The results demonstrate associations between the concentrations of serum proteins and the thyroid panel undergoing changes in consecutive trimesters of pregnancy. Significant associations were demonstrated between the concentrations of albumin, alpha-2- and beta-1-globulins separated by a preliminary serum protein electrophoresis and the levels of TSH, fT4, fT3, and the fT3/fT4 ratio. Four proteins (ALB, TRF, CER and AMG) were selected as representative for specific electrophoretic fractions and used in further immunoassays. These representative proteins were selected from 22 most abundant serum proteins, with well characterized electrophoretic localizations and biological properties described in the literature [3, 11, 12, 13, 14]. Serum concentrations of ALB (albumin fraction), TRF (beta-1-globulin fraction), and CER and AMG (alpha-2-globulin fraction) demonstrated different rates of change from trimester to trimester which may reflect differences in their involvement in metabolic processes associated with consecutive stages of pregnancy.
The amount of TSH in the blood is a parameter of thyroid function routinely used to identify thyroid disorders , but its association with the concentrations of serum proteins was weak, with the exception of the alpha-2- and beta-1-globulin fractions in the third trimester only.
No correlation was established throughout pregnancy between the levels of TSH and changing concentrations of ALB, TRF, CER and AMG.
Interestingly, the associations observed for the concentrations of fT4 vs ALB and fT4 vs TRF, maintained in the three trimesters of pregnancy, move in opposite directions.
The finding we report of a positive association between fT4 and ALB serum concentrations may provide novel, clinically useful information about the underlying physiological mechanisms of some pregnancy-associated processes or the causes of abnormalities. ALB, along thyroxine-binding globulin (TBG) and transthyretin, is a major transport protein transporting thyroid hormones in the vascular bed. TBG concentration is negatively correlated with ALB concentration which means that a significant increase in the concentration of TBG in pregnancy leads to decreases in the concentrations of ALB [2, 15]. A slight decrease in the serum concentration of ALB in pregnancy is considered normal , but significantly lower ALB levels in the third trimester of pregnancy were associated with increased maternal and neonatal mortality and morbidity . In pregnancy, serum albumin levels may be decreased in cases of renal insufficiency with proteinuria, pre-eclampsia, gestational hypertension and gestational edema. Therefore, there is a supposition, consistent with other authors, that proteinuria and low serum ALB may lead to low fT3 and fT4 levels with adverse effects on fetal development [12, 15, 17, 18, 19, 20, 21].
A negative correlation of fT4 with TFR we observed is consistent with the finding that iron metabolism and thyroid functions are interdependent . Iron deficiency has been linked to hypothyroidism and high TRF and vice versa, low TRF reflects excess iron stores in hyperthyroidism. TRF delivers iron to tissues, due to its iron-binding capacity is considered an important regulator of iron levels in the body and it maintains oxidant/anti-oxidant balance [23, 24].
The mechanism of the negative association of fT4 with TFR levels found in this study in sera from pregnant women remains unclear. Reports by other authors point to high iron stores as a risk factor of type 2 diabetes mellitus and gestational diabetes mellitus (GDM) which may develop as early as the first trimester. GDM is a very common metabolic disorder in pregnancy but the mechanism underlying a relationship between excess body iron and GDM has not been elucidated . The authors of a population-based cohort study suggest that hypothyroidism may be associated with risk of type 2 diabetes while gradually increased fT4 levels decrease the risk . The findings in the present study allow the conclusion that a negative correlation of increased fT4 with decreased TRF may indicate their shared biological role producing a combined protective effect against GDM associated with excess body iron. A question arises whether the evaluation of fT4 vs TFR would provide additional arguments to solve an existing controversy  over routine prophylactic iron supplementation in all pregnant women without earlier laboratory investigations to confirm its actual need.
The above observations suggest novel laboratory investigations to assess maternal health in pregnancy, using the association of serum fT4 concentrations with changes in iron levels and its metabolism, and ALB concentrations. According to the literature, in hypothyroidism, low fT4 may suggest the effect of iron depletion leading to the increased serum concentrations of TRF [23, 26]. Low fT4 levels have been also linked to low ALB, which is widely used to determine nutritional status [27, 28, 29]. The association of low ALB levels and high TRF levels with low fT4 levels in maternal serum we observed may serve as a diagnostic panel to confirm altered nutritional status in malnutrition.
A negative correlation was established in the second trimester of pregnancy between serum fT3 and alpha-2-globulins. The negative correlations of fT3 with CER and AMG, high-abundance proteins located in this fraction may indicate their interaction in ongoing metabolic processes. As shown in the results we present, AMG concentrations changed only slightly across pregnancy, although the presence in pregnant women of pregnancy zone protein (PZP) which is part of the alpha-2-globulin fraction might have contributed to the observed increases. AMG, a 720 kDa tetramer and PZP, a 360 kDa dimer are strongly homologous glycoprotein proteinase inhibitors of human plasma . AMG functions as a major endoprotease inhibitor and its concentrations are decreased in hypothyroidism [1, 7, 31]. CER is an intravascular antioxidant and can function as a free radical scavenger. Thyroid hormones are involved in both production and elimination of reactive oxygen species (ROS). In hyperthyroidism, oxidative stress increases proportionally to the degree of thyroid overactivity while in hypothyroidism decline in ROS generation is associated with antioxidant activity. GDM has been identified as an inductor of oxidative stress and ROS production [32, 33].
In conclusion, changes in the thyroid panel observed during pregnancy demonstrate the association of thyroid hormones with the concentrations of proteins in the albumin and alpha-2- and beta-1-globulin fractions. ALB, TRF, CER and AMG are individual proteins in these fractions and are specifically associated with changes in serum levels in thyroid hormones in pregnancy. Published studies emphasize potential effects of even mild subclinical thyroid dysfunction on detrimental pregnancy outcomes. A novel panel of serum proteins specifically associated with thyroid hormones may be an alternative method to diagnose changes in thyroid function in pregnancy and a potential source of novel biomarkers to identify the effects on maternal metabolism during pregnancy.