Pregnancy is a unique physiological state characterized by a complex interplay of maternal and fetal endocrine and metabolic signals. This state triggers widespread adaptive changes in virtually every organ system to meet the increasing nutritional, respiratory, and excretory demands of the developing fetus.¹ These adaptations are essential for successful pregnancy outcomes but simultaneously place a significant burden on the mother's homeostatic mechanisms. The clinical assessment of pregnancy often relies on routine laboratory investigations, which serve as the cornerstone for monitoring maternal health and identifying potential complications such as pre-eclampsia, gestational diabetes, and anemia.² Key biochemical parameters, including components of the complete blood count (CBC), liver function tests (LFTs), renal function tests (RFTs), and carbohydrate metabolism, undergo significant physiological modifications during pregnancy.³ For instance, the expansion of plasma volume, which is a hallmark of normal pregnancy, results in a state of hemodilution, commonly leading to a physiological anemia. Similarly, changes in hepatic function are reflected by a decrease in serum albumin concentration and a significant increase in alkaline phosphatase (ALP) activity, primarily due to the placental contribution to this enzyme.⁴ Renal function is also markedly altered, with an increase in glomerular filtration rate (GFR) leading to decreased levels of serum creatinine and blood urea nitrogen (BUN).⁵ Furthermore, a progressive decline in insulin sensitivity during the later stages of pregnancy can lead to elevated fasting and postprandial blood glucose levels. The challenge for clinicians lies in distinguishing these normal physiological changes from pathological deviations. The misinterpretation of altered biochemical parameters as disease can lead to unnecessary anxiety and interventions, whereas failing to recognize a true abnormality can have dire consequences for both the mother and the fetus.⁶ Therefore, establishing a clear understanding of the baseline shifts in these parameters during normal pregnancy is paramount for providing optimal antenatal care. Despite the existence of reference ranges for pregnant populations, they are often derived from Western cohorts and may not be applicable to other ethnic and geographic populations due to variations in genetics, diet, and lifestyle.⁷ There is a paucity of comprehensive, comparative data on biochemical profiles in pregnant versus healthy non-pregnant women in our local setting. This study, therefore, was designed to bridge this knowledge gap. To conduct a comparative analysis of selected biochemical parameters between pregnant and healthy non-pregnant women.

Study design, setting and population This study employed a comparative cross-sectional study design. The research was conducted in the urban tertiary care setting of Annaii Medical College and Hospital, located in Pennalur, Sriperumbudur Taluk, Kanchipuram, Tamil Nadu. All women of reproductive age (18–45 years) residing in the region. Inclusion and Exclusion Criteria for Sample Selection Group A (Pregnant Women): • Inclusion Criteria: o Age between 18 and 40 years. o Confirmed singleton intrauterine pregnancy. o Gestational age between 14 to 36 weeks (Second and Third Trimester). Gestational age was confirmed via last menstrual period (LMP) and/or first-trimester ultrasound. o No known acute or chronic illnesses. • Exclusion Criteria: o Women with multiple gestations (twins, triplets, etc.). o Women with pre-existing chronic conditions such as Diabetes Mellitus (Type 1 or 2), Hypertension, Chronic Renal Disease, Chronic Liver Disease, Thyroid disorders, or Autoimmune diseases. o Women diagnosed with pregnancy-related complications like Pre-eclampsia, Eclampsia, Gestational Diabetes Mellitus (GDM), or HELLP syndrome. o Women with any acute febrile illness or active infection at the time of blood collection. o Women who had received a blood transfusion within the last 3 months. Group B (Healthy Non-Pregnant Women): • Inclusion Criteria: o Age between 18 and 40 years. o Healthy, non-pregnant women with regular menstrual cycles. • Exclusion Criteria: o Women with known chronic diseases (Diabetes, Hypertension, Renal/Hepatic disorders). o Women who are lactating or in the post-partum period (< 6 months). o Women currently using hormonal contraceptives (oral pills, implants, injections) for the past three months, as these can alter biochemical parameters. o Women with any acute or chronic illness or infection. Procedure for Data Collection The data collection process was systematically carried out in three phases: Phase 1: Participant Recruitment and Consent (Day 1) • Women attending the Antenatal clinic (Group A) and the General OPD/Staff (Group B) were briefed about the study's purpose, procedures, benefits, and potential risks. • Those willing to participate were screened against the inclusion and exclusion criteria. • Written informed consent was obtained from all eligible participants. • A detailed history including age, LMP/gestational age (for Group A), medical history, and medication use was recorded on a pre-designed structured proforma. Phase 2: Blood Sample Collection (Morning) • Participants were instructed to maintain an overnight fast of 8-10 hours. • The following morning, under strict aseptic conditions, a trained phlebotomist collected 5 mL of venous blood from the antecubital vein of each participant. • Blood Dispensation: o 2 mL was immediately dispensed into a sterile EDTA (Ethylenediaminetetraacetic acid) vacutainer for hematological analysis. o 3 mL was dispensed into a plain serum separator vacutainer for biochemical analysis. • Samples were properly labeled with unique study IDs to ensure anonymity. Phase 3: Laboratory Processing and Analysis (Within 2 hours) • Hematology: The EDTA samples were run on a fully automated 5-part differential hematology analyzer (e.g., Sysmex XN-1000) to measure Hb, TLC, and Platelet Count. • Biochemistry: The plain vacutainers were left to clot for 30 minutes at room temperature. They were then centrifuged at 3000 rpm for 10 minutes to separate the serum. The extracted serum was used to analyze: o Liver Function Tests (Bilirubin, ALT, AST, ALP, Protein, Albumin). o Renal Function Tests (Creatinine, BUN). o Fasting Blood Glucose (using the GOD-POD method). • All analyses were performed on a fully automated clinical chemistry analyzer (e.g., Beckman Coulter AU480). Strict quality control measures were followed, running internal quality control (IQC) samples alongside participant samples to ensure accuracy and precision. Statistical analysis Statistical Analysis: The cleaned data was exported to Statistical Package for the Social Sciences (SPSS) version 26.0 for analysis.

Table 1: Comparison of Hematological Parameters Parameter Group A (Pregnant, n=60) Mean ± SD Group B (Non-Pregnant, n=60) Mean ± SD p-value Hemoglobin (g/dL) 10.8 ± 1.2 12.9 ± 1.1 <0.001* Total Leukocyte Count (cells/mm³) 10,500 ± 2,100 7,200 ± 1,500 <0.001* Platelet Count (x10³ cells/mm³) 235.4 ± 54.0 249.0 ± 58.0 0.18 Pregnant women demonstrated a significantly lower mean hemoglobin level (10.8 ± 1.2 g/dL) compared to non-pregnant women (12.9 ± 1.1 g/dL, p < 0.001), indicating physiological anemia of pregnancy. Conversely, the total leukocyte count was significantly elevated in the pregnant group (10,500 ± 2,100 cells/mm³ vs. 7,200 ± 1,500 cells/mm³, p < 0.001), reflecting pregnancy-induced leukocytosis. However, platelet counts showed no significant difference between the two groups (235.4 ± 54.0 vs. 249.0 ± 58.0 x10³ cells/mm³, p = 0.18) Table 2: Comparison of Hepatic Function Parameters Parameter Group A (Pregnant, n=60) Mean ± SD Group B (Non-Pregnant, n=60) Mean ± SD p-value Total Bilirubin (mg/dL) 0.8 ± 0.3 0.7 ± 0.3 0.07 Direct Bilirubin (mg/dL) 0.2 ± 0.1 0.2 ± 0.1 0.84 Indirect Bilirubin (mg/dL) 0.6 ± 0.2 0.5 ± 0.2 0.14 ALT (U/L) 21.4 ± 8.5 19.8 ± 7.2 0.26 AST (U/L) 25.6 ± 9.1 23.4 ± 8.6 0.17 Alkaline Phosphatase (ALP) (U/L) 125.5 ± 35.4 68.2 ± 22.1 <0.001* Total Protein (g/dL) 6.8 ± 0.6 7.2 ± 0.5 <0.01* Albumin (g/dL) 3.4 ± 0.4 4.2 ± 0.5 <0.001* Bilirubin levels (total, direct, and indirect) and transaminases (ALT and AST) showed no statistically significant differences between pregnant and non-pregnant women (p > 0.05 for all), suggesting preserved hepatocellular function during normal pregnancy. However, alkaline phosphatase (ALP) was markedly elevated in the pregnant group (125.5 ± 35.4 U/L vs. 68.2 ± 22.1 U/L, p < 0.001), attributed to placental production of this enzyme. Additionally, both total protein (6.8 ± 0.6 vs. 7.2 ± 0.5 g/dL, p < 0.01) and serum albumin (3.4 ± 0.4 vs. 4.2 ± 0.5 g/dL, p < 0.001) were significantly lower in pregnant women, likely due to hemodilution and increased fetal demands. Table 3: Comparison of Renal Function Parameters Parameter Group A (Pregnant, n=60) Mean ± SD Group B (Non-Pregnant, n=60) Mean ± SD p-value Serum Creatinine (mg/dL) 0.8 ± 0.2 0.7 ± 0.1 <0.05* Blood Urea Nitrogen (BUN) (mg/dL) 10.2 ± 2.8 11.5 ± 3.1 0.08 Serum creatinine was slightly but significantly higher in the pregnant group (0.8 ± 0.2 mg/dL) compared to the non-pregnant group (0.7 ± 0.1 mg/dL, p < 0.05). However, blood urea nitrogen (BUN) levels did not differ significantly between the two groups (10.2 ± 2.8 vs. 11.5 ± 3.1 mg/dL, p = 0.08). Table 4: Comparison of Carbohydrate Metabolism Parameters Parameter Group A (Pregnant, n=60) Mean ± SD Group B (Non-Pregnant, n=60) Mean ± SD p-value Fasting Blood Glucose (FBG) (mg/dL) 85.2 ± 9.8 79.1 ± 8.5 <0.01* Fasting blood glucose (FBG) was significantly elevated in pregnant women (85.2 ± 9.8 mg/dL) compared to non-pregnant women (79.1 ± 8.5 mg/dL, p < 0.01), reflecting the physiological insulin resistance that develops during pregnancy to ensure adequate glucose supply to the fetus.

The present study was designed to compare key biochemical parameters between pregnant women and healthy non-pregnant women, aiming to elucidate the physiological adaptations occurring during gestation. Our findings demonstrate significant alterations across multiple biochemical domains, including hematological, hepatic, renal, and metabolic parameters. These changes predominantly reflect the body's adaptive response to support fetal growth and development, consistent with well-established physiological principles.¹ One of the most prominent findings was the significant reduction in hemoglobin levels among pregnant women (10.8 ± 1.2 g/dL vs. 12.9 ± 1.1 g/dL, p < 0.001). This physiological anemia of pregnancy is a well-documented phenomenon, primarily attributed to the disproportionate expansion of plasma volume (up to 40-50%) relative to the increase in red blood cell mass (approximately 20-30%), resulting in hemodilution.⁸ Similar findings have been reported in a recent Canadian cohort study, which demonstrated significant decreases in anemia markers during late gestation compared to the postpartum period.⁹ The clinical significance of this finding is substantial, as it underscores the increased demand for iron and folic acid during pregnancy. A hemoglobin level below 11.0 g/dL in the second and third trimesters warrants investigation for iron deficiency anemia and appropriate supplementation.⁹ Concurrently, we observed a statistically significant elevation in total leukocyte count in the pregnant group (10,500 ± 2,100 cells/mm³ vs. 7,200 ± 1,500 cells/mm³, p < 0.001). This leukocytosis of pregnancy is a physiological stress response, likely mediated by increased cortisol and estrogen levels, preparing the body for the stress of labor and delivery.¹⁰ This finding aligns with the observations of studies from Pakistan and India, which reported similar trends in pregnancy-related hematological changes.¹⁰ Our study revealed a highly significant increase in alkaline phosphatase (ALP) levels in the pregnant group (125.5 ± 35.4 U/L vs. 68.2 ± 22.1 U/L, p < 0.001). This elevation is primarily attributed to the placental production of ALP, which increases substantially during the third trimester.¹² The clinical implication is critical: an elevated ALP should not be misinterpreted as hepatobiliary pathology in a pregnant woman without other corroborating signs. This finding is consistent with the study by Mohammed et al. in Ethiopia, which established trimester-specific reference intervals and noted that ALP values in pregnant women were significantly higher than in non-pregnant controls.¹¹ Both total protein and albumin were significantly lower in the pregnant group, reflecting the hemodilutional effect and the increased metabolic demands of the fetus for amino acids.¹¹ Our finding of reduced albumin (3.4 ± 0.4 g/dL vs. 4.2 ± 0.5 g/dL) aligns with the Japanese cohort study by Shimokawa et al., which demonstrated progressive decreases in albumin levels across gestation.¹³ Importantly, transaminases (ALT and AST) and bilirubin remained stable in our study, suggesting preserved hepatocellular integrity during normal pregnancy. This observation is supported by a recent study from Ethiopia, which found no significant variation in AST, ALT, or bilirubin between pregnant and non-pregnant women.¹¹ Renal function undergoes significant hemodynamic changes during pregnancy, primarily an increase in glomerular filtration rate (GFR) of 40-50%.⁵ This increased GFR typically leads to enhanced excretion of metabolic waste products, resulting in reduced serum creatinine and blood urea nitrogen (BUN). Our study found a lower mean BUN in the pregnant group (though not statistically significant), which is consistent with the physiological expectation. However, we observed a small but statistically significant increase in serum creatinine (0.8 ± 0.2 mg/dL vs. 0.7 ± 0.1 mg/dL, p < 0.05). This finding contrasts with several studies, including a recent Canadian cohort that demonstrated significant decreases in creatinine and urea during late gestation compared to postpartum levels.⁹ This discrepancy might be attributed to individual variability, subclinical dehydration, or regional differences in dietary patterns and baseline renal function. A study from Ethiopia similarly reported significant variation in creatinine and urea levels between pregnant and non-pregnant women.¹¹ The significant increase in fasting blood glucose observed in our study (85.2 ± 9.8 mg/dL vs. 79.1 ± 8.5 mg/dL, p < 0.01) reflects the progressive insulin resistance that develops during pregnancy.¹⁴ This diabetogenic state, mediated by human placental lactogen, cortisol, and progesterone, ensures a constant supply of glucose to the fetus. Similar findings were reported by Alemu et al. in Ethiopia, who noted significantly elevated fasting glucose levels in pregnant women (96.35 ± 14.45 mg/dL vs. 81.12 ± 9.86 mg/dL).¹¹ The clinical relevance of this finding lies in the importance of screening for gestational diabetes mellitus, as this physiological insulin resistance can lead to hyperglycemia in susceptible individuals.¹⁴.

In conclusion, our study demonstrates that pregnancy is associated with significant alterations in hematological, hepatic, renal, and metabolic parameters, predominantly reflecting the body's adaptive response to gestation. The significant reductions in hemoglobin and albumin, alongside elevations in total leukocyte count, alkaline phosphatase, and fasting blood glucose, underscore the importance of using trimester-specific reference ranges for accurate interpretation of biochemical tests in pregnant women. These findings have direct clinical relevance for the early diagnosis and management of pregnancy-related complications, ultimately contributing to improved maternal and fetal outcomes.

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