Measurement of serum C-Reactive protein levels in early second trimester as a predictor of preterm delivery

MEASUREMENT OF SERUM C-REACTIVE PROTEIN LEVELS IN EARLY SECOND TRIMESTER AS A

PREDICTOR OF PRETERM DELIVERY

THE TAMIL NADU DR.M.G.R.UNIVERSITY

INSTITUTE OF OBSTETRICS AND GYNECOLOGY MADRAS MEDICAL COLLEGE

CHENNAI.

M.S. OBSTETRICS AND GYNECOLOGY

APRIL 2018

CERTIFICATE

This is to certify that this dissertation titled “MEASUREMENT OF SERUM C- REACTIVE PROTEIN LEVELS IN EARLY SECOND TRIMESTER AS A PREDICTOR OF PRETERM DELIVERY” is the bonafide work carried out by Dr.S.DHAMAYANTHI at the Institute of Obstetrics and Gynaecology, Madras Medical College.

Prof . Dr.D.TAMIL SELVI M.D, D.G.O., Institute of Social Obstetrics and

Kasturba Gandhi Hospital Madras Medical College,

Chennai-8

PLACE:Chennai DATE:

CERTIFICATE

This is to certify that this dissertation titled “MEASUREMENT OF SERUM C- REACTIVE PROTEIN LEVELS IN EARLY SECOND TRIMESTER AS A PREDICTOR OF PRETERM DELIVERY” is the bonafide work carried out by Dr.S.DHAMAYANTHI at the Institute of Obstetrics and Gynaecology, Madras Medical College.

Prof . Dr.PREMA ELIZABETHM.D, D.G.O.,

Institute of Obstetrics and Gynecology, Madras Medical College,

Chennai-8

PLACE:Chennai DATE:

DECLARATION

I solemnly declare that this dissertation entitled, “A STUDY OF MEASUREMENT OF SERUM C-REACTIVE PROTEIN LEVELS IN EARLY SECOND TRIMESTER AS A PREDICTOR OF PRETERM DELIVERY” was prepared by me under the guidance and supervision of Dr.

Prema Elizabeth, M.D, D.G.O., Professor, Department of Obstetrics and Gynecology, Instititue of Obstetrics and Gynecology, Egmore, Chennai.

This dissertation is submitted to The Tamil Nadu Dr.M.G.R Medical University, Chennai in partial fulfilment of the University regulations for the award of the degree of M.S. (Obstetrics and Gynecology).

Place: Chennai Date:

DR.S.DHAMAYANTHI

CERTIFICATE

This is to certify that this dissertation titled “MEASUREMENT OF SERUM C- REACTIVE PROTEIN LEVELS IN EARLY SECOND TRIMESTER AS A PREDICTOR OF PRETERM DELIVERY” is the bonafide work carried out by Dr.S.DHAMAYANTHI at the Institute of Obstetrics and Gynaecology, Madras Medical College.

DR.R.NARAYANA BABU M.D.D.C.H.

PLACE: DEAN, DATE: MADRAS MEDICAL COLLEGE, CHENNAI 600003.

ACKNOWLEDGEMENT

It is a privilege to express my feelings of deep gratitude to my esteemed teacher and my guide Prof . Dr.PREMA ELIZABETHM.D, D.G.O Institute of

Obstetrics and Gynaecology who has not only helped and guided me with her valuable suggestions and advice ,but has also been a constant source of knowledge and inspiration during my entire course of study.

I gratefully acknowledge and sincerely thank our Director Prof Dr. D.TAMIL SELVI M.D DGO Director of Kasturba Gandhi Hospital chennai for her guidance and encouragement given in

fulfilling my work.

I also extend my thanks to all Associate and Assistant Professors of the department for their unrelenting support and encouragement throughout my course.

I am thankful to the Dean Madras Medical college,Chennai for allowing my thesis in this institute .

Finally , I am thankful to all the patients who are willingly co operated with me during the study.

CONTENTS

1. INTROUCTION

2. AIM OF THE STUDY

3. REVIEW OF LITERATURE 4. MATERIALS AND METHODS 5. OBSERVATIONS AND RSULTS 6. DISCUSSION

7. CONCLUSION 8. BIBILOGRAPHY 9. ANNEURES

PROFORMA

MASTER CHART

INTRODUCTION

Preterm labour is defined as one where the labour starts before the 37^th completed weeks or <259 days, counting from the last day of menstrual period .The lower limit of gestation is not uniformly defined; whereas in developed countries it has been brought down to 20 weeks, in developing countries it is 28 weeks.

It is the leading cause of perinatal morbidity and mortality . Babies who survive are also at high risk of neurological disability , further breathing, feeding, visual and hearing problems.

Despite improvements in perinatal care in the last two decades allowing for increased survival of preterm infants, changes in obstetric practice have not had an appreciable impact on preterm delivery rates. One of the main reasons for this is that there is a very limited understanding of the physiology and causes of preterm delivery.

Iatrogenic (medical) causes of preterm delivery due to materna1 or fetal factors account for 18.7% to 28.8% of preterm deliveries. Preterm premature rupture of membranes varies from 7.1% to 5 1.2% (average of 25%) of all preterm deliveries. Idiopathic preterm labour is the most common initiator of preterm delivery accounting for approximately 50%'- Up to 40% of women who have spontaneous preterm birth do not have observable known risk factors for preterm labor.

In order to address the problem of preterm delivery, identification of those at risk is an important step. Several strategies have been suggested , including risk scoring systems, biochemical markers of inflammation, and screening for various infections. It is important to evaluate these potential predictors alone and in

combination in the asymptomatic population and consider the economic impact of these screening tests.

C-reactive protein (CRP) is a sensitive marker of inflammation that remains stable in serum . Elevated concentrations of CRP in peripheral circulation has been associated with the presence of intrauterine infection . The maternal concentration of CRP has been studied as an aid to diagnose subclinical infections. In this study the association between maternal serum CRP in early second trimester and

subsequent preterm delivery among a cohort of singleton pregnant women was studied.

AIM OF THE STUDY

To measure the levels of maternal CRP levels in early second trimester of pregnancy and to correlate its association with preterm delivery.

REVIEW OF LITERATURE

Preterm birth is defined, as those infants who delivered ,prior to completion of 37 weeks of pregnancy. There are subcategories preterm birth, based on gestational age ;

 Extremely preterm (< 28 weeks)

 Early preterm (28-32 weeks)

 Moderate preterm (32-34 weeks)

 Late preterm (34-37 weeks).

TYPES OF PRETERM LABOUR

THREATENED PRETERM LABOUR

If the uterine contractions are perceived in te absence of cervical changes ,it is termed as threatened preterm labour.

SUSPECTED PRETERM LABOUR

Suspected preterm labour is when patient had symptoms of preterm labour and

has had a clinical assessment (including a speculum or digital vaginal examination)

that confirms the possibility of preterm labour but rules out established labour.

ESTABLISHED PRETERM LABOUR

If there is 4 regular contractions in 20 minutes or 8 in 60 minutes with progressive cervical changes ,effacement of 80% and dilatation greater than 1 cm.

INCIDENCE

Globally ,15 million babies are born preterm each year and 1.1 million dies as a result 0f complication (WHO 2012).the majority (60%) of preterm births occur in Africa and south asia( Beck et al 2010). Rate of preterm births ranges from 5%-18% World wide. NHP 2016 state that ;In India ,out of 27 million babies born every year (2010 data), 3.5 million babies born are premature (24%).

ETIOLOGY

Etiology of preterm labour is multifactorial; Van der Pool found that nearly 30%

preterm birth associated with ruptured membranes.25% preterm cases are due to intrauterine infections with/without intact membranes ( Gonclaves et al).

Lamon , concludes that infection is responsible in 40% cases.

Wright et al found UTI as a significant risk factor contributing to 7% of preterm;

According to Cram et al asymptomatic bacteriurea, gonococcal cervicitis and bacterial vaginosis are strongly associated with preterm labor .

IDIOPATHIC: In majority of preterm cases,the cause is unknown.

INFECTIONS;

CHORIOAMNIONITIS Bacterial vaginosis UTI

Systemic infection like malaria,listeria Dental caries, Periodontal disease FETAL FACTORS:

Multiple pregnancy

Congenital malformations

IATROGENIC: Indicated preterm birth Preeclampsia (40%)

Fetal distress (25%) IUGR (10%)

Placental abruption (7%) Uncontrolled diabetes

Decompensated heart failure .

RISK FACTORS

OBSTECTRIC :

With every previous history of preterm delivery , there is two fold increase in risk of preterm birth in present pregnancy ; the risk is inversely related to the GA of previous preterm labour .Mercer and colleagues reported that women with previous preterm deliveries had a 2.5–fold increased risk in their next pregnancy.

The risk of another preterm birth, is inversely related to the gestational age of the previous preterm birth. Persistent or recurrent intrauterine infections probably explain many repetitive spontaneous preterm births.

History of induced or spontaneous abortions and second trimester pregnancy loss will increase the risk. This may be due to cervical incompetence because of

repeated evacuations or aggresive dilatation of cervix in surgical termination of pregnancy resulting cervical damage.

Conditions which produce overdistension of uterus like multiple pregnancies and polyhydramnios predispose to preterm labour. A critical volume of uterus increased in these cases probably initiates labour.Intrauterine bleeding as occurred in APH like placenta previa , abruptio placenta which may stimulate cytokines release will trigger preterm process . Similarly first and second trimester bleeding have high association with preterm birth.

Intepregnancy interval less than 6 months

Uterine anomalies

MATERNAL CHARACTERISTICS

Diallo et al found that young and old age of mother (7.75% and 5.31%),poor and rich women(7.3%and 3.8%) are the important risk factors. They state that efficient prenatal care could control 85% of maternal causes.

Begum et al state that weight less than 45 kg(Or4.9),height <150 cm(OR 3.4)BMI

<19 (OR2.9),monthly income <2000rs(5.05),birth interval less than 12 months(OR6.39) are significant risk factors for preterm labor.

Maternal medical disorders, such as thyroid disease, asthma, diabetes, and hypertension, are associated with increased rates of preterm delivery, many of which are indicated because of maternal complications.Murphy et al claimed that a disease in mother has an influence on preterm birth in 25% cases.Goldenberg et al observed more significant preterm birth in women suffering from diabetes and hypertension.

INFECTIONS

The main reason for premature deliveries is an intrauterine infection.

Microorganisms can gain access to the amniotic cavity by:

(1) ascending from the vagina and the cervix;

(2) haematogenous dissemination through the placenta;

(3) accidental introduction at the time of invasive procedures; and (4) by retrograde spread through the fallopian tubes .

The most common pathway is the ascending route.

Reron et al showed increased percentage of pathogenic bacterial flora in

preterm labour patients with ruptured membrane compared with intact membranes indicates bacterial vaginosis is a significant risk factor for premature births.

Mikhov et al bacterial vaginosis patients had 2.5 times more risk of preterm delivery .

CERVICAL INCOMPETNCE

Romero, et al. concluded that the shorter the CL, the higher the risk of spontaneous PTB.There are three main mechanisms that have been associated with the development of an asymptomatic short CL. 13

First, the most obvious hypothesis is that a short CL is caused by an intrinsic

weakness of the cervix or cervical insufficiency . This cervical insufficiency is due in most cases to traumatic or surgical damage, or much more rarely, a congenital disorder or a connective tissue disease.

Second, another hypothesis is that a short CL is due to an inflammatory or infectious process as there is a strong association between a shortCL on TVU and infection.

Third, recent studies have shown that the majority of

asymptomatic women with CL less than 25 mm before 24 weeks have some contractions, more than controls with a normal cervix.

Ochedalski reported that if cervix length is below 10^th percentile of gestational age,the risk of prematurity increases to 6 times.If the length of cervix in 24 weeks gestation is found to be <25mm on ultrasound, the risk of preterm delivery

increased. The causes of cervical incompetence are congenital collagen disorders,cervical trauma due to forceful dilatation of cervix during surgical abortions , surgical procedures like conization, LEEP,LETZ.

PSYCOSOCIAL FACTORS

Copper et al reported, stress doubles the risk of preterm delivery.

Mothers experiencing high levels of psychological or social stress are at increased risk of preterm birth (generally <2–fold) even after adjustment for the effects of sociodemographic, medical, and behavioural risk factors.Although the mechanism underlying the association between psychological or social stress and increased risk of preterm birth is unknown, a role for corticotropin releasing hormone has been proposed. Women exposed to stressful conditions also have increased serum concentrations of inflammatory markers—such as C-reactive protein, These

findings suggest that systemic inflammation might be a pathway by which stress could increase the risk of preterm birth.

Haram et al. found a high correlation between domestic violence and major social and lifestyle risk factors for spontaneous preterm labour.The EUROPOP

multicentre study found that specific working conditions affect the risk of preterm birth. A moderate excess risk (adjusted) was observed among those who worked more than 42 hours a week (OR = 1.33, CI 1.1–1.6), stood more than 6 hours a day (OR = 1.26, CI 1.1–1.5) or were dissatisfied with their job (OR = 1.27, CI 1.1–1.5).

DIETARY FACTORS AND SUBSTANCE ABUSE

Tobacco use increases the risk of preterm birth (<2–fold) .both nicotine and carbon monoxide are powerful vasoconstrictors, and are associated with placental damage and decreased uteroplacental blood flow. Both pathways lead to fetal growth

restriction and indicated preterm births. Smoking is also associated with a systemic inflammatory response and can increase spontaneous preterm birth through that Pathway.

Windham et al sowed that mothers who smoked over 10 cigarettes a day delivered preterm. The risk of premature delivery related to smoking was also confirmed by Burguet et al. (2004) and Resende et al..

Parazzini et al. showed that having more than 2 drinks a day is related to preterm delivery.

ART

Perri et al showed increased risk of preterm delivery in cases of singleton pregnancies due to assisted reproductive techniques.The risk is related to greater chances of obstetrical complications like gestational diabetes , preeclampsia.

GENETIC PREDISPOSITION

It has been observed that women who were born prematurely are in the risk group of preterm delivery. The lower the gestational age the mother was born in is, the higher the risk.

Chaves et al [62] showed that the carrier-state of allele 2 of this

gene(IL1RN*) is related to the increased preterm delivery risk.Kalinka & Bitner observed that when a mother is a carrier of the polymorphic allele 2 of IL-1

(IL1RN*) receptor antagonist-coding gene, the risk of shorter gestation is increased.

PATHOGENESIS

The exact cause of preterm birth is unsolved. The cause of 50% of preterm births is never determined. Four different pathways have been identified that can result in preterm birth.One or more than one pathway can be activated at the same time.

It may be a physiological process which occur soon or pathological due to abnormal stimulus.If it occurs early most probably pathological because of infection.There are numerous theories proposed ; Of these ascending infection, withdrawal of progesterone,premature decidual activation, oxytocin withdrawal are important ones.

Important common pathways leading to preterm birth include stress, systemic or maternal genital tract infections, placental ischemia or vascular

lesions, and uterine overdistension. These pathways differ in their initiating factors and mediators, but ultimately, they share many common features that result in preterm uterine contractions and birth.

Premature activation of the maternal or fetal hypothalamic-pituitary-adrenal axis Amniochorionic-decidual systemic inflammation

Decidual Hemorrhage

Abnormal Uterine Distension

For preterm delivery to occur, the cervix undergoes considerable change, related to collagen breakdown and altered proteoglycan and water content, allowing effacement and dilation. The hypertrophied upper uterine segment

switches to fundally dominant contractions, which coordinate to expel the fetus.

This is a gradual process, often over a number of weeks, allowing both biophysical (e.g., cervical ultrasound) and biochemical (e.g., fetal fibronectin) tests to predict delivery.These tests have greater predictive value if performed in

combination. Clinically identified factors such as contractions or cervical dilation occur late in the process, partly explaining the poor performance of tocolysis in improving outcome. The lower segment also stretches, and there is an increase in inflammatory mediators and prostaglandins.

Cervical change is mediated by the influx of inflammatory cells releasing matrix metalloproteinases. Increased contractility of the upper segment is associated with expression of prostaglandin and oxytocin receptors and gap

junction proteins and other signalling pathways. Progesterone withdrawal is not seen in humans prior to labour, but progesterone therapy may be anti-inflammatory, and thus has a plausible mechanism as a prophylactic treatment.

Infection also causes inflammation, and ascending micro-organisms through a deficient cervix can stimulate an inflammatory response through the innate immune system.

Other routes include haematogenous spread, iatrogenic introduction, and retrograde spread through the fallopian tubes. These activate prostaglandins, inflammatory cytokines, and phospholipase A2 and can result in ruptured membranes or contractions. This explains the increased risk of premature labour with genital tract infection.



PREDICTORS OF PRETERM

Primary predictors of preterm are those known before the onset of pregnancy,secondary predictors are those may presnt only after the

pregnancy .secondary predictors are found during the examinations done on current pregnancy; allows a more accurate assessment of risk of preterm birth.

These may used to intervene in prevention of preterm in high risk group.

The major secondary predictors are detection of fetal fibronectin in cervicovaginal secretions and shortened cervix diagnosed by transvaginal ultrasonography

Current screening tests for the prediction of spontaneous preterm labour can be divided into three general categories: (i) risk factor assessment, (ii) cervical measurement, and (iii) biochemical markers;

RISK FACTOR ASSESSMENT:

Clinical risk factors for preterm birth include (i) demographic characteristics such as low socioeconomic status, poor antenatal care, extremes of maternal age, or malnutrition, (ii) behavioural factors including smoking, illicit drug use, alcohol consumption, or heavy physical work, (iii) obstetric history including familial (genetic) predisposition, uterine malformation, previous preterm labour or preterm PROM, previous cone biopsy or cervical surgery, and (iv) aspects of the current pregnancy such as multifetal gestation, genital tract bleeding and/or infection, fetal malformation, preterm rupture of membranes, shortened cervix, and other

pregnancy complications including preeclampsia and gestational diabetes mellitus . A previous preterm birth before 34 weeks’ gestation is among the

strongest risk factors for subsequent preterm birth with a relative risk of 13.56 However, insofar as nulliparous women have no past obstetric history to call upon, any such previous history risk factor-based assessment is inapplicable in their situation. Overall risk factor assessment alone is unreliable, as over 50 percent of pregnancies that deliver preterm will fail to be identified .

CERVICAL LENGTH MEASUREMENT

Cervical length is normally distributed and remains relatively constant in pregnancy until the third trimester.Cervical length is inversely related to the risk of preterm birth in asymptomatic women.In some women, a shortened cervical length can be due to natural biological variation. In other cases early cervical shortening or effacement may be due to haemorrhage or infection leading to inflammation, or due to biophysical effects of uterine overdistension (e.g., multifetal gestation) or subclinical contractions..

Cervical length is a better predictor of preterm birth in women at increased risk, such as those with a history of spontaneous preterm birth, than in asymptomatic women at low risk. In studies of women with a history of preterm birth, using a cervical length cut-off of 25 to 30 mm to predict preterm birth is 60%

to 80%, positive predictive value is 55% to 70and negative predictive value is 89%

to 94%.

For ease of clinical use, 25 mm has been chosen as the ‘cut off’ at above which a cervix can be regarded as normal, and below which can be called short .A cervix that is less than 25 mm may be indicative of preterm birth.A cervical length

< 25 mm before 28 weeks gestation is abnormal and associated with a higher incidence of PTB, women with a cervical length < 25 mm and contractions have twice the incidence of PTB than women with a cervical length < 25 mm but no contractions.

BIOCHEMICAL MARKERS

SALIVARY ESTRIOL

Estriol, estradiol, and estrone, all three play an important role in the preparation of uterine tissue, which signals the parturition. It is observed that a rise in estriol is there preceding the labor.

Soghra et al. in Iran revealed that there was a significant relation between salivary estriol level and PTL. Hussein et al. Studied on 338 pregnant women suggested that salivary estriol and progesterone can both be useful for the detection of PTL.

Fetal Fibronectin

Fetal fibronectin (FFN), a glycoprotein produced by the fetal membrane, is one of the biomarkers for the detection of PTL. Its presence within the CVF is said to be an indicator of the presence of PTD. The release of FFN is thought to be due to mechanically mediated damage or inflammatory-mediated damage to membrane or placenta before birth. Generally FFN is not present during 24-37 weeks gestation period. Its presence in the CVF is a signal of spontaneous preterm birth within 7 days.

In 2002, Honest et al. published a systematic review on the predictive capacity of fibronectin for PTD. They identified 40 studies which were conducted on total 26,876 women in total and concluded that cervicovaginal FFN was the most accurate test in predicting spontaneous preterm birth within 7-10 days of testing. In practice, it is used as a negative marker. Absence of FFN suggests that the patient is at a low risk of PTD and thus, the patient can be discharged,

minimizing unnecessary hospital stay and unwanted treatment.

Another study conducted by Goldenberg et al. included 2,929 women for determining the levels of vaginal and cervical FFN during 24-30 weeks and its association with spontaneous PTD. It was observed that positive vaginal and cervical FFN test were directly proportional to spontaneous preterm birth.

The FFN levels, along with the cervical length measurement, are used as a sensitive method of the detection for PTD.

Placental alpha microglobulin-1

Placental alpha microglobulin-1 (PAMG-1) has been the subject of several

investigations ;evaluating its ability to predict imminent spontaneous preterm birth in women with signs, symptoms, or complaints suggestive of preterm labor. In one investigation comparing ,this test to fetal fibronectin testing and cervical length measurement through transvaginal ultrasound, the test for PAMG-1 ( the PartoSure test) has been reported to be the single best predictor of imminent

spontaneous delivery within 7 days of a patient presenting with signs,

symptoms, or complaints of preterm labor. Specifically, the PPV, orpositive

predictive value, of the tests were 76%, 29%, and 30% for PAMG-1,FFN and CL, respectively (P < 0.01).

Pregnancy-associated plasma protein A (PAPP-A) is a metalloprotease, which is routinely measured in the maternal serum as a part of the first-trimester screening. PAPP-A expression has been shown to be regulated by inflammatory cytokines such as TNF-a and IL-1b. Thus, PAPP-A is shown to be implicated as an acute reactant in inflammatory processes, one of which may be

PTL.

Granovsky et al. in 2007 conducted a prospective cohort study on 26 women and reported that PAPP-A levels ≤30,000 mUl at admission were associated with increased risk for preterm birth of ≤7 days.

Pregnancy-Associated Plasma Protein A

Adrenocorticotropic Hormone

Pituitary adrenocorticotropin is the major regulator of adrenocortical zona fasciculata and reticularis function and is a significant growth factor for normal adrenocortical cells. The actions of ACTH are mediated by its specific membrane receptor.

Singh et al., found that there was a rise in the

ACTH level in PTD as compared to the control group. This rise during the gestation period may be due to multiple factors such as placental synthesis and release of biologically active corticotropin-releasing hormone (CRH) and ACTH, pituitary desensitization to cortisol feedback, or enhanced pituitary responses to corticotropin-releasing factors such as vasopressin and CRH.

Makrigiannakis et al., 79 pregnant women were observed for plasma ACTH level. They found that ACTH level was significantly higher in women who delivered preterm compared to full term.

BETA HCG

Ramos et al., they monitored beta hCG in CVF as a marker for PTD in 86 women.

They concluded that beta hCG can be a useful predictor of preterm birth in symptomatic women.

Gurbuz et al. studied that , Increased concentration of hCG in CVF was found in women with PTD.

C REACTIVE PROTEIN

CRP is a phylogenitically , highly conserved plasma protein,that participates in systemic response to inflammation.

HISTORY

In 1930,Oswald Avery’s lab CRP was discovered during the studies of patients infected with Strep.Pneumonia.Sera from these patients in acute phase of illness had a protein which precipitates the ‘C’ polysaccharide of pneumococcal cell wall.Once disease subsided,the C substance became undetectable.Although it was discovered 85 years ago,it is an ancient one; with homologous protein present in species limulus polyphemus which is a horseshoe crab appeared 50 million years ago . These organisms rely on phagocytosis and CRP like molecule for defense against infection.

STRUCTURE

CRP gene located in short arm of chromosome 1. Determination of 3D structure by X ray crystallography. It consists of five identical non covalently associate 23kDa protomer arranged symmetrically around a central pore. It belongs to member of Pentraxin family of proteins.

SYNTHESIS

It is primarily synthesised in liver, as a acute phase response. IL-6 and IL-1 are the major inducer of CRP production. It was synthesised as monomers and gets

assembled as pentamers. CRP as pleotropic effect that is having both proinflammatory and antiinflammatory activity.

FUNCTIONS

CRP is a member of acute phase reactants,its level dramatically increased during inflammatory process.This increment is due to ,rise in plasma concetration of IL- 6 ,which is produced predominantly by macrophages and adipocytes.CRP binds to phosphocholine on the surface of dying cells and some bacteria finally activates complement, binds to Fc receptors and acts as an opsonin for various pathogens.

Interaction of CRP with Fc receptors leads to the generation of pro-inflammatory cytokines that enhance the inflammatory response. Unlike IgG, which specifically recognizes distinct antigenic epitopes, CRP recognizes altered self and foreign molecules based on pattern recognition. Thus, CRP is though to act as a

surveillance molecule for altered self and certain pathogens.

Another proinflammatory function of CRP includes the induction of cytokines and tissue factor in monocytes . However, its main function is antiinflammatory by decreasing neutrophil migration to the site of inflammation, preventing adhesion of neutrophils to endothelial cells , and affecting clearance of nuclear antigens

released from apoptotic or necrotic cells.The function of CRP is felt to be related to its role in innate immune system. Alpha interferon inhibits its synthesis which explain the lower level of CRP in viral infection compared to bacterial infection.

Elevated CRP as a marker of low grade inflammation found to be associated with increased risk of neonatal complications like preterm , IUGR low birth weight.The explanations is, low grade inflammation is associated with endothelial dysfunction leading to vascular dysfunction and suboptimal placental development. It predicts the future cardiovascular events if elevated chronically.Elevated maternal CRP in early pregnancy associated with preterm labour and those who deliver preterm have high risk for subsequent

cardiovascular events.

Apart from infections, inflammation and trauma, factors associated with increased levels of CRP include; obesity, cigarette smoking, hormonal use, metabolic syndrome and cardiovascular disease . Moderate

alcohol consumption, increased physical activity and medication use (particularly statins, fibrates, and niacin) are associated with reduced CRP levels .

Unlike ESR, CRP measurement does not change in the presence of anemia, polycythemia, spherocytosis, macrocytosis,congestive heart failure, or

hypergammaglobulinemia.

CRP is predominantly secreted by the liver and starts four to six hours after the stimulus; it duplicates every eight hours,and peaks within 36 to 50 hours. CRP has a plasma half-life of 19 hours, and even after a single stimulus, as in a trauma Or surgery, it may take several days to return to the baselines.

CRP during normal pregnancy

CRP does not cross the placental barrier and therefore, will be useful in diagnosing infections in newborns.Recently, it has been shown that CRP is present in amniotic fluid and fetal urine, and the elevated levels are associated with adverse pregnancy outcome. These results demonstrate that the human placenta produces and releases CRP, like other placental proteins, mainly into the maternal circulation.

CRP and cardiovascular risk

The association between CRP and cardiovascular risk is driven predominantly by systemic inflammation. CRP is unlikely to contribute directly to cardiovascular disease as a pathogenic factor. Similar conclusions were drawn from recent Mendelian randomization studies. Using widely available high-sensitivity assays, CRP levels of 1, 1 to 3, and 3 mg/L have been classified as low, moderate, and high-risk groups for future cardiovascular events. Individuals with LDL cholesterol below 130 mg/dl and CRP levels of 3 mg/dl represent a high-risk group. The

conversion of plasma CRP (pCRP) to monomeric CRP (mCRP) has been described as being mediated by activated platelets, which are associated with cardiovascular risks.

CRP and cancer

CRP levels have been used to predict the risk of cancer, detect cancer recurrence, and in prognosis. CRP is a biomarker of inflammation and indicator of the immune response to tumors.Its role as a predictor of survival has been shown in multiple myeloma, melanoma, lymphoma, ovarian, renal, pancreatic, and gastrointestinal tumors. Recent evidence has also associated CRP elevation with the progression of melanoma, ovarian, colorectal and lung cancers, and recurrence of cancer after surgery in certain situations .

CRP and infection

CRP is an important factor in determining the etiology of infection. The level of CRP can be significantly higher in bacterial infections. A value higher than 100mg/L strongly suggests bacterial infections, whereas that below 10 mg/L indicates viral infection. In tuberculosis, it is often found to be between 10 to 100 mg/L.Additional determination of procalcitonin can add specificity in the case of bacterial infections.The above information is also helpful to distinguish infection from an autoimmune flare. Similarly, the rate of change in CRP levels can

differentiate tuberculosis from bacterial pneumonia.

CRP and inflammatory diseases

In the case of inflammatory diseases, CRP level represents the disease activity.

Studies have suggested direct correlations of CRP with RA and inflammatory bowel diseases like Crohn’s disease. In contrast, in conditions like SLE, CRP is not significantly elevated.

CRP and obesity

CRP concentrations are elevated, predominantly in obese individuals who are insulin resistant, and are in line with the weight loss-associated improvements in insulin resistance. The relation between CRP concentrations and insulin resistance is independent of obesity.

CRP and diabetes

Elevated levels of CRP and IL-6 predict the development of type 2 diabetes. This association supports a possible role for inflammation in diabetogenesis. CRP is a powerful independent predictor of diabetes, after adjustment for obesity, clinical risk factors, and fasting insulin levels. Minor increase in CRP level has also been reported to be associated with a number of medical conditions that do not appear to be associated with inflammation. Elevated CRP is also observed with several

genetic polymorphisms of the CRP and other genes, ethnicity, dietary patterns and obesity.

CRP AND PRETERM LABOUR

Lohsoonthorn et al. on 1796 women in the USA suggested that elevated maternal serum CRP levels in early pregnancy are positively associated with PTD risk. They noted no association between elevated CRP and risk of preterm premature rupture of membranes and little evidence of an association between maternal serum CRP concentrations and risk of delivery between 34 weeks and 36 weeks of gestation (i.e., moderate PTD). However, elevated CRP concentrations were associated with an increased risk of delivery prior to the completion of 34 weeks gestation.

Hastie et al. in Scotland concluded that the women who undergo the indicated PTD are at an increased risk of raised CRP in later life.

Dhok et al. in 2011 indicated that increased serum high-sensitivity CRP (hsCRP) levels in early second trimester primigravidae were significantly associated with adverse pregnancy outcomes such as PTD and pregnancy-induced hypertension

Halder et al. showed that high levels of CRP in early pregnancy in the absence of any medical/surgical or obstetric complication was associated with nearly a

twofold increased risk of PTD. It was also shown that neonatal outcomes such as preterm, low birth weight, septicemia, birth asphyxia, and others are more likely in such patients.

Ghezzi et al. looked into whether the amniotic fluid CRP level at the time of genetic amniocentesis is a marker for spontaneous PTD before 34 weeks and 37 weeks of gestation the amniotic fluid CRP concentration of >110 ng/mL had a sensitivity of 80.8% and a specificity of 69.5% in the prediction of spontaneous PTD at <34 weeks.

Hvilsom et al reported high CRP levels at the beginning of a pregnancy was associated with a nearly twofold increased risk of PTD.

DIAGNOSIS OF PRETERM LABOUR

SYMPTOMS

Menstrual like cramps Dull low back ache Abdominal cramping

Changes in vaginal discharge CRITERIA

UTERINE CONTRACTIONS OF 4 IN 20 MIN OR 8 IN 60 MIN PLUS PROGRESSIVE CHANGES IN CERVIX

- CERVICAL DILATATION GREATER THAN 1 CM - CERVICAL EFFACEMENT OF 80% OR GREATER DIAGNOSIS

 CLINICAL EXAMINATION

 ULTRASONOGRAM

 TOCOCARDIOGRAPHY

NEONATAL COMPLICATIONS IMMEDIATE COMPLICATIONS Hypothermia

Hypoglycemia Hypocalcemia

Respiratory difficulties

Intra-ventricular hemorrhage (IVH) Liver immaturity

Increased susceptibility to infections Necrotizing enterocolitis (NEC) Patent ductus arteriosus

Feeding problems Anemia of prematurity Retinopathy of prematurity

Metabolic bone diseases of prematurity

LONG TERM COMPLICATIONS Cerebral palsy

Mental retardation

Visual and Hearing impairments Poor health and growth

Behavioral and social-emotional problems Learning difficulties

Increased risk of conditions such as Attention Deficit-Hyperactivity Disorder (ADHD)

The study group patients are divided into two groups as group A and group B depending upon CRP levels .The diagnosis of preterm delivery was made using ACOG guidelines. The women who delivered before 37 weeks gestational age were taken as preterm and >37 weeks as term by using the detailed information collected during the time of delivery from the medical records.

Neonatal outcome of all the babies were studied, 1 minute and 5 minute APGAR and neonatal complications like RDS,neonatal sepsis, intraventricular hemorrhage were noted.

RDS were diagnosed based on clinical symptoms like tacypnea , grunting, subcostal retraction,cyanosis and X ray findings such as reticular granularity and air bronchogram.

Neonatal sepsis used to present with diminished activity, poor sucking, apnea, bradycardia, temperature instability, vomiting, diarrhoea, abdominal distension, seizure and jaundice.Diagnosis is clinical and based on culture reports.

MATERIALS AND METHODS INCLUSION CRITERIA

All cases of singleton gestation (confirmed by early second trimester ultrasound),

gestational age between 14-20 weeks (by LMP and confirmed by ultrasound) Intact amniotic membranes,

.

EXCLUSION CRITERIA Multiple gestation, Pre-eclampsia GDM

Polyhydramnios

History of Pre-term labor in prior pregnancy

Medical conditions like SLE,Rheumatoid arthritis,H/O recent bacterial or viral infections,Heart disease

Gestational age less than 13 weeks or more than 20 completed weeks.

Women not sure of LMP and having a dating scan

METHOD

Generally all, healthy pregnant women attending the antenatal clinic are to be enrolled and the serum C-Reactive protein levels to be measured at GA of 14 To 20 weeks pregnancy and followed up to delivery and then they will be allocated to preterm and non-preterm groups based on the GA at delivery.

Gestational age was calculated based on last menstrual period and confirmed by ultrasound report of 11-14 weeks.If both the LMP and USG EDD are within 14 days ,gestational age is based on LMP, if >14 days USG EDD can be taken into account.

A detailed history regarding the maternal socioeconomic status, anthropometry, lifestyle habits, medical and surgical illness was taken.

SAMPLE COLLECTION:

Venous blood samples were obtained from patient to measure serum c-reactive protein concentrations between 14 and 20 weeks' gestation. Samples were

collected in a test tube without anticoagulant and allowed to clotted .serum removed from the clot as soon as possible to avoid hemolysis and kept frozen until tested by lab.The levels of C-Reactive protein measured through a

quantitative highly sensitive immunoassay test (ELISA).

The reference value is 1.5 mg/dl. Thus, high maternal serum c-reactive protein in pregnancies were defined as those in which maternal serum level was above 1.5mg/dl..

Association between high maternal serum c-reactive protein and preterm labour.

the two outcome variables were tested using chi-square. All significance are reported at P<0.05.

OBSERVATIONS AND RESULTS AGE DISTRIBUTION

Age of patients in this study ranged from 18-38 years . The mean age of the patients in this study 25 years. The age distribution was as follows:

AGE_GROUP No. Of patients

Percent

18-21 YEARS 61 30.5

22-24 YEARS 48 24.0

25-28 YEARS 47 23.5

29-32 YEARS 30 15.0

ABOVE 32

YEARS 14 7.0

Total 200 100.0

GESTATIONAL AGE AT SAMPLE COLLECTION

The mean gestational age in this study was 16.5 weeks. The minimal gestational age was 14+1 weeks and maximal was 19+3 weeks. The gestational age

distribution was as follows :

GESTATIONAL AGE AT SAMPLE COLLECTION

GA GROUP Frequency Percent(%)

14 - 14.6 16 8.0

15-15.6 68 34.0

16-16.6 82 41.0

17-17.6 25 12.5

18-18.6 8 4.0

19-19.6 1 .5

Total 200 100.0

GESTATIONAL AGE AT SAMPLE COLLECTION - DISTRIBUTION

PARITY IN SAMPLE COLLECTION

Totally 104 primi (52%) and 96 multiparous (48%) women were studied. The parity distribution was as follows;

PARITY Frequency Percent

PRIMI 104 52.0

MULTI 96 48.0

Total 200 100.0

DISTRIBUTION OF TERM AND PRETERM DELIVERY

In this study 42 patients(21%) had preterm delivery and 158 patients (79%) delivered in Term

TERM Frequency Percent

<37WEEKS 42 21.0

>37WEEKS 158 79.0

Total 200 100.0

DISTRIBUTION OF TERM AND PRETERM DELIVERY

MODE OF DELIVERY

In this studied population 57 patients (28.5%) had LSCS and the remaining 143 patients (71.5%) delivered vaginally.

MODE DELIVERY

Frequency Percent

LSCS 57 28.5

Vaginal 143 71.5

Total 200 100.0

DIVISION OF GROUPS

A Total of 200 patients were included in the study , these patients were divided into two groups based on their serum CRP levels. A total of 52 (26%) had CRP levels >1.5 mg/dl belonged to group B and 148 (74%) had CRP level <1.5 mg/dl as group A

CRP GROUP No. of patients Percent(%)

0-1.5 GROUP A 148 74

ABOVE 1.5

GROUP B 52 26

Total 200 100.0

CRP LEVELS AND INCIDENCE OF PRETERM DELIVERY

A total of 200 patients included in this study ; 158patients had CRP<1.5mg/dl ;of these 136 patients (91.9%) delivered term ; 12 patients (8.1%) had preterm delivery. Remaining 42 patients had CRP>1.5 ; of these 30patients (57.7%) delivered preterm, 22 patients(42.3%) had term delivery.

TERM * CRP_GROUP Crosstabulation

CRP_GROUP Total GROU

PA0- 1.5

GROUP B .

>1.5

TERM

<37WEEKS

Count 12 30 42

% within

CRP_GROUP 8.1% 57.7% 21.0%

>37WEEKS

Count 136 22 158

% within

CRP_GROUP 91.9% 42.3% 79.0%

Total

Count 148 52 200

% within CRP_GROUP

100.0

% 100.0% 100.0

% Pearson Chi-Square=57.026** P<0.001

CRP GROUP

Frequency Percent

0-1.5 148 74.0

ABOVE 1.5 52 26.0

Area Under the Curve Test Result Variable(s): CRP

Area Std.

Error^a

Asymptotic Sig.^b

Asymptotic 95%

Confidence Interval Lower

Bound

Upper Bound

.880 .027 .000 .827 .932

The test result variable(s): CRP has at least one tie between the positive actual state group and the negative actual state group. Statistics may be biased.

a. Under the nonparametric assumption b. Null hypothesis: true area = 0.5

Coordinates of the Curve Test Result Variable(s): CRP

Positive if Greater Than or Equal To^a

Sensitivi ty

1 - Specificity

1.5000 0.714 0.139

IF WE SELECT THE CRP VALUE IS 1.5 THEN SENSITIVITY WILL BE 71.4% WITH specificity WILL BE 86% TO PREDICT THE PRE TERM.

CRP LEVELS AND RESPIRATORY DISTRESS SYNDROME

Of the total 42 preterm babies , 4 from group A (33.3%) and 11 from group B(36.6%) developed RDS

GROUP A

No. Of patients (%)

GROUP B

No. Of patients (%)

RDS 4 (33.3%) 11 (36.6%)

There is no statistical significant difference in development of RDS between preterm infants of 2 groups.

CRP AND NEONATAL SEPSIS

Of the total 42 preterm babies , 1 from group A (8.1%) and 8 from group B(26.6%) developed Sepsis and were treated with IV antibiotics

GROUP A

No. Of patients (%)

GROUP B

No. Of patients (%)

SEPSIS 1 (8.3%) 8 (26.6%)

P <0.05, hence there was statistical significant difference in development of

neonatal sepsis between preterm infants of 2 groups.with infants born to increased CRP group there is increased chance of neonatal sepsis.

DISCUSSION

CRP is an acute phase reactant protein synthesised primarily in liver cells in response to proinflammatory cytokines incluing IL-6 and alpha TNF. C reactive protein in maternal peripheral circulation is associated with presenc of intrauterine infection.

In this study, 200 pregnant women of singleton pregnancy between the gestational age of 14 to 20 weeks were enrolled . After detailed history taking , maternal serum CRP levels were estimated by ELISA method.These women were divided into two groups according to CRP levels .Incidence of preterm delivery, mode of delivery was noted in both the groups.The incidence of neonatal morbidity was compared in both groups to ascertain if measured maternal CRP levels has any association with neonatal morbidity.

The mean age of the patients in this study 25 years. The mean gestational age was 16.5 weeks. The minimal gestational age was 14+1 weeks and maximal was 19+3 weeks. In this study 42 patients(21%) had preterm delivery.Of these 30 patients had CRP >1.5mg/dl; 12 had CRP < 1.5 mg/dl.

In CRP increased group , 30patients (57.7%) delivered preterm, 22 patients(42.3%)

had term .

It was found that increased levels of maternal serum CRP in early pregnancy were associated with increased incidence of preterm delivery.According to this study if the CRP value 1.5 the sensitivity will be 71.4% with specificity will be 86% to predict the preterm.

Neonatal complications like respiratory distress syndrome has no significant association with CRP levels.

Of the total 42 preterm babies, 1 from group A (8.1%) and 8 from group B(26.6%) developed Sepsis and were treated with IV antibiotics.there was

statistical significant difference in development of neonatal sepsis between preterm infants of 2 groups.with infants born to increased CRP group there is increased chance of neonatal sepsis.This result is similar to the study of Shabrin et al .

CONCLUSION

Elevated maternal serum CRP concentration in early second

trimester was associated with increased incidence of preterm delivery and showed a positive correlation with neonatal sepsis.

BIBILIOGRAPHY

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PROFORMA

NAME:

ADDRESS:

IP NO:

ADDRESS:

OBSTETRIC FORMULA:

MARITAL HISTORY:

MENSTRUAL HISTORY:

LMP:

EDD:

HEIGHT:

WEIGHT:

BMI:

HISTORY OF PRESENTING ILLNESS FAMILY HISTORY:

PAST HISTORY:Diabetes, Hypertension, Renal disease,SLE,Rheumatoid arthritis,Cardiac disease.

OBSTETRIC HISTORY:

PERSONAL HISTORY:

GENERAL EXAMINATION:

SYSTEMIC EXAMINATION:

Cardiovascular, Respiratory ,Central Nervous System OBSTETRIC EXAMINATION:

Inspection Palpation Auscultation SFH

INVESTIGATIONS

USG

LABOUR Mode of labour Date of delivery Baby weight Apgar

INFORMATION SHEET

• We are conducting a study on “MEASUREMENT OF SERUM C- REACTIVE PROTEIN LEVELS IN EARLY SECOND TRIMESTER AS A PREDICTOR OF PRETERM DELIVERY” among patients attending Egmore Maternity Hospital, Chennai and for that your clinical details may be valuable to us.

• We are selecting certain patients and if you are found eligible, we may be using your clinical details in such a way so as to not affect your final report or management.

• The privacy of the patients in the research will be maintained throughout the study. In the event of any publication or presentation resulting from the research, no personally identifiable information will be shared.

• Taking part in this study is voluntary. You are free to decide whether to participate in this study or to withdraw at any time; your decision will not result in any loss of benefits to which you are otherwise entitled.

• The results of the special study may be intimated to you at the end of the study period or during the study if anything is found abnormal which may aid in the management or treatment.

Signature of investigator Signature of participant

Date:

CONSENT FORM

STUDY TITLE : “MEASUREMENT OF SERUM C-REACTIVE PROTEIN LEVELS IN EARLY SECOND TRIMESTER AS A PREDICTOR OF PRETERM DELIVERY”

STUDY CENTRE : Institute of Obstetrics and Gynecology, Egmore Maternity Hospital, Chennai-8.

PARTICIPANT NAME : AGE: SEX: MRD.NO:

I confirm that I have understood the purpose of procedure for the above study, I have the opportunity to ask the question and all my questions and doubts have been answered to my satisfaction.

I have been explained about the possible complications that may occur during the procedure, I understand that my participation in the study is voluntary and that I am free to withdraw at any time without giving any reason.

I understand that investigator, regulatory authorities and the ethics committee will not need my permission to look at my health records both in respect to the current study and any further research that may be conducted in relation to it, even if I withdraw from the study. I understand that my identity will not be revealed in any information released to third parties of published, unless as required under the law. I agree not to restrict the use of any or results that arise from the study.

I hereby consent to participate in this study of “MEASUREMENT OF SERUM C-REACTIVE PROTEIN LEVELS IN EARLY SECOND TRIMESTER AS A PREDICTOR OF PRETERM DELIVERY”

Signature of Investigator: Place : Chennai Date :