Association of 2-Methoxyestradiol (2ME) Plasma Levels with Clinical Severity Indices and Biomarkers of Preeclampsia

Abstract

Introduction

Preeclampsia is an important cause of maternal and perinatal morbidity and mortality. The pathophysiology of preeclampsia has been extensively studied. 2ME seems to maintain placental homeostasis necessary for appropriate cytotrophoblast invasion of the maternal deciduas. We plan this study to compare its value in the plasma of normotensive and preeclamptic women and studying its correlation with the severity indices of preeclampsia.

Aim and Objectives

The aim of this study was to estimate plasma levels of 2ME in preeclamptic and normotensive pregnant women and correlate it with the severity in terms of clinical indices, laboratory investigations, and fetomaternal outcome.

Materials and Methods

This study was a case–control study conducted in KGMU, Lucknow, August 2015–2016. Sample size was 90 pregnant women including 30 normotensive, 30 non-severe preeclampsia, and 30 severe preeclampsia patients. Plasma 2ME levels were analyzed using ELISA kit and the outcome was compared in terms of systolic and diastolic blood pressure, proteinuria, liver and renal function tests, serum LDH, uric acid, plasma 2ME levels, and fetomaternal outcome.

Results

There was a significant (p = 0.0001) difference in 2ME plasma levels among the groups (normotensive, non severe and severe preeclampsia patients). 2ME plasma levels significantly negatively correlated with SBP (r = − 0.62, p = 0.0001) and DBP (r = − 0.63, p = 0.0001). With one-unit increase in SBP, 2ME will decrease by 2445.14 units, and in one-unit increase in DBP, 2ME will decrease by 1807.28 units. The sensitivity and specificity of 2ME for preeclampsia were found to be high.

Conclusion

It was seen that there was a statistically significant difference between plasma 2ME levels among the three groups. The ROC curve obtained showed that sensitivity was predicted to be 98.3%, specificity 76.7%, positive predictive value of 89.4%, negative predictive value of 95.8% and accuracy of 91.1%.

Introduction

Hypertensive disorders of pregnancy and specially preeclampsia are the leading causes of maternal and perinatal morbidity and mortality in developing and developed countries [1]. The pathophysiology of preeclampsia has been extensively studied over the past decades. Recent research has been focused on the role of catechol-O-methyltransferase (COMT) and its product 2-methoxyestradiol (2ME) [2] in pathophysiology of preeclampsia. 2ME is a naturally occurring metabolite of 17 β estradiol generated via enzyme catechol-O-methyl transferase (COMT) [3].

A role for 2ME in the pathophysiology of PE seems likely because this estrogen appears to be a key factor in maintaining placental homeostasis necessary for appropriate cytotrophoblast invasion of the maternal decidua [3].

Other researchers have also studied 2ME for its role in preeclampsia, yet large RCT have not been done to establish 2ME in the pathophysiology and prognosis of the disease. Hence we plan this study to test the hypothesis that 2ME may be a key factor in the pathophysiology of preeclampsia, by comparing its value in the plasma of normotensive and preeclamptic women and studying its correlation with the severity indices of preeclampsia.

Methods and Material

The present study was a cross-sectional case–control study conducted in Deptt of Obstetrics and Gynaecology, Queen Mary hospital, Lucknow, in collaboration of Department of Biochemistry, King George Medical University, Lucknow.

Duration of study was 1 year.

Sample size was 90 (n = [16 σ2/d2] + 1): Cases—60 (Study group 1—30; Study group 2—30); Controls—30

In accordance with American College of Obstetrics and Gynaecology (ACOG) 2013 preeclampsia was defined as blood pressure elevation with proteinuria after 20 weeks of gestation. Blood pressure elevation defined as BP ≥ 140/90 mm of Hg, measured on 2 occasions 4 h apart in women previously known to be normotensive.

Proteinuria defined as ≥ 300 mg protein in 24-h urine sample or protein/creatinine ratio ≥ 0.3 both values in mg/dl or proteinuria of + 1 or more by Dipstick test.

Preeclampsia with severe disease or severe preeclampsia constituted (ACOG) blood pressure greater than 160/110 mm of Hg systolic or greater than or equal to 110 mm of Hg diastolic, or BP ≥ 140/90 mm of Hg plus proteinuria may or may not be present plus one or more of the following:

• Thrombocytopenia—platelet count less than 100,000/microliter.

• Renal sufficiency.

• Serum creatinine concentration greater than 1.1 mg/dl.

• Doubling of the serum creatinine concentration in the absence of other renal diseases.

• Impaired liver function—elevated blood concentration of liver transaminases to twice the normal limit (SGOT/SGPT ≥ 80 IU/ml).

• Pulmonary edema.

• Cerebral or visual symptoms.

There was no selection bias as all the cases were chosen as per the above defined criteria.

Participants Allocated in Three Groups

Study Group 1

Comprised of 30 pregnant women of gestational age ≥ 34 weeks with non-severe preeclampsia (late onset).

Study Group 2

Comprised of 30 pregnant women of gestational age ≥ 34 weeks with severe preeclampsia (late onset).

Controls

Comprised of 30 pregnant women, with normal pregnancy and gestational age of ≥ 34 weeks.

Inclusion criteria for cases—Comprised of 60 pregnant women of gestational age ≥ 34 weeks with non-severe preeclampsia (study group 1 = 30) and severe pre eclampsia (study group 2 = 30).

Inclusion criteria for controls—Comprised of 30 pregnant women, with normal pregnancy and gestational age of ≥ 34 weeks.

Exclusion criteria for cases and controls—Chronic hypertension with or without any anti hypertensives, preeclampsia superimposed on chronic HTN, DM, chronic renal or liver disorders, maternal or fetal infection, fetal congenital anomalies, multiple pregnancies.

Any chronic illness.

Methodology

After taking informed consent, all the women were subjected to the detailed history of present pregnancy including past history, family history, obstetrical history socioeconomic history, and complaints of all subjects were noted. Thereafter, general examination, systemic examination, obstetrical examination, and various investigations pertinent to the case were sent.

Blood Pressure Measurements

Using an appropriate-sized cuff, BP was measured in the right arm sitting position with the arm kept at the level of the heart, using a sphygmomanometer. All patients were relaxed at the time of BP assessment. Two readings were taken 4 h apart by two different observers, thus eliminating observation bias.

Investigations

Routine Antenatal Investigations

Hemoglobin, ABO Rh typing, VDRL, blood sugar screening, viral markers, USG (obstetrical), and urine routine microscopy were carried out.

Specific Investigations for Study Group Only

Hemogram, liver function tests, kidney function test, serum electrolytes, serum uric acid, and serum LDH were performed.

Special Investigation

Plasma levels of 2-Methoxyestradiol were measured.

Collection of Sample

• After obtaining ethical clearance and informed consent, blood samples were taken from antecubital veins into tubes containing sodium citrate.

• Samples were kept cool in normal domestic refrigerator at 4 C until they were processed in the laboratory at Department of Biochemistry.

• Samples were then centrifuged at room temperature at 1000 gm for 15 min. The aliquots of plasma were stored at – 80 C at Department of Biochemistry KGMU, Lucknow, until analysis.

Analysis of Samples

Kit used Plasma 2ME levels were measured using ELISA kit from Cayman Chemical Company. According to Cayman chemical company, this assay has a range from 13.1 to 8000 pg/ml and a sensitivity of (80%) of about 40 ng/ml. The kit was designed to check 100 samples and cost Rs 48,000. It was a self-funded project. The kits required to check 2ME levels are not commercially available, making them costly.

Statistical Tools Employed

The statistical analysis was done using SPSS (Statistical Package for Social Sciences) version 15.0 statistical Analysis Software. The values were represented in number (%) and mean ± SD.

The following Statistical formulas were used:

Mean; standard deviation; Chi-square test; Student’s t test; odds ratio; ROC curve

The Outcome of Study were Compared in Terms of

• Systolic blood pressure values.

• Diastolic blood pressure values.

• Values of proteinuria.

• Liver and renal function tests.

• Serum LDH and uric acid levels.

• Levels of 2-Methoxyestradiol in plasma of both cases and controls.

• Fetomaternal outcome.

Results

The results of the present study were as follows:

The three groups were matched for demographic variables (Table 1).

Table 1.

Comparison of demographic variables among the groups

	Non-severe Preeclampsia Gropu 1	Severe Preeclampsia Group 2	Control Group 3
Age (mean ± SD)	25.67 ± 2.86	25.40 ± 2.45	25.40 ± 2.45
Occupation
Housewife	96.7 %	100 %	86.7 %
Service	3.3 %	0 %	13.3 %
Dietary habits
Vegetarian	56.7 %	56.7 %	53.3 %
Non-vegetarian	43.3 %	43.3 %	46.7 %
Parity (gravida)
1	26.7	30	36.7
2	36.7	33.3	30.0
3	23.3	26.7	26.7
> 3	13.3	10	6.7
POG
34−35 week 6 day	13.3	16.7	16.7
36−37 week 6 day	43.3	66.7	43.3
38−39 week 6 day	36.7	16.7	33.3
> 40	6.7	0	6.7

Figure 1 shows the comparison of 2ME among the groups. Analysis of variance showed that there was significant (p = 0.0001) difference in 2ME among the groups. Post hoc tests showed that 2ME was significantly (p = 0.0001) different in each other groups. The mean value of 2ME in the control group was 1860.79 ± 613.62 pg/ml; in the study group 1, it was 648.00 ± 268.28 pg/ml, and in study group 2, it was 312.07 ± 168.26 pg/ml.

Fig. 1.

Comparison of 2ME among the groups

2ME plasma levels significantly negatively correlated with SBP (r = − 0.62, p = 0.0001) and DBP (r = − 0.63, p = 0.0001). Regression analysis revealed that in one-unit increase in SBP, 2ME will decrease by 2445.14 units. Similarly, with one-unit increase in DBP, 2ME will decrease by 1807.28 units (Table 2).

Table 2.

Correlation of 2ME with blood pressure among the cases

Blood pressure in mm of Hg	2ME in pg/ml
	Correlation coefficient	p value	Regression equation
SBP	− 0.62	0.0001*	2ME = 2457.52 − 12.38 × SBP
DBP	− 0.63	0.0001*	2ME = 1819.76 − 12.56 × SBP

*Significant

2ME plasma levels correlated moderately with ALP, mildly with platelet count and poorly with proteinurea (Table 3).

Table 3.

Correlation of 2ME with LFT, HB, and platelet count among the cases

	2ME
	Correlation coefficient	p value	Regression equation
Urea (mg/dl)	− 0.35	0.005*	2ME = 795.12 − 6.24 × Urea
Creatinine (mg/dl)	− 0.19	0.13	2ME = 661.27 − 90.26 × Creatinine
Total bilirubin (mg/dl)	− 0.26	0.03*	2ME = 725.61 − 164.06 × Total billirubin
Direct bilirubin (mg/dl)	− 0.32	0.01*	2ME = 650.16 − 173.22 × Direct billirubin
Indirect bilirubin (mg/dl)	− 0.32	0.01*	2ME = 764.14 − 375.72 × Indirect billirubin
AST (IU/Lt)	− 0.20	0.11	2ME = 665.45 − 1.12 × AST
ALT (IU/Lt)	− 0.23	0.07	2ME = 680.30 − 1.13 × ALT
ALP (IU/Lt)	− 0.51	0.001*	2ME = 8.13.58 − 0.79 × ALP
Uric acid (mg/dl)	− 0.48	0.001*	2ME = 1094.74 − 89.32 × Uric acid
Hb (gm %)	0.15	0.22	2ME = 310.45 + 28.03 × Hb
Platelet count (Lakh/cumm)	0.28	0.02*	2ME = 390.43 + 108.37 × PC
Proteinuria (mg/dl)	0.12	0.51	2ME = 366.87 + 23.71 × Proteinurea

*Significant

The sensitivity and specificity of 2ME for preeclampsia was found to be high (Fig. 2). At a 2ME level of 1284 pg/ml the sensitivity was 98.3%, specificity was 76.7%, positive predictive value of 89.4% and negative predictive value of 95.8% with an accuracy of 91.1%.

Fig. 2.

Receiving operating curve showing the sensitivity and specificity of 2ME for preeclampsia AUC (95% CI) = 0.95 (0.90–1.00), p = 0.0001

Discussion

The study was conducted with an aim of comparing levels of 2-Methoxyestradiol in the plasma of normotensive pregnant women and preeclamptic women and finding a correlation between 2-Methoxyestradiol plasma levels of preeclamptic patients and biomarkers and clinical severity indices of preeclampsia. To the best of our knowledge, ours is the first article aiming to study 2ME and its role in pregnancy pertaining to Indian population, and no such article has been published in the literature so far.

The subjects were matched for demographic variables.

Subjects were analyzed in accordance with the clinical symptoms they presented with. Although pedal edema is no longer required in making a diagnosis of preeclampsia, significant amount of pedal edema was present in the study group 1 (4 subjects had + 2 edema, 7 subjects had + 3 edema) and study group 2 (2 subjects had + 3 edema, 11 had + 2 edema and 12 had + 1 edema) as compared to the control group which had no subjects with pedal edema (p value of 0.0001).

In our study also, we found a statistically significant negative correlation between 2ME levels in all the three groups. The control group had a mean value of 1860.79 ± 613.62 pg/ml. The study group 1 had a mean value of 2ME in the range 648.00 ± 268.28 pg/ml. The study group 2 on the other hand had a mean value of 312.07 ± 168.26 pg/ml.

Miriam pertegal et al. [4] conducted a similar study to evaluate the correlation of 2ME plasma levels with clinical severity indices of preeclampsia. He included a total of 126 pregnant women (53 PE and 73 control, normal pregnancies) in his study Control individuals (n ¼ 66) had significantly higher 2ME plasma levels than women with PE (n ¼ 50; 2906.43 + 200.69 pg/mL vs. 1818.41 + 189.25 pg/mL; p < 0.001). Increased plasmatic 2ME was observed to have a significant effect (p < 0.001) and was associated with a decreased risk of PE. The adjOR was 0.05 [95% confidence interval (CI) 0.01–0.25; n ¼ 113].

Since our results have been consistent with previous studies done over the years, it can be inferred that 2ME levels correlate well with the severity of preeclampsia, being lowest in those suffering from severe form of the disease and can be considered as a marker of the disease severity.

We found a linear negative correlation between systolic blood pressure values (SBP) and the plasma levels of 2ME in study group 1 and study group 2. The correlation coefficient was found to be − 0.62. The correlation was significant (p value = 0.0001). With decreasing values of 2ME systolic blood pressure was found to increase among the subjects. This result was similar to Miriam Pertegal et al. [4] who found that the 2ME values significantly and negatively correlated with systolic peak blood pressure, correlation coefficient of − 0.283, and p = 0.47.

Similarly we found a linear negative correlation between diastolic blood pressure values (DBP) and the plasma levels of 2ME in study group 1 and study group 2. The correlation coefficient was found to be − 0.63. The correlation was significant (p value = 0.0001). With decreasing values of 2ME diastolic blood pressure was found to increase among the subjects. However, this result was different than Miriam Pertegal et al. [4] who found that the 2ME values did not correlate with diastolic peak blood pressure values, p = 0.66.

Our findings were also consistent with the study conducted by Zang et al. [5] according to whom plasma 2ME level was significantly lower in early onset severe PE patients than normal pregnant controls, plasma 2ME level correlated negatively with blood pressure only in women with PE and plasma 2ME level did not correlate with proteinuria, plasma levels of E2, sFLT-1, ET-1 or NO in PE patients. Their study also provided clinical evidences that 2ME deficiency is strongly related to hypertension in early onset severe PE patients.

Perez-Sepulveda et al. [6] in his study involving 2ME, its metabolic pathway and its role in preeclampsia, found that during pregnancy, 2ME levels increase significantly when compared to levels during the menstrual cycle; however, plasma levels of 2ME are lower in patients with clinical PE when compared to normotensive pregnancies.

Based on our assessment, plasma 2ME levels were evaluated in normotensive healthy pregnant women, those suffering from non-severe preeclampsia and severe preeclampsia, it was seen that there was statistically significant difference between the three groups. The ROC curve thus obtained showed area under the curve 0.95 for the plasma 2ME cutoff of ≤ 1284 pg/ml (95% CI 0.90–1.00) at this cutoff, the sensitivity test was predicted to be 98.3%, specificity 76.7%, positive predictive value of 89.4%, negative predictive value of 95.8%, and accuracy of 91.1%. Ours was a cross-sectional study, and further prospective studies are needed to accurately predict the period of gestation and frequency with which the 2ME levels should be investigated, monitored, and repeated.

Conclusion

In summary, our study proved that 2ME levels decrease with the severity of preeclampsia, being the lowest in those suffering from severe preeclampsia with increasing levels in non-severe preeclampsia and normotensive healthy pregnant women. We also provided clinical evidence that the levels of 2ME correlate negatively and significantly with the systolic and diastolic blood pressures of those suffering from preeclampsia. 2ME may not only be used as a prognostic and predictive marker but also a therapeutic agent for the patients of preeclampsia, but further studies and clinical trials involving larger population are needed to study the same.

Dr. Vartika Tripathi

is currently working in the Department of Obstetrics and Gynaecology KGMU, Lucknow, as a senior resident. She has completed her MBBS from Kasturba Medical College, Manipal, and has passed her M.D. with gold medal, from KGMU, Lucknow, in 2017. She has presented various papers and posters in National and State level conferences. High-risk pregnancy is among the many academic interests that she has. She plans to strive and continue her work in the field of high-risk pregnancy specially preeclampsia.

Compliance with Ethical Standards

Conflict of interest

There are no conflicts of interest in the study.

Ethical approval

Ethical clearance for the concerned study "Association of 2 Methoxyestradiol plasma levels with clinical severity indices and biomarkers of preeclampsia" has been obtained from the institutional ethics committee.

Human and Animal Participants

Being a research involving human participants, all ethical guidelines were followed.

Informed Consent

Informed consent was taken from the patients.