Association of whole blood copper and preeclampsia: A Cohort study in China

Preeclampsia is a leading cause of maternal and fetal morbidity and mortality. The associations between whole blood copper and pre-eclampsia were controversial. Our study was aimed to investigate the association between whole blood copper concentration and preeclampsia in pregnant woman in China. Methods This retrospective cohort study was conducted between August 1, 2019 to December 31, 2019 in a comprehensive tertiary hospital in Foshan city Guangdong Province, China. We measured copper concentration in maternal whole blood in 12–27 (+ 6) week of pregnancy, using ame atomic absorption spectrometer. We ascertained preeclampsia diagnosis from electronic medical records system. Covariates included demographic, offspring characteristics, serum biomarkers. Logistic regression was applied to explore the association between the concentration of trace element and preeclampsia. are presented as median and interquartile range (IQR) compared categorical variables, normally and non-normally distributed variables between Normal and Pre-eclampsia women, used Pearson Chi-square, ANOVA and Kruskal-Wallis Rank sum test. Scatter plot were used to display the median, IQR, and range of four trace element, and Kruskal-Wallis Rank sum test were used to determine differences in their distributions in Normal and Pre-eclampsia women.


Background
Preeclampsia (PE) is a pregnancy-speci c complication chie y characterized by new-onset hypertension and proteinuria after 20 weeks of gestation, and accompanied by signs of damage to other organ systems, such as hemolysis, elevated liver enzymes, and low platelet count. [1,2] [1,2]Preeclampsia affects 5% to 7% of pregnant women worldwide and it persists as a leading cause of maternal and fetal morbidity and mortality. [3][4][5] And Hypertensive disorders including preeclampsia of pregnancy are the direct cause of death of about 30000 women annually [6],or approximately 14% of maternal death [7],most of which occur in low-income countries and have higher maternal mortality rates [8].
Strategies to prevent preeclampsia have been studied extensively [9], but only low-dose aspirin treatment may be effective in preventing preeclampsia in high-risk women. [10] Timely delivery of the fetus and placenta still remains the only de nitive treatment. However, unfortunately, preeclampsia can persist after delivery and, in some cases, can develop de novo in the postpartum period. [11] Beside, there is accumulating evidence that preeclampsia and eclampsia predisposes to long-term cardiovascular risk. [12,13] The etiology of preeclampsia remains ambiguous, albeit, reports that implicated placental defects and oxidative stress early during pregnancy in affected pregnancies. [14]Therefore, identifying modi able risk factors for preeclampsia is an important priority for reducing morbidity and maternal mortality from preeclampsia.
Copper is an essential trace element ,which is a co-factor of antioxidant enzymes, thus appropriate copper is necessary for health and reducing the risk of PE. Many studies attempted to explore the relationships between copper level in pregnant women and PE, but the results varied greatly. Some studies found signi cantly lower levels of copper in PE patients than the control group. [15][16][17] However, Some studies report signi cantly higher levels of copper in PE patients. [18,19] Meanwhile, some studies nd no associations. [20] Furthermore, to our knowledge, little information is available in the association between copper level during early or mid-term pregnancy and preeclampsia and separately by its subtype. Therefore, research on copper level during early or mid-term pregnancy and preeclampsia is of paramount importance for clinician. In light of these literature gaps, we examined the associations of copper level with pre-eclampsia in women from the China Cohort, using concentrations measured in maternal whole blood in 12-27 (+6) week of pregnancy. We hypothesized that higher copper concentration are associated with lower risk of preeclampsia after appropriate adjustment.

Study design, Setting and Participants
This was a retrospective pregnant women cohort study, studying association of copper level with preeclampsia. The study was conducted in obstetrics department of Foshan Chancheng Central Hospital, Foshan city Guangdong province, China, which obtain the Joint Commission International (JCI) Hospital Accreditation 6th ed. Pregnant women with induced labour, fetal death in uterus, serious birth defects, age at delivery Less than 18 years or more than 42 years were not included in the cohort study.
The population of this study was consecutive participants, which pregnancy registered, checked up and delivered in our hospital among on August 1, 2019 to November 30, 2019. The Hospital Ethics Committee approved this study and waived informed consent because data were unidenti ed. The study protocols were registered in Chinese Clinical Trial Registry (Registration number: ChiCTR2000029643).All reporting followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines. [21] 2512 women were enrolled in the study. 335 were excluded for the following reasons: 281 had missing information on all four trace element,5 had chronic hypertension prior to pregnancy,3 had T2DM prior to pregnancy,20 had twin pregnancy,1 had BMI missing,19 had BMI≥30,1 age<18,5 age>42.2177 participants were remained quantity. We excluded the extreme value of copper ≤1%(9.32-13.08,n=22)and > 99% (21.13-25.98,n=21).A total of 2134 participants were included in the nal analysis( Figure 1).

Data Collection
We extracted socio-demographic characteristics and delivery from electronic medical records system. Information on prepregnancy height and weight, smoking and drinking history,education level, past medical history, last menstrual period, parity history, method of conception were gathered by the physicians at the rst visit for pregnancy registered. And the rst visit for pregnancy registered usually opens at 8-12weeks of gestation in this city. In Guangdong province, China, the typical schedule of routine antenatal visits are at weeks 8-12,week 16,week 20,week 24,week 28 and thereafter every second week until birth.
Prepregnancy height and weight refers to the height and weight at the rst visit for pregnancy registered in 8-12weeks of gestation, which self-reported by pregnant women. Prepregnancy body mass index (BMI) was calculated as weight (kg) divided by height squared(m2).Smoking and drinking refer to no smoking and no drinking in pregnancy, which self-reported by pregnant women at admission to the hospital and before delivery. In this study, all women self-reported no smoking and drinking in pregnancy. Education level included middle school or below, high school, college or above. Parity is divided into nulliparous and multiparous. Method of conception included natural conception, assisted reproductive technology (ART) conception. Gestational age (weeks) was calculated from the rst day of last menstrual period (LMP) until the date of delivery. Women with irregular menstrual cycles or unsure of their LMP, we based on their rst trimester ultrasound examination to corrected gestational age.

Copper sampling and measurement
Copper concentration were measured in maternal whole blood in 12-27(+6) week of pregnancy, from women declaring themselves to be fasting. In this study, copper concentrations were measured prior to the date of diagnosis pre-eclampsia. Blood samples were collected from the antecubital vein, using heparin anti-coagulation Vacuum blood vessel collection. Whole blood copper concentration were measured by ame atomic absorption spectrometer (BH5100S, BOHUI, Beijing, China) with copper -zinc composite element hollow cathode lamp at 3 mA current.
Both before and after the detection, we have internal quality control. Related reagents (trace element calibration solution, batch numbe1070719) were provided by BOHUI, Beijing, China. We respectively conducted calibration before and after testing the sample, with two concentrations of the calibration solution. Copper concentration of the calibration solution were 4.6mg/L and 1mg/L, respectively.Correlation coe cients for calibration curves of copper were greater than 0.999. All measurement on copper concentration in maternal whole blood were performed in the Center for Clinical laboratory of Foshan Chancheng Central Hospital. The respective reference intervals of copper concentration in our laboratory included were 7.12-21.29umol/L.

Outcomes
Preeclampsia was the outcome of this study, obtained directly from electronic medical records system. Preeclampsia was de ned according to report of the American College of Obstetricians and Gynecologists' Task Force on Hypertension in Pregnancy. [22]Preeclampsia was de ned as newly diagnosed hypertension and proteinuria occurring after 20 weeks of gestation. Hypertension was de ned as systolic≥140mmHg or diastolic≥90mmHg,2 occasions,4h apart in previously normotensive woman. Proteinuria was de ned as≥300 mg/24 hour urine collection, or protein/creatinine≥0.3,or or dipstick reading =1+. Severe preeclampsia case were considered if they had at least one of the following symptoms: 1) Systolic blood pressure≥160mmHg or diastolic≥110mmHg, 2 occasions, 4h apart on bedrest;2)Thrombocytopenia(blood platelet<100×10 9 /L);3)Liver function tests ≥ 2×normal or severe persistent right upper quadrant or epigastric pain;4)Serum creatinine concentration >1.1 mg/dL or doubling of creatinine in the absence of other renal disease;5)Pulmonary edema;6)New-onset cerebral or visual symptoms. All other cases were diagnosed mild pre-eclampsia. Early onset and late onset preeclampsia de ned as delivery before 34 and≥34weeks, respectively. In this study, all cases of preeclampsia occurred after 28 weeks, with 5 cases of early onset and 52 cases of late onset.

Statistical Analysis
Frequency and percentage were used in categorical variables, such as education level, parity, and method of conception, delivery way. Normally distributed variables are presented as means and standard deviations (SD). Non-normally distributed variables are presented as median and interquartile range (IQR) .We compared categorical variables, normally and non-normally distributed variables between Normal and Pre-eclampsia women, used Pearson Chi-square, ANOVA and Kruskal-Wallis Rank sum test. Scatter plot with bar were used to display the median, IQR, and range of four trace element, and Kruskal-Wallis Rank sum test were used to determine differences in their distributions in Normal and Pre-eclampsia women.
We used Logistic regression analyses to estimate odds ratios and 95% con dence intervals (CIs) for preeclampsia in relation to trace element, unadjusted model and multivariable adjusted model. Copper concentrations were treated as continuous variables scaled to per1 SD increase and also as categorical variables by quintiles to explore potential nonlinear dose-response relationships on preeclampsia. P values for trend were obtained by coding concentration categories as ordinal variables in the regression models. To assess potential confounders, we adjusted for age at delivery (smooth),prepregnancy body mass index(smooth),education level (middle school or below, high school, college or above),parity(nulliparous, multiparous), and method of conception(natural conception, ART conception). We also evaluated subgroup analysis of copper with preeclampsia. Furthermore, age at delivery, prepregnancy BMI, education level, parity and method of conception were considered as potential effect measure modi ers. We conducted interaction test of copper with preeclampsia and showed P values for interaction under multivariable adjusted model.
We did sensitivity analysis to investigate the robustness of the ndings in line with missing data. Nearly all clinical factors were similar in 2231 patients with available data on copper and the 281 patients with missing data on copper (Table S1 in the Supplementary Appendix).It shows that the missing data has little effect on the overall research conclusion to some extent. Given the differences in the baseline characteristics between eligible participate in the two groups, propensity-score matching was used to identity a cohort of patients with similar age at delivery and prepregnancy BMI. Matching was performed with the use of a 1:4 matching protocol Without replacement (greedy-matching algorithm), with a caliper width equal to 0.01 of the standard deviation of the logit of the propensity score. The results were compared in the two different samples, which show that the results are relatively robust (Table S2 in

Characteristics of Participants
Of the 2134 women included in the analysis, 57(2.67%) developed preeclampsia.
Women with preeclampsia were more likely to have older, higher prepregnancy body mass index and lower natural conception than women without preeclampsia (Table 1).Babies born to preeclampsia mothers were more likely to have shorter gestation, higher abdominal delivery, lower birth weight and length. Figure 1S in the Supplementary Appendix, indicated that women with preeclampsia had no statistically signi cantly than normal participants.

Subgroup analysis of copper with Preeclampsia
In subgroup analysis, The odds ratios for preeclampsia in relationship to per 1 SD increment in copper were consistent among subgroup of age at delivery, prepregnancy BMI, education level, parity, method of conception ( Figure 3).

Interaction test of copper with Preeclampsia
Furthermore, all P values for interaction test on copper with preeclampsia were not statistically signi cant, suggesting that the association with copper and preeclampsia was not affected by the interaction between age at delivery, prepregnancy BMI, education level, parity, method of conception (Figure 3).

Discussion
In this cohort of pregnant women with low burden of preeclampsia, higher copper levels (measured in whole blood at 12-27 (+ 6) week of pregnancy) were associated with lower preeclampsia risk in a doseresponse fashion. Because the risk factors like age, parity, and Body Mass Index (BMI) are associated with preeclampsia [1,23], we controlled these confounders in this study. And these associations persisted after controlling for potential confounders and were consistent across levels of age at delivery, prepregnancy BMI, education level, pregnancy, parity, abortion and method of conception.
In recent years, many studies had been performed to evaluate the relationship between blood copper level and PE. Our study founds were consistent with the results of earlier similar studies. [24,25] Keshavarz P [26]reported in their case-control study that compared with healthy pregnant women, plasma levels of copper in women with preeclampsia were signi cantly reduced. Similarly, Acikgoz, S also observed signi cantly low copper levels in preeclampsia patients,compared to healthy pregnant women. [27] Whereas, in contrast, a recently published meta-analysis [28],included nine case-control studies and seven cross-sectional in Asians, reported that the serum copper level was signi cantly higher in the PE patients [SMD (95% CI): 1.05 (0.34, 1.77), Z = 2.88, P for Z = 0.004; I2 = 96.9%, P for I2 < 0.0001)] as compared to the healthy pregnancy control, similarly to another meta-analysis [29].However, studies in two meta-analysis were case-control studies and cross-sectional, which is not su ciently powerful to demonstrate a causal relationship. Moreover, confounding factors across studies were different in two meta-analysis, which could affect the observed associations. Therefore, the association with Cu and preeclampsia is controversial, needed more cohort study.
To our best knowledge, our study is the rst cohort study to discuss the association between copper level and PE. Our ndings showed that higher copper levels were associated with lower preeclampsia risk, consistent after controlling for potential confounders. A major strength of our study is the level of copper were measured in whole blood at 12-27 (+ 6) week of pregnancy, prior to the date of diagnosis preeclampsia, which is of great clinical signi cance to prevent the occurrence of preeclampsia.
However, our study also has limitations of the current study. First, misclassi cation of preeclampsia could exist, since the diagnosis was extracted directly from electronic medical records. It is reasonable to consider the misclassi cation as independent and nondifferential, which would bias the observed association towards the null. Second, because there are only 5 cases of early onset preeclampsia in our cohort, we were unable to distinguish between early onset and late onset preeclampsia, which may differ with respect to pathogenesis and clinical managements [30].Therefore, we cannot speci cally discern whether copper level have different associations with early onset and late onset preeclampsia in our study.
Third, we excluded people with self-reported chronic hypertension prior to pregnancy, T2DM prior to pregnancy, BMI ≥ 30, and twin pregnancy,So our ndings do not apply to this population. Fourth, missing data were unavoidable as we did a retrospective study. Considering that the missing data were caused by non-human factors and many were from the same individuals, patients with missing data were not included in the subsequent analysis. The statistical power might be reduced as exclude patients with missing information on copper. There might also be an effect on the ndings and increased bias, while the relatively large sample in this study could compensate for this. Additionally, nearly all clinical factors were similar in 2231 patients with available data on copper and the 281 patients with missing data on copper(Table S1 in the Supplementary Appendix).Finally, because our study is observational, we cannot rule out the possibility of residual or unmeasured confounding of the reported associations. Future studies including a larger cohort with standardised data collection is necessary to further validate these results.

Conclusion
Women with higher blood concentration of copper may associated with lower preeclampsia. More well designed studies are needed for the interpretation of these ndings.

Declarations Ethical Approval and Consent to participate
The Hospital Ethics Committee approved this study and waived informed consent because data were unidenti ed (approval number: CYIRB2020001PJ20200316).

Consent for publication
Not applicable.

Availability of supporting data
The data, analytic methods, and study materials that support the ndings of this study available from Dr Huan Ying Xu (843857263@qq.com) on reasonable request.     The odds ratios and 95 % CI for preeclampsia in relationship to quintiles of copper. Models adjusted for adjusted for age at delivery (smooth),prepregnancy body mass index (smooth) , education level (middle school or below, high school, college or above), parity (nulliparous, multiparous), and Method of conception(natural conception, ART conception).