False Positive Values of Biomarkers of Prenatal Screening on Chromosomopathy as Indicators of a Risky Pregnancy

False Positive Values of Biomarkers of Prenatal Screening on Chromosomopathy as Indicators of a Risky Pregnancy Genetic screening on chromosomopathy has been performed on 2000 pregnant women in their first trimester of pregnancy by determining Pregnancy associated plasma protein-A and free-beta HCG biomarkers in maternal serum. After obtaining a normal fetal karyotype, the pathological values of the biomarkers have been correlated with other pregnancy disorders, and the possible causes of the positive genetic screening have been tested. 340 false positive biomarkers (17%) have been detected. The increased free-beta HCG (48.24%) had a significant influence. A significant correlation (p > 0.01) between the increased free-beta HCG and bleeding during pregnancy has been established. Complications occurred in 78.52% pregnancies with pathological biomarkers, MISSed in 13.82%, miscarriages in 10.88%, induced pregnancy terminations caused by fetal anomalies in 8.82% and births with disturbed fetal vitality in 45%. The research results have shown a significant correlation (p > 0.01) between the increased value of the free-beta HCG biomarkers and fetal hypoxia. The false positive genetic screening, caused by the increased free-beta HCG, can indicate placental dysfunction and fetal vitality disruption. Lažno Pozitivne Vrednosti Biomarkera Prenatalnog Skrininga na Hromozomopatije Kao Pokazatelji Rizične Trudnoće Kod 2000 trudnica urađen je u prvom trimestru trudnoće genetski skrining na hromozomopatije određivanjem biomarkera Pregnancy associated plasma protein-A i free-beta-HCG u maternalnom serumu. Posle dobijanja normalnog kariotipa fetusa, patološke vrednosti biomarkera su korelisane sa drugim poremećajima trudnoće kako bi se ispitali mogući uzroci pozitivnog genetskog skrininga. Otkriveno je ukupno 340 lažno pozitivnih nalaza biomarkera (17%). Značajan udeo imao je povišeni free-beta-HCG (48,24%). Utvrđena je značajna povezanost (p>0,01) povišenog free-beta-HCG i krvarenja u trudnoći. U 78,52% trudnoća sa patološkim biomarkerima nastale su komplikacije: 13,82% MISSed, 10,88% spontani pobačaj, 8,82% indukovani prekid trudnoće zbog anomalija ploda i 45% porođaja sa poremećajem fetalnog vitaliteta. Rezultati istraživanja su pokazali veoma značajnu povezanost (p>0,01) između povećane vrednosti biomarkera free-beta-HCG i fetalne hipoksije. Lažno pozitivan genetski skrining uzrokovan povišenim free-beta-HCG može da bude pokazatelj placentarne disfunkcije i poremećaja fetalnog vitaliteta.


Introduction
Genetic screening on chromosomopathy is performed in the first trimester of pregnancy, between the 11 th and 14 th week, by determining the nuchal tran s lucency of a fetus (of the cervical crease -NT) with the ultrasound and the fetopla cental Pregnancy associated plasma protein-A (PAPP-A) and free human chorionic gonadotropin (free-beta HCG) biomarkers in maternal serum. PAPP-A is a glycoprotein synthe sized in the trophoblastic placental tissue. In Summary: Genetic screening on chromosomopathy has been performed on 2000 pregnant women in their first trimester of pregnancy by determining Pregnancy associated plasma protein-A and free-beta HCG biomarkers in maternal serum. After obtaining a normal fetal karyo type, the pathological values of the biomarkers have been correlated with other pregnancy disorders, and the possible causes of the positive genetic screening have been tested. 340 false positive biomarkers (17%) have been detected. The increased free-beta HCG (48.24%) had a significant influence. A significant correlation (p > 0.01) between the increased free-beta HCG and bleeding during pregnancy has been established. Complications occurred in 78.52% pregnancies with pathological biomarkers, MISSed in 13.82%, miscarriages in 10.88%, induced pregnancy terminations caused by fetal anomalies in 8.82% and births with disturbed fetal vitality in 45%. The research results have shown a significant correlation (p > 0.01) between the increased value of the free-beta HCG biomarkers and fetal hypoxia. The false positive genetic screening, caused by the increased free-beta HCG, can indicate placental dysfunction and fetal vitality disruption.
Keywords: prenatal screening, chromosomopathy, risky pregnancy, false positive values trisomy 21, 18 and 13, concentrations in maternal serum are more than 40% lower than in normal pregnancy. Free-beta HCG is a hormone which produces syncytiotrophoblast. Its increased level in pregnancy is considered the most sensitive marker in detecting trisomy 21, while in trisomy 18 and 13 the values are much lower. The value of each biomarker is calcu lated in conventional units and in relation to a median (MOM -multiple of median). The sensitivity of ge netic screening has been improved with the use of software which adapts the values of fetal biomarkers in the serum according to the mother's age, weight, ethnic group, diabetic status, the number of smoked cigarettes and gestational age calculated with the ultrasound by measuring the crown-rump length (CRL). By combining the biomarkers with the ultrasound marker NT, an individual risk expressed as 1:n can be obtained. As a cut-off risk indicating prenatal karyotyping, 1:270 is used which corresponds to a pregnant woman aged 35.
It has been estimated that the rate of false positive values of genetic screening is about 5%, which has resulted in the increased number of invasive diagnostic procedures of prenatal karyo typing in risk free pregnant women with respect to age, but also in the negative implications on the psychological con dition of a pregnant woman (12)(13)(14). The causes of the false positive results of genetic screening have lately been increasingly investigated and the model for the reading of fetal biomarkers as well as for the interpretation of pathological values has been sought for.
Through the prospective and partly retrospective analysis of genetic screening on chromosomopathy in the first trimester of pregnancy, the frequency of false positive results has been tested. In normal fetal karyotyping, the correlation between the pathological values of biomarkers and other pregnancy disorders has been sought out. Is false genetic screening worthless after the prenatal karyotyping and normal fetal karyotyping? Are the pathological values of the biomarkers indicators of other possible risks in pregnancy?

Material and Methods
2000 pregnant women have been tested. In the first trimester of their pregnancy the genetic screening on chromosomopathy has been performed, the nuchal translucency of the fetus (NT) has been measured, and the PAPP-A and free-beta HCG biomarkers in maternal serum have been determined. From every pregnant woman a detailed personal, gynecological, hereditary and teratogenic anamnesis has been taken, while monitoring their respective health status during pregnancy, the outcome of the pregnancy and the health status of the neonate. PAPP-A and free-beta HCG biomarkers have been read with IMMULITE 2000 SIEMENS which operates on the principle of chemiluminescence, using the original reagents (15). The processing of data and deter mination of the risk of trisomy 21 and 18 have been done with PRISCA 4 SOFTWARE (16).
For the statistical processing of the data in a dichotomous manner, i.e. to establish whether a marker is normal or pathological, whether there is a risk or not, the table of contingency (chi-quadrant test) has been used.

Results
Out of 2000 pregnant women tested, there were 138 (6.9%) false positive screening results with the final risk in PRISCA software higher than 1:250 indicating prenatal karyotyping, while 202 pregnant women (10.1%) had a final risk higher than the initial age risk. 340 pregnant women with bad genetic screening results were submitted to the fetal karyotyping test and a normal karyotype of the fetus was obtained. In pregnancies terminated in misca rriages the cytogenetic analysis of the miscarried sample of the chorion and fetal tissue has been performed and a normal karyotype has been obtained which proved the false positive result of genetic screening.
Only 5.3% of the false positive results had a pathological NT marker, while in 94.7% false results were caused by the pathological values of PAPP-A and free-beta HCG biomarkers. Increased free-beta HCG had the largest influence on false positive results (48.24%).
The connection between the increased value of free-beta HCG over 2 MOM and the pregnancies accompanied by bleeding and maintained by progesterone drugs has been examined. There is a significant connection between the increased value of free-beta HCG over 2 MOM and the maintained pregnancy ( Table I).
The outcome of pregnancies with false positive genetic screening has been monitored. 33.53% of preg nancies terminated in a miscarriage in the second trimester ( Table II).
The vitality of a newborn has been examined in 66.47% pregnancies that ended in labour (Table III).  The connection between increased free-beta HCG over 2 MOM and fetal hypoxia has been analysed. A significant connection was established between the increased value of free-beta HCG over 2 MOM and fetal hypoxia (Table IV).

Discussion
The free-beta HCG biomarker is the most frequent cause of false positive results of genetic screening with values higher than 2 MOM, thus its correct interpretation and correlation with the pregnancy pathology is necessary.
The increased free-beta HCG over 2 MOM is particularly noticeable in pregnant women who have suffered from threatened miscarriage, bleeding in the early pregnancy and the maintained pregnancy supported by progesterone pills. Of 140 maintained pregnancies with bleeding, the increased free-beta HCG over 2 MOM was noticed in 60 (47.14%). Freebeta HCG is the product of placental syncytio trophoblast. It is considered that the level of the HCG serum biomarker can also represent a placental marker. Retrospectively, the correlation between beta HCG and placental vascular disruption has been observed, so that the high level of beta HCG markers over 2.5 MOM can indicate a risk of preeclampsia (17,18). The increased level of free-beta HCG biomarkers is the result of placental trophoblast hyperplasia. In these conditions, the mechanism for the rise of free-beta HCG lies in the focal necrosis of syncytiotro phoblast and the rise of mitotic activity with cellular cytotrophoblast proliferation which is transformed into syncytiotrophoblast. In the transformed syncytio trophoblast there is an increased production of free-beta HCG.
Out of 340 pregnancies with the false positive genetic screening, 47 (13.82%) ended in MISSed, 37 (10.88%) in miscarriage, 30 (8.82%) by induced pregnancy termination. Among the indicated preg nancy terminations there were 3 cases of anencephaly, 8 of cystic hygroma, 3 of omphalocele, 4 of heart defect, 1 of a diaphrag matic hernia, 3 of Dandy-Walker Syndrome, 2 of poly cystic kidney disease, 1 of spina bifida, 3 of multiple anomaly and 2 pregnan cies terminated because of teratogenic effects. Fetal karyotyping has been done in all pregnancies before or after the termination and there were no chromosomal aberrations. 226 (66.47%) pregnancies ended in labour, 184 (54.12%) in spontaneous labour, 38 (11.18%) in a Caesarean section and 4 (1.17%) in stillbirth.
Based on the health status of neonates, two groups were formed: healthy and vital babies made up one group, diseased and non-vital babies with normal karyotypes formed the other group. Among the diseased babies were the ones with fetal respiratory distress, asphyxiation, intracranial hemor rhage and convulsions, all resulting from hypoxia. The connection between the level of HCG markers in maternal serum on the one hand and complications in pregnancy on the other has been mentioned in various reference books (19)(20)(21)(22). In this research, the condition of fetal hypoxia has been statistically much more often observed in pregnancies with the increased level of HCG markers over 2 MOM than in the control group (p > 0.01). Inadequate perfusion leads to reduced placental oxy genation. The con dition of hypoxia leads to syncy tiotropho blastic hyperplasia which produces increa sed HCG and these changes in the placenta are connected to the fatal outcome of pregnancy.
Although the primary risk was low, compli ca tions occurred in 78.52% of pregnancies with patho logical genetic screening, MISSed in 13.82%, miscar riages in 10.88%, induced pregnancy termination in 8.82% and births with disturbed fetal vitality in 45%.  screening and to the determination of the level of risk in a pregnancy. Pathological genetic screening with normal fetal karyotyping can indicate placental dysfunction and fetal vitality disruption. When the perfor med prenatal cytogenetic analysis has confirmed the fetal karyotype as normal and the genetic screen as falsely positive, it should not be for gotten that the fetoplacental biomarker values were pathological; instead we should look for the cause of their abnormal production. Such pregnan cies should be treated as risky and should be clinically and laboratory controlled. In the third trimester there is the possibility of determining other biomarkers indicating fetoplacental vitality, such as human placental lacto gen (HPL), glucose tolerance tests, preeclampsia risks, regular ultrasound controls of fetal biometry, estimation of biophysical profile, doppler test of utero placental and fetoplacental circulation.

Conflict of interest statement
The authors stated that there are no conflicts of interest regarding the publication of this article.