During pregnancy, maternal lipid metabolism undergoes several transformations originated by hormonal changes [1, 2]. There are two distinct phases: an initial phase characterized by an accumulation of fatty acids, resulting from the deposition of triglycerides (TG) in maternal adipose tissue, and the late phase characterized by the accelerated catabolism of adipose tissue, with the increased availability of fatty acids and glycerol in the circulation, generating hepatic very-low-density lipoprotein (VLDL) secretion and hypertriglyceridemia [3]. Maternal hyperlipidemia, especially hypertriglyceridemia, may be associated with the increased hepatic synthesis of VLDL originated by a high concentration of estrogen during gestation and the reduction of TG-rich lipoproteins catabolism due to low activity of lipoprotein lipase (LPL) and hepatic lipase (LH) [4-6].

Hypertriglyceridemia during pregnancy is responsible for modifying different pathways of lipid and lipoprotein metabolism. Besides increased cholesteryl ester transfer protein (CETP) activity, some studies have shown alterations in the composition and size of lipoproteins, such as higher TG content and the formation of smaller and denser particles [7, 8]. The accumulation of small and dense low-density lipoprotein (LDL) during gestation increases the risk of endothelial damage and oxidative stress [2].

The evaluation of high-density lipoprotein (HDL) functionality, together with serum HDL-cholesterol (HDL-C), is essential for a cardiovascular risk assessment [9, 10]. These protective characteristics can be altered in the presence of pro-inflammatory conditions and diverse modifications, such as oxidative changes [11]. A considerable cardiovascular risk may exist even with conditions, such as a high HDL cholesterol concentration, and therefore, the analysis of HDL metabolism biomarkers, such as apolipoprotein (apo) A-I, and HDL particle size, must be performed [11].

Maternal hyperlipidemia may be associated with the development of maternal-fetal complications (preterm birth, preeclampsia, vascular complications) and the development of cardiovascular diseases in the long term [3, 4]. The evolution of pregnancy represents an early opportunity to identify the presence of cardiovascular risk biomarkers [12]. Studies suggest that gestation constitutes a “stress test” for the metabolic pathways of carbohydrates and lipids. Besides causing inflammatory biomarkers hyperregulation, it also changes vascular function. Thus, this period may be an adequate time for the long-term prediction of hemodynamic complications [13-15].

The purpose of the present study was to evaluate the lipoprotein modulators, lipoprotein particle quality, and functionality during uncomplicated pregnancy.

The general characteristics and socioeconomic descriptive data from the study participants are shown in Table 1. Fig. (1) shows the observed frequency of the main risk factors for CAD development before becoming pregnant.

Fig. (1). The observed risk factors for CAD in the study participants at the first and third trimesters of gestation. Frequency was obtained from the analysis of the data by interviewing the study participants.

Table 2 shows the biochemical and hematological parameters in the first and third trimesters of gestation, performed to evaluate the pregnant women’s health status. The levels of urea and CRP increased (p<0.001), while ALT, hemoglobin, and hematocrit decreased (p<0.0001) during the gestation.

Table 3 shows the lipid profile and the serum concentration of apolipoproteins in the first and third trimesters of gestation. Data show the development of dyslipidemia in the first and third trimesters, represented by increased LDL-C and triglycerides levels (p<0.0001). There was also a significant increase in the number of antiatherogenic and atherogenic particles, addressed by the apolipoprotein values (p<0.0001).

Table 4 shows the calculated cardiovascular risk ratios used to estimate lipoprotein remodeling and composition and the number of circulating particles as suggestive markers of cardiovascular risk. Apo B/apo A-I ratio increased (p<0.01), and HDL-C/apo A-I decreased (p<0.0001) in the third trimester of gestation.

As shown in Fig. (2), the CETP activity significantly increased in the first and third trimesters of gestation (56.6±5.4 to 70.5±9.6 pmol/µL/h, p<0.0001). On the other hand, the PLTP activity did not show significant differences between the evaluated periods (28.9 ± 6.8 and 27.2 ± 9.1 pmol/µL/h, respectively).

Fig. (3) shows the linear correlation analysis between serum TG and CETP activity in the first (r2 = 0.70, Pearson correlation test, p<0.0001) and third trimesters of gestation (r2 = 0.33, Pearson correlation test, p=0.0209).

As shown in Fig. (4), there were no significant differences between HDL particle size in the first and third trimesters of gestation (11.78±1.2 and 11.8±1.0 nm).

To evaluate the mean size of circulating LDL particles, the TG/HDL-C ratio was used. A value above 2.0 is an indicator of small and dense LDL particle predominance [22]. TG/HDL-C ratio in the first (1.3; 1.2-2.0) and third (2.7; 2.0-3.8) trimesters of gestation shows significant differences (p<0.0001) (Fig. 5).

Fig. (2). Activity of cholesterol ester transfer protein (CETP) (left), and the phospholipid transfer protein (PLTP) (right) of the study participants at the first and third trimesters of gestation. n=40. D´Agostino and Pearson linear correlation tests, paired t-test. Data are shown as mean±SD. Not significant (ns), *** p<0.0001.

Fig. (3). Linear correlation between serum triglycerides (TG) concentration and cholesterol ester transfer protein (CETP) activity in the study participants at the first (A) and third trimesters (B) of gestation. n= 40. Pearson linear correlation tests, one-way analysis.

Fig. (4). HDL particle size at the study participants at the first and third trimesters of gestation. n=40. D´Agostino and Pearson linear correlation tests, paired t-test. Data are shown as mean ± SD. ns: not significant.

Fig. (6) shows the PON-1 antioxidant activity. There was a significant decrease in PON-1 activity from the third trimester of gestation (88.3 [61.3 -114.5] and 65.4 [46.5-105] nMol of p-nitrophenol/min/mL, p=0.0037).

Here, we evaluated the health status of pregnant women through the analyses of renal, hepatic, and inflammatory profiles, glucose, hematocrit, and hemoglobin levels. Among the findings, although some parameters differed significantly before and after, all results were inside the reference values. Most women start the gestational period healthily, but some may have a silent kidney or liver disease, subclinical, without adverse health effects [23, 24].

Importantly, in our study, the lipid profile of pregnant women shows marked alterations. According to Geraghty et al. [25], lipid metabolism during pregnancy is completely altered. By the final of gestation, all lipid biomarkers, such as lipoprotein particles carrying cholesterol and triglyceride, may increase. Lipids are essential for fetal development, as they act

Fig. (5). LDL particle size, estimation by TG/HDL-C ratio at the study participants at the first and third trimesters of gestation. n=40. D´Agostino and Pearson linear correlation tests, Wilcoxon test. Data are shown as median and IQR (25%-75%). *** p< 0.0001.

Fig. (6). Paraoxonase-1 (PON-1) activity (nMol/p-nitrophenol/min/mL) at the study participants at the first and third trimesters of gestation. n= 40. D´Agostino and Pearson linear correlation tests, Wilcoxon test. Data are shown as median and IQR (25%-75%). ** p=0.0037.

as a source of energy and as constituents of the cell membrane. Cholesterol and phospholipids increase moderately, and triglycerides increase sharply [26]. Hypertriglyceridemia is one of the main alterations in the lipid profile noticed at the end of gestation. However, it is linked to VLDL content modification, which also modifies LDL and HDL structures by fulfilling it with higher triglyceride content [27].

In the present study, the total cholesterol, lipoprotein cholesterol fractions, and triglycerides results were significantly increased during the gestational trimesters, except for HDL-C, which remained unaltered. According to Emet et al. [3], LDL-C, triglycerides, and HDL-C were significantly higher from the third trimester of gestation. Other studies have shown that the lipid profile becomes more atherogenic at the end of gestation [26, 28-30]. Vrijkotte et al. [31] found that hypertriglyceridemia in pregnancy is associated with maternal-fetal complications. It can compromise endothelial function and contribute to the development of atherosclerotic vascular disease, preeclampsia, and oxidative stress promotion [32, 33].

We noted that apo A-I and apo B increase from the third trimester of gestation. In the same way, the apo B/apo A-I ratio showed the same profile during this period. The study by Piechota and Staszewski [34] shows that apo A-I and apo B increased in the second and third trimesters, in which apo A-I increased by 32% and apo B by 56%. Belo et al. [35] conducted a longitudinal study to evaluate apolipoprotein concentration during the gestational period. They observed an apo B increasing from the first to the third trimesters of gestation, while apo A-I showed a slight reduction in the third trimester. Rymer et al. [36] concluded that in pregnant women, apo A-I and B concentrations were higher than those of non-pregnant women. The apo B/apo A-I ratio at the third trimester of gestation reflects the balance between two distinct processes: cholesterol transport to extrahepatic tissues and reverse transport to the liver. The increase of the apo B/apo A-I ratio, indicating the higher proportion of atherogenic lipoproteins in the plasma, is an index that reflects the dyslipidemia progression and endothelial dysfunction [37, 38].

Other cardiovascular risk ratios were evaluated in our study, such as HDL-C/apo A-I and LDL-C/apo B, which reflected lipoprotein remodeling, composition, and particle number [37]. The significantly reduced HDL-C/apo A-I ratio from the third trimester of gestation is suggestive of an HDL particle number increase with lower cholesterol content, suggesting an alteration in HDL particle composition and size. The LDL-C/apo B ratio did not change significantly during gestation, perhaps because the increase in cholesterol content of LDL is proportional to the increase in the apo B-containing particle number. A study by Tani et al. [39] showed that values ≤1.2 for the LDL-C/apo B ratio are suggestive of small and denser LDL particle predominance.

In our study, the CETP activity was different between the first and third trimesters of gestation, showing more frequent high activity at the end of gestation. The CETP activity influences the esterified cholesterol concentration and its distribution between lipoprotein classes, altering the lipoprotein composition, and thus influencing the lipoprotein profile atherogenicity [27, 40, 41]. In addition, the CETP increased activity plays a decisive role in the formation of small and denser LDL [42]. Patients with dyslipidemias, such as hypertriglyceridemia and hypercholesterolemia, with diabetes, obesity, or venous thrombosis have increased esterified cholesterol transfer from HDL to apo B-containing lipoproteins, as a result of an increased CETP activity, which contributes to a rise in the atherosclerosis progression and cardiovascular risk [27, 43]. Zhang et al. [44] have shown that endogenous estrogen can affect the CETP concentration in pregnant women´s plasma. It is known that in addition to hormonal status and metabolic conditions, lifestyle habits, such as diet and physical activity, can also influence CETP activity [45].

In the present study, a positive correlation between serum triglyceride and CETP activity in the first and third trimesters of gestation was found. The CETP activity did not increase proportionally as triglycerides from the third trimester, probably due to CETP enzymatic catalytic saturation, which may have masked the expected increase in CETP activity. Another significant finding was that CETP has a higher affinity for esterified cholesterol substrate than triglycerides as a function of the steric impediment since triglyceride molecule is approximately 1.5 times greater than esterified cholesterol [46]

In our study, the PLTP activity did not change significantly during pregnancy. PLTP contributes to HDL metabolism because it is associated with lipoprotein remodeling and reverse cholesterol transport [47]. The increase in PLTP activity leads to increased cholesterol efflux from the endothelial cell membranes to HDL, permitting the formation of larger and anti-inflammatory HDL particles [47]. According to Jiang et al. [48], the adipose tissue produces modulators that can interfere with the performance of PLTP activity. Furthermore, it is noteworthy that apo E plays an essential role in PLTP regulation, as an activator or just increasing its activity [49].

When evaluating the lipoprotein remodeling, there was no significant difference in HDL particle size compared to the first and third trimesters of gestation. Several HDL subpopulations show different qualitative properties that directly affect its activity [50]. HDL particles are quite heterogeneous structurally and functionally [51, 52]. Among the HDL subpopulations, the larger lipoprotein particles (the cholesterol-riches) are inversely related to cardiovascular risk, and the smaller particles (probably inefficient to remove cholesterol) show a cardiovascular-risk positive correlation [53-55].

LDL particles are also heterogeneous and have different physical and chemical characteristics [2]. In the present study, the LDL particle size was estimated by the TG/HDL-C ratio. From the third trimester of gestation, the values suggest a predominance of smaller, denser, more atherogenic particles. A study conducted by Jayanta et al. [56] showed that in the group of uncomplicated and non-hypertensive pregnant women, the TG/HDL-C ratio value from the third trimester of gestation was 3.6 higher than that found in our study. Meyer et al. [7] showed that in pregnant women, from the second and third trimesters, the proportion of small and dense LDL particles increased when compared to women in the first trimester of gestation and the postpartum period. This proportion was higher in obese pregnant women (40.7%) than those with normal weight (21.9%). Zeljkovic et al. [57], in a longitudinal study, concluded that LDL particle size reduced, but the proportion of small and dense LDL increased during gestation. The increase in the proportion of small and dense LDL particles is indicative of an atherogenic phenotype, which has been associated with the development of hepatic steatosis and has also been observed in women with gestational diabetes and preeclampsia [32, 58, 59].

Regarding the oxidative profile during pregnancy, the present study showed a decrease in PON-1 activity in the third trimester. A significant PON-1 activity decrease was observed in previously performed studies [60-62]. Stefanovic et al. [61] showed, in a longitudinal study, that a significant PON-1 activity decreased in the third trimester (32 weeks) of gestation. In addition, the lipid profile became more atherogenic. Similar results were found in the study conducted by Ferre et al. [60], where the PON-1 activity decreased in different moments (145.8 U/L at 8-20 weeks; 111.1 U/L at 32 weeks, and 100.4 U/L during labor). Ferretti et al. [63] evaluated the relationship between obesity and oxidative stress in pregnancy. Their data showed a decrease in PON-1 plasma activity in obese pregnant women, indicating that mothers and newborns are susceptible to greater tissue oxidative damage. Changes in lipid profile, body mass index, hormonal profile, and oxidative stress lead to PON-1 activity changes, mainly at the end of gestation. This process represents a decrease in antioxidant capacity and may be an additional reason for a cardiovascular risk increase in pregnant women [61]. Also, our findings from a previously published systematic review, an animal model [64], showed that an offspring of parents, mother, and father, exposed to a high-fat diet exhibited a variety of up and down gene expressions, influencing changes in the status of glycemia, insulinemia, pancreatic islet tissues transcriptome, levels of triglycerides, and lipogenic genes expression.

The development of dyslipidemia, mainly hypertriglyceridemia, during the gestational period associated with lipoproteins' structural and functional changes, such as CETP activity, increased apo B/apo A-I ratio, and decreased HDL antioxidant capacity, argues for the presence of an atherogenic lipid profile, with less LDL protection. Although the HDL particle remodeling does not change, the predomination of small and denser LDL particles at the end of gestation points to important lipoproteins' structure and function changes. The observed lipoprotein modifications in women with uncomplicated pregnancy alert for close monitoring of metabolic predisposing conditions that can aggravate cardiovascular risk.