The Combined Screening Test in the First Trimester of Pregnancy and Preeclampsia

Introduction: The combined screening test in the first trimester of pregnancy and determination of biochemical markers, pregnancy-associated plasma protein-A (PAPP-A) and free beta subunit of human chorionic gonadotropin (free ß-hCG) were measured to assess the risk for developing hypertensive disorders during pregnancy after 20 weeks of gestation. Preeclampsia (PE) is the leading cause of maternal and fetal morbidity and mortality. Aim of the study: To present obtained results of biochemical markers (PAPP-A and free ß-hCG) taken from a serum of pregnant women with preeclampsia in the first trimester of pregnancy and to examine the correlation between biochemical markers, age of the pregnant woman and body mass index (BMI) with preeclampsia. Patients and methods: This retrospective study included 70 pregnant women in two groups: (1) preeclampsia group (n=40), (2) control group (n=30). Results: In the preeclampsia group, the mean value of free ß-hCG was 37.18±20.64 IU/L and for PAPP-A 2711.15±1788.60 mIU/L with a statistical significance of p=0.007, compared to the control group. The mean value of body mass index (BMI) was 26.18±4.93 kg/m² and for age 31.53±5.00 years. In the control group, the mean value of free ß-hCG was 29.97±10.39 IU/L and for PAPP-A 3411.30±1227.59 mIU/L. The mean value of body mass index (BMI) was 23.47±4.09 kg/m² and for age of pregnant women 28.10±4.57 years. Conclusion: There is a significant association between the low level of biochemical marker PAPP-A in serum and the risk for PE. The difference of values of free ß-hCG between the groups tested was not statistically significant. Maternal age and body mass index (BMI) are confirmed risk factors for development of PE. The combined screening test contributes to the prevention of PE, with the goal of improving health of pregnant women.


Introduction
Preeclampsia is the dominant, representative form of hyper tensive disease in the pregnancy, characterized by the presence of high blood pressure after 20 weeks of gestation, appearance of edema and proteinuria, with the completed healing process in the first six weeks after birth. The incidence of preeclampsia is in the range of 2-8%, depending on diagnostic criteria. The predisposing risk factors for preeclampsia are: pregestational diabe tes mellitus, chronic kidney disease, pregestational hypertension -existing hypertension, obesity, nulliparous, pregnant women younger than 18 and older than 35 years, preeclampsia in previous pregnancy, molar pregnancy, multi ple pregnancy, thrombophilia and fetal hydrops. Some risk factors may appear before or during pregnancy.¹ Beside regular controls of a pregnant woman (blood pressure, body weight and other risk factors) and fetus, the prevention of PE is presented by the specific activities and measures in accordance with the recommendations of the Guideline for the health care of women during pregnancy. [1][2][3] Owing to preventive measures, we would be able to reduce the incidence of preeclampsia and accompanying complications, which would contribute to improving health of pregnant women.

Aim of the study
The aims of the study are: 1. to determine the results of biochemical markers (PAPP-A and free beta-hCG) from serum of pregnant women with preeclampsia in the first trimester of pregnancy, 2. to determine the correlation between biochemical markers (PAPP-A and free beta-hCG), age and body mass index (BMI) in pregnant women with preeclampsia.

Materials and Methods
This paper represents a retrospective analysis which included 70 pregnant women classified in two groups. The first group consisted of pregnant women with preeclampsia (n = 40), with the average age of 31.53 ± 5.00, and the second, control, group consisted of healthy pregnant women (n = 30), with the average age of 28.10 ± 4.57.
The criteria for inclusion of pregnant women in our study were defined by the Guideline for the health care of women during pregnancy and it was confirmed by clinical and laboratory measurings. 1 The combined screening test is used for the early detection of risk for chromosomal abnormalities (trisomies 21, 18 and 13) in prenatal diagnosis and includes measuring of biochemical markers PAPP-A and free beta-hCG from the serum, and ultrasound parameters (nuchal translucency thickness, NT, and crown-rump length, CRL ). [4][5][6] In the group with preeclampsia, the youngest pregnant wo man was 21 and the oldest 46 years old. In the control group, the youngest pregnant woman was 20 and the oldest 38 years old. Biochemical markers (PAPP-A and free beta-hCG) were measured from the sample of serum by using Electrochemiluminescen immunoassay (ECLIA) -"sandwich" technique, on Roche Cobas E601 analyzer. La ter, based on the clinical examination and laboratory analysis, the first group of pregnant women (n = 40) was diagnosed with preeclampsia.
BMI within combined screening test in the first trimester of pregnancy was defined for all pregnant women. Obtained results of biochemical markers PAPP-A and free beta-hCG, BMI and age of pregnant women are presented with mean values ± standard deviation (SD).
Statistical descriptive analysis of results and testing of variables in two groups of pregnant women were performed using Excel and SPSS program version 22.0. with nonparametric Spearman correlation coefficient (rho) and nonparametric tests (Mann-Whitney test and Kolmogorov-Smirnov test ).
Using logistic regression analysis we examined the influence of individual variables on the occurrence of preeclampsia. ROC curve analysis (Receiver Operating Characteristic) was conducted for biochemical markers PAPP-A and free beta-hCG, based on which the sensitivity and specificity of the tests are estimated.

Results
Descriptive statistics of analyzed parameters (concen trations of biochemical markers of the combined screening test in the first trimester of pregnancy PAPP-A and free beta-hCG, maternal age and BMI) are presented in Table 1 by the groups (a group of pregnant women with preeclampsia (PE) and the control group of healthy pregnant women).
Results of examined variables obtained by statistical data analysis between two groups of pregnant women, using nonparametric Mann-Whitney test and comparing their medians, were ranked and there was a statistically significant difference (p < 0.05) for variables such as age, BMI and biochemical marker PAPP-A. Values of variables are presented with Z -approximation and medium effect size r (r = Z / √n).
Mann-Whitney test did not found a statistically significant difference for the values of free beta-hCG between the group of pregnant women with preeclampsia and the control group of healthy pregnant women. The level of significance for free beta-hCG is p = 0.291, statistically not significant for the result Z (Table 2.). With the combination of biochemical markers and confirmed risk factors of preeclampsia, such as age and body mass index (BMI), by using the model of logistic regression their effect and a statistical significance change.
Biochemical markers PAPP-A and free beta-hCG were examined using ROC curve analysis (Receiver Operating Characteristic ) based on which the ratio of sensitivity and specificity of the test at different decision limits (cut-off values) is estimated. Diagram 1. ROC curve for free beta-hCG

Diagram 1. ROC curve for free beta-hCG
The area under ROC curve for free beta-hCG is 0.574, p = 0.291. This shows that the test is bad and not statistically significant (Diagram1.).
The area under ROC curve for PAPP-A is 0.691, p = 0.007. This shows that the test is statistically significant and classifies as a poor test (Diagram 2.). The ROC curve analysis for biochemical marker PAPP-A showed that the optimal cut-off presents the value of 2530.00 mIU/L, has sensitivity of 80% and specificity of 63%.

Discussion
Preeclampsia is a complex multisystematic and multifactorial syndrome specific for pregnancy. It is the leading cause of maternal and fetal morbidity and mortality. 7 On a sample of 70 pregnant women, 40 (57.1%) of them represents a group of pregnant women with preeclampsia and 30 of them (42.9%) makes the control group of healthy pregnant women. Obtained results of biochemical markers during the combined screening test in the first trimester of pregnancy (free beta-hCG and PAPP-A) were presented in a form of concentration for the markers, while numerous studies that we used for comparison with our results describe parameters through the multiples of median (MoM), making the interpretation of markers difficult. We decided to use this way of presentation of results because of the nonhomogeneous and nonlinear distribution for the markers and because by using the SsdwLab v5,0 programme for the combined screening test, the corrected multiple of median is calculated according to the data on pregnancy (e.g. age of pregnant woman, body mass index, presence of diabetes type I) and ultrasound markers for fetus (nuchal translucency thickness, NT, crown-rump length, CRL, gestational age of the fetus, GA).
Descriptive statistical data analysis of variables examined showed that pregnant women in the group with preeclampsia were older than healthy pregnant women from the control group (31.53 ± 5.00 vs. 28. 10  Considering the absence of normal and linear distribution for the variables examined, we used, except for maternal age, non-parametric statistical tests, which do not take distribution into account. 8,9 The youngest among pregnant women was 20 and the oldest 46 years old. The Mann-Whitney test confirmed that, for age, there is a statistically significant difference be tween the groups, Z = -2.689, r = 0.321, p = 0.007. By conducting statistical analysis of the age of pregnant women and examining correlations with other variables, we found a statistically significant correlation with body mass index (BMI) p = 0.008 and the occurrence of preeclampsia p= 0.006. In the control group, the age of pregnant women had positive and moderate correlation with free beta-hCG (p = 0.021) and negative and moderate correlation with marker PAPP-A (p=0,011), with the level of statistical significance p = 0.05.
Our results are in accordance with the data from the literature stating that hypertension in pregnancy is more common in older pregnant women and, by the model of logistic regression of individual variables, this risk factor is very important for the development of preeclampsia. It was only for age, as an examined variable, that we had a normal distribution, which is in accordance with other authors. 1,3 Numerous studies specify obesity as a risk factor for preeclampsia. 10,11 In our results, using correlation data analysis, body mass index (BMI) was confirmed as a statistically significant (p < 0.05). In the preeclampsia group there was a negative, moderate and statistically significant correlation between BMI and biochemical markers, PAPP-A (p= 0.028) and free ß-hCG (p = 0.014) while in the control group of healthy pregnant women there was a marginally significant and negative correlation with marker PAPP-A (p = 0.057).
Mann-Whitney test confirmed a statistically significant difference for the body mass index and that it is more important in relation to other examined variables, Z = -3.205, r = 0.383, p = 0.001. According to Kolmogorov-Smirn ov test, the BMI has a statistical significance, p = 0.003.
The model of logistic regression of individual variables showed the age of pregnant women as more important risk factor for the prediction in comparison with the body mass index. New studies suggest the prevention of obesity prior to conceiving, that is, planning of pregnancy, because PE can be a risk factor for cardiovascular, cerebrovascular and/or other chronic diseases later in life. 11,12 During the combined screening test in the first trimester of pregnancy between the 12th and 14th week of gestation, biochemical markers (free ß-hCG and PAPP-A) secreted by the trophoblastic cells of placenta were determine from the serum of pregnant women.
Free ß-hCG often has false positive values greater than 2.0 MoM, and it requires a necessary correct interpretation and determination of the correlation between its value and the state of pathological pregnancy. [4][5][6] Data from the literature show that increased levels of hCG, regardless if the total or free ß-hCG subunit is being measured, are indicators of fetal hypoxia. Higher levels of free ß-hCG (> 2.5 MoM) can also indicate the risk of preeclampsia. 6 In our results, free ß-hCG in the preeclampsia group of pregnant women was increased but it was not statistically significant, according to Mann-Whitney test (p=0.294) and Kolmogorov-Smirnov test (p = 0.397).
The ROC curve analysis (Receiver Operating Characteristic) for the free ß-hCG marker found that the test was bad and not statistically significant (p = 0.291) because of the small area under the ROC, AUC=0.574.
Numerous studies have examined the effect of biochemical marker PAPP-A on the assessment of the course and outcome of pregnancies with a diagnosed threatened preterm labour and PE. Some studies describe connection between the low values of biochemical marker PAPP-A, less than 10%, with SGA (Small for Gestational Age ) and PE. 12,13 A study conducted by Lončar, et. al When we take all this into account, we could expect that pregnant women with low values of biochemical marker PAPP-A, measured during the combined screening test in the first trimester of pregnancy, should be, apart for the risk of trisomies 21, 18 and 13 for which screening the test is being conducted, followed up as high-risk pregnancies for the development of PE and its complications (threatened preterm labour, PE, placental abruption, intrauterine growth restriction). 11,12,16 These pregnant women should be controlled every four weeks after the 20th week, until the 30th week of pregnancy even more often, if there are signs of preeclampsia. 15 With an appropriate prevention we could reduce the incidence of morbidity and mortality of pregnant women and fetus, that is, pregnancy complications. 7,16 The use of selected markers is also cost-effective in clinical application when it comes to the price and providing the most optimal diagnostic information. 17 The application of a well-planned screening test in the prevention of disease is cheaper in comparison to the costs of treating already affected patients.
Given the results of our study and confirmed correlations between variables, a combination of examined risk factors such as age, body mass index (BMI), together with the combined screening test in the first trimester of pregnancy and the use of Doppler blood vessels of the uterus, contributes to the prevention of PE and improves maternal health. 1,3,12,18.

Conclusion
The age of pregnant women is a statistically significant risk factor for the development of PE with a negative and medium significant correlation with PE. The BMI has a predictive value for the occurrence of PE and we found negative and moderately significant correlation with biochemical markers and PE. Prevention of obesity as a risk factor for the development of PE is an integral part of the overall prevention of this disease. Statistical significance between the groups examined was not confirmed for free ß-hCG. Our results showed significantly lower values of marker PAPP-A in the group of pregnant women with PE compared to the control group and also confirmed the correlation between the low PAPP-A value and the risk of developing PE. The combined screening test in the first trimester of pregnancy for the early detection of chromosomal abnormalities trisomies 21, 18 and 13 is of great importance in the prevention and early detection of high-risk pregnancy for preeclampsia.