
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

71986
10.1038/s41598-024-71986-z
Article
Mass spectrometry of water-soluble vitamins to establish a risk model for predicting recurrent spontaneous abortion
Wu Bitao 12
Li Zhenghao 12
Peng Bi 3
Yang Qiang 12
Jiang Wenqiang 12
Ma Ying lizza2222@126.com

12
Tang Jie tjandbetty@sc-mch.cn

12
Yang Yuwei yyw318@sc-mch.cn

12
1 grid.54549.39 0000 0004 0369 4060 Mianyang Central Hospital, affiliated to School of Medicine, University of Electronic Science and Technology of China, Mianyang, China
2 grid.490255.f 0000 0004 7594 4364 NHC Key Laboratory of Nuclear Technology Medical Transformation (Mianyang Central Hospital), Mianyang, China
3 grid.452803.8 Sichuan Mental Health Center, The Third Hospital of Mianyang, Mianyang, China
6 9 2024
6 9 2024
2024
14 208309 4 2024
2 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
The adverse pregnancy outcomes, including recurrent spontaneous abortion (RSA), are strongly correlated with water-soluble vitamins, but how to predict RSA occurrence using them remains unsatisfactory. This study aims to investigate the possibility of predicting RSA based on the baseline levels of water-soluble vitamins tested by ultra-liquid chromatography-tandem mass spectrometry. A total of 918 pregnant women was consecutively enrolled in this cross-sectional study. According to the miscarriage numbers, they were divided into normal first pregnancy (NFP, n = 608), once spontaneous abortion (OSA, n = 167), and continuous spontaneous abortion (CSA, n = 143) groups. The Cox proportional-hazards regression model was employed to establish a risk model for predicting RSA. The RSA occurrence was 6.54% in overall pregnant women, with a prevalence of 12.57% in the OSA group and 27.27% in the CSA group. Significant differences were observed in baseline deficiencies of vitamin B3, B5, B6, and B9 among NFP, OSA, and CSA groups (χ2 = 12.191 ~ 37.561, all P < 0.001). Among these vitamins, B9 (HR = 0.89 and 0.88, all P < 0.001) and B6 (HR = 0.83 and 0.78, all P < 0.05) were identified as independent factors in both the OSA and CSA groups; whereas B5 was identified as an additional independent factor only in the CSA group (HR = 0.93, P = 0.005). The Cox proportional-hazards model established using these three vitamins exhibited poor or satisfactory predictive performance in the OSA (Sen = 95.2%, Spe = 39.0%) and CSA (Sen = 92.3%, Spe = 60.6%) groups, respectively. However, B5, B6, and B9 compensatory levels were not associated with RSA occurrence (all P > 0.05). Our study presents a highly sensitive model based on mass spectrometry assay of baseline levels in B vitamins to predict the RSA occurrence as possible.

Keywords

Recurrent spontaneous abortion
Water-soluble vitamins
Baseline
Mass spectrometry
Risk model
Subject terms

Biomarkers
Diseases
Risk factors
http://dx.doi.org/10.13039/501100004829 Science and Technology Department of Sichuan Province 2019YFS0416 2019YJ0701 Ma Ying Yang Yuwei Mianyang Central Hospital, School of Medicine, University of Electronic Science and Technology of China2022FH005 Tang Jie issue-copyright-statement© Springer Nature Limited 2024
==== Body
pmcIntroduction

Spontaneous abortion (SA), an accidental miscarriage occurring without external intervention prior to 20 ~ 24 weeks gestational age, affects more than 20% of clinically recognized pregnancies1. It can subdivide into threatened, inevitable, incomplete, missed, septic, and complete abortions1,2, and manifest imperceptible embryo loss even life-threatening shock during a pregnancy course2. When consecutive spontaneous pregnancy losses occur, it is referred to as recurrent spontaneous abortion (RSA), which affects approximately 1 ~ 5% of reproductive women3,4. The definition of RSA in relation to the number of miscarriages varies among different countries. The European Society of Human Reproduction and Embryology and the American Society for Reproductive Medicine define RSA as two or more accidental miscarriages, while the Royal College of Obstetricians and Gynaecologists defines it as three or more4,5. Additionally, recent epidemiological surveys provide evidence to suggest that the COVID-19 pandemic has potentially accelerated the occurrence of RSA, with a prevalence rate reaching up to 6.5 ~ 9.5%6,7.

Although the etiology of RSA can often be attributed to fetal, maternal, or external factors, in many cases, the specific etiology remains never to be identified. Numerous studies have demonstrated a significant association between water-soluble vitamins and adverse pregnancy outcomes, including anemia, nausea and vomiting, and malnutrition in pregnant women, and/or fetal neural tube defects, other central system disorders, and poor embryonic development8–10. The water-soluble vitamins include nine compounds, that is, eight B vitamins (B1, B2, B3, B5, B6, B7, B9, and B12) and one vitamin C11. Of them, B vitamins have been found to potentially induce chromosomal abnormalities12,13, as well as a broad spectrum of endocrine, immune, and metabolic disorders14,15, thereby impeding proper embryo development during pregnancy, potentially increasing abortion risk. The systematic review and Meta-analysis on vitamin and mineral Supplementation during pregnancy have demonstrated a significant impact of vitamin B9 supplementation on enhancing pregnancy outcomes16–18, while the role of other vitamin supplementation remains a subject of controversy19–22.

Up to now, the progress in predicting and preventing RSA is unencouraging, despite the relative simplicity of its diagnosis3. The lack of standardized definitions, the uncertainties surrounding pathogenesis, and the highly variable clinical presentation, as well as the compensatory vitamin levels, and the accuracy of the measurement technique, all contribute to challenges in predicting RSA. The isotope-dilution liquid chromatography tandem mass spectrometry offers a highly sensitive, selective, and specific testing method for water-soluble vitamins23. Therefore, based on this technique for assessing the baseline levels of water-soluble vitamins in pregnant women, this study aims to investigate the potential for predicting RSA. According to the number of accidental miscarriages, we defined once SA (OSA) as only once occurrence, and continuous SA (CSA) as twice or more occurrences. Our OSA definition is consistent with the definition of RSA in most countries.

Methods

Participants

The study enrolled pregnant women with a SA history who underwent long-term follow-up at Mianyang Central Hospital, School of Medicine, University of Electronic Science and Technology of China from Jan. 2021 to Dec. 2023. Inclusion criteria: (1) pregnant women with clinically recognized pregnancies at 4 and 6 gestational weeks; (2) their baseline levels of water-soluble vitamins were tested at 4–6 gestational weeks; (3) with well-documented pregnancy outcomes. Exclusion criteria: (1) pregnant women who had previously undergone an induced abortion or were deciding an artificial abortion based on personal choice; (2) pregnant women who had taken complex or individual B vitamins, excluding vitamin 9, prior to conception and prior to the examination of water-soluble vitamins; (3) pregnant women requiring termination of their pregnancies due to severe illness; (4) pregnant women who were lost to follow-up. During the same period, pregnant women with a first pregnancy further a successful delivery were consecutively enrolled referring to the inclusion and exclusion criteria for pregnant women with SA history.

Finally, a total of 918 pregnant women was enrolled in this study (See Supplementary Fig. 1). According to the SA experience, all participants were divided into three groups: normal first pregnancy (NFP, pregnant women with a first pregnancy further a successful delivery, n = 608), once spontaneous abortion (OSA, pregnant women with once SA history, n = 167), and continuous spontaneous abortion (CSA, pregnant women with two or more SA history, n = 143). After conducting baseline testing, the OSA and CSA groups were administered with a low dosage of B complex vitamin supplementation until 24 gestational weeks. The daily dosage of B complex vitamin supplementation was as follow: 1.4 mg of B1, 1.4 mg of B2, 36 mg of B3, 4 mg of B5, 7 mg of B6, 30 μg of B7, 360 μg of B9, and 6 μg of B12.

Sample collecting and processing

Aliquots of 3.0 ml fasting blood were collected in an SST-II vacuum tubes (BD, USA) during 4 ~ 6 gestational weeks and after 4 weeks of vitamin supplementation. Serum was separated within 30 min via centrifuging at approximately 1500 × g for 10 min, and used for the quantification of water-soluble vitamin levels within a timeframe of 3 days. Prior to quantification, the serum was stored at − 20 °C.

Before mass spectrometry, the thawed serum was preprocessed using a water-soluble vitamin assay kit (CAT.NO. YS20020009, Shandong Yingsheng Biotechnology Co., Ltd., CHN). The sample releaser III from the kit was mixed with the freshly dissolved internal standard concentrate at a volume ratio of 200:1. 60 μl of this mixture was combined with 60 μl of serum and vortexed for 5 min, followed by centrifugation at 12000 × g for an additional 5 min. Subsequently, 70 μl of supernatant was transferred into a microplate, and sealed for chromatographic analysis.

Chromatography-tandem mass spectrometry assay of water-soluble vitamin

Nine water-soluble vitamins were measured on a EXT9050MD ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) (Shandong Yingsheng Biotechnology Co., Ltd., CHN). The chromatography conditions were as follows: C18 column at 40 °C, 30 μl injection volume, and density gradient elution with 0.5 ml/min flow rate by mobile phase A of 0.1% formic acid in deionized water and mobile phase B of 0.1% formic acid in methanol. The percentage of mobile phase B was 1% from 0 to 1 min, 98% from 1 to 2 min, 98% from 2 to 3.5 min, 1% from 3.5 to 4.5 min, 0% from 4.5 to 5.0 min.

The mass spectrometry conditions were as follows: positive ion mode of electrospray ion trap, electrospray voltage 3500 V, ionization temperature 350 °C, sheath gas (GS1) 50psi, aux gas 10psi. The cluster removal voltages and collision voltages were 30 V and 21 V for vitamin B1, 60 V and 31 V for vitamin B2, 45 V and 28 V for vitamin B3, 40 V and 21 V for vitamin B5, 48 V and 29 V for vitamin B6, 37 V and 23 V for vitamin B7, 46 V and 25 V for vitamin B9, 45 V and 55 V for vitamin B12, 25 V and 14 V for vitamin C, respectively. Mass spectrometry scan used a multiple reaction monitoring mode with the range from 150 to 500 m/z, and the rate of 1000 Da/s. Chromatograms and process data were analyzed using TraceFinder™ modern data visualization software (version 4.1, Thermo). Via monitoring the paired Q1/Q3 ion mass, vitamin B1, B2, B3, B5, B6, B7, B9, B12, and C were distinctly separated and identified with the retention time of 1.47 min, 5.14 min, 1.81 min, 3.70 min, 2.92 min, 5.22 min, 4.30 min, 5.26 min, and 0.81 min, respectively (Supplementary Fig. 2).

Cut-off definitions of B vitamins during early pregnancy

The water-soluble vitamin level was determined using the isotope-dilution internal standard method. The chromatographic peak area ratio was calculated between the sample (or standard) and the internal standard. For each water-soluble vitamin, five standards with gradient concentration were used to draw a “peak area ratio-concentration” curve, which was used to quantify the concentration in sample.

The cut-off values for vitamin B9 and B12 deficiencies during early pregnancy adopted the BOND project and WHO recommendation24–27, which were < 10 ng/mL and < 20 ng/dL respectively. While the cut-off values for vitamin B1, B2, B3, B5, B6, B7, B9, and B12, and C deficiencies adopted the lower reference limit provided by the kit, which were < 0.5 ng/mL, < 3 ng/mL, < 12 ng/mL, < 10 ng/mL, < 0.5 ng/mL, and < 6 μg/mL, respectively.

Statistical analysis

Statistical analysis was performed in the MedCalc software v20.1 (MedCalc, Belgium) and SPSS software v22.0 (SPSS, USA). The levels of water-soluble vitamins were presented as median (P25, P75) [min, max]. The Kruskal–Wallis test was employed to analyze the differences among groups, and POST-HOC analysis was conducted for pairwise comparisons using the adjusted P value (Padj) to determine statistical significance. The deficiency rates were presented as n (%), and χ2 test was used to compare the group difference. The Kaplan–Meier curves were used to depict the occurrence of miscarriage among the entire population of pregnant women, as well as within the OSA and CSA groups. The Cox proportional-hazards regression was used to examine the hazard ratio of each vitamin and establish a risk model. A significance level of α = 0.05 was used for a two-tailed test.

Results

The baseline levels of nine water-soluble vitamins in three pregnant groups

The median, P25, P75, min, and max of eight vitamin levels in the three pregnant groups were listed in Table 1. There were significant differences in vitamin B1, B5, B6, B9, and B12 levels among three pregnant groups (χ2 = 9.445 ~ 65.634, all P < 0.01). Pairwise comparisons found that vitamin B1, B5, B6, B9, and B12 levels were lower in the CSA group than in the NFP group (z = 2.915 ~ 8.008, all Padj < 0.05), while only vitamin B1, and B5 levels were lower in the CSA group than in the OSA group (z = 2.656 and 4.368, both Padj < 0.05). Additionally, vitamin B5, B6, and B9 levels were lower in the OSA group than in the NFP group (z = 2.680 ~ 3.355, all Padj < 0.05). Except for vitamin B7, the levels of the other eight vitamins ranged widely within each pregnant group, with the exceptionally high individual outliers. Table 1 Nine water-soluble vitamin levels in three pregnant groups [Median (P25, P75)].

Vitamin	NFP Group (n = 608)	OSA Group (n = 167)	CSA Group (n = 143)	χ2	P	
VitB1 (ng/mL)	1.74 (1.27, 2.30)

{0.41, 14.09}

	1.75 (1.21, 2.38)

{0.44, 7.69}

	1.47 (1.14, 2.01) ▲☺

{0.44, 11.97}

	9.445	0.009	
VitB2 (ng/mL)	7.20 (4.50, 11.58)

{1.48, 37.2}

	8.20 (5.34, 13.93)

{1.90, 31.08}

	7.69 (4.44, 10.97)

{1.13, 29.13}

	4.977	0.071	
VitB3 (ng/mL)	17.04 (12.54, 23.45)

{4.48, 75.13}

	17.81 (13.83, 22.31)

{3.36, 66.21}

	16.57 (10.71, 21.81)

{4.73, 75.59}

	5.185	0.053	
VitB5 (ng/mL)	26.68 (21.83, 38.1) {9.61, 101.45}	23.96 (17.41, 42.31)▲

{7.07, 77.68}

	17.68 (14.50, 30.16)▲☺

{6.09, 71.10}

	65.634	< 0.001	
VitB6 (ng/mL)	2.78 (2.08, 4.27)

{0.75, 22.41}

	2.69 (1.43, 3.95)▲

{0.83, 15.25}

	2.26 (1.82, 3.00)▲

{0.62, 10.10}

	28.653	< 0.001	
VitB7 (ng/mL)	0.59 (0.55, 0.65)

{0.12, 1.91}

	0.59 (0.55, 0.65)

{0.20, 0.92}

	0.59 (0.54, 0.67)

{0.37, 1.64}

	0.096	0.953	
VitB9 (ng/mL)	23.50 (17.44, 29.02)

{3.21, 40.86}

	21.23 (18.25, 25.37)▲

{5.08, 35.28}

	19.20 (14.15, 24.31)▲

{2.35, 35.57}

	31.421	< 0.001	
VitB12 (ng/dL)	14.00 (11.49, 19.19)

{2.04, 118.00}

	13.72 (11.03, 20.20)

{9.80, 70.29}

	12.74 (10.78, 17.64)▲

{4.08. 75.46}

	10.253	0.006	
VitC (μg/mL)	8.80 (6.73, 11.35)

{0.59, 27.91}

	8.24 (6.52, 10.60)

{1.37, 25.57}

	8.29 (6.52, 11.25)

{1.70, 24.21}

	3.609	0.165	
NFP, normal pregnancy; OSA, once spontaneous abortion; CSA, continuous spontaneous abortion. ▲ vs. NFP group, Padj < 0.05; ☺ vs. OSA group, Padj < 0.05. Results suggested that all eight vitamin levels ranged widely with notably high individual outliers, while only five B vitamins (B1, B5, B6, B9, and B12) existed significant differences in the median levels among the three pregnant groups.

The prevalence of nine water-soluble vitamins deficiencies

According to the lower reference limit provided by the kit, or the BOND project and WHO recommendation, we analyzed the prevalence of nine water-soluble vitamins deficiencies. Vitamin B12 (76.7%), B6 (26.8%), B3 (22.8%), C (18.6%), B2 (8.0%) and B9 (7.8%) deficiencies exceeded 5% among overall pregnant women, moreover vitamin B5 (7.7%) and B9 (17.5) deficiencies were also significant in CSA group (Table 2). Only vitamin B3, B5, B6 and B9 deficiencies were significantly different among the three groups (χ2 = 12.191 ~ 37.561, all P < 0.001). Pairwise comparisons found that B3 deficiency was higher in the CSA group than in the NFP and OSA groups (χ2 = 9.202 and 9.927, both P = 0.002), while B5 and B6 deficiencies were higher in the OSA and CSA groups than in the NFP group (χ2 = 13.637 ~ 41.669, all P < 0.001). Table 2 The deficiencies of nine water-soluble vitamins in three pregnant groups [n(%)].

Vitamin	Overall
(n = 918)	NFP Group
(n = 608)	OSA Group
(n = 167)	CSA Group
(n = 143)	χ2	P	
VitB1	5 (0.5)	2 (0.3)	1 (0.6)	2 (1.4)	2.456	0.293	
VitB2	73 (8.0)	55 (9.0)	6 (3.6)	12 (8.4)	5.367	0.068	
VitB3	209 (22.8)	131 (21.5)	30 (18.0)	48 (33.6) ▲☺	12.191	0.002	
VitB5	17 (1.9)	1 (0.2)	5 (3.0) ▲	11 (7.7) ▲	37.561	< 0.001	
VitB6	246 (26.8)	126 (20.7)	69 (41.3) ▲	51 (35.7) ▲	35.114	< 0.001	
VitB7	20 (2.2)	12 (2.0)	5 (3.0)	3 (2.1)	0.645	0.724	
VitB9	4 (0.4)	1 (0.2)	0 (0.0)	3 (2.1)▲	10.869	0.004	
VitB12	48 (5.2)	26 (4.3)	10 (6.0)	12 (8.4)	4.194	0.123	
VitC	171 (18.6)	107 (17.6)	35 (21.0)	29 (20.3)	1.281	0.527	
Note: NFP, normal pregnancy; OSA, once spontaneous abortion; CSA, continuous spontaneous abortion. ▲ vs. NFP group, P < 0.05; ☺ vs. OSA group, P < 0.05.

Univariate risk analysis of each water-soluble vitamin to RSA

The RSA occurrence in overall pregnant women was 6.54% (60/918), with a prevalence of 12.57% (21/167) in the OSA group and 27.27% (39/143) in the CSA group. Fetal survival curve showed that the RSA occurrence was higher in the CSA group than in the OSA group (χ2 = 10.627, P = 0.001) (Fig. 1). The risk analysis of each water-soluble vitamin demonstrated that vitamin B5, B6, and B9 were the risk factors to RSA occurrence among both overall pregnant women (HR = 0.95, 0.87, and 0.87, all P < 0.001) and the CSA group (HR = 0.87, 0.71, and 0.85, all P < 0.01), while vitamin B6 and B9 were the risk factors to RSA occurrence among the OSA group (HR = 0.82 and 0.91, all P < 0.05) (Table 3). These results suggest that the miscarriage rate in the CSA group significantly increased, which may be involved in more influencing factors including vitamin B5 deficiency.Fig. 1 The fetal survival curve in overall pregnant women, OSA and CSA groups. Note OSA, once spontaneous abortions; CSA, continuous spontaneous abortions. The default duration for fetal survival in any surviving fetus was set at 40 gestational weeks. The corresponding cumulative miscarriage rate in each group is highlighted using a dot.

Table 3 The hazard ratio of each water-soluble vitamin to RSA occurrence.

Vitamin	Overall (n = 918)	OSA vs. NFP (n = 775)	CSA vs. NFP (n = 751)	
HR (95%CI)	Wald	P	HR (95%CI)	Wald	P	HR (95%CI)	Wald	P	
VitB1	0.94 (0.73,1.22)	0.229	0.632	1.12 (0.83, 1.51)	0.575	0.448	0.79 (0.53, 1.16)	1.452	0.786	
VitB2	1.00 (0.96, 1.04)	0.001	0.983	1.01 (0.95, 1.08)	0.233	0.629	0.99 (0.95, 1.04)	0.040	0.841	
VitB3	0.98 (0.95, 1.01)	1.926	0.165	0.96 (0.91, 1.02)	1.844	0.175	0.98 (0.95, 1.02)	0.565	0.452	
VitB5	0.95 (0.92, 0.97)	15.881	< 0.001	0.99 (0.96, 1.03)	0.028	0.867	0.87 (0.83, 091)	31.773	< 0.001	
VitB6	0.87 (0.74, 0.95)	11.595	< 0.001	0.82 (0.67, 0.98)	6.336	0.019	0.71 (0.62, 0.89)	7.517	0.009	
VitB7	1.18 (0.22, 6.39)	0.038	0.845	0.60 (0.02, 15.21)	0.098	0.597	1.37 (0.21, 9.18)	0.107	0.743	
VitB9	0.87 (0.84, 0.90)	58.130	< 0.001	0.91 (0.86, 0.96)	11.032	< 0.001	0.85 (0.81, 0.89)	45.946	< 0.001	
VitB12	1.00 (0.98, 1.02)	0.002	0.963	0.99 (0.95, 1.04)	0.038	0.844	1.00 (0.97, 1.03)	1.198	0.274	
VitC	0.99 (0.92, 1.06)	0.161	0.688	1.00 (0.89, 1.13)	0.001	0.999	0.97 (0.89, 1.06)	0.436	0.509	
NFP, normal pregnancy; OSA, once spontaneous abortion; CSA, continuous spontaneous abortion. Significance values are shown in bold.

Comprehensive risk assessment of water-soluble vitamin deficiency to RSA occurrence

Under the confounding effect of age and the interaction of each water-soluble vitamin (Table 4), vitamin B9 was an independent factor among overall pregnant women, OSA, and CSA groups (HR = 0.87, 0.89, and 0.88, all P < 0.001); B6 was an independent factor among OSA, and CSA groups (HR = 0.83 and 0.78, all P < 0.05); while B5 was an independent factor only among CSA group (HR = 0.93, P = 0.005). These results suggest that the risk of water-soluble vitamin deficiencies contributing to miscarriage varies among pregnant women with different SA history, indicating the need for tailored treatment approaches. Table 4 The hazard ratio of water-soluble vitamin to RSA occurrence under a confounding effect.

Vitamin	Overall (n = 918)	OSA vs. NFP (n = 775)	CSA vs. NFP (n = 751)	
HR (95%CI)	Wald	P	HR (95%CI)	Wald	P	HR (95%CI)	Wald	P	
Age	1.06 (0.99–1.12)	2.977	0.084	1.07 (0.96–1.19)	1.612	0.204	1.06 (0.98–1.15)	2.066	0.151	
VitB1	1.05 (0.91–1.22)	0.449	0.503	1.13 (0.88–1.44)	0.905	0.342	0.99 (0.82–1.19)	0.021	0.885	
VitB2	1.01 (0.98–1.05)	0.445	0.505	1.02 (0.96–1.08)	0.255	0.614	1.00 (0.95–1.05)	0.000	0.988	
VitB3	0.99 (0.96–1.01)	1.009	0.315	0.96 (0.91–1.02)	1.667	0.197	1.00 (0.97–1.04)	0.079	0.778	
VitB5	0.98 (0.95–1.01)	1.896	0.169	1.01 (0.98–1.05)	0.402	0.526	0.93 (0.88–0.98)	7.843	0.005	
VitB6	0.89 (0.71–1.10)	1.233	0.267	0.83 (0.67–0.99)	4.922	0.027	0.78 (0.66–0.94)	4.885	0.028	
VitB7	1.83 (0.37–9.15)	0.540	0.463	0.50 (0.01–9.64)	0.142	0.707	1.59 (0.27to 9.51)	0.257	0.612	
VitB9	0.87 (0.84–0.91)	46.430	< 0.001	0.89 (0.83–0.94)	14.076	< 0.001	0.88 (0.84–0.93)	22.985	< 0.001	
VitB12	1.00 (0.98–1.03)	0.053	0.818	1.00 (0.96–1.05)	0.009	0.926	1.00 (0.97–1.04)	0.004	0.952	
VitC	1.07 (1.00–1.15)	2.159	0.121	1.12 (0.99–1.26)	3.317	0.069	1.03 (0.95–1.12)	0.475	0.491	
NFP, normal pregnancy; OSA, once spontaneous abortion; CSA, continuous spontaneous abortion. Significance values are shown in bold.

Predictive power of water-soluble vitamins on RSA occurrence

According to the above analysis, we utilized the data of overall pregnant women to establish a Cox proportional-hazards model for predicting the RSA occurrence based upon vitamin B5, B6, and B9 levels: Prognostic index (PI) =  − 0.020 × B5 − 0.143 × B6 − 0.123 × B9 (χ2 = 71.327, P < 0.001; C-index = 0.777, 95% CI = 0.722 ~ 0.831) (See supplementary Table 1 for the details about the baseline cumulative hazard). The cumulative RSA risks at the average levels of vitamin B5 (29.92 ng/mL), B6 (3.45 ng/mL), and B9 (22.17 ng/mL) were observed to range from 0.1 ~ 3.5% (Fig. 2A). The performance of this model for predicting miscarriage in the OSA group was relatively low, with an AUC of 0.704, a sensitivity of 95.2%, and a specificity of 39.0% (Fig. 2B). In contrast, the model exhibited a relatively high performance for predicting miscarriage in the CSA group, with an AUC value of 0.825, a sensitivity of 92.3%, and a specificity of 60.6% (Fig. 2C). Overall, the model demonstrated quite high sensitivity in predicting RSA for both OSA and CSA groups, with no significant difference observed between the two groups (χ2 = 1.134, P = 0.287). However, the model was more suitable for predicting RSA occurrence in the CSA group, as it exhibited significantly higher AUC (z = 2.056, P = 0.040) and specificity (χ2 = 14.549, P < 0.001).Fig. 2 The Cox proportional-hazards model based on baseline levels of vitamin B5, B6, and B9 to predict miscarriage risk. Note (A) Survival curve at mean baseline level of vitamin B5, B6, and B9, which cumulative RSA risks was 3.5% at 23 gestational weeks; (B) ROC curve of OSA vs. NFP, with sensitivity of 95.2% and specificity of 39.0% when maximum prediction accuracy; (C) ROC curve of CSA vs. NFP, with sensitivity of 92.3% and specificity of 60.6% when maximum prediction accuracy. NFP, normal first pregnancy; OSA, once spontaneous abortions; CSA, continuous spontaneous abortions.

Correlation of water-soluble vitamins with RSA occurrence after supplementation therapy

After 4 weeks of vitamin supplementation, only in the CSA group, the pregnant women with miscarriage exhibited significantly lower levels in vitamin B5 (z = − 2.828, P = 0.005) and B9 (z = − 2.453, P = 0.014) compared to the pregnant women with non-miscarriage (Table 5). However, their B5, B6, and B9 levels were not associated with RSA occurrence (all P > 0.05). Table 5 Compensatory essential vitamin levels with RSA occurrence.

Vitamin	Median (P25, P75)	z	P	HR (95%CI)	Wald	P	
Non-miscarriage	Miscarriage	
OSA group	
VitB5	32.16 (24.78, 52.65)	32.17 (24.64, 58.88)	− 0.130	0.896	0.99 (0.97, 1.02)	0.408	0.523	
VitB6	5.33 (3.47, 7.21)	5.59 (2.97, 8.54)	− 0.191	0.849	1.08 (0.98, 1.20)	2.435	0.119	
VitB9	24.81 (20.99, 30.45)	25.46 (22.84, 29.66)	0.738	0.460	1.03 (0.97, 1.10)	0.951	0.330	
OSA group	
VitB5	28.27 (22.09, 48.08)	24.33 (18.23, 30.27)	− 2.828	0.005	0.97 (0.94, 1.01)	2.346	0.126	
VitB6	3.61 (2.90, 4.84)	3.27 (2.74, 3.88)	− 1.929	0.054	0.88 (0.67, 1.15)	0.891	0.345	
VitB9	24.66 (19.39, 29.04)	19.65 (14.53, 27.04)	− 2.453	0.014	0.98 (0.94, 1.02)	0.828	0.363	
OSA, once spontaneous abortion; CSA, continuous spontaneous abortion. Significance values are shown in bold.

Discussion

This study focused on pregnant women who had experienced one or more SA. Using UHPLC-MS/MS, their baseline water-soluble vitamin levels were measured at the time of clinically recognized pregnancy. Our study revealed significant heterogeneity in baseline levels of almost all water-soluble vitamins, and evident deficiencies in multiple B vitamins and vitamin C among pregnant women, as well as risks of baseline levels in vitamin B5, B6 and B9 to the RSA occurrence. The most notable highlight was the successful development of an RSA prediction model based on these risky vitamins, which demonstrated a sensitivity exceeding 90% for predicting RSA in either OSA or CSA groups. However, this model displayed a lower specificity of 39.0% in the OSA group compared to a higher specificity of 60.6% in the CSA group. The prediction of miscarriage using water-soluble vitamins has posed a significant puzzle in this research field. Our achievement on the initial attempt may be attributed to the use of a UHPLC-MS/MS technique for precise quantification, the detection of baseline levels in water-soluble vitamins, the distinctiveness of the pregnant women group being focus on, and subgrouping the RSA pregnant women by number of miscarriages. In addition, the value of compensatory essential vitamin levels in predicting RSA occurrence is limited.

Numerous studies have reported the relationship between vitamin B9 and adverse pregnancy28,29. Vitamin B9, also known as folic acid, plays a crucial role in various cellular metabolic activities. Its deficiency can result in the occurrence of macrocytic anemia, mucositis, infertility, muscular weakness, cardiovascular disease, neurological disorders, cancer, other related conditions30. Over the past two decades, numerous studies have consistently demonstrated that vitamin B9 supplementation significantly enhances pregnancy outcomes16–18, and continuous use of vitamin B9 during pregnancy also improved perinatal depression31. Despite some conflicting evidence32, vitamin B9 supplementation is now widely recommended and universally practiced among reproductive women for preconception planning or during pregnancy in many countries33–35. In this study, only 0.4% of the pregnant women exhibited vitamin B9 levels below the lower limit of reference (4.0 ng/mL), thereby confirming that a majority of pregnant women had initiated vitamin B9 supplementation prior to conception. However, owing to the heightened demand during pregnancy36, there can still be a relative deficiency in vitamin B9. Referring to the recommended optimal serum folate threshold for neural tube defect prevention37,38 and the BOND project and WHO recommendation24–26, we set a threshold of 10 ng/mL, and identified significantly relative deficiencies of vitamin B9 during pregnancy, especially in the CSA group of 17.5% (25/143). Taken together, adequate pre-pregnancy supplementation of vitamin B9 may serve as a potent intervention in facilitating the growth and development of embryos and fetuses. It is noteworthy that recent evidence suggested excessive vitamin B9 as potential risks of adverse genomic and epigenomic alterations39, thereby necessitating a consideration for continuous monitoring of maternal vitamin B9 levels during pregnancy.

Our study also revealed a significant insufficiency of vitamin B6 in pregnant women with different SA histories (ranging from 20.7 to 41.3%), and further it emerged as a crucial predictor for RSA. Vitamin B6, also known as pyridoxine, serves as a crucial cofactor regulating approximately 150 metabolic reaction processes of protein, glucose, lipids, neurotransmitters, and DNA40. Numerous studies have demonstrated the involvement of vitamin B6, B9, and B12 in methionine metabolism, resulting in the accumulation of homocysteine41,42. Elevated levels of homocysteine can lead to the occurrence of neural tube defect43, preeclampsia44,45, intrauterine growth retardation or fetal death44,45, and gestational diabetes mellitus46,47, as well as other factors contributing to miscarriages. Some scholar has suggested the preventive administration of vitamin B6 to reduce these complications48. But its role in preventing miscarriage remains controversial48,49. Our study findings indicated that vitamin B6 deficiency increase the risk for RSA in pregnant women with a history of at least one miscarriage, highlighting the importance of considering supplementation prior to planning subsequent pregnancies.

As for vitamin B5, also known as pantothenic acid, is essential for the synthesis of Coenzyme A, which plays a crucial role in various physiological processes such as energy metabolism from fats, carbohydrates, and proteins50. In general, the abundant dietary sources and infrequent deficiencies of vitamin B5 contribute to limited research on this nutrient, as well as a dearth of significant findings. It has been reported that the levels of vitamin B5 in the blood significantly decrease during pregnancy51, which is associated with a higher risk of low birth weight in offspring52. Although clinical evidence is extremely lacking, vitamin B5 deficiency has been observed to be associated with miscarriage in an old animal study53. Interestingly, our study revealed that vitamin B5 deficiency was extremely rare among overall pregnant women, but occurred to a certain extent in the CSA group (7.7%); so, it still posed a risk for the CSA group. Regarding the potential link between vitamin B5 deficiency and miscarriage in the CSA group, it could be attributed to factors such as fetal underdevelopment53, as well as maternal depression54,55, anxiety55, and severe malnutrition53. These conditions are commonly observed among reproductive women with continuous SA history1. Moreover, it has been indicated that pregnant women should maintain only the average level of vitamin B5, as an increased intake of more than 5.6 mg/day may lead to genome instability and subsequent teratogenicity54.

To the best of our knowledge, we successfully addressed the puzzle of utilizing B vitamins to predict RSA. Our prediction models, constructed using B5, B6, and B9, exhibited exceptional sensitivity and were deemed suitable for primary screening of RSA. Consequently, regular prenatal screening for B vitamins, particularly B5, B6, and B9, is imperative for ensuring pregnancy safety. However, the utilization of mass spectrometry for vitamin detection is not prevalent. Therefore, it is also imperative to explore alternative detection techniques that offer greater convenience compared to mass spectrometry.

The limitation of our study: (1) Our study retained only the NFP group as a control without women who experienced a miscarriage in their first pregnancy, which may have introduced bias into the conclusions. (2) The failure to investigate the exact etiology, which was unknown in most miscarriages, might introduce potential bias in the model applicability. (3) Although the proposed model demonstrated excellent sensitivity in predicting RSA, its unsatisfactory specificity resulted in significant false positives that cannot be disregarded, especially in OSA group.

Conclusion

Taken together, we developed for the first a Cox proportional-hazards model based on baseline levels of vitamin B5, B6, and B9 to predict miscarriage risk in pregnant women with a history of SA. On one hand, the model may facilitate the identification of pregnant women at high risk for RSA, thereby timely implementing individualized treatment and intervention. On the other hand, for the pregnant women at low risk for RSA identified by the model, the excessive vitamin supplementation should be avoided to prevent vitamin toxicity-related adverse events.

Supplementary Information

Supplementary Figure 1.

Supplementary Figure 2.

Supplementary Legends.

Supplementary Table 1.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-71986-z.

Author contributions

Y.Y., J.T. and Y.M. proposed the conceptualization and obtained funding support. B.W. and Z.L. wrote the main manuscript text. B.P. and Y.M. followed up the patients. B.W. and W.J. performed the investigation and conducted the data curation. Y.Y. graphed all figures and supplementary figures. All authors reviewed the manuscript. B.W., Z.L., and B.P. contributed equally to this work.

Funding

This study was financially supported by the Basic Application Project of Science & Technology Department of Sichuan Province [2019YJ0701, and 2019YFS0416], and the Incubation Project of Mianyang Central Hospital, School of Medicine, University of Electronic Science and Technology of China [2022FH005]. The authors have no other relevant affiliations or financial involvement with any organization apart from those disclosed.

Data availability

The datasets used and analyzed are available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.

Ethical approval

This study was conducted in accordance with the Declaration of Helsinki (as revised in 2013), and approved by the Medical Ethics Committee of Mianyang Central Hospital, School of Medicine, University of Electronic Science and Technology of China (No. S2018085, and S2018093). All participants signed informed consents.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Bitao Wu, Zhenghao Li and Bi Peng.
==== Refs
References

1. Griebel CP Halvorsen J Golemon TB Day AA Management of spontaneous abortion Am. Fam. Physician 2005 72 7 1243 1250 16225027
Griebel, C. P., Halvorsen, J., Golemon, T. B. & Day, A. A. Management of spontaneous abortion. Am. Fam. Physician 72(7), 1243–1250 (2005).16225027
2. McBride WZ Spontaneous abortion Am. Fam. Physician 1991 43 1 175 182 1986486
McBride, W. Z. Spontaneous abortion. Am. Fam. Physician 43(1), 175–182 (1991).1986486
3. Dimitriadis E Menkhorst E Saito S Kutteh WH Brosens JJ Recurrent pregnancy loss Nat. Rev. Dis. Primers. 2020 6 1 98 10.1038/s41572-020-00228-z 33303732
Dimitriadis, E., Menkhorst, E., Saito, S., Kutteh, W. H. & Brosens, J. J. Recurrent pregnancy loss. Nat. Rev. Dis. Primers. 6(1), 98. 10.1038/s41572-020-00228-z (2020).33303732 10.1038/s41572-020-00228-z
4. Green DM O'Donoghue K A review of reproductive outcomes of women with two consecutive miscarriages and no living child J. Obstet. Gynaecol. 2019 39 6 816 821 10.1080/01443615.2019.1576600 31006300
Green, D. M. & O’Donoghue, K. A review of reproductive outcomes of women with two consecutive miscarriages and no living child. J. Obstet. Gynaecol. 39(6), 816–821. 10.1080/01443615.2019.1576600 (2019).31006300 10.1080/01443615.2019.1576600
5. Potdar N Iyasere C Early pregnancy complications including recurrent pregnancy loss and obesity Best Pract. Res. Clin. Obstet. Gynaecol. 2023 90 102372 10.1016/j.bpobgyn.2023.102372 37451193
Potdar, N. & Iyasere, C. Early pregnancy complications including recurrent pregnancy loss and obesity. Best Pract. Res. Clin. Obstet. Gynaecol. 90, 102372. 10.1016/j.bpobgyn.2023.102372 (2023).37451193 10.1016/j.bpobgyn.2023.102372
6. Trostle ME Penfield CA Roman AS Adjustment of the spontaneous abortion rate following COVID-19 vaccination Am. J. Obstet. Gynecol. MFM. 2022 4 1 100511 10.1016/j.ajogmf.2021.100511 34656732
Trostle, M. E., Penfield, C. A. & Roman, A. S. Adjustment of the spontaneous abortion rate following COVID-19 vaccination. Am. J. Obstet. Gynecol. MFM. 4(1), 100511. 10.1016/j.ajogmf.2021.100511 (2022).34656732 10.1016/j.ajogmf.2021.100511
7. Jacoby VL Murtha A Afshar Y Gaw SL Asiodu I Tolosa J Risk of pregnancy loss before 20 weeks' gestation in study participants with COVID-19 Am. J. Obstet. Gynecol. 2021 225 4 456 457 10.1016/j.ajog.2021.06.080 34175267
Jacoby, V. L. et al. Risk of pregnancy loss before 20 weeks’ gestation in study participants with COVID-19. Am. J. Obstet. Gynecol. 225(4), 456–457. 10.1016/j.ajog.2021.06.080 (2021).34175267 10.1016/j.ajog.2021.06.080
8. Hanna M Jaqua E Nguyen V Clay J B Vitamins: Functions and uses in medicine Perm. J. 2022 26 2 89 97 10.7812/tpp/21.204 35933667
Hanna, M., Jaqua, E., Nguyen, V. & Clay, J. B Vitamins: Functions and uses in medicine. Perm. J. 26(2), 89–97. 10.7812/tpp/21.204 (2022).35933667 10.7812/tpp/21.204
9. Chawla J Kvarnberg D Hydrosoluble vitamins Handb. Clin. Neurol. 2014 120 891 914 10.1016/b978-0-7020-4087-0.00059-0 24365359
Chawla, J. & Kvarnberg, D. Hydrosoluble vitamins. Handb. Clin. Neurol. 120, 891–914. 10.1016/b978-0-7020-4087-0.00059-0 (2014).24365359 10.1016/b978-0-7020-4087-0.00059-0
10. Fejzo MS Trovik J Grooten IJ Sridharan K Roseboom TJ Vikanes Å Nausea and vomiting of pregnancy and hyperemesis gravidarum Nat. Rev. Dis. Primers. 2019 5 1 62 10.1038/s41572-019-0110-3 31515515
Fejzo, M. S. et al. Nausea and vomiting of pregnancy and hyperemesis gravidarum. Nat. Rev. Dis. Primers. 5(1), 62. 10.1038/s41572-019-0110-3 (2019).31515515 10.1038/s41572-019-0110-3
11. Said HM Nexo E Gastrointestinal handling of water-soluble vitamins Compr. Physiol. 2018 8 4 1291 1311 10.1002/cphy.c170054 30215865
Said, H. M. & Nexo, E. Gastrointestinal handling of water-soluble vitamins. Compr. Physiol. 8(4), 1291–1311. 10.1002/cphy.c170054 (2018).30215865 10.1002/cphy.c170054
12. Bascom JT Stephens SB Lupo PJ Canfield MA Kirby RS Nestoridi E Scientific impact of the national birth defects prevention network multistate collaborative publications Birth Defects Res. 2024 116 1 e2225 10.1002/bdr2.2225 37492989
Bascom, J. T. et al. Scientific impact of the national birth defects prevention network multistate collaborative publications. Birth Defects Res. 116(1), e2225. 10.1002/bdr2.2225 (2024).37492989 10.1002/bdr2.2225
13. Fenech MF Bull CF Van Klinken BJ Protective effects of micronutrient supplements, phytochemicals and phytochemical-rich beverages and foods against DNA damage in humans: A systematic review of randomized controlled trials and prospective studies Adv. Nutrit. (Bethesda, Md). 2023 14 6 1337 1358 10.1016/j.advnut.2023.08.004
Fenech, M. F., Bull, C. F. & Van Klinken, B. J. Protective effects of micronutrient supplements, phytochemicals and phytochemical-rich beverages and foods against DNA damage in humans: A systematic review of randomized controlled trials and prospective studies. Adv. Nutrit. (Bethesda, Md). 14(6), 1337–1358. 10.1016/j.advnut.2023.08.004 (2023).10.1016/j.advnut.2023.08.004
14. Zhu J Chen C Lu L Shikany JM D'Alton ME Kahe K Folate, Vitamin B6, and Vitamin B12 status in association with metabolic syndrome incidence JAMA Netw. open. 2023 6 1 e2250621 10.1001/jamanetworkopen.2022.50621 36630134
Zhu, J. et al. Folate, Vitamin B6, and Vitamin B12 status in association with metabolic syndrome incidence. JAMA Netw. open. 6(1), e2250621. 10.1001/jamanetworkopen.2022.50621 (2023).36630134 10.1001/jamanetworkopen.2022.50621
15. Sechi G Sechi E Fois C Kumar N Advances in clinical determinants and neurological manifestations of B vitamin deficiency in adults Nutrit. Rev. 2016 74 5 281 300 10.1093/nutrit/nuv107 27034475
Sechi, G., Sechi, E., Fois, C. & Kumar, N. Advances in clinical determinants and neurological manifestations of B vitamin deficiency in adults. Nutrit. Rev. 74(5), 281–300. 10.1093/nutrit/nuv107 (2016).27034475 10.1093/nutrit/nuv107
16. Oh C Keats EC Bhutta ZA Vitamin and mineral supplementation during pregnancy on maternal, birth, child health and development outcomes in low- and middle-income countries: A systematic review and meta-analysis Nutrients 2020 10.3390/nu12020491 33322005
Oh, C., Keats, E. C. & Bhutta, Z. A. Vitamin and mineral supplementation during pregnancy on maternal, birth, child health and development outcomes in low- and middle-income countries: A systematic review and meta-analysis. Nutrients10.3390/nu12020491 (2020).33322005 10.3390/nu12020491
17. Mousa A Naqash A Lim S Macronutrient and micronutrient intake during pregnancy: An overview of recent evidence Nutrients 2019 10.3390/nu11020443 31783602
Mousa, A., Naqash, A. & Lim, S. Macronutrient and micronutrient intake during pregnancy: An overview of recent evidence. Nutrients10.3390/nu11020443 (2019).31783602 10.3390/nu11020443
18. Santander Ballestín S Giménez Campos MI Ballestín Ballestín J Luesma Bartolomé MJ Is supplementation with micronutrients still necessary during pregnancy? A review Nutrients 2021 10.3390/nu13093134 34579011
Santander Ballestín, S., Giménez Campos, M. I., Ballestín Ballestín, J. & Luesma Bartolomé, M. J. Is supplementation with micronutrients still necessary during pregnancy? A review. Nutrients10.3390/nu13093134 (2021).34579011 10.3390/nu13093134
19. Bhowmik B Siddiquee T Mdala I Quamrun Nesa L Jahan Shelly S Hassan Z Vitamin D3 and B12 supplementation in pregnancy Diabetes Res. Clin. Practice. 2021 174 108728 10.1016/j.diabres.2021.108728
Bhowmik, B. et al. Vitamin D3 and B12 supplementation in pregnancy. Diabetes Res. Clin. Practice. 174, 108728. 10.1016/j.diabres.2021.108728 (2021).10.1016/j.diabres.2021.108728
20. Gaskins AJ Chavarro JE Diet and fertility: A review Am. J. Obstet. Gynecol. 2018 218 4 379 389 10.1016/j.ajog.2017.08.010 28844822
Gaskins, A. J. & Chavarro, J. E. Diet and fertility: A review. Am. J. Obstet. Gynecol. 218(4), 379–389. 10.1016/j.ajog.2017.08.010 (2018).28844822 10.1016/j.ajog.2017.08.010
21. Avram C Bucur OM Zazgyva A Avram L Ruta F Vitamin supplementation in pre-pregnancy and pregnancy among women-effects and influencing factors in romania Int. J. Environ. Res. Public Health. 2022 10.3390/ijerph19148503 35886354
Avram, C., Bucur, O. M., Zazgyva, A., Avram, L. & Ruta, F. Vitamin supplementation in pre-pregnancy and pregnancy among women-effects and influencing factors in romania. Int. J. Environ. Res. Public Health.10.3390/ijerph19148503 (2022).35886354 10.3390/ijerph19148503
22. Ali MA Hafez HA Kamel MA Ghamry HI Shukry M Farag MA Dietary Vitamin B complex: Orchestration in human nutrition throughout life with sex differences Nutrients 2022 10.3390/nu14193940 36615817
Ali, M. A. et al. Dietary Vitamin B complex: Orchestration in human nutrition throughout life with sex differences. Nutrients10.3390/nu14193940 (2022).36615817 10.3390/nu14193940
23. Lamers Y Indicators and methods for folate, vitamin B-12, and vitamin B-6 status assessment in humans Curr. Opin. Clin. Nutrit. Metab. Care. 2011 14 5 445 454 10.1097/MCO.0b013e328349f9a7 21832901
Lamers, Y. Indicators and methods for folate, vitamin B-12, and vitamin B-6 status assessment in humans. Curr. Opin. Clin. Nutrit. Metab. Care. 14(5), 445–454. 10.1097/MCO.0b013e328349f9a7 (2011).21832901 10.1097/MCO.0b013e328349f9a7
24. Raiten DJ Namasté S Brabin B Combs G Jr L'Abbe MR Wasantwisut E Executive summary–biomarkers of nutrition for development: Building a consensus Am. J. Clin. Nutrit. 2011 94 2 633s s650 10.3945/ajcn.110.008227 21733880
Raiten, D. J. et al. Executive summary–biomarkers of nutrition for development: Building a consensus. Am. J. Clin. Nutrit. 94(2), 633s-s650. 10.3945/ajcn.110.008227 (2011).21733880 10.3945/ajcn.110.008227
25. WHO Serum and red blood cell folate concentrations for assessing folate status in populations 2015 World Health Organization
WHO. Serum and red blood cell folate concentrations for assessing folate status in populations (World Health Organization, 2015).
26. Allen LH Miller JW de Groot L Rosenberg IH Smith AD Refsum H Biomarkers of nutrition for development (BOND): Vitamin B-12 review J. Nutrit. 2018 148 1995s 2027s 10.1093/jn/nxy201 30500928
Allen, L. H. et al. Biomarkers of nutrition for development (BOND): Vitamin B-12 review. J. Nutrit. 148, 1995s–2027s. 10.1093/jn/nxy201 (2018).30500928 10.1093/jn/nxy201
27. Wilson RD O'Connor DL Guideline No. 427: Folic acid and multivitamin supplementation for prevention of folic acid-sensitive congenital anomalies J. Obstet. Gynaecol. Can. 2022 44 6 707 19.e1 10.1016/j.jogc.2022.04.004 35691683
Wilson, R. D. & O’Connor, D. L. Guideline No. 427: Folic acid and multivitamin supplementation for prevention of folic acid-sensitive congenital anomalies. J. Obstet. Gynaecol. Can. 44(6), 707–19.e1. 10.1016/j.jogc.2022.04.004 (2022).35691683 10.1016/j.jogc.2022.04.004
28. George L Mills JL Johansson AL Nordmark A Olander B Granath F Plasma folate levels and risk of spontaneous abortion Jama. 2002 288 15 1867 1873 10.1001/jama.288.15.1867 12377085
George, L. et al. Plasma folate levels and risk of spontaneous abortion. Jama. 288(15), 1867–1873. 10.1001/jama.288.15.1867 (2002).12377085 10.1001/jama.288.15.1867
29. Qin XY Ha SY Chen L Zhang T Li MQ Recent advances in folates and autoantibodies against folate receptors in early pregnancy and miscarriage Nutrients 2023 10.3390/nu15234882 38140344
Qin, X. Y., Ha, S. Y., Chen, L., Zhang, T. & Li, M. Q. Recent advances in folates and autoantibodies against folate receptors in early pregnancy and miscarriage. Nutrients10.3390/nu15234882 (2023).38140344 10.3390/nu15234882
30. Shulpekova Y Nechaev V Kardasheva S Sedova A Kurbatova A Bueverova E The concept of folic acid in health and disease Molecules (Basel, Switzerland) 2021 10.3390/molecules26123731 34207319
Shulpekova, Y. et al. The concept of folic acid in health and disease. Molecules (Basel, Switzerland)10.3390/molecules26123731 (2021).34207319 10.3390/molecules26123731
31. Jin X Cheng Z Yu X Tao Q Huang R Wang S Continuous supplementation of folic acid in pregnancy and the risk of perinatal depression-A meta-analysis J. Affect. Disorders 2022 302 258 272 10.1016/j.jad.2022.01.080 35066009
Jin, X. et al. Continuous supplementation of folic acid in pregnancy and the risk of perinatal depression-A meta-analysis. J. Affect. Disorders 302, 258–272. 10.1016/j.jad.2022.01.080 (2022).35066009 10.1016/j.jad.2022.01.080
32. Lassi ZS Salam RA Haider BA Bhutta ZA Folic acid supplementation during pregnancy for maternal health and pregnancy outcomes Cochrane Database Syst. Rev. 2013 2013 3 CD006896 10.1002/14651858.CD006896.pub2 23543547
Lassi, Z. S., Salam, R. A., Haider, B. A. & Bhutta, Z. A. Folic acid supplementation during pregnancy for maternal health and pregnancy outcomes. Cochrane Database Syst. Rev. 2013(3), CD006896. 10.1002/14651858.CD006896.pub2 (2013).23543547 10.1002/14651858.CD006896.pub2
33. Njiru H Njogu E Gitahi MW Kabiru E Effectiveness of public health education on the uptake of iron and folic acid supplements among pregnant women: A stepped wedge cluster randomised trial BMJ Open 2022 12 9 e063615 10.1136/bmjopen-2022-063615 36691203
Njiru, H., Njogu, E., Gitahi, M. W. & Kabiru, E. Effectiveness of public health education on the uptake of iron and folic acid supplements among pregnant women: A stepped wedge cluster randomised trial. BMJ Open 12(9), e063615. 10.1136/bmjopen-2022-063615 (2022).36691203 10.1136/bmjopen-2022-063615
34. Caniglia EC Zash R Swanson SA Smith E Sudfeld C Finkelstein JL Iron, folic acid, and multiple micronutrient supplementation strategies during pregnancy and adverse birth outcomes in Botswana Lancet Global Health 2022 10 6 e850 e861 10.1016/s2214-109x(22)00126-7 35561720
Caniglia, E. C. et al. Iron, folic acid, and multiple micronutrient supplementation strategies during pregnancy and adverse birth outcomes in Botswana. Lancet Global Health 10(6), e850–e861. 10.1016/s2214-109x(22)00126-7 (2022).35561720 10.1016/s2214-109x(22)00126-7
35. Zhang S Yang M Hao X Zhang F Zhou J Tao F Peri-conceptional folic acid supplementation and children's physical development: A birth cohort study Nutrients 2023 10.3390/nu15061423 38201975
Zhang, S. et al. Peri-conceptional folic acid supplementation and children’s physical development: A birth cohort study. Nutrients10.3390/nu15061423 (2023).38201975 10.3390/nu15061423
36. Greenberg JA Bell SJ Guan Y Yu YH Folic Acid supplementation and pregnancy: More than just neural tube defect prevention Rev. Obstet. Gynecol. 2011 4 2 52 59 22102928
Greenberg, J. A., Bell, S. J., Guan, Y. & Yu, Y. H. Folic Acid supplementation and pregnancy: More than just neural tube defect prevention. Rev. Obstet. Gynecol. 4(2), 52–59 (2011).22102928
37. Rahimi S Martel J Karahan G Angle C Behan NA Chan D Moderate maternal folic acid supplementation ameliorates adverse embryonic and epigenetic outcomes associated with assisted reproduction in a mouse model Human Reproduction (Oxford, England) 2019 34 5 851 862 10.1093/humrep/dez036 30989206
Rahimi, S. et al. Moderate maternal folic acid supplementation ameliorates adverse embryonic and epigenetic outcomes associated with assisted reproduction in a mouse model. Human Reproduction (Oxford, England) 34(5), 851–862. 10.1093/humrep/dez036 (2019).30989206 10.1093/humrep/dez036
38. Fothergill A Crider KS Rose CE Bose B Guetterman HM Johnson CB Estimating the serum folate concentration that corresponds to the red blood cell folate concentration threshold associated with optimal neural tube defects prevention: A population-based biomarker survey in Southern India Am. J. Clin. Nutrit. 2023 117 5 985 997 10.1016/j.ajcnut.2023.01.016 37137617
Fothergill, A. et al. Estimating the serum folate concentration that corresponds to the red blood cell folate concentration threshold associated with optimal neural tube defects prevention: A population-based biomarker survey in Southern India. Am. J. Clin. Nutrit. 117(5), 985–997. 10.1016/j.ajcnut.2023.01.016 (2023).37137617 10.1016/j.ajcnut.2023.01.016
39. Movendane Y Sipalo MG Chan LCZ Advances in folic acid biosensors and their significance in maternal, perinatal, and paediatric preventive medicine Biosensors 2023 10.3390/bios13100912 37887105
Movendane, Y., Sipalo, M. G. & Chan, L. C. Z. Advances in folic acid biosensors and their significance in maternal, perinatal, and paediatric preventive medicine. Biosensors10.3390/bios13100912 (2023).37887105 10.3390/bios13100912
40. Mascolo E Vernì F Vitamin B6 and diabetes: Relationship and molecular mechanisms Int. J. Molecular Sci. 2020 10.3390/ijms21103669
Mascolo, E. & Vernì, F. Vitamin B6 and diabetes: Relationship and molecular mechanisms. Int. J. Molecular Sci.10.3390/ijms21103669 (2020).10.3390/ijms21103669
41. Kataria N Yadav P Kumar R Kumar N Singh M Kant R Effect of vitamin B6, B9, and B12 supplementation on homocysteine level and cardiovascular outcomes in stroke patients: A meta-analysis of randomized controlled trials Cureus 2021 13 5 e14958 10.7759/cureus.14958 34123655
Kataria, N. et al. Effect of vitamin B6, B9, and B12 supplementation on homocysteine level and cardiovascular outcomes in stroke patients: A meta-analysis of randomized controlled trials. Cureus 13(5), e14958. 10.7759/cureus.14958 (2021).34123655 10.7759/cureus.14958
42. Liampas I Siokas V Mentis AA Aloizou AM Dastamani M Tsouris Z Serum homocysteine, pyridoxine, folate, and vitamin B12 levels in migraine: Systematic review and meta-analysis Headache 2020 60 8 1508 1534 10.1111/head.13892 32615014
Liampas, I. et al. Serum homocysteine, pyridoxine, folate, and vitamin B12 levels in migraine: Systematic review and meta-analysis. Headache 60(8), 1508–1534. 10.1111/head.13892 (2020).32615014 10.1111/head.13892
43. D'Souza SW Glazier JD Homocysteine metabolism in pregnancy and developmental impacts Front. Cell Dev. Biol. 2022 10 802285 10.3389/fcell.2022.802285 35846363
D’Souza, S. W. & Glazier, J. D. Homocysteine metabolism in pregnancy and developmental impacts. Front. Cell Dev. Biol. 10, 802285. 10.3389/fcell.2022.802285 (2022).35846363 10.3389/fcell.2022.802285
44. Ogawa S Ota K Takahashi T Yoshida H Impact of homocysteine as a preconceptional screening factor for in vitro fertilization and prevention of miscarriage with folic acid supplementation following frozen-thawed embryo transfer: A hospital-based retrospective cohort study Nutrients 2023 10.3390/nu15173730 37686762
Ogawa, S., Ota, K., Takahashi, T. & Yoshida, H. Impact of homocysteine as a preconceptional screening factor for in vitro fertilization and prevention of miscarriage with folic acid supplementation following frozen-thawed embryo transfer: A hospital-based retrospective cohort study. Nutrients10.3390/nu15173730 (2023).37686762 10.3390/nu15173730
45. Chaudhry SH Taljaard M MacFarlane AJ Gaudet LM Smith GN Rodger M The role of maternal homocysteine concentration in placenta-mediated complications: Findings from the Ottawa and Kingston birth cohort BMC Pregnancy Childbirth. 2019 19 1 75 10.1186/s12884-019-2219-5 30782144
Chaudhry, S. H. et al. The role of maternal homocysteine concentration in placenta-mediated complications: Findings from the Ottawa and Kingston birth cohort. BMC Pregnancy Childbirth. 19(1), 75. 10.1186/s12884-019-2219-5 (2019).30782144 10.1186/s12884-019-2219-5
46. Malaza N Masete M Adam S Dias S Nyawo T Pheiffer C A systematic review to compare adverse pregnancy outcomes in women with pregestational diabetes and gestational diabetes Int. J. Environ. Res. Public Health 2022 10.3390/ijerph191710846 36078559
Malaza, N. et al. A systematic review to compare adverse pregnancy outcomes in women with pregestational diabetes and gestational diabetes. Int. J. Environ. Res. Public Health10.3390/ijerph191710846 (2022).36078559 10.3390/ijerph191710846
47. Liu YH Lu LP Yi MH Shen CY Lu GQ Jia J Study on the correlation between homocysteine-related dietary patterns and gestational diabetes mellitus: A reduced-rank regression analysis study BMC Pregnancy Childbirth 2022 22 1 306 10.1186/s12884-022-04656-5 35399065
Liu, Y. H. et al. Study on the correlation between homocysteine-related dietary patterns and gestational diabetes mellitus: A reduced-rank regression analysis study. BMC Pregnancy Childbirth 22(1), 306. 10.1186/s12884-022-04656-5 (2022).35399065 10.1186/s12884-022-04656-5
48. Ramakrishnan U Grant F Goldenberg T Zongrone A Martorell R Effect of women's nutrition before and during early pregnancy on maternal and infant outcomes: A systematic review Paediatric Perinatal Epidemiol. 2012 26 Suppl 1 285 301 10.1111/j.1365-3016.2012.01281.x
Ramakrishnan, U., Grant, F., Goldenberg, T., Zongrone, A. & Martorell, R. Effect of women’s nutrition before and during early pregnancy on maternal and infant outcomes: A systematic review. Paediatric Perinatal Epidemiol. 26(Suppl 1), 285–301. 10.1111/j.1365-3016.2012.01281.x (2012).10.1111/j.1365-3016.2012.01281.x
49. Balogun OO da Silva LK Ota E Takemoto Y Rumbold A Takegata M Vitamin supplementation for preventing miscarriage Cochrane Database Syst. Rev. 2016 2016 5 CD004073 10.1002/14651858.CD004073.pub4 27150280
Balogun, O. O. et al. Vitamin supplementation for preventing miscarriage. Cochrane Database Syst. Rev. 2016(5), CD004073. 10.1002/14651858.CD004073.pub4 (2016).27150280 10.1002/14651858.CD004073.pub4
50. Miallot R Millet V Galland F Naquet P The vitamin B5/coenzyme a axis: A target for immunomodulation? Eur. J. Immunol. 2023 53 10 e2350435 10.1002/eji.202350435 37482959
Miallot, R., Millet, V., Galland, F. & Naquet, P. The vitamin B5/coenzyme a axis: A target for immunomodulation?. Eur. J. Immunol. 53(10), e2350435. 10.1002/eji.202350435 (2023).37482959 10.1002/eji.202350435
51. Adams JB Kirby JK Sorensen JC Pollard EL Audhya T Evidence based recommendations for an optimal prenatal supplement for women in the US: Vitamins and related nutrients Maternal Health Neonatol. Perinatol. 2022 8 1 4 10.1186/s40748-022-00139-9
Adams, J. B., Kirby, J. K., Sorensen, J. C., Pollard, E. L. & Audhya, T. Evidence based recommendations for an optimal prenatal supplement for women in the US: Vitamins and related nutrients. Maternal Health Neonatol. Perinatol. 8(1), 4. 10.1186/s40748-022-00139-9 (2022).10.1186/s40748-022-00139-9
52. Watson PE McDonald BW The association of maternal diet and dietary supplement intake in pregnant New Zealand women with infant birthweight Eur. J. Clin. Nutrit. 2010 64 2 184 193 10.1038/ejcn.2009.134 19920847
Watson, P. E. & McDonald, B. W. The association of maternal diet and dietary supplement intake in pregnant New Zealand women with infant birthweight. Eur. J. Clin. Nutrit. 64(2), 184–193. 10.1038/ejcn.2009.134 (2010).19920847 10.1038/ejcn.2009.134
53. Pregnancy and pantothenic acid deficiency in the guinea pig. Nutrition reviews. 1966;24(6):169–70. 10.1111/j.1753-4887.1966.tb08408.x
54. Fenech M Baghurst P Luderer W Turner J Record S Ceppi M Low intake of calcium, folate, nicotinic acid, vitamin E, retinol, beta-carotene and high intake of pantothenic acid, biotin and riboflavin are significantly associated with increased genome instability–results from a dietary intake and micronucleus index survey in South Australia Carcinogenesis 2005 26 5 991 999 10.1093/carcin/bgi042 15705599
Fenech, M. et al. Low intake of calcium, folate, nicotinic acid, vitamin E, retinol, beta-carotene and high intake of pantothenic acid, biotin and riboflavin are significantly associated with increased genome instability–results from a dietary intake and micronucleus index survey in South Australia. Carcinogenesis 26(5), 991–999. 10.1093/carcin/bgi042 (2005).15705599 10.1093/carcin/bgi042
55. He, Y. et al. Common mental disorders and risk of spontaneous abortion or recurrent spontaneous abortion: A two-sample Mendelian randomization study. J. Affect. Disord. 354, 258–266. 10.1016/j.jad.2024.03.026 (2024).
