71,72], and multiple sclerosis [73,74], whereas the allele A of this polymorphism has a protective role in local inflammation of tissue damage [75]. Lee et al. revealed that elevated expression of myeloperoxidase may be involved in the pathogenesis of PID and could be useful for the diagnosis of PID [76]. There was no significant difference in the genotype distribution of myeloperoxidase between PID patients and controls. However, the plasma myeloperoxidase concentration was significantly elevated in PID patients with the G/A allele of the myeloperoxidase gene compared to those with the G/G allele.

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4.2. Stromal cell-derived factor 1 Stromal cell-derived factor 1 (SDF-1), the C-X-C (Cys-X-Cys, where X is any amino acid) subfamily of proinflammatory chemokines [77], is produced by stromal cells and primarily targets neutrophils, monocytes, T-lymphocytes, and basophils [78]. SDF-1 has been implicated to have roles in cell migration, cell adhesion, neutrophil activation, and the process of inflammation [79–82]. The SDF-1 gene is located on chromosome 10, and a SNP (G → A) at position 801 of the 3′-untranslated gene region leads to an SDF-1 chemokine gene polymorphism [83,84]. The SDF-1–3′A (3′A/3′A) homozygous genotype has been suggested to be associated with the alteration of SDF-1 protein production [85,86]. Moreover, the SDF-1 genotype for the 3′A allele was reported to be implicated in the pathogenesis of chronic myelogenous leukemia [87]. Tsai et al. found that the level of plasma SDF-1α is elevated in patients with PID compared to normal controls and decreased significantly after treatment [88]. There was no significantly different distribution of SDF-1 genotypes between patients with PID and normal controls. PID patients with the SDF-1–3′ A allele were associated with significantly elevated plasma stromal SDF-1α concentration compared to patients with the G/G homozygous genotype. 4.3. E-cadherin Epithelial (E)-cadherin, a member of the cadherin family, is the most important cell-cell adhesion molecule in a variety of epithelial cells and is necessary in tissue architecture maintenance [89,90]. Impaired maintenance of epithelial integrity is a chief process in the development of inflammation [91,92]. Diminished E-cadherin, resulting in disarray in epithelial features, has been found among patients with inflammatory bowel diseases, including active Crohn's disease and active ulcerative colitis [93]. Two SNPs of E-cadherin have been identified in its promoter region [94]. One is E-cadherin SNP −160 C/A, and the other is SNP −347 G/GA; both are on chromosome 16. Genetic epidemiologic studies recently showed that the A allele of E-cadherin −160 C/A SNP is more frequent in pancreatic carcinoma, and patients with the A/A genotype have poor differentiation and worse disease-specific survival compared to patients with the CC/CA genotype [95]. Shin et al. found that individuals with the GA genotype have a higher risk of developing sporadic colorectal cancer compared to those with the G genotype of E-cadherin − 347 SNP [96]. Tsai et al. demonstrated that elevated soluble E-cadherin levels in plasma expression are involved in the pathogenesis of PID and useful for the diagnosis of PID [97]. However, there was no association between E-cadherin genotypes and PID. Additionally, there was no significant relationship between plasma concentrations of E-cadherin and their genetic polymorphisms. We found that myeloperoxidase, SDF-1 and E-cadherin can be used to detect PID (Table 2). However, E-cadherin has the highest sensitivity and SDF-1 has best likelihood ratio for a positive result of these three markers. Additionally, myeloperoxidase SNP − 463 G/A and SDF-1 SNP 801 G/A may affect the aggravated expression of these biomarkers in PID, although these SNPs do not seem to be associated with the development of PID. 4.4. Urokinase-type plasminogen activator (uPA) and soluble urokinase-type plasminogen activator receptor (suPAR) The plasminogen activation system has been reported to play an important role in mediating fibrin degradation in inflammatory processes [98,99]. Plasminogen activator (PA) binds to a cellular receptor and then converts the inactive plasminogen to the active enzyme plasmin [100,101]. Two plasminogen activators mediating inflammatory processes include urokinase (uPA) and tissue type (tPA). Urokinase plasminogen activator and its receptor (uPAR) are thought to regulate pericellular matrix degradation, cell migration and cell adhesion during the inflammatory process [102–104]. uPAR may become cleaved at its

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Table 2 The sensitivity, specificity and likelihood ratio for a positive result (LR+) of biomarkers for the prediction of pelvic inflammatory disease (PID) and the correlation among PID susceptibility and their gene single nucleotide polymorphisms (SNPs) and expression.a Biomarkers

Sensitivity

Specificity

LR+b

SNP and the correlation of genotype or allele with PID

Elevated biomarker levels for genotype or allele in PID

Myeloperoxidase [76]

61.4%

84.0%

3.84

Stromal cell-derived factor 1(SDF-1) [88]

77.3%

88.0%

6.44

−463 G/A, Elevated myeloperoxidase for G/A compared to G/G 801 G/A, 3′-untranslated gene region Elevated SDF-1 for G/A or A/A compared to G/G

E-cadherin [97]

81.3%

80%

4.06

−463 G/A no 801 G/A, 3′-untranslated gene region no −160 C/A, −347 G/GA no

a b

−160 C/A, −347 G/GA no

The data were collected or calculated from the cited references. LR+ = sensitivity/1-specificity.

cell surface anchor to form a free soluble receptor (suPAR) in blood, and increased plasma suPAR concentrations have been noted in patients with rheumatoid arthritis [102]. Single nucleotide polymorphisms in uPA system genes, such as uPA SNP 1583 C/T located at the untranslated region-3 on chromosome 10 and uPAR SNP −516 T/C on chromosome 19, have been demonstrated to affect gene expression [105]. Yeh et al. found that the levels of plasma uPA and suPAR are significantly increased in PID patients compared to normal controls, but only the suPAR level is decreased significantly after treatment in the same patients [106]. The lack of association between PID susceptibility and gene polymorphisms of uPA or suPAR, as well as between gene polymorphisms of uPA or suPAR and their gene expression, may indicate that these gene variants do not have a strong impact on the susceptibility of patients to PID. 4.5. Other biomarkers The recruitment and activation of monocytes and T-lymphocytes through activation of the monocyte chemotactic protein 1 (MCP-1), a potent chemotactic factor for monocytes, play a pivotal role during inflammatory processes [107]. MCP-1 may be an important mediator in chronic inflammation [108,109]. An SNP of the G allele of MCP-1 − 2518 G/A in the regulatory region of chromosome 17 has been found to increase MCP-1 gene expression [110]. Although plasma MCP-1 has been reported to be significantly elevated in patients with PID compared to that in normal controls and decreased significantly compared to that in patients with PID after they received treatment [111], there is no association between the MCP-1 −2518 G/A SNP and its gene expression levels and PID susceptibility. Concerning osteopontin SNPs, no significant difference has been found in the genotype or allele distribution of osteopontin SNPs, T/C (located at coding region in the exon 7 of osteopontin gene on chromosome 4) [112] or C/A (located at 3′-untranslated region in the exon 7 of osteopontin gene on chromosome 4) [112–114], between patients with PID and normal controls [54]. Plasma osteopontin concentration is also not associated with osteopontin polymorphisms. 5. Conclusions Pentraxin 3, E-cadherin, myeloperoxidase, SDF-1 and the MMP-9/ MMP-2 ratio may detect PID with a better likelihood ratio for a positive result [26,30,76,88,97]. In addition to the clinical manifestations of PID, they may be added as adjuvant tools for the diagnosis of PID. Because PID is often subtle, it should be diagnosed early and treated in time to prevent severe sequelae such as tubal infertility. High sensitivity PID detection methods reduce the probability of not diagnosing patients that have PID. sAxl, the soluble tyrosine kinase receptor of Gas 6, in combination with Gas6 may elevate the sensitivity to 92%, which is the highest sensitivity among all of the markers examined, including CRP [46]. Therefore, Gas6/sAxl should be suggested to detect PID to prevent severe sequelae. Moreover, PTX3 and YKL-40 concentrations can

predict TOA and the clinical course of PID [26,62]. Myeloperoxidase SNP − 463 G/A and SDF-1 SNP 801 G/A 3′-untranslated gene regions are not correlated with the development of PID [76,88]. However, concentrations of plasma myeloperoxidase are elevated for SNP −463 G/A compared to G/G in patients with PID [76]. Additionally, levels of SDF-1 are increased for SNP 801 G/A or A/A in comparison with G/G in PID patients [88]. Although gene SNPs of the biomarkers mentioned above do not seem to be associated with the development of PID, myeloperoxidase SNP − 463 G/A and SDF-1 SNP 801 G/A may affect the aggravated expression of these biomarkers in PID.

Acknowledgments This article was supported by grants from the Taiwan National Science Council (NSC 102-2314-B-040-014-MY3; approved by the Chung Shan Medical University Hospital Institutional Review Board, CSMUH IRB CS12219) and Chung Shan Medical University Hospital (CSH-2012-D-003, CSH-2013-D-001; approved by the Chung Shan Medical University Hospital Institutional Review Board, CSMUH IRB CS12218).

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