Investigation of maternal serum cardiotrophin-1 concentrations in pregnant women with preeclampsia; a prospective case-control study

Abstract Objective We aimed to investigate the cardiotrophin-1 (CT-1) concentrations in the serum of pregnant women with preeclampsia Methods This cross-sectional study was conducted with 88 pregnant women who applied to the Umraniye Training and Research Hospital Gynecology and Obstetrics Clinic between May 2022 and September 2022. The preeclampsia group consisted of 44 pregnant women diagnosed with preeclampsia, and the control group consisted of 44 healthy pregnant women matched with the preeclampsia group in terms of age and body mass index. Demographic characteristics, ultrasound and laboratory findings, perinatal outcomes, and maternal serum CT-1 concentrations were recorded. Results Both groups were similar in terms of demographic features and the gestational week at blood sampling for CT-1. Preeclampsia and control groups were compared in terms of maternal serum CT-1 concentrations and no significant difference was found between the two groups (2061.4 pg/ml, 2168.5 pg/ml, respectively, p = .516). The preeclampsia group was divided into subgroups as mild and severe preeclampsia according to the severity of the disease and early-onset and late-onset preeclampsia according to the time of onset and compared with the control group in terms of maternal serum CT-1 concentration, no significant difference was found between the groups (p > .005, for all). Conclusion The serum CT-1 concentration of women whose pregnancy was complicated with preeclampsia was found to be similar to that of healthy controls. Although it has been shown in the literature that high serum CT-1 concentrations are associated with hypertensive heart diseases, its role in the pathophysiology of preeclampsia remains unclear.


Introduction
Preeclampsia, which affects 3-5% of pregnancies, is characterized by de novo hypertension accompanied by proteinuria or renal failure, liver involvement, and some hematological and neurological complications [1].While preeclampsia is still the main cause of maternal and neonatal morbidity and mortality, especially in underdeveloped countries, the management of patients whose pregnancy was complicated with preeclampsia poses a challenge for obstetricians, even at experienced tertiary obstetric care centers.
Although the pathophysiology of the disease has not yet been clarified, it is thought that there is impaired placental development due to maternal-fetal immune incompatibility during early pregnancy.This impaired placental development leads to the release of some vasoactive inflammatory cytokines and free radicals from the placenta into the maternal circulation.These mediators cause endothelial dysfunction and hemodynamic changes in the expectant mother, resulting in clinical signs of preeclampsia [2].
Cardiotrophin-1 (CT-1), was first identified in 1995 as a protein of 21.5 kDa and 203 amino acids in length.Although the amino acid sequence of CT-1 shows similarity with leukemia inhibitory factor and ciliary neurotrophic factor, studies showed that CT-1 was a new member of the interleukin-6 (IL-6) cytokine family [3].One year after the protein isolation, the CT-1 gene was determined to be located on chromosomes 16p11.1-16p11.2[4].
In the human body, CT-1 is expressed in adult heart tissue, skeletal muscle, colon, ovary, testis, prostate, and fetal kidney and lung [4].It has been found that CT-1 exerts its effect through the combination of b receptor glycoprotein 130 (gp130) and b receptor leukemia inhibitory factor (LIFR) signaling pathway in the tissues [5].In different studies, CT-1 has been shown to support cardiac myocyte survival through the activation of the gp130/LIFR-b heterodimer complex resulting in cardiomyocyte proliferation and cardiac hypertrophy [6,7].It has also been shown that CT-1 stimulates the secretion of endothelin-1, a vasoconstrictor mediator, in vascular endothelial cells via the gp130 receptor.Thus, it is thought that CT-1 also affects vascular tone and remodeling [8].
After the discovery of its biological functions, many studies have been conducted to investigate the relationship between CT-1 and hypertensive cardiac diseases [9,10].It is now known that CT-1 plays an important role in the regulation of the cardiovascular system, but its relation with preeclampsia remains unclear.In light of this information, we aimed to investigate the CT-1 concentrations in the serum of pregnant women diagnosed with preeclampsia.

Materials and methods
This prospective case-control study was conducted with 88 pregnant women who applied to Umraniye Training and Research Hospital, Gynecology and Obstetrics Clinic, Istanbul, Turkey, between May 2022 and September 2022 and had their pregnancy followup and delivery in our hospital.44 pregnant women between the ages of 18 and 39 whose pregnancy was complicated by preeclampsia formed the preeclampsia group, and the control group consisted of 44 healthy pregnant women who were matched with the preeclampsia group in terms of age and body mass index (BMI).
Smokers, multiple pregnancies, those who conceive with in vitro fertilization method, with a history of pregestational hypertension and diabetes mellitus, and pregnant women with known vascular disease, thrombophilia, autoimmune disease, and congenital uterine anomaly were not included in the study.Those who had COVID-19 or another infectious disease during pregnancy were also not included in the study.
Participants' age, BMI, weight gain during pregnancy, obstetric history, laboratory findings, and perinatal outcomes were recorded.All participants underwent fetal ultrasound examination by a single obstetrician on the same ultrasound device (Hitachi Aloka Prosound F37).Ultrasonographic measurements were made by the recommendations of the ISUOG guideline [11].Fetal biometry, amniotic fluid index, and umbilical artery Doppler velocimetry values were evaluated.
The diagnosis of preeclampsia was made according to the criteria of the American College of Obstetricians and Gynecologists [12].According to these criteria, preeclampsia was diagnosed after the 20th week of pregnancy when the systolic blood pressure was !140 mmHg or the diastolic blood pressure was !90 mmHg at least in two measurements made 4 h apart and the presence of proteinuria or if systemic involvement of at least one organ was detected in addition to hypertension in the absence of proteinuria.Proteinuria was defined as the detection of 300 mg or more protein in a 24-h urine sample or a protein/creatinine ratio (both in mg/dL) of 0.3 or more in a single voided urine sample.The following findings were accepted as indicative of systemic involvement; thrombocytopenia (platelet count <100 000/lL), impaired liver function (elevated blood concentration of liver transaminases to twice normal concentration), renal insufficiency (serum creatinine concentration >1.1 mg/dL or doubling of serum creatinine concentration in the absence of any kidney disease), pulmonary edema, new onset headache not responding to medication and not explained by alternative diagnoses or visual symptoms.
Before receiving any treatment, approximately 5 ml of blood samples were drawn from the participants at the time of initial diagnosis.Blood samples were gathered into laboratory tubes containing no anticoagulants and the tubes were left at room temperature for 2 h to allow fibrin formation.The samples were then centrifuged at 1000 rpm for 20 min.After centrifugation, the supernatant was separated from the residue and stored at À80 C. CT-1 concentrations in blood samples from all participants were studied with the Human CT-1 Elisa Kit (Wuhan Fine Biotech Co., Ltd., Wuhan, Hubei, China, Lot number: H1055H087) using the Enzyme-Linked Immunosorbent Assay (ELISA) method by the manufacturer's recommendations.For the CT-1 kit used in the study, a measurement value between 15.625-1000 pg/ml and a sensitivity of 9.375 pg/ml was determined.
When CT-1 concentrations were determined with the Human CT-1 Elisa Kit in serum samples taken from the participants, it was observed that some of the absorbance values determined in the first raw data were above the highest standard range (1000 pg/ml).Therefore, serum samples were diluted at a ratio of 1/8, and serum CT-1 concentrations were studied again, and the results were calculated according to this dilution ratio.
As the primary outcome of the study preeclampsia and control groups were compared in terms of maternal serum CT-1 concentrations. The

Statistical analysis
Power analysis was performed using the G Ã Power (v3.1.9)program to determine sample sizes.The power of the study is expressed as 1-b (b ¼ Type II error probability) and has 80% power.Assuming that the effect size (d ¼ 0.593) will be observed according to the effect size coefficients determined by Cohen, it was determined that the required number of patients should be 88 (44 for the preeclampsia group and 44 for the control group).
Statistical analysis was performed with the Statistical Package for the Social Sciences (SPSS) version 25.0.The Kolmogorov-Smirnov test was used to determine whether the data were distributed normally or not.Descriptive statistical methods (mean, standard deviation, median, IQR, frequency, ratio) were used while evaluating the data.Independent t-test was used for comparison of two groups showing parametric distribution, One Way ANOVA was used for comparisons of more than two groups.Mann Whitney U test was used for the comparison of two groups showing non-parametric distribution, and the Kruskal Wallis test was used for comparisons of more than two groups.Significant differences as a result of comparisons of more than two groups were examined by Tamhane and Tukey tests.The Chi-square test was used in the comparison of the groups in terms of categorical data.Statistical significance was accepted at p < .05for all values.

Results
In this study, 44 pregnant women diagnosed with preeclampsia and 44 healthy pregnant women in the control group were compared in terms of maternal serum CT-1 concentration.
Both groups were similar in terms of age, BMI, weight gain during pregnancy, gravida, and parity (p > .05,for each).While 4 patients in the preeclampsia group had a history of preeclampsia, none of the patients in the control group had a history of preeclampsia (Table 1).
Systolic and diastolic blood pressure and mean arterial pressure were significantly higher in the preeclampsia group than in the control group (p ¼ .000,for each).In the preeclampsia group, the median creatinine level was 95 mg/dl, the median protein level was 63.3 mg/dl, and the median protein/creatinine ratio was 1.084 in the spot urine sample.While AST and ALT levels were significantly higher in the preeclampsia group, the hematocrit level was lower (p ¼ .002,p ¼ .010,p ¼ .030,respectively).Hemoglobin level and platelet count were similar in both groups (p > .05for each).The umbilical artery pulsatility index (PI), resistance index (RI), and systole/diastole (S/D) ratio were significantly higher in the preeclampsia group compared to the control group (p ¼ .000,for each).Oligohydramnios was detected in 8 patients in the preeclampsia group and intrauterine growth restriction (IUGR) was detected in 18 patients (Table 2).
In the preeclampsia group, visual symptoms developed in 14 patients, placental abruption in 4 patients, HELLP syndrome in 2 patients, and eclampsia in 1 patient.26 patients in the preeclampsia group received antihypertensive treatment until delivery, and 26 patients received MgSO4 therapy at the time of delivery.The number of emergency cesarean sections and total cesarean deliveries in the preeclampsia group was significantly higher than in the control group (p ¼ .000,for each).Gestational age at birth, birth weight, and 1st, and 5th minute Apgar scores were significantly lower in the preeclampsia group than in the control group, while NICU admission was higher (p ¼ .000,for each).The gender of the newborn and the number of newborns whose amniotic fluid was stained with meconium were similar in both groups (p > .005)(Table 3).
Gestational week at blood sampling for CT-1 was similar for the two groups (p ¼ .151).The median maternal serum CT-1 level was found to be 2061.4pg/ml in the preeclampsia group, while it was determined as 2168.5 pg/ml in the control group (p ¼ .516)(Table 4).
When we divided the preeclampsia group into two groups as mild and severe, the median CT-1 concentration was found to be 2098.1 pg/ml in the mild preeclampsia group and 1982.9pg/ml in the severe preeclampsia group.When these groups were compared with the control group, the three groups were similar in terms of CT-1 concentrations (p ¼ .729)(Table 5).
When we divided the preeclampsia group into two groups early-onset and late-onset, the median CT-1 concentration was found to be 2166 pg/ml in the early-onset preeclampsia group and 1956.8 pg/ml in the late-onset preeclampsia group.When these groups were compared with the control group, the three groups were similar in terms of CT-1 concentrations (p ¼ .541)(Table 6).Finally, Spearman correlation analysis was performed to understand the relationship between maternal serum CT-1 concentration and laboratory parameters associated with preeclampsia.Accordingly, no significant relationship was found between preeclampsia-related laboratory parameters and CT-1 (Table 7).

Discussion
In this study, contrary to expectations, we found the serum concentration of the CT-1, whose high serum concentrations are thought to be associated with hypertensive heart diseases, was lower in the preeclampsia group than in the control group.
The CT-1 molecule was first isolated as a protein that stimulates cardiomyocyte growth and was named 'cardiotrophin' because of this effect [3].In later studies, it was revealed that CT-1 was not specific to the cardiomyocytes only and was also expressed in the skeletal muscle, lung, kidney, pancreas, small intestine, thymus, prostate, testis, and ovary [4].
Asai et al. demonstrated that the human heart itself secretes CT-1 directly, based on a significant increase in the plasma concentration of CT-1 in plasma samples obtained directly from the aorta and coronary sinus in those undergoing diagnostic cardiac catheterization for angina pectoris [13].On the other hand, vascular endothelial cells and adipocytes have also been shown to contribute to circulating CT-1 by expressing CT-1 [8,14].Although the mechanism regulating CT-1 synthesis in the human body has not yet been fully elucidated, several different possible mechanisms have been discussed so far.Mechanical stretching of cardiac myocytes, atrial natriuretic peptide, brain/B-type natriuretic peptide, hypoxia, and reactive oxygen species have been shown to stimulate CT-1 synthesis [15][16][17].
Studies have shown that CT-1 not only induces hypertrophy in cardiac myocytes but also plays an active role in various hematopoietic and neuronal developmental processes and that it exerts this effect through the LIF receptor and gp130 signaling subunit [5,18].Although it has been shown to affect the developmental processes of different tissues and organs, research on CT-1 has mostly focused on hypertensive heart diseases.
In 2014, Song et al. published a meta-analysis of studies investigating the relationship between the CT-1 molecule and cardiovascular disease.This meta-    analysis showed that the serum CT-1 concentration was higher in patients with hypertension than in the control population.Also, patients with left ventricular hypertrophy with hypertension had a higher plasma CT-1 concentration than those without left ventricular hypertrophy.According to the subgroup analysis, it was stated that the plasma CT-1 concentration was significantly higher in patients diagnosed with heart failure compared to healthy controls.Song et al. concluded that increased plasma CT-1 level is associated with an increased risk of left ventricular hypertrophy and heart failure in patients with hypertension [9].
Based on its hypertrophic effect on cardiac myocytes in individuals suffering from hypertension, the effect of CT-1 on the structure of the vascular wall was also investigated.In an animal study, it was shown that CT-1 induces proliferation, hypertrophy, and extracellular matrix production of vascular smooth muscle cells, and this effect of CT-1 is upregulated in hypertension and by aldosterone stimulation [19].In another animal study, it has been shown that CT-1 accelerates the development of atherosclerotic lesions in the vessels by stimulating the inflammatory response in macrophages, foam cell formation, and collagen-1 production in vascular smooth muscle [20].
Jougasaki et al. showed that CT-1 is expressed in vascular endothelial cells and stimulates ET-1 synthesis and secretion in endothelial cells through the gp130 signaling pathway [8].Since ET-1 plays an important role in the regulation of vascular tone, it is conceivable that CT-1 acts as an endothelial-derived biological factor, possibly involved in the regulation of vascular tone, in normal physiological conditions or secondary to pathological processes.
In light of all this information presented in the literature, we hypothesized that the CT-1 molecule may somehow be involved in the pathogenesis of preeclampsia, and we investigated CT-1 concentration in the serum of expectant mothers whose pregnancy was complicated by preeclampsia.At the beginning of the study, we expected maternal serum CT-1 concentrations to be higher in the group whose pregnancy was complicated with preeclampsia compared to the controls.However, contrary to expectations, the median maternal serum CT-1 concentration in the preeclampsia group was lower than in the control group.Again, contrary to expectations, when we performed subgroup analysis, the lowest median CT-1 concentration was found in the severe preeclampsia group.Therefore, in our study, high maternal serum CT-1 concentration was not found to be associated with the severity of preeclampsia.
In this study, the BMI values of both groups at the time of blood sampling for CT-1 were above the normal values and although there was no significant difference, the BMI of the preeclampsia group was higher than the control group (30.7 kg/m2, 28.9 kg/m2, respectively, p ¼ .096).In a study published by Hung et al. it was shown that serum CT-1 level is inversely associated with obesity in nondiabetic individuals [21].Also, Jung et al. found that the serum CT-1 concentration was lower in overweight adolescents than in normal-weight adolescents, although there was no significant difference [22].One reason for the lower median serum CT-1 concentration in the preeclampsia group in our study compared to the control group may be the effect of the participants' BMI, as stated in the studies above.Another reason why CT-1 was not higher in the preeclampsia group than in the control group is that CT-1 and the gp130 signaling pathway, which have been shown to be associated with hypertension in non-pregnant individuals, may not be the dominant pathway responsible for hypertension in preeclampsia.
The small number of participants and the fact that CT-1 expression in the placental tissue was not evaluated are the most important limitations of this study.In addition, CT-1 concentration in participants was assessed only once at the time of initial diagnosis.Weekly changes in serum CT-1 concentrations until delivery and the fact that the placenta's contribution to these potential changes was not investigated are other limitations of our study.
To the best of our knowledge, this is the first study in the literature to examine maternal serum CT-1 concentrations in pregnant women diagnosed with preeclampsia.
In conclusion, this study investigating serum CT-1 concentrations in preeclampsia is a preliminary study.We found that the serum CT-1 concentration of women whose pregnancy was complicated by preeclampsia was similar to that of healthy controls.To clearly understand the role of the CT-1 molecule in the pathophysiology of preeclampsia, there are many issues still to be investigated, including the expression of the CT-1 in the placental tissue, its contribution to the serum CT-1 concentration, and the changes in the serum CT-1 concentrations throughout the pregnancy.
Local Ethics Committee of Umraniye Training and Research Hospital, Istanbul, Turkey has approved this study (Ethics Committee Approval No: B.10.1.TKH.4.34.H.GP.0.01/158).The study protocol was maintained by the Declaration of Helsinki, and informed consent was obtained from all the participants.

Table 1 .
Demographic characteristics of control and preeclampsia groups.
b Chi-Square test; BMI, body mass index.

Table 2 .
Comparison of control and preeclampsia groups in terms of laboratory and ultrasound findings.Independent t-test; b Man Whitney U test; c Chi-Square test.AST: aspartate aminotransferase; ALT: alanine aminotransferase; IUGR: intrauterine growth restriction.Bold values are statistically significant at p < .05.

Table 3 .
Comparison of control and preeclampsia groups in terms of perinatal outcomes.Square test; b Man Whitney U-test; c Independent t-test.NICU: neonatal intensive care unit.Bold values are statistically significant at p < .05.

Table 4 .
Comparison of control and preeclampsia groups in terms of maternal serum cardiotrophin-1 concentration.Man Whitney U-test; b Independent t-test.

Table 5 .
Comparison of control, mild preeclampsia, and severe preeclampsia groups in terms of maternal serum cardiotrophin-1 concentration.

Table 6 .
Comparison of control, early-onset preeclampsia, and late-onset preeclampsia groups in terms of maternal serum cardiotrophin-1 concentration.Kruskal Wallis test; b One Way ANOVA test.Bold values are statistically significant at p < .05.