This study was conducted at Suzhou Ninth People's Hospital and retrospectively analyzed the clinical data of 122 pregnant women who underwent prenatal check-ups between May 2023 and April 2024. They were divided into three groups according to the actual dose of calcium and vitamin D3 supplementation: the control group (n = 48), the Calcium 600-Vitamin D group (n = 41), and the Calcium 1,200-Vitamin D group (n = 33). The study protocol was approved by the Ethics Committee of Suzhou Ninth People's Hospital (KY-2023004). All subjects signed the consent form before participation in the study.

Inclusion criteria: healthy pregnant women; aged 20–40 years; with a singleton pregnancy of at least 20 weeks gestation; no severe complications or chronic diseases; no calcium or vitamin D metabolic disorders; and the ability to comply with the treatment protocol. Exclusion criteria: multiple pregnancies; Women with pre-existing hypertension/diabetes or complications diagnosed before supplementation initiation were excluded to isolate supplement effects on incident outcomes; a history of severe pregnancy complications; use of diuretics or calcium channel blockers; and allergies to the study medications.

Participants in the control group did not receive any calcium or vitamin D supplements. Those in the Calcium 600-Vitamin D group were given 600 mg of calcium and 1,000 IU of vitamin D daily, while participants in the Calcium 1,200-Vitamin D group received 1,200 mg of calcium and 1,000 IU of vitamin D daily. The supplementation continued from enrollment until delivery. Compliance with the supplementation regimen was monitored through regular follow-ups. The three regimens reflected: (1) Standard care (control), (2) WHO-recommended pregnancy dose (600 mg/1,000 IU), and (3) High-dose for women with baseline insufficiency [serum 25(OH)D < 30 nmol/L or calcium < 2.1 mmol/L]. Allocation was based on baseline nutritional status.

Clinical baseline data were collected, including age, gestational age, parity (nulliparous or multiparous), smoking status (non-smoker or smoker), weight gain during pregnancy (categorized as inadequate, adequate, or excessive), and physical activity levels before and during pregnancy (classified as none, low, moderate, or high intensity).

Maternal outcomes were assessed by recording the incidence of gestational diabetes, preeclampsia, gestational hypertension, and weekly frequency of leg cramps. Blood pressure [Systolic Blood Pressure (SBP) and Diastolic Blood Pressure (DBP)], blood calcium levels, and vitamin D levels were measured at baseline and at delivery.

Pregnancy and neonatal outcomes were thoroughly documented, including miscarriage rates, live birth rates, mode of delivery (vaginal delivery or cesarean section), preterm birth, premature rupture of membranes, chorioamnionitis, polyhydramnios, gestational age, neonatal sex, birth weight, birth length, head circumference, 1- and 5-minute Apgar scores, neonatal asphyxia, neonatal infection, and Neonatal Intensive Care Unit (NICU) admissions. The Apgar scores, which evaluate the health of newborns at 1 and 5 min after birth, were assessed based on five criteria: heart rate, respiratory effort, muscle tone, reflex response, and color, with each criterion scoring between 0 and 2, and a total score ranging from 0 to 10 (12).

The occurrence of adverse effects such as fever, diarrhea, rash, joint pain, and dizziness/headache was monitored throughout the study period. These adverse effects were recorded based on self-reports from participants and verified through clinical assessments.

Statistical analyses were conducted using SPSS 24.0 (SPSS Inc., Chicago, IL, USA). Continuous variables were expressed as mean ± SD and compared using ANOVA or the Kruskal–Wallis test, based on Shapiro–Wilk normality results. Post-hoc comparisons were performed using the Dunnett test. Categorical variables were analyzed using the Chi-square or Fisher's exact test. Multivariable logistic regression assessed associations between supplementation and binary outcomes (e.g., GDM, gestational hypertension, NICU admission), adjusting for maternal age, parity, gestational weight gain, smoking, and physical activity. Results were presented as odds ratios (ORs) with 95% confidence intervals, and P < 0.05 was considered significant.

The baseline characteristics of the participants are summarized in Table 1. There were no significant differences among the three groups regarding age, gestational age, parity, smoking status, weight gain during pregnancy, physical activity levels before and during pregnancy, Dietary Calcium, and Dietary Vitamin D (P > 0.05 for all comparisons).

As shown in Table 2, the supplementation of calcium and vitamin D3 had significant effects on several maternal outcomes. The incidence of gestational diabetes was significantly lower in both the Calcium 600-Vitamin D group (7.3%) and the Calcium 1,200-Vitamin D group (6.1%) compared to the control group (22.9%) (P = 0.035). Similarly, preeclampsia rates were reduced to 2.4% and 0% in the Calcium 600-Vitamin D and Calcium 1,200-Vitamin D groups, respectively, vs. 12.5% in the control group (P = 0.032). Gestational hypertension was significantly lower in the supplemented groups, with rates of 9.8% and 0%, compared to 16.7% in the control group (P = 0.047). The frequency of leg cramps per week was also significantly reduced in the Calcium 600-Vitamin D and Calcium 1,200-Vitamin D groups (P < 0.001 for both comparisons). There were no significant differences in baseline SBP and DBP among the groups (P > 0.05), but at delivery, both SBP and DBP were significantly lower in the Calcium 600-Vitamin D and Calcium 1,200-Vitamin D groups (P < 0.05 for both).

The effects of calcium and vitamin D3 supplementation on pregnancy and neonatal outcomes are detailed in Table 3. The miscarriage rate was significantly lower in both the Calcium 600-Vitamin D (2.4%) and Calcium 1,200-Vitamin D (3.0%) groups compared to the control group (14.6%) (P = 0.049). The mode of delivery also differed significantly, with a higher rate of vaginal deliveries in the Calcium 600-Vitamin D (87.5%) and Calcium 1,200-Vitamin D (84.4%) groups compared to the control group (65.9%) (P = 0.039). The preterm birth rate was significantly lower in the supplemented groups (5.0% and 3.1%) compared to the control group (19.5%) (P = 0.029). The incidence of polyhydramnios was reduced to 2.5% and 3.1% in the Calcium 600-Vitamin D and Calcium 1,200-Vitamin D groups, significantly lower than the control group's 19.5% (P = 0.011). Other pregnancy complications, such as premature rupture of membranes and chorioamnionitis, did not show significant differences among the groups (P > 0.05). Regarding neonatal outcomes, birth weight was significantly higher in the Calcium 600-Vitamin D (3,413.53 ± 251.29 g) and Calcium 1,200-Vitamin D (3,561.78 ± 230.64 g) groups compared to the control group (3,179.54 ± 409.81 g) (P < 0.001). Additionally, the 1-minute Apgar score was significantly higher in the supplemented groups (P = 0.001), though the 5-minute Apgar score and the incidence of neonatal asphyxia did not differ significantly (P > 0.05).

To assess the independent effect of supplementation, multivariable logistic regression was performed, adjusting for maternal age, parity, gestational weight gain, smoking status, and physical activity. After adjustment, Calcium 1,000-Vitamin D supplementation was significantly associated with reduced odds of gestational diabetes (OR = 0.423, 95% CI: 0.123–0.869, P = 0.043) and gestational hypertension (aOR = 0.342, 95% CI: 0.126–0.875, P = 0.012), compared to the control group. The Calcium 600–Vitamin D group showed similar trends, with a borderline association for gestational diabetes (OR = 0.136, 95% CI: 0.018–1.022, P = 0.053). Both supplementation groups were also significantly associated reduced weekly leg cramp frequency (Calcium 600: OR = 0.507, 95% CI: 0.355–0.723; Calcium 1,200: OR = 0.512, 95% CI: 0.256–0.985; P < 0.001). No significant associations were observed for preeclampsia, 1-minute Apgar score, neonatal infection, or NICU admission, although the effect estimates generally favored supplementation. Full results are presented in Table 4.

In addition to clinical efficacy, the tolerability and adverse effects of the interventions were assessed. The adverse effects of calcium and vitamin D3 supplementation are summarized in Table 5. There were no significant differences in the incidence of fever, diarrhea, rash, joint pain, and dizziness/headache among the control, Calcium 600-Vitamin D, and Calcium 1,200-Vitamin D groups (P > 0.05 for all comparisons). The occurrence of these adverse effects was low and comparable across all groups, indicating that calcium and vitamin D3 supplementation was well-tolerated by the participants. Notably, constipation—a frequently reported adverse effect of calcium supplementation—was comparably low across groups (Control: 8.3% vs. Calcium 600: 7.3% vs. Calcium 1,200: 6.1%, P = 0.928), indicating the regimens were well-tolerated.