Indicators of angiogenesis and hormonal profile in pregnant women with chronic hypertension in the first trimester

The role of angiogenesis factors and hormonal profile in pregnant women with arterial hypertension has not been detailed studied, and their interaction in such patients has not been determined at present time. The objective: to determine the features of angiogenesis factors and hormonal profile in pregnant women with arterial hypertension of the 1and 2 grade. Materials and methods. A prospective study was conducted in 88 pregnant women, which included: analysis of clinical and anamnestic characteristics and the study of pregnancy characteristics in women with chronic arterial hypertension (CAH) in the I trimester of pregnancy (11–12 weeks). The open prospective controlled study involved 61 pregnant women with CAH of the 1 and 2 stages which consisted the main group. The control group included 27 healthy pregnant women with physiological pregnancy. Concentrations of human chorionic gonadotropin (hCG), progesterone (PG), estradiol (E) and markers of angiogenesis – placental growth factor (PlGF) as a proangiogenic factor and soluble fms-like tyrosine kinase (sFlt-1) as an antiangiogenic factor were determined in the blood serum of all patients, sFlt-1/PGF ratio was also estimated (K). Statistical analysis was performed using «STATISTICA® for Windows 13.0». Results. In the I trimester of pregnancy in women with CAH, a shift in the balance between pro- and anti-angiogenic fac- tors is determined, which is manifested by a statistically significant (p<0.05) increase sFlt-1 level (1700.9 pg/ml) and a decrease PlGF level in 3.7 times (9.1 pg/ml) and, accordingly, an increase K coefficient (sFlt-1/PlGF) by 5.3 times (184.5). The mean concentrations of E, PG and hCG in pregnant women of the main group did not differ statistically significantly from those in the control group. However, according to the results of the correlation analysis, PlGF and PG indicators have feedback in pregnant women with CAH (r=-0.29; p<0.05). When calculating the correlation of PlGF and hormones, it was found that in pregnant women with CAH there is their growth according to the control group, namely the indicator hCG correlates with PlGF (51.8 units vs. 14.8 units) 3.5 times, E correlates with PlGF (309.7 units vs. 70.0 units) 4.4 times and PG correlates with PlGF (4.8 units vs. 1.0 units) 4.8 times (p<0.001 for all indicators). These changes indicate the presence of angiogenesis disorders in pregnant women with chronic hypertension, starting from the I trimester of gestation. Conclusions. Pregnant women with CAH in the I trimester have disturbances in balance between pro-angiogenic and anti-angiogenic factors with prevalence of sFlt-1 and decrease of PlGF in blood serum. Because of such imbalance in pregnant women with CAH of the 1 and 2 grade, the interaction of angiogenesis factors and hormones changed from direct to inverse. It may lead to the development of placental dysfunction in the future, so treatment and prevention should be provided for the pregnant women with CAH in the I trimester.

The activity of placental angiogenesis is controlled by a spectrum of growth factors with pro-angiogenic and anti-А К У Ш Е Р С Т В О angiogenic properties [8,9,10,11]. The placental growth factor (PlGF) in the first trimester of pregnancy stimulates the synthesis of trophoblastic DNA, increases the number of trophoblast cells and improves the conditions for its infestation [12,13,14,15]. During the third trimester of pregnancy PlGF expression reaches a maximum of 28-30 weeks of gestation, gradually increasing from the first to the second trimester of pregnancy [16,18]. Antiangiogenic factors include placental soluble fms-like tyrosine kinase (sFlt-1) [17]. It counteracts the action of PlGF on specific receptors [19]. The imbalance between pro-and anti-angiogenic factors contributes to placental insufficiency [9,11,19].
Estradiol (E) is the main hormone of pregnancy. Reduced E, its constantly low concentration, or insufficient growth indicate a violation of the fetoplacental complex [22,23]. Chorionic gonadotropin (CHG) appears in the blood of the mother on 8-9 days after fertilization of the egg. During the first trimester of pregnancy, CHG levels are rapidly increasing, doubling every 2-3 days, reaching its maximum at 8-10 weeks of gestation. After that, its content is somewhat reduced and remains constant during the second half of pregnancy [24,25,26,27]. Progesterone (PG) improves the conditions for trophoblast migration in 1-2 gestational trimesters by stimulating the growth and remodeling of the coiled arteries of the uterus, increasing the expression of the vascular endothelial growth factor and its receptor [28].
The role of the factors of angiogenesis and hormones of pregnancy in pregnant women with hypertension has been studied inadequately and their interaction in such patients is not fully elucidated at present time.
The objective: to detect peculiarities of angiogenesis factors and hormonal profile in pregnant women with arterial hypertension 1 and 2 grade.

MATERIALS AND METHODS
Criteria for inclusion in the study: pregnancy, the presence of CAH of 1-2 grades. Criteria for exclusion from the study: CAH of 3 grade, diabetes mellitus, multiple pregnancy, chromosomal and genetic disorders, thrombophilia, perinatal infections, systemic connective tissue diseases, heart disease (heart`s defects, myocarditis), anemia of moderate to severe degrees, diseases of the lungs, oncological diseases, pregnancy that comes with assisted reproductive technology.
Conducting a prospective study of 88 pregnant women, which included: analysis of clinical and anamnestic characteristics and study of the peculiarities of pregnancy in women with chronic hypertension in the first trimester of pregnancy (11-12 weeks). In the dynamics of pregnancy, all patients were examined by a physician. According to the indications of pregnant women, consultations of specialists of other specialties were carried out and additional instrumental research methods were performed.
The open prospective controlled study involved 61 pregnant women with CAH 1-2 degrees, who were included in the first -the main group (mean age was 27.7 ± 1.7 years). The second control group included 27 healthy pregnant women with physiological pregnancies (mean age -27.9 ± 1.4).Anamnesis, general clinical examination, measured of blood pressure, standard obstetric and gynecological examination according to clinical protocols were done in all cases. The evaluation of the outcome of the accouchement was performed on the assessment of the fetal condition on the Apgar scale, weight of the fetus. AH was diagnosed according to the existing clinical protocols.
The following hormones were determined in blood serum: chorionic gonadotropin (HCG), progesterone (PG), estradiol (E). Among the markers of angiogenesis in blood serum, the following were determined: the placental growth factor (PGF), as a pro-angiogenic factor and placental soluble fms-like tyrosine kinase (sFlt-1) was determined as an anti-angiogenic factor, sFlt-1/PGF ratio was also estimated. Research of hormones and factors of angiogenesis was performed on the basis of the Educational Medical Laboratory Center (the head is professor A.V. Abramov) of the Zaporizhzhya State Medical University. For this purpose, the full-wave enzyme-linked enzyme analyzer Sirio-S (Seac, Italy) was used. Indicators of hormones and factors of angiogenesis were performed using immuno-enzymatic methods in accordance with the relevant instructions using the appropriate sets of reagents: CHG, PG, E (Monobind Inc, USA); PGF and sFlt-1 (R&D systems, Inc, USA&Canada).
All stages of research were carried out with the assurance of the rights and freedoms of patients provided for by the Declaration of Helsinki (Declaration of Helsinki1964 -2000) of the International Conference on Harmonization (ICH) and compliance with the standards of good clinical practice (GCP), the Convention of the Council of Europe on the protection of human rights and dignity in connection with the use of achievements of biology and medicine (from 04.04.1997).
Statistical analysis was done by using «STATISTI-CA® for Windows 13.0». Normality of data`s distribution in groups was determined by Shapiro-Wilk method. Results were presented as mean ± error of mean (M±m). Differences between groups were estimated by Student's criterion. To determine the relationship between the indicators, the correlation coefficient was calculated using the Spirmen method; statistically significant results were considered with a coefficient of more than 0,3 and with a level of p<0,05.

RESULTS AND THEIR DISCUSSION
We could say that assessing the average levels of angiogenic factors, found a statistically significant difference between the groups, р>0,05 (tab. 1). It was found that the level of PlGF was significantly reduced in persons of group I 3.7 times: 9.1 (3.8; 19.2) pg / ml against 33.6 (26.8; 45.6) pg / ml in women of the II group, р<0,001. The rate of sFlt-1, in contrast, was higher in pregnant women with CAH: 1700.9 (1315.6; 2005.6) pg / ml against 1419.7 (1060.3; 1673.5) pg / ml, p<0, 05. Given the changes in the markers of angiogenesis, it was decided to calculate the ratio of these factors (soluble fmslike tyrosine kinase to placental growth factor) to each other. The ratio of sFlt-1 to PlGF was denoted by the coefficient K. Accordingly, K was significantly increased in persons of group I in 5. Assessing hormone levels in pregnant women (progesterone, estradiol, human chorionic gonadotropin), no sta-tistically significant difference was found between groups, p>0.05 (tab. 2).
The situation was somewhat different when comparing the ratios of PlGF and pregnancy hormones with each other (tab. 3).
Thus, the rate of CHG / PlGF was 3.5 times higher in women with CAH than the corresponding rate of pregnant women with a physiological course of pregnancy. The E / PlGF index exceeded 4.4 times, and the PG / PlGF-4.8 times, respectively, p<0.001 for all indicators.
Correlation analysis between angiogenesis and pregnancy hormones in group I of women with CAH revealed that PlGF and CHG had a weak force feedback (R = -0.29), PG and E levels -a direct link of medium strength (R = +0.50). Statistically significant correlations were not determined between other data (tab. 4).
The corresponding analysis in women of the control group showed that PlGF and CHG had a direct relationship of medium strength (R = + 0.43) (p<0.05).     Table 5 The results of the correlation analysis between angiogenesis and pregnancy hormones in the control group Note. * -р<0.05.
Thus, it was found that in the first trimester of pregnancy (11-12 weeks of gestation) in women with CAH is determined by a shift in the balance between proand antiangiogenic factors, manifested by statistically significant (p<0.05) increase in sFlt-1 levels (1700,9 pg/ ml) and a decrease in PlGF levels in 3.7 times (9.1 pg/ml) and, accordingly, an increase in the levels of the coefficient K in 5.3 times (sFlt-1 / PlGF) (184.5).
The mean values of estradiol, progesterone and chorionic gonadotropin levels in pregnant women with CAH did not differ statistically significantly from those of the control group (p>0.05). However, according to the results of correlation analysis, the indicators of PlGF and PG have feedback in pregnant women with CAH (r=-0.29; p<0.05).
When calculating the correlation of PlGF and hormones, it was found that in pregnant women with CAH there is their growth according to the control group, namely the indicator CHG correlates with PlGF (51.8 units vs. 14.8 units) 3

CONCLUSIONS
1. Pregnant women with CAH in 1 trimester have disturbances in balance between pro-angiogenic and antiangiogenic factors with prevalence of sFlt-1 and reducing of PlGF in serum.
2. Due to such disbalance in pregnant women with CAH of 1 and 2 grade changed cooperation between angiogenesis factors and hormones from direct to inverse.
3. It may lead to development of placental insufficiency in future. That's why treatment/prophylaxis of it should be administered to pregnant women with CAH in 1 trimester.