This prospective, observational, case-control study was conducted in the Department of Biochemistry and Department of Obstetrics and Gynaecology, Jaipur National University Institute of Medical Sciences and Research Centre (JNUIMSRC), Jaipur, Rajasthan, India, for a period of one year (February 2019-January 2020). The study was approved by Institutional Ethics Committee (IEC) vide reference no. (JNUIMSRC/IEC/2019/74) dated 21-1-2019. The ethical approval was in accordance with Helsinki Guidelines.

Sample size: A minimum sample size of 100 patients in each group was calculated using effect size of 0.4 and with user constant type I error α=0.05 and type II error β=0.2 by using G power software (3.1.9.7).

The study protocol and the objectives of the study were explained to the enrolled subjects and their written informed consent was obtained from all the participants. After a thorough clinical examination, a total of 200 pregnant females were selected: 100 in pre-eclampsia group (case group); 100 in normotensive group (control group), in the third trimester and between 18-45 years, visiting Gynaecology Out Patient Department (OPD) or admitted in JNUIMSRC were enrolled in the study. Equal number of primigravid and multigravid women (age and gestational age matched) were included in both case and control groups. The demographic details, obstetric history and relevant medical history of all the study subjects were recorded in a predesigned performa. Investigations were conducted in third trimester of pregnancy.

All the enrolled study participants received oral elemental iron (60 mg/day) from the beginning of the second trimester and supplement continued throughout the gestation as a measure for preventing iron deficiency anaemia.

Inclusion criteria:

Exclusion criteria: Pregnant women, transferred with pack red blood cells in last 30 days, previously diagnosed with haemochromatosis, and/or complications like diabetes mellitus, chronic hypertension, chronic renal disease, chronic liver disease, cardiovascular disease, endocrinal disorder, autoimmune disease, hyperuricaemia, genital malignancies, blood discrasias, acute infections, and those on anticonvulsant drugs were excluded from the study. Among the multigravida any history of hypertension, trophoblastic disease, placental abruption, malformed foetus, history of pre-eclampsia, metabolic disorders or convulsions were ruled out. Also, child-bearing women with history of substance abuse or smoking were excluded.

Procedures: The digital sphygmomanometer was used for measuring BP from the left arm in sitting position after five minutes rest. The systolic and diastolic readings were recorded by calculating an average of three readings taken after a gap of five minutes.

Sample collection: Blood samples were collected from the subjects and were coded to avoid possible bias. A total of five mL venous blood was collected from each subject under aseptic conditions in a sterile syringe. Thereafter, blood sample was transferred into clean and dry vial without anticoagulant, was allowed to clot spontaneously and then centrifuged at 3000 revolution per minutes (rpm) for 15 minutes to extract serum. The extracted serum was collected in microcentrifuge tubes and was used for estimation of following biochemical parameters: serum iron, serum ferritin, sTFR and TIBC.

Freshly voided, random, midstream urine sample (15-20 mL) was collected on two different occasion’s at least (four hours apart) in clean, wide mouthed, leak proof containers for urinalysis.

Biochemical investigations: Among the biochemical parameters; serum iron, TIBC and sTFR were analysed using a fully automated analyser by RANDOX, Crumlin, UK, and serum ferritin was analysed using a fully automatic analyser by Johnson and Johnson, VITROS^®ECiQ, Immunodiagnostic system, Ortho Clinical Diagnostics, New Jersey, USA.

Proteinuria was measured by Combur10 test^® M strips by Roche and analysed by using semi-automatic Cobas u 411 urine analyser by Roche diagnostics, Germany, and the samples were graded as 1+, 2+ or 3+.

The statistics analysis was conducted using Statistical Package for the Social Sciences (SPSS) 26.0. Kolmogorov-Smirnov test (N=200) was used to assess normality of parameters concerned. None of the parameters followed normal distribution and thus were expressed as Median IQR and compared by using Mann-Whitney U test. The correlation was assessed by Spearman’s Rank correlation and categorical variables were assessed by Chi-square tests. The p-value <0.05 was considered statistically significant.

Demographic details and the general characteristics of case and control groups are compared in [Table/Fig-1]. Both the groups; case group as well as control group, were comparable regarding age, gestational age and parity. Median (IQR) of systolic and diastolic BP of the cases were found to be significantly higher than controls (p<0.0001, respectively). [Table/Fig-1] also depicts socio-economic, demographic as well as dietary habits of pre-eclamptic cases and normotensive controls. There was no significant difference with the cases and controls with respect to the aforesaid parameters.

[Table/Fig-2] depicts the median (IQR) values of serum iron, TIBC, serum ferritin and sTFR among both the study group. The difference of median (IQR) of serum iron levels and TIBC between cases and controls were statistically insignificant. Median IQR levels of sTFR (mg/dL) in cases were significantly low in comparison to controls (8.5 (7-9) vs 8.9 (8-10, p=0.023). Also, median IQR of serum ferritin levels (ng/mL) of the cases were found to be significantly higher than the controls (41(30-70) vs. 24 (17-44), p<0.001). Moreover, participants with serum ferritin levels >40 (ng/mL) were associated with three times increased risk of PE (Odds ratio: 3, p=0.004, 95% Cl=1.571-5.157) [Table/Fig-3].

Serum ferritin levels in pre-eclamptic cases significantly correlated positively with systolic BP (r=0.37, p=0.001). However ferritin levels in controls did not show significant correlation with systolic BP (r=0.043, p=0.67) [Table/Fig-4,5 and 6]. TIBC in case group showed significant negative correlation with diastolic BP (r=-0.238, p<0.05) and insignificant correlation was observed in controls (r=-0.057, p=0.57) [Table/Fig-7]. The sTFR in pre-eclamptic cases and controls showed insignificant negative correlation with systolic and diastolic BP [Table/Fig-4]. In controls, ferritin, TIBC and sTFR did not show significant correlation with either systolic/diastolic BP [Table/Fig-5] whereas in cases ferritin failed to show any significant correlation with diastolic BP and TIBC with systolic BP [Table/Fig-4].

[Table/Fig-6]:

Serum ferritin levels were observed to correlate positively with systolic Blood Pressure (BP). Observed correlation was significant in pre-eclamptic cases (r=0.37, p=0.001). No significant correlation was observed between serum ferritin and systolic BP in control subjects (r=0.043, p=0.67).

[Table/Fig-7]:

TIBC levels were observed to correlate negatively with diastolic Blood Pressure (BP). Observed correlation was significant in pre-eclamptic cases (r=-0.23, p=0.017). No significant correlation was found between TIBC and diastolic BP in control subjects (r=-0.057, p=0.57).

Iron deficiency is one among the most common causes of anaemia in pregnancy and hence in many countries, particularly in developing nations, routine iron supplementation to pregnant women has been a public health intervention for a long time [22]. There are many studies supporting the benefit of this practice, however, some reports suggest that prophylactic iron supplementation may be harmful to pregnant women who are not iron deficient [10]. Thus, iron intake, transport, distribution and storage needs to be organised accurately to balance these double characteristics of essentiality and toxicity, which is critical for normal body homeostasis [10,23]. Also, the central organ regulating pregnancy, the placenta, acts as one of the chief sources of iron to the foetus. Due to its potential redox activity, iron can be toxic as it catalyses the formation of ROS, which eventually can result in a state of oxidative stress, mainly in the second trimester of pregnancy. Excess iron supplementation can result in saturation of proteins to which it binds (ferritin and transferrin), leaving iron in its free form in the bloodstream which can lead to oxidative stress with potentially damaging effects in mother and the newborn.

In the present study, serum iron levels were observed to be similar in cases and controls. Similar findings were reported by Gutierrez-Aguirre CH et al., [5]. However, contradictory observations have been reported by Shaji Geetha N et al., and Jana SM et al., where serum iron levels were observed at increased levels in cases [18,20]. These differences could indicate compromised iron intake in the population under present study.

Present study results regarding TIBC levels in pre-eclamptic cases were similar to controls. Similar findings were observed in the study conducted by Jana SM et al., [20]. However, the reliability of TIBC as an indicator of iron availability to the tissues is questionable since its levels show fluctuations in physiological conditions like diurinal rythm [19].

In the present study, serum ferritin levels were higher in pre-eclamptic cases. Similar finding were also observed in the studies of Shaji Geetha N et al., Elshahat AM et al., and Mandal D et al., [18,19,24]. The serum ferritin levels <15 ng/mL serve as an indicator of depleted iron stores [25]. However, in the present population, serum ferritin levels were comparatively high thus ruling out the possibility of iron deficiency anaemia.

Increased serum ferritin can also be due to acute phase response, a phenomenon observed in pre-eclamptic cases. However, past studies have revealed that acute phase response is not the sole cause for increased ferritin levels in pre-eclampsia [9]. This complication lead us to sTFR that serves as a more reliable indicator in determining iron status as its levels are not altered because of acute phase response [26]. Present study results show lower sTFR levels in cases. This further indicates that iron supplementation could be a possible cause of elevated serum ferritin levels in pre-eclampsia.

Previous studies have shown increased risk of complications like preterm birth among pre-eclamptic women having serum ferritin levels >40 ng/mL [5]. In the view of above facts, in present study subgroup analysis considering the participants having serum ferritin >40 ng/mL. Present study results revealed greater number of participants in case group having serum ferritin levels >40 ng/mL (50% vs 26%). Moreover, the study conducted by Elshahat AM et al., evaluated a cut-off value of serum ferritin >43 ng/mL as a criteria for prediction of pre-eclampsia [19]. In this context, present study revealed that participants having serum ferritin levels >40 ng/mL had about three times increased risk of pre-eclamptic occurrence.

A positive correlation of serum ferritin with BP and a negative correlation of TIBC with BP further strengthen the association between iron status and the contributions it can make in aetiopathogenesis of pre-eclampsia. Similar findings were also observed in a study conducted by Biswas S et al., [27]. Contrary to these findings Kanagal DV et al., did not find any significant correlation of the above paramaters with systolic or diastolic BP among pre-eclamptic cases [17]. A small sample size of their study could have been the possible cause attributed to this non correlation.

This study, with larger sample size, can be extended in the form of multicentric study, in order to include other populations with demographic variations. Also, similar studies could be performed on non anaemic population, population with predominant iron deficiency anaemia and that with iron mediated toxicity. These case-control studies are also needed to be supplemented with more cohort studies in order to establish the iron contribution in pre-eclamptic aetiopathogensis.

Higher serum ferritin levels and lower sTFR values were observed in cases. A correlation of BP with ferritin and that with TIBC is also established. Present study results show lower sTFR levels in cases. These observations indicate that iron supplementation could lead to enhanced iron status in pre-eclamptic cases compared to controls. Although, none of the iron related parameters in this study could be related to iron overload, yet an unchecked and unmonitored iron supplementation could further increase a possibility of iron mediated added complications in pre-eclampsia. Present study results, in combination with previous studies from others, hereby suggest that cautionary tests for estimation of serum ferritin and sTFR levels should be conducted to assess iron status in pre-eclampsia before prescribing or continuing with iron supplementation. This would help to prevent any possibility of iron overload in pre-eclamptic patients. The assessment would further help clinicians to restrict/lower the prescribed dose of iron supplements. Authors however, also believe that in situations where screening for serum ferritin and sTFR levels is not possible, iron supplementation during pregnancy should be kept continued, especially in population with predominant iron deficiency.