The number of pregnancies through assisted reproductive technology (ART) has been on the rise significantly since the mid-1980s when in vitro fertilization (IVF) became available in China (1). Following this, it is important to check the possible dangers to mothers and their offspring. Numerous studies have found that compared to women who conceive spontaneously, women who conceive though IVF appear at a higher risk of obstetric and perinatal complications (2–5). One of the most common and important pregnancy complications is gestational diabetes mellitus (GDM). The latest systematic review and meta-analysis showed a higher risk of GDM in women whose pregnancy is through ART than spontaneous (6). However, the study on the relationship between ART and OGTT blood glucose levels remains to be examined.

Maternal advanced age, obesity, multiple pregnancies, and polycystic ovary syndrome (PCOS) are the major risk factors for GDM. They are also frequently present in women who received ART, suggesting a potential association between GDM and ART (7–10). Some modifiable risk factors during pregnancy, in addition to BMI, include vitamin supplements. B vitamins are necessary for the One Carbon Cycle (C1) and are crucial for numerous metabolic pathways and methylation processes. Folate interacts closely with vitamin B6 and B12 for functional effects on the C1 network (11). Meanwhile, folate and vitamin B6 and B12 are crucial enzymatic cofactors in the synthesis of methionine from homocysteine (12, 13), and their lacking increases homocysteine concentrations in the blood (14, 15). Since elevated homocysteine is associated with insulin resistance, it leads to cause the risk of GDM (16, 17). Hence, our study investigated the effect for folate and vitamin B6 and B12 on GDM.

Although the previous study showed that folate supplementation and vitamin B12 were related to a higher probability of live birth among women who underwent ART (18, 19), the combined effect of B vitamins (folate, vitamin B6, and vitamin B12) and women who received ART on GDM has not been investigated till now. To address this gap, this study aimed to determine whether women who underwent ART are an independent risk factor for the development of GDM from a prospective cohort study. Moreover, it is further hypothesized that maternal B vitamins (folate, vitamin B6, and vitamin B12) intake from pre-pregnancy to the first trimester would alter the relationship between ART and GDM and maternal blood glucose levels.

Between 2019 and 2022, 3,252 eligible singleton pregnancies were among 3,980 women. 333 (10.24%) of these women underwent ART, while 2,919 (89.76%) conceived spontaneously. Table 1 shows the characteristics of the study population. Distinctions exist between the ART and spontaneous conception groups. Women in the ART groups were likelier to have lower education level, nulliparous, no occupation and higher BMI. Regarding nutrient intake, the ART group possibly received higher doses of supplemental B6, total B6, supplemental B12, total B12, and total folate. Similarly, OGTT 1-h and OGTT 2-h blood glucose levels were greater than the spontaneous conception group (Table 1).

As shown in Table 2, in the unadjusted analysis, women who conceived ART exhibited almost double the odds of GDM than the spontaneous conception women (OR = 1.78, 95% CI: 1.37–2.31). After adjusting for maternal education, occupation, smoking, passive smoking, age, pre-pregnancy BMI, abortion history, PCOS, GDM history, macrosomia history, and family history of diabetes and gestational diabetes, the odds of GDM remained significantly higher in the ART group (aOR = 1.59, 95% CI: 1.08–2.34).

Fasting blood glucose levels between the ART and the spontaneous conception groups were not statistically different adjusting for variables. Neither were the regression coefficients in the multivariable linear regression models. However, OGTT 1-h and OGTT 2-h blood glucose levels were significantly higher in the ART group before adjustment, continuing the same trend after adjustment for covariates. The regression coefficients (β) of 1-h blood glucose (β = 0.43, 95% CI: 0.19–0.68) and 2-h blood glucose levels (β = 0.40, 95% CI: 0.19–0.60) in multiple linear regression are also statistically significant (Figure 2).

Comparing the highest to the lowest quartile, a significant relationship between dietary vitamin B6 and GDM (aOR = 1.60, 95% CI: 1.13–2.27) has been found. Similar results were observed for total vitamin B6 (aOR = 1.33, 95% CI: 1.00–1.77), including supplemental vitamin B6 sources. Both dietary vitamin B12 and total vitamin B12 exhibited a significant correlation with GDM in the highest quartile (aOR = 1.88, 95% CI: 1.34–2.64, and aOR = 1.52, 95% CI: 1.14–2.02) (P_trend < 0.05). The association between total vitamin B6 and GDM risk was entirely driven by dietary vitamin B6, similar to vitamin B12. Dietary folate and total folate intake were positively associated with GDM risk. The aORs of GDM for dietary folate and total folate intake were 1.66 (95% CI: 1.19–2.32) and 1.35 (95% CI: 1.02–1.77) (P_trend < 0.05). The aORs of GDM by supplemental folate intake were 0.93 (95% CI: 0.73–1.20) for 400–800 μg/day and 1.30 (95% CI: 1.01–1.68) for > 800 μg/day compared to intake < 400 μg/day (P_trend = 0.033) (Table 3).

Since only the interaction between supplemental folate and the ART on GDM and OGTT blood glucose levels was statistically significant (P for interaction < 0.05), we further stratified by supplemental folate: inadequate folate < 400 μg/day, adequate folate (400–800) μg/day, and excessive folate > 800 μg/day, and found that women with ART and excessive supplemental folate (> 800 μg/day) showed a slightly higher risk of developing GDM than women with spontaneous conception (aOR = 1.62, 95% CI: 1.39–3.39) (Table 4). Moreover, the increase in 1-h OGTT blood glucose risk (β = 0.76, 95% CI: 0.39–1.13) as well as 2-h OGTT blood glucose risk (β = 0.60, 95% CI: 0.29–0.92), were statistically significant only in women with excessive supplemental folate (> 800 μg/day) and ART (Table 5). All multivariable models adjusted for maternal age, education, occupation, smoking, passive smoking, pre-pregnancy BMI, abortion history, PCOS, GDM history, macrosomia, dietary vitamin B6, dietary vitamin B12, family history of diabetes and family history of gestational diabetes.

This study demonstrated that women-conceived ART appears to be a risk factor for GDM and elevated blood glucose levels. We also found that ART was linked with GDM and higher blood glucose concentrations 1- and 2-h after OGTT in the 24–28 weeks, especially in women using excessive supplemental folate (> 800 μg/day).

Ashrafi et al. carried out a similar study and found that women who underwent ART had a higher risk of GDM than those who were spontaneously pregnant. However, they did not observe significant differences in blood glucose levels. The primary cause of the discrepancy is the various GDM judging standards. Their study followed the guidelines set forth by the American Diabetes Association (35), which called for two-step screening pregnant women at 24–28 weeks of gestation with a 50 g, 1-h oral glucose challenge test. If the screening test’s results were abnormal (glucose ≥ 7.8 mmol/l or 140 mg/dl), a 100 g 3-h oral glucose tolerance test (OGTT) was then carried out in the 1–2 weeks that followed. Our study used one-step screening (i.e., a glucose-tolerance test in which the blood glucose level was obtained after the oral administration of a 75 g glucose load in the fasting state). It has been indicated that the two-step strategy led to lower diagnoses of GDM than the one-step approach (35). Another reason is that their sample size is relatively small, only including 360 women (36). Cai et al. found that IVF was associated with an increase in fasting blood glucose concentration in the second and third trimesters of pregnancy 2-h after OGTT, but only in women with a BMI of 25 kg/m² or higher (37). However, this study lacked a 1-h post-OGTT plasma glucose sample. Szymanska et al. observed an increase in fasting blood glucose levels during the first trimester of an IVF pregnancy but no increase in blood glucose levels during the second trimester. However, the study was limited to women with GDM (38).

Additionally, the results of the OGTT blood glucose test were examined in our study, and it was shown that women underwent ART would experience an increase in 1-h and 2-h blood glucose levels with no effect on fasting blood glucose. Gestational abnormal glucose metabolism is associated with a remarkably increased risk of adverse perinatal outcomes, even when levels are below GDM thresholds (39–41), and it is worth noting to observe the magnitude of these effects.

According to previous research, the risk of GDM may be increased by the relative deficiency of vitamin B6 and the comparatively low intake of vitamin B12 (42, 43). However, a positive correlation has been found between total vitamin B6 and total vitamin B12 in the highest quartile and the risk of GDM. This may be due to the fact that the supplements’ contents were primarily the focus of the earlier research. However, our study’s association between total vitamins B6 and B12 on GDM risk was entirely driven by dietary vitamins B6 and B12.

Both dietary folate and supplemental folate are statistically associated with GDM. Nevertheless, 10 years cohort study showed no statistically significant association between dietary folate before pregnancy and the prevalence of GDM (44). Given that sufficient dietary folate (> 400 μg/day) is expected to be rare in our sample, only 13.10% of our sample exhibited dietary folate intakes meet the recommended daily allowance (45). It is predicted that different associations between dietary folate and GDM in the study may be due to the different folate intakes of various populations. According to the USPSTF and WHO recommendations (31, 32), in this investigation, supplemental folate of > 800 μg/day is considered to be excessive, revealing that excessive folate supplementation would increase the incidence rate of GDM. Consistent with our study results, a cohort conducted in China showed that periconceptional folate supplement use of more than 800 μg/day from pre-pregnancy through mid-pregnancy was related to elevated GDM risk (34). The existing research on the relationship between B vitamins (folate, vitamin b6, and vitamin b12) and GDM is still controversial, more in-depth studies across multiple populations are needed to verify these results.

Previous studies have also considered the folate impact on women with ART, showing that folate can improve total and mature oocyte counts (46), embryo quality (47), pregnancy rates (48) and increase the likelihood of embryo survival (19, 49) after infertility treatment. However, these studies did not consider the effect of folate supplementation on perinatal diseases in pregnant women received ART. Numerous studies have demonstrated that folate supplementation, particularly excessive supplemental folate (> 800 μg/day), can alter the incidence of GDM in spontaneous conception women (34, 44, 50). Our research enriches fields related to ART and folate supplementation and showed that pre-pregnancy to first trimester folate supplements could modify the effect of ART on GDM risk and blood glucose levels.

The strengths of our study include a unique design and aim, specifically evaluating whether women on B vitamins exhibit a higher risk of GDM after ART. Moreover, using of a prospective cohort, which reduces the effects of selection or recall bias. To avoid information bias, women with hypertension or diabetes mellitus, as well as major influences of GDM were excluded. The E-value for the association between the women who underwent ART and GDM risk was 1.83, suggesting that a potential confounder must have relatively strong associations with both women who underwent ART and GDM risk to fully explain the association between supplemental folate intake and GDM risk.

This study has some limitations. The plasma homocysteine levels of B vitamins in pregnant women were not measured to support our conclusions. As a result, there is a possibility of misclassification. However, significant efforts were made to ensure reliable vitamin B data were collected on time by trained medical personnel with meticulous follow-up. Furthermore, self-reported folate from supplements was correlated with plasma folate and was regarded as a reliable indicator of folate exposure (51). Also, a relatively small sample size limits our ability to investigate the relationship between B vitamins and ART at different levels, and the generalizability of the findings to other regions needs further validation. Therefore, it is necessary to perform such study on a larger population.

ART appears to be an independent risk factor for GDM and elevated blood glucose levels. Our findings reinforce the need to advise women contemplating ART to avoid excessive folate supplements (>800 μg/day) to reduce their risk of GDM and hyperglycemia-related adverse outcomes. Women who seek fertility therapy should be counseled about this potential risk, and efforts should be taken to regulate the dose of folate supplementation throughout pregnancy. Pregnant women who underwent ART should have their health care during pregnancy, focusing more on blood glucose fluctuations.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethical approval for this study has been obtained from the Ethics Committee of Qingdao Women’s and Children’s Hospital (Number: 019–2019-FEKY). The patients/participants provided their written informed consent to participate in this study.

ML and HW conceived, designed, and supervised the cohort study. YW and YC conceptualized idea for the manuscript. ML, HW, and XD were involved in design of questionnaire and protocol as well as supervision of data collection, analyzed, and interpreted the data. GL contributed to the preparation of the manuscript. All authors critically revised the article for intellectual and scientific content and approved this submission for publication.