Analyses of Maternal Plasma Cadmium, Lead and Vanadium Levels in the Diagnosis and Severity of Late-onset Preeclampsia; A Prospective and Comparative Study From Turkey
Conditions: Preeclampsia
Sponsor: Cengiz Gokcek Women's and Children's Hospital

Full study protocol and statistical 
analysis plan 
Official Title of the study: 
Analyses of maternal plasma cadmium, lead and vanadium levels in the diagnosis and 
severity of late-onset preeclampsia; A prospective and comparative study from Turkey 
 
Date of the document: 
December 2019. 
Contents: 
1. Participant Flow 
2. Baseline Characteristics 
3. Outcome Measures 
4.Endpoints of the study 
5.Limitations and Strengths 
6. Certain Agreements 
7. Results Point of Contact  
 
1. Participant Flow 
Recruitment Details 
This observational case-control study was conducted at the Department of Obstetrics and 
Gynecology, Cengiz Gokcek Public Hospital, Gaziantep, Turkey, between March 2018 and June 2019. 
The protocol was approved by the Ethics Committee for Clinical Research of Gaziantep University 
(reference no: 2019/36). The investigators included subjects consisted of women with a singleton 
pregnancy who were diagnosed as having late-onset preeclampsia between 34+0 and 41+0 weeks of 
gestation. Pregnant women with uncomplicated pregnancies were randomly selected to serve as 
controls. The study was conducted with 46 late-onset preeclampsia patients as study group and 46 
patients with normal pregnancies as control group. All participants included in the study gave oral and 
written informed consent. 
Pre‐assignment Details 
The authors firstly were assessed the recruited people to ensure meeting the inclusion and 
exclusion criteria. 
The inclusion criteria were maken according to the official documents. 

Arm/Group Information * 
 
There are two groups in the study. 
 
Arm/Group Title * 
 
Preeclampsia group 
Control group 
2. Baseline Characteristics 
To detect significant difference between groups according to lead levels with a moderate 
effect size (Cohen's d =0.62), minimum required sample size was estimated as 42 for each 
group (α=0.05, 1-β=0.80). Power analysis was performed by using G power package version 
3.1. Kolmogorov Smirnov and Shapiro Wilk tests were used to test the normal distribution of 
data. For comparing groups (L-PrE/control) the student t-test was used for variables that have 
a normal distribution, and the Mann Whitney U test was used for variables that have not a 
normal distribution. The ROC analysis was applied for the determination of cut-off point for 
vanadium variable. Moreover, Spearman correlation test was used for the relationship of 
between variables. SPSS for Windows 22.0 and Medcalc programs were used for statistical 
analysis. p<0.05 was accepted as statistical significance. 
3. Outcome Measures 
 
Every pariticipant in the study population underwent obstetric ultrasound examination and 
fetal-maternal assesment was carried out by one of the investigators. The obstetric anamnesis 
were obtained from all pariticipants. The demographic data like age, gravidity, parity, body 
mass index (BMI) and gestational age were recorded. The gestational age was determined by 
calculation from the last menstrual period and supported by the ultrasonography 
measurements at first trimester of gestation. Maternal venous plasma samples were taken for 
measurement of cadmium, lead and vanadium levels after the diagnosis of L-PrE in outpatient 
clinic. These samples quickly centrifuged at 1,500 g for 10 min, plasma samples were 
seperated and stored at -80 C until the day of measurement. All patients with L-PrE were also 
hospitalized and their pregnancies were terminated. The samples of the control groups were 
obtained during the routine obstetrical care examination in the third trimester of pregnancy. 
Then, these pregnant women were followed-up until the delivery. Both groups were compared 
in terms of maternal age, BMI, week of gestation, gravida, parity, live born, systolic/diastolic 
blood pressure, total protein in spot urine sample, hemoglobin, hematocrit, platelet count, 
white blood cell count, urea,  uric acid, albümin, blood urea nitrogen, creatinine, liver 
function tests (AST, ALT), lactic acid dehydrogenase, cadmium, lead and vanadium 
concentrions and infant weight at delivery. Small for gestational age (SGA) neonate was 
defined as birth weight <10th percentile for gestational age with Turkey's national nomogram 
as the reference for fetal growth. The samples were transferred within boxes in ice molds to 
Yozgat Bozok University Science and Technology Application and Research Center 
(Occupational and Environmental Toxicology Laboratory) for measuring heavy metal and 
trace element levels. The frequency of seafood consumption was divided into four categories: 
2-3 per week, 1-2 per week, 1-2 per month, and rare. Smoking was classified into 3 
groups (none, formerly smoker, and active smoking). Passive smoking was also categorized 
into 2 groups (no and yes). The education levels was classified into 4 groups  (illiterate, 

primary education, high school, and university). The residences of women was classified as 
rural and urban. Furthermore, the residences of women have divided either living near a busy 
street (around 100 metres) or not. 
Venous blood sampled from the antecubital veins for measuring plasma concentration of 
cadmium, lead and vanadium levels.  The three metals (cadmium, lead and vanadium) were 
measured using inductively coupled plasma-mass spectrometry (Thermo Scientific ICAPQc, 
USA). 
 
 
4. Endpoints of the study:  
The primary outcome in these analyses was to compare cadmium, lead and vanadium levels in 
L-PrE group and control group.  
The secondary outcome was to compare the cadmium, lead and vanadium levels in mild L-
PrE group and severity L-PrE group.  
Tertiary outcome was to compare the cadmium, lead and vanadium levels in SGA group and 
non-SGA group. 
5.Limitations and strengths: 
 
Our study has some limitations. Firstly, none of these patients had ever been occupationally exposed 
to these three metals. Further, the city in which the study was performed is far from the coast. 
Therefore, the participants’ seafood consumption was very low or absent because of the low socio-
economic level. Furthermore, our population in this study are more illiterate than others. Because of 
these reasons, the sample size of the population may not reflect the population in general. Second, 
the blood samples in the study were only collected at a single time point which may reflect the three 
metals exposure levels only recent exposure, not before and during pregnancy, and also limiting the 
ability to establish temporality. Third, it could be even better to measure urinary levels of the metals. 
Lastly, because our study is observational, we could not eliminate the possibility of unmeasured 
and/or residual confounding of the reported associations. On the contrary, some confounding factors 
which are considered as risk factors for PrE and metals exposure are either nearly equally distributed 
or absent in our patient population. For example, patients with pre-pregnancy systemic diseases, 
advanced maternal age, multifetal gestation were excluded. Nulliparity, increased body mass index 
and sociodemographic characteristics such as smoking and traffic exposure of the two groups are 
similiar. 
 
6. Certain Agreements 
 
The authors declare that they have no conflict of interest. 
 
7. Results Point of Contact 
Corresponding Author: Ali Ovayolu, Department of Obstetrics and Gynecology, Cengiz 
Gokcek Women's and Children's Hospital, Gaziantep, Turkey, drovayolu@yahoo.com 

Address: Osmangazi Mahallesi, Cengiz Gokcek Kadın Hastaliklari ve Dogum Hastanesi, 
27010 Gaziantep, Turkey 
GSM: +90 (532) 640 40 60 
Tel.: +90.342 360 08 88 
Fax: +90.342 360 02 90