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PRETERM: PREDICTIVE MARKERS TO DIAGNOSE
Le Gia Vinh*, Nguyen Viet Trung**
Nguyen Duy Bac**, Dao Nguyen Hung**
ABSTRACT
There has been some improvement in the treatment of preterm infants, which helped to increase their chance of survival. However, the rate of premature birhts is still globally increasing. Infants with preterm are most at risk of developing severe medical conditions that can affect the respiratory, gastrointestinal, immune, central nervous, auditory and visual systems. That can also lead to long-term conditions, such as cerebral palsy, mental retardation, learning difficulties, including poor health and growth. So many people believe that a better understanding of why preterm births occur, how do diagnose and a strategic focus on prevention? will help to improve the health of infants and reduce healthcare costs.
I. INTRODUaiON
Preterm delivery is one of the most common causes of perinatal morbidity and martality in developed countries. Preterm birth is those that occur at less than 37 week' gestational age, however, the low-gestational age cutoff, or that used to distinguish preterm birth from spontaneous abortion, varies by location [1]. According to the Worid Health Organisation, worldwide in 2010, preterm deliveries accounted for 1 to 10 births [1]. In the USA, the preterm delivery rate is 12-13%; in Europe and other developed coimtries, reported rates are generally 5-9% [5].
Preterm births account for 75% of perinatal mortality and more than half the long-term morbidity [1]. Preterm births can occur for three different reasons. According to, roughly one- third are medically indicated or induced; delivery is brought forward for the best interest of the mother or baby. Another third occurs because the membranes rupture, perior to labour, called preterm pemature rupture of membranes. Lastly, spontanous contractions can develop.
In clinical practice, the women who experience regular contractions, increased vaginal discharge, pelvic pressure labour. While this is a good measure, Mangham et al., suggest that clinical methods for diagnosing preterm labour are insufficient [4].
Predicting preterm birth and diagnosing preterm labour cleariy have important consequences, for both health and the economy. Effective predictive markers of prerterm births could contribute to improving prevention, through appropriate medical and lifestyle interventions.
II. CERVICAL ASSESSMENT II. CERVICAL ASSESSMENT
Traditionally, the assessment of symptomatic patients for preterm delivery has involved digital examination of the cervix and monitoring of contractions. The classic digital examination assesses the position, effacement, softness, and dilatation of the cervix. Bishop
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score is a digital scoring system. The total score is achieved by assessing the five components on vaginal examination, cervical dilatation, cervical effacement, cervical consis-tency, cervical position and fetal station. It is traditionally used to determine how successfiil an induction of labor might be but in some countries it is also used to predict preterm birth.
Cervical sonography
The most accurate and reproducible method of cervical evaluation is transvaginal ultrasonography (TVU). Sonographic measurement of cervical length is an established predictor for preterm birth. The shorter the sonographic cervical length, the higher the risk of spontaneous preterm delivery. However there is no agreement on what a sonographic short cervix is. Some investigations have proposed a cutoff of 25 mm while others prefer a cutoff of 15 mm. When using a cutoff of 5 mm at 22 weeks of gestation a subgroup com-prising about .5 % of the female population at particularly high risk for early preterm delivery was identified [4]. Cervical length measurement is a useful tool but it requires a change in current ultrasound practices. Measurement of cervical length takes about 5 min and it could be carried out at the time of the second scan [6]. Sonographic cervical length is not a comprehensive screening test for spontaneous preterm delivery due to the limitations in the sensitivity of the method. The sensitivity is greater than 50 %, usually 60-80 % [7] meaning that not all women who will have a spontaneous preterm birth have a short cervix at mid-trimester.
III. BIOCHEMICAL MARKERS
The term biochemical marker is not well defined. The following definition has been suggested: "a parameter which can be measured in a biological sample and which provides information on an exposure, or on the actual or potential effects of that exposure in an individual or in a group."
The clinical utility of biochemical markers is that they can give objective results when measured with an accurate test.
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Figure I. Association between ultrasonographic cervical length measured at 23 weeks and preterm birth (>32 weeks). [8]
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Fetalfibronectin
Fetal fibronectin (fFN), an isoform of fibronectin, is a complex adhesive glycoprotenin with a molecular weight of approximately 500000 daltons. Matsuura and co-workers have described a monoclonal antibody called FDC-6, which specifically recognizes III-CS, the region defining the fetal isoform of febronectin. Immunohistochemical studies of placentae have shown that fFN is confined to the extracellular matrix of the region defining the juncfion of the maternal and fetal units within the uterus.
Fetal fibronectin can be detected in cervicovagina! secretion of women throughout pregnancy by use of a monoclonal andbody based immunoassay. Fetal fibronectin is elevated in cervicovaginal secertions during eariy pregnancy but is diminished from 22 to 35 weeks in normal pregnancies. The significance of its presence in the vagina during the early weeks of pregnancy is not understood. However, it may simply reflect the normal growth of the extravillous trophoblast population and the placenta. Detection of fFN in cervicovaginal secretions between 22 weeks, 0 day and 34 weeks, 6 days gestation is reported to be associated with preterm delivery in symtomatic and between 22 weeks, 0 days and 30 weeks, 6 days in asymptomatic pregnancy women [22].
Ph-IGFBP-1
Phosphorylated insulin-like growth factor binding protein (phlGFBP-) is a protein secreted by decidual cells. When the delivery is approaching, fetal membranes begin to detach from the decidua and phlGFBP-leaks into cervical secretions. Detection of phlGFBP- in cervical secretions of symptomatic patients is a marker for prediction of preterm delivery.
phlGFBP can be measured with a commercial rapid immunochromatographic test (Actim Partus, Medix Biochemica). The test result is not affected by urine or seminal plasma but matemal blood may interfere with the assay. The strength of the test is the high negative predictive value (NPV). A negative phlGFBP-indicates a very low risk of PTD and thus that there is no need for additional care beyond the antenatal care program.
Estriol
Estriol (E3) is a naturally occurring derivative of the hormone estrogen. There is a surge in salivary estriol several weeks prior to the onset of spontaneous preterm labor. Therefore, measurement of salivary estriol has been explored as a risk predictor for preterm labor.
SalEST^" is a laboratory technique for measuring salivary estriol levels. The method is not satisfactory. For example, AGOG does not recommend the salivary estriol test as a screening test for premature labor because it produces a high percentage of false positive results and could potentially add significant costs and unnecessary interventions to prenatal care [7]. The positive predictive value of a salivary estriol test is only about 20%, (the negative predictive value is about 98%) [10].
IV. COMBINATION OF MARKERS
Many markers have been reported to be associated with preterm birth. However, few have demonstrated sufficiently good predictive value when used alone. Today's data strongly suggests that a single predictive marker effectively ac-counting for a large proportion of
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premature delivery is imlikely to be found simply because of the multifactorial etiology of PTB. Combination of non-over-lapping biochemical markers may considerably improve the success of any indi-vidual marker to predict PTB. Combination of biochemical markers, cervical length and previous obstetric history may provide a useful screening tool [3] (Figure 2). Several studies have tested the effect of a combination of potential markers for predicting PTB and some of them have shown improved performance.
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Figure 2. Risk of spontaneous PTB at various gestational ages with various combinations of risk factors in nulHparas and multiparas [11].
V. POTENTIAL FUTURE BIOMARKERS Cytokines
Cytokines have been investigated as protein biomarkers of impending prema-ture delivery.
Cytokines are low-molecular weight glycoproteins that mediate the activation of immune cells and coordinate the production and secretion of antibodies and other cytokines. They also stimulate uterine contractions via the production of prostaglandins and perhaps cause preterm cervical ripening and PPROM via stimulation of metalloproteinases [13]. Elevation of pro- inflamma-tory cytokines in maternal serum and cervicovaginal fluids during infection and before parturition has been extensively described. Pro-inflammatory cyto-kines such as IL-Ip, IL-6, IL-8 and TNF-a protect the host against invading micro-organisms [12]. In addition to protective effects this response can also be harm-flil to the host. For this reason the pro- inflammatory immune cascade is tightly regulated. Resolution of the inflammatory process and healing occurs through the activity of anti-inflammatory cytokines such as interleukin receptor antagonist. An imbalance between pro- and anti-inflammatory response has been implicated in the pathogenesis of infection related premature birth [14].
There are compelling evidence that infection gives rise to an inflammatory response and that tiiis inflammation contributes to neonatal tissue injury including brain injury [23]. The mechanisms responsible for the initiation of preterm labor in the setting of infection have been
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the subject of intensive investigation. It has been proposed that the host, fetus and/or mother, signal the onset of labor by activation of pro-inflammatory cytokines such as IL-1, TNF-a, IL- 6, and IL-8. These cytokines are capable of inducing a complex set of biological actions including triggering of prostaglandin biosynthesis, degradation of extracellular matrix and neuroendocrine activation leading to uterine contractions and cervical ripening, described above, culminating in preterm birth [24] (Figure 3).
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TNF-o IL-6 IL-8 Cervical npening
PGE2 ^ ^. ^ PGF2ct PreEerm birth
Figure 3: Pro-inflammatory cytokines interact and induce synthesis of prostaglandins (PGE2 and PGF2a ) which start uterine contractions. Interleukin-8 (IL-8) attracts leucocytes to cervix and hereby induces cervical ripening. The contractions and cervical ripening lead to preterm birth.
Matrix metalloproteinases
Matrix metalloproteinases (MMPs) are responsible for the turnover and degrada-tion of connective tissue proteins. MMPs affect the activity of various cytokines, indicating dual role for MMPs in the activation and inactivation of the inflam-matory system. MMPs are involved as proteolytic enzymes in preterm labor and PPROM as well as in pre-eclampsia, intrauterine growth restriction, chronic lung disease, necrotizing enterocolitis, intraventricular hemorrhage, cystic periventricular leucomalacia and retinopathy of prematurity [15]. MMP-9 is involved in the degradation of the basement membrane and other extracellular matrix components and increase in humans at the time of parturition [16]. Matemal serum MMP-9 concentration raises 24 h before the initiation of labor. Such late prediction is of little value in allowing initiation of preventive steps, but can aid in understanding the mechanisms of PTB [21]. Recent studies sug-gest that increased fetal levels of MMP-9 are involved with PPROM and differ-entiate fetuses with PPROM from those undergoing premature labor with intact membranes [15]. Thus, it has been questioned whether PPROM is simply an ac-cident or whether a sick fetus may initiate preterm parturition by activating the mechanisms responsible for rupture of membranes. The teleologic advantage of the latter would be a more rapid exit from a hostile environment. Alterations in the concentrations of other MMPs (MMP-, -2, -3, 7 _8^ -2, -3 and -4) have alsobeen associated with preterm birth. Since MMPs are involved in
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the perinatal complications of prematurity they are thus potential targets for therapeutic in- tervention [15].
Relaxin
Relaxin is « collagenolytic hormone that causes increased production of tiie ma-trix metalloproteinases. Decidual expression of relaxin is increased in patients with PPROM. Relaxin causes increase in expression MMP-, MMP-3 and MMP-9. Local relaxin coidd therefore cause activation of specific enzyme cascade re-sulting in degradation of membranes [7].
Stress related biomarkers
Empirical evidence, based on prospective population-based studies provide sub-stantial support for the premise that infants of mothers experiencing high levels of psychological or social stress during pregnancy are at significantly increased risk for preterm birth and low birth weight. Stress is known to evoke a variety of adaptational responses, including stimulation of the hypothalamic-pituitary-adrenal (HPA) axis. Several studies have shown elevated levels of placental corticotrophin releasing hormone, CRH to be implicated in spontaneous preterm birth. Stress-related physiological responses may thereby contribute to adverse birth outcomes. Evidence suggests that chronic stress is associated with immu- nosuppression. Matemal stress and infection are risk factors in premature birth. However, the nature of the stress-immune system relationship has been littie studied in human pregnancy.
Women at risk for spontaneous preterm delivery may possibly be identified solely by stress related biomarkers e.g., CRH, Cortisol and urocortin, obtained from matemal blood in early pregnancy. Urocortin is a member of the CRH family and according to recent studies its measurement in matemal plasma suggests it may have potential as a new biochemical marker of PTD [16].
Endocannabinoids
Endocaimabinoids are substances produced within the body, which activate cannabinoid receptors. Anandamide (N-arachidonoylethanolamine) was the first endocannabinoid discovered. In recent years it has been demonstrated that high circulating levels of this endogenous cannabinoid, as a result of low expression of its metabolizing enzyme, fatty acid amide hydrolase (FAAH), may contribute to spontaneous miscarriage [17].
PAPP-A
Pregnancy associated plasma protein A (PAPP-A) is a glycoprotein which is se-creted from the trophoblastic tissues of the placenta. Decreased levels of PAPP-A can be seen in pregnancies affected with Down syndrome in the first trimester of the pregnancy. Low levels of matemal serum PAPP-A have been shown to be associated, in the absence of an abnormal karyotype, with an increased risk of preterm or early preterm delivery [18]. Low matemal serum PAPP-A levels during the first trimester may reflect a trophoblast invasion defect in the matemal fetal interface, resulting in subsequent preterm delivery, particularly in the case of PPROM [20].
Genetic polymorphism
Well-established observations support a genetic influence on the risk of pre-mature birth.
Many studies have found associations between single nucleotide polymorphisms (SNP) in
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cytokine genes responsible for inflammation in women who experienced premature birth.
However most studies lack sufficient power to make strong associations. Polymorphisms in the TNF-a, IL-6 and lL-4 genes as well as other inflammatory mediators have been reported.
Fetal poly-morphisms have also been evaluated. Candidate gene studies can be used in premature birth to understand the contribution of DNA polymorphism/genetic predisposition to disease risk. SNP analysis may reveal opportunities even for pre-pregnancy testing for premature birth. Besides developing assays for these SNP genotypes one of the major challenges is to investigate further the molecular mechanisms behind already established SNPs associated with premature birth.
Cervicovaginal markers
A case-control proteomic study identified several proteins with greater differen-tial expression than fFN when comparing an asymptomatic group and a symp-tomatic group.
Among these proteins were calgranulins, annexins, SOO calcium binding protein A7, a-acid glycoprotein, and a- antitrypsin precursor. Further characterization of these markers in a large cohort of subjects may provide the basis for a new test for early, noninvasive prediction of PTB. [19]
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