Maternal obesity during pregnancy leads to derangements in one-carbon metabolism and the gut microbiota: implications for fetal development and offspring wellbeing

A healthy diet before and during pregnancy is beneﬁcial in acquiring essential B vitamins involved in 1-carbon metabolism, and in maintaining a healthy gut microbiota. Each play important roles in fetal development, immune-system remodeling, and pregnancy-nutrient acquisition. Evidence shows that there is a reciprocal interaction between the one-carbon metabolism and the gut microbiota given that dietary intake of B vitamins has been shown to inﬂuence the composition of the gut microbiota, and certain gut bacteria also synthesize B vitamins. This reciprocal interaction contributes to the individual’s overall availability of B vitamins and, therefore, should be maintained in a healthy state during pregnancy. There is an emerging consensus that obese pregnant women often have derangements in 1-carbon metabolism and gut dysbiosis owing to high intake of nutritiously poor foods and a chronic systemic inﬂammatory state. For example, low folate and vitamin B 12 in obese women coincide with the decreased presence of B vitamin-producing bacteria and increased presence of inﬂammatory-associated bacteria from approximately mid-pregnancy. These alterations are risk factors for adverse pregnancy outcomes, impaired fetal development, and disruption of fetal growth and microbiota formation, which may lead to potential long-term offspring metabolic and neurologic disorders. Therefore, preconceptional and pregnant obese women may beneﬁt from dietary and lifestyle counseling to improve their dietary nutrient intake, and from monitoring their B vitamin levels and gut microbiome by blood tests and microbiota stool samples. In addition, there is evidence that some probiotic bacteria have folate biosynthetic capacity and could be used to treat gut dysbiosis. Thus, their use as an intervention strategy for obese women holds potential and should be further investigated. many knowledge gaps concerning the relationship between one-carbon metabolism and the gut microbiota, and future research should focus on intervention strategies to counteract B vitamin deﬁciencies and gut dysbiosis in obese pregnant women, commencing with the use of probiotic and prebiotic supplements.


Introduction
The World Health Organization recommends pregnant women to adhere to a healthy diet, exercise regularly, and take 0.4-mg folic acid supplement per day to ensure a healthy pregnancy and improve pregnancy outcomes. 1 These guidelines are strongly recommended because, during pregnancy, there is a high demand for essential vitamins required to support basic cellular processes involved in developmental programming of fetal and maternal tissues. 2 These include B vitamins involved in one-carbon metabolism (eg, vitamin B 9/11 [folate] and vitamin B 12 [cobalamin]). 3 Dietary intake of B vitamins is known to influence and maintain a healthy maternal gut microbiota during pregnancy, which plays a major role in nutrient acquisition and absorption, 4 immune remodeling, 5,6 protection against infections, 7 and shaping the fetal immune system and gut microbiota. 8 In addition, certain strains of gut bacteria have been shown to biosynthesize B vitamins, 9,10 which demonstrates that one-carbon metabolism and the gut microbiota are involved in pregnancy health. Derangements in one-carbon metabolism, as a result of vitamin deficiencies, are a risk factor for developmental disorders and adverse pregnancy outcomes, including neural-tube defects (NTDs), miscarriages, preterm birth, and low birthweight. 11e13 Similarly, perturbations of the maternal gut microbiota (termed dysbiosis) are also a risk factor for adverse pregnancy outcomes, such as miscarriage and intrauterine growth restriction, and recent evidence highlights potential long-term effects of maternal dysbiosis on offspring metabolic and neurologic health, resulting in obesity and mental health disorders. 14e16 The prevalence of obesity (body mass index [BMI] >30 kg/m 2 ) among pregnant women is increasing worldwide, 17 and obese pregnant women are more likely to adhere to an unhealthy diet, exercise less, and be less compliant with daily folic acid supplementation. 18 Therefore, it is common to find onecarbon metabolism deficiencies and gut dysbiosis in obese pregnant women, which expose the mother and fetus to the aforementioned health risks. 19 However, little is known about the interaction between the gut microbiota and onecarbon metabolism during pregnancy in obese and non-obese women.
Taking these issues into account, this article aims to: (1) summarize the evidence of interactions between obesity, one-carbon metabolism, and the gut microbiota; (2) provide evidence that gut dysbiosis mediates derangements in one-carbon metabolism in obese individuals; and (3) report the risks for offspring health in obese pregnancies. Understanding interactions between the gut microbiota and one-carbon metabolism in obese and non-obese women will favor more targeted interventional approaches to improve treatment during pregnancy, prevent adverse pregnancy outcomes, and improve offspring health.

One-Carbon Metabolism
One-carbon metabolism is composed of the interlinked folate and methionine cycles and the transsulfuration pathway, which are central to cellular function, providing substrates for DNA synthesis and epigenetic regulation (Figure). Folate from dietary, synthetic, or bacterial sources enters the folate cycle once reduced to its biologically active form, tetrahydrofolate (THF). THF is irreversibly reduced to 5methyl-THF via B 6 and/or B 2 vitamin cofactors, providing purines for DNA repair. 5-methyl-THF can only enter the methionine cycle by donating a one-carbon unit for the remethylation of homocysteine to methionine via vitamin B 12 . In the methionine cycle, methionine is converted to S-Adenosyl methionine, which serves as a universal methyl donor for biosynthetic and epigenetic processes. Lastly, homocysteine is remethylated to methionine if there is demand for tissue methyl donors, or enters the transsulfuration pathway where it is irreversibly transformed to cystathionine. Elevated homocysteine levels occur when there is insufficient folate or vitamin B 12 to allow the conversion of homocysteine to methionine; therefore, homocysteine is perceived as a sensitive marker for derangements in one-carbon metabolism. 20e23 B vitamins that drive the folate and methionine cycles are essential vitamins that cannot be produced by any cell of the human body. Consequently, sources of B vitamins arise from dietary components such as green vegetables, red meat, dairy, or synthetic supplementation or from the local gut microbes such as Bacteroidetes, Proteobacteria, and Fusobacteria (Table).
One-carbon metabolism during the periconceptional period and pregnancy One-carbon metabolism plays a key role during the periconceptional period and
Rubini. Maternal obesity during pregnancy leads to derangements in one-carbon metabolism and the gut microbiota. Am J Obstet Gynecol 2022.
ajog.org Expert Reviews pregnancy by providing substrates for the biosynthesis of DNA, RNA, proteins, and lipids, which are necessary for developmental processes such as cell replication and differentiation. 24e26 Adequate periconceptional folate and vitamin B 12 levels increase the probability of fertilization success, 27 improve preimplantation embryo quality, 28 increase implantation rates, reduce the incidence of NTDs, and improve live birth rates. 29 In addition, folate requirements are higher in pregnancy because of uterine-and placentalcell proliferation, fetal-tissue differentiation and growth, and increased erythrocyte production. 30 Folate and/or vitamin B 12 deficiencies can lead to adverse pregnancy outcomes. It is well established that folate deficiency is a severe risk factor for fetal NTDs and other congenital anomalies, 30e32 whereas vitamin B 12 deficiency during early pregnancy is a risk factor for preterm birth and adverse offspring neurodevelopment. 11,12 Moreover, elevated homocysteine is associated with miscarriages, gestational hypertension, and preterm birth. 13 Therefore, pregnant women are strongly recommended to consume micronutrient-rich foods and use folic acid supplementation from as early as 5 to 6 months before conception up to 12 weeks of gestation, preferably continuing until the end of pregnancy, to enhance fertility, prevent pregnancy complications, and reduce the risk of impairments to fetal development. 24,33e35 Obesity and one-carbon metabolism during pregnancy Obesity is characterized by high adiposity, associated with a wide range of health risks including insulin resistance, cardiovascular diseases, and metabolic and endocrine imbalances, and contributes to a chronic inflammatory physiological state. 36,37 One of the etiologic factors that induces obesity is Expert Reviews ajog.org excessive caloric input owing to consumption of an unbalanced diet predominantly composed of nutritionally poor and high-caloric food, which often leads to micronutrient deficiencies. 38 Consequently, derangements in onecarbon metabolism are more common in obese individuals. 39 In particular, low levels of vitamin B 12 and folate are associated with high BMI, 40e42 and it is known that, despite regular folic acid supplement use, obese women retain low fasting serum folate levels and have a lower folate absorption phase than normal-weight women. 19,43 This is also observed in early, mid, and late pregnancy, when obese women tend to have lower serum vitamin B 12 and folate concentrations, and higher odds of vitamin B 12 and folate deficiency than non-obese pregnant women. 40,44e52 In fact, offspring of obese women have a 2fold higher risk of being born with NTDs despite maternal folic acid supplement use. 53 With regard to homocysteine, increased levels during mid-pregnancy are observed with increasing prepregnancy BMI. 46,48 The emerging consensus is that the requirements for one-carbon metabolites during all stages of pregnancy are not met in obese women 19 despite micronutrient supplementation. 40 Therefore, levels of onecarbon metabolites should be monitored carefully and, if necessary, be improved by intervention strategies that involve lifestyle counseling with a focus on diet. For the time being, although the evidence is disputable, obese women are recommended to take higher doses of folic acid during pregnancy than nonobese women. 19 The Gut Microbiota The human gut microbiota consists of a complex and dynamic mixture of microorganisms, including bacteria, viruses, and fungi, which coexist in the gastrointestinal tract. 54 The gut microbiota is shaped in early life and more definitively established in adulthood, depending on age, ethnicity, antibiotics, medication, and, importantly, diet. 55 For the purpose of the current review, only bacterial microbiota will be addressed because of the availability of literature and our current understanding of its relationship to maternal one-carbon metabolism.
Bacterial microbiota perform essential activities such as food digestion, release of short-chain fatty acids for energy and metabolic processes, immunomodulation, and antimicrobial protection, and they are important suppliers of essential vitamins including intermediates of onecarbon metabolism. 56,57 An overview of the most important bacterial phyla and genera in the human gut and their primary roles in health and disease is provided in the Table. In general, healthy gut microbiota has a stable and balanced composition of Firmicutes and Bacteroidetes and, to lesser extent, Actinobacteria, Proteobacteria, and Verrucomicrobia. 56 Firmicutes are mostly responsible for carbohydrate metabolism and energy extraction, whereas Bacteroidetes have more mixed functions including degradation of polysaccharides, activation of the immune system, regulation of pathogenic gut bacteria, and transformation of toxic compounds. 58,59 Actinobacteria, Proteobacteria, and Verrucomicrobia are involved in decomposition of organic compounds, supporting the immune system, and mucus degradation. 59 Dysbiosis occurs when there is a disruption in the composition of the residential bacterial community, either in number or in phyla or genera predominance, resulting in loss of gut homeostasis and bacterial function, which is associated with inflammatory bowel diseases and metabolic disorders such as obesity and diabetes mellitus type 2. 60 The maternal gut microbiota during pregnancy A healthy maternal gut microbiota is essential during pregnancy because it contributes to obstetrical outcomes and long-term health sequelae for the mother and child. Various studies have shown that the gut microbiota remodels and fluctuates during pregnancy depending on gestational age. In particular, a substantial shift in bacteria phylogenies occurs from the second to third trimester, characterized by a reduction in bacterial richness (alpha diversity), with predominance of Proteobacteria and Actinobacteria, contributing to the inflammatory response in pregnancy, and a decrease in butyrateproducing bacteria, which have antiinflammatory effects (Table). 61e64 Rodent studies report the same phylogenetic patterns but a dominance of Proteobacteria in early pregnancy. 6,63,65 Other experimental studies have shown that transplantation of third-trimester microbiota into germ-free mice increases fat deposition, inflammation, and insulin sensitivity compared with transplantation of first-trimester microbiota into germ-free mice. 61,66 This suggests that the gut microbiota may adapt to sustain the superior metabolic demands of pregnancy and to support the immune system. 5,7 Recently, it has been shown that changes in the gut microbiota correlate to changes in the maternal immune response in pregnant mice, suggesting a role for the maternal microbiota in adapting the maternal immune response to pregnancy to protect the semiallogeneic fetus. 6 In contrast to studies showing changes in the maternal microbiota, there are also studies that report a relatively stable microbiota throughout gestation given that pregnant and nonpregnant women have a similar core microbiota of Firmicutes and Bacteroidetes, which are responsible for the provision of nutrition and vitamins to the host and other gut microbiota (Table). 67,68 Therefore, further research of the impact of pregnancy on maternal microbiota during pregnancy is necessary.
Obesity and the maternal gut microbiota during pregnancy The composition of gut microbiota in obese pregnant women differs from that of normal-weight pregnant women. In obese women, dysbiosis is represented by an increase in Firmicutes, resulting in a high gut Firmicutes-to-Bacteroidetes ratio. 69 Many genera of the Firmicutes have enzymes that metabolize carbohydrates to extract energy from food, thus a high prevalence of Firmicutes may increase calorie absorption, predispose to weight gain, and is additionally a symptom of gut inflammation and intestinal permeability. 69e72 Gomez et al 73 (2016) and Zacarias et al 74 (2018) report that obese pregnant women have a high Firmicutes-to-Bacteroidetes ratio, an increase in Actinobacteria in mid and late pregnancy, and a decrease in bacterial diversity in the third trimester of pregnancy compared with non-obese women. 73,74 According to this evidence, obesity during pregnancy can lead to a change in bacterial phyla that is not observed in non-obese pregnancies. Such microbial shifts are also observed in animal models including rodents. 65,74e77 Various studies have shown a causal role for the gut microbiota in obese individuals regarding weight gain and metabolic disturbances. Turnbaugh et al 78 (2006) demonstrated that germ-free mice inoculated with gut microbiota of obese mice, consisting of a high Firmicutes-to-Bacteroidetes ratio, gain more body fat and acquire metabolic disturbances. In addition, Ridaura et al 79 (2013) observed that transplanting human fecal microbiota from obese individuals to germ-free mice on a low-fat diet caused mice to develop increased fat mass and obesity-associated metabolic phenotypes. This was confirmed when microbiota from lean donors infused to recipients with metabolic syndrome (BMI >30 kg/m 2 and fasting plasma glucose >5.6 mmol/L) restored insulin sensitivity and increased butyrateproducing intestinal bacteria, exerting immunomodulatory and antiinflammatory properties. 80 More research is required to determine if this shift in bacterial populations is a consequence or cause of maternal obesity, and to determine its possible positive or negative impact on fetal growth and development.

Linking One-Carbon Metabolism to The Microbiota of Obese Pregnant Women
One-carbon metabolism and the gut microbiota are strongly connected. The reciprocal interaction between B vitamins of one-carbon metabolism and gut bacteria contributes to the overall availability of B vitamins and gut homeostasis. Despite diet being the best-known source of essential vitamins, several bacterial phyla produce B vitamins in quantities that match dietary intake. 81,82 One of the most prevalent phyla in the gut, Bacteroidetes, is the predominant producer of vitamin B 2 , B 6 , and B 12 (Table). Furthermore, most B vitamins required for one-carbon metabolism play a role in supporting bacterial survival and fitness in the gut. 83 Animal and human studies report that vitamin B 6 , folate, and B 12 deficiencies contribute to disturbances of bacterial gut homeostasis. 10, 84,85 Gut dysbiosis and derangements in one-carbon metabolites occur more often in obese than non-obese pregnant women; however, it is not known if this is the cause or consequence of maternal obesity. Often, consumption of diets poor in micronutrients and rich in energy-dense food groups is associated with lower blood concentrations of folate and vitamin B 12 . 86 This may be exacerbated by the changes in patterns of bacterial populations associated with such dietary regimes. 10,87 Low serum folate and vitamin B 12 in pregnant obese women coincide with the decreased presence of B vitamin-producing Bacteroidetes and an increased prevalence of Firmicutes from mid-pregnancy onward. 73 Consequently, folate and vitamin B 12 may be synthesized to a lesser extent because of the loss of bacterial richness and diversity. Despite this reasoning, given that there are many bacterial strains that synthesize onecarbon metabolic intermediates, it is hard to define the exact roles of each bacteria genus in pregnancy.

Offspring Health
Derangements of maternal one-carbon metabolism and maternal gut dysbiosis, present in obese pregnancies, represent a risk for fetal and offspring health. Low serum folate concentrations in obese women during late pregnancy are associated with an increased prevalence of fetal macrosomia, preterm birth, increased placental weight, and increased offspring BMI. 88,89 In addition, gut dysbiosis in obese women may be strongly associated with offspring predisposition to metabolic and neurodevelopmental abnormalities, and debate continues on whether or not this is mediated by the offspring microbiota. 90,91 Current literature seems to support that the fetal microbiota is colonized by the maternal bacteria via transplacental transport. 92 These observations are supported by rodent studies, which reveal that high-fat diets lead to gut dysbiosis and offspring with altered birthweight, fat mass, and percentage of body fat. 6,90,93,94 In conclusion, although not much is known about the effects of derangements in one-carbon metabolism and maternal gut dysbiosis on offspring health in obese pregnancies, these factors seem to play an important role in offspring weight and body fat content, and should therefore be rigorously monitored as early in pregnancy as possible to avoid dysbiosis and micronutrient deficiencies known to occur from mid-pregnancy.

Clinical Implications
It is clinically relevant to know that B vitamin availability depends on multiple factors, such as diet, metabolism and gut microbiota, which opens the window for multiple treatment strategies. In this case, if vitamin supplementation is insufficient to restore B vitamin levels in obese women, microbiota homeostasis can be considered as a second target for treating the deficiency. Currently, there is no effective intervention strategy for (obese) pregnant women with derangements in one-carbon metabolism and/or gut dysbiosis. Lifestyle care, including a combination of dietary advice and folic acid/multivitamin supplement use, is currently the most used treatment approach. 95,96 However, its effectiveness is either variable or unknown. Alongside lifestyle care tailored to obese pregnant (or preconceptional) women, we would advise to integrate microbiota analyses of stool samples and blood tests for one-carbonerelated metabolites/cofactors throughout pregnancy, particularly homocysteine plasma levels given that this is a sensitive marker of derangements in one-carbon metabolism. Currently, several stool sampling methods are used to analyze the gut microbial population, and they could be Expert Reviews ajog.org integrated in clinical practice during pregnancy visits. 97 The 2 must be independently but simultaneously monitored because a healthy microbiota does not exclude derangements in onecarbon metabolism and vice versa.
Furthermore, the field is now focusing on the use of probiotics and/or prebiotics as an intervention strategy in the treatment of gut dysbiosis. Probiotic bacteria (most common are Lactobacillus and Bifidobacterium species) confer longlasting health benefits when administered in adequate quantities, such as when treating chronic bowl diseases and infections. 98,99 Lactobacillus and Bifidobacterium species have folate biosynthetic capacity and are used to restore the Firmicutes-to-Bacteroidetes ratio in gut dysbiosis. 100 In addition, adding probiotics to the diet can favor an increase in bacterial diversity, which counteracts the loss of diversity seen in gut microbiomes of obese patients. Conversely, prebiotics are substrates (mostly nondigestible carbohydrates) that are selectively utilized by gut microorganisms to nourish probiotic bacteria and other gut bacteria. 101 Indeed, the consumption of prebiotic-rich foods has been shown to support probiotic bacteria activity, particularly by increasing gut microbiota concentrations of Lactobacillus and Bifidobacterium species. 102 Currently, there is contradictory evidence on the efficacy of probiotic and prebiotic interventions during pregnancy, particularly because the doses have to be tailored to the composition of the host microbiota, which is individualspecific. 103 However, both have been demonstrated to be safe for use during pregnancy. 103e105 The use of probiotics and prebiotics during pregnancy in obese women holds potential and should be investigated because there is currently no evidence that they confer substantial benefits for obese pregnant women.

Future Studies
From the foregoing discussion, we can conclude that there are numerous knowledge gaps concerning the relationship between one-carbon metabolism and the gut microbiota in nonobese and obese pregnancies, although it is known that both play a major role in pregnancy health and fetal development, and the subsequent long-term health of offspring. It would be important to know which specific bacteria are associated with low serum folate and vitamin B 12 during pregnancy. Future research should focus on intervention strategies, such as blended periconception lifestyle care approaches, 106 to detect and counteract B vitamin deficiencies and gut dysbiosis in obese pregnant women, starting with the use of probiotic supplements. Lastly, given that current knowledge is based on maternal obesity only, further studies need to address other potential mechanisms and the relationship between one-carbon metabolism and gut bacteria in other circumstances prone to vitamin deficiencies, such as in patients with metabolic disorders, those who have a family history of pregnancy complications, or those who harbor genetic polymorphisms in any one of a number of one-carbon metabolism genes.

Conclusion
In this review, we propose that the association between one-carbon metabolism and the gut microbiota contributes to the overall availability of B vitamins and to gut homeostasis of the individual, which subsequently determines maternal and fetal health during pregnancy. Despite limited evidence, it seems that dietary patterns in obese pregnancies are associated with derangements in one-carbon metabolism, aggravated by changes in patterns of bacterial populations associated with poor diets. These are risk factors for adverse pregnancy outcomes, impaired fetal development, and disruption of fetal growth and microbiota formation, leading to potential long-term offspring metabolic and neurologic disorders. Further studies are required to find effective intervention strategies for derangements in one-carbon metabolism and gut dysbiosis tailored to obese pregnant women. Disturbances in metabolism that are detected during early pregnancy or even during the preconceptional period create a window of opportunity for clinicians to provide lifestyle care and to prevent adverse pregnancy outcomes.