Steroids and One-Carbon Metabolism: Clinical Implications in Endocrine Disorders

Abstract

Background

One-carbon metabolism (OCM) and steroid metabolism are fundamental biochemical pathways that regulate essential cellular processes and physiological functions. OCM is involved in DNA synthesis, methylation, and redox balance, while steroid metabolism governs the production and degradation of steroid hormones, which notably influence growth, reproduction, and stress responses. Despite their distinct roles, emerging evidence suggests a strong interplay between these two pathways.

Summary

This review examines the bidirectional relationship between OCM and steroid metabolism, emphasizing their shared intermediates and cofactors. Folates and methionine, key intermediates of OCM, influence steroid biosynthesis, while the methylation status regulated by OCM affects the expression of steroidogenic enzymes. Conversely, steroid hormones modulate OCM by altering the activity of enzymes in folates and methionine cycles. Disruptions in either pathway are linked to diseases such as cancer, cardiovascular disorders, and neurodegenerative conditions. This narrative review examines the clinical and preclinical data on the interactions between OCM and sex steroids, in both women and men, adrenal steroids and vitamin D metabolism.

Key Messages

The interdependence between OCM and steroid metabolism highlights the need to consider both pathways in disease pathogenesis and therapeutic strategies. Their crosstalk plays a crucial role in maintaining cellular homeostasis and responding to metabolic stress. Targeting this metabolic interplay offers new opportunities for treating metabolic disorders, with potential clinical applications in modulating one pathway to influence the other for more integrated disease management.

Plain Language Summary

Our bodies rely on many chemical processes to function properly. Two important ones are one-carbon metabolism (OCM) and steroid metabolism. OCM helps with DNA production, gene regulation, and maintaining balance in our cells. Steroid metabolism is responsible for producing hormones that control growth, reproduction, and how we respond to stress. Although they seem separate, research shows that these two processes are closely connected. This review explores how OCM and steroid metabolism influence each other. Certain nutrient-related components, like folates and methionine, which are part of OCM, help the body produce steroid hormones. At the same time, steroid hormones can affect how OCM works by changing how specific enzymes function. When there are malfunctions in either of these systems, it can lead to diseases such as cancer, heart disease, and brain disorders. This review looks at studies on how OCM interacts with sex hormones in both men and women, as well as hormones produced by the adrenal glands and vitamin D metabolism. Since OCM and steroid metabolism are so closely linked, it is important to study them together when looking at diseases and possible treatments. Their interaction helps keep our bodies in balance and allows us to respond to stress and environmental changes. Understanding this connection could lead to better treatments for metabolic disorders. By adjusting one of these pathways, we may be able to influence the other, offering new ways to manage health conditions more effectively.

Introduction

Background of the Review

Since the past decade, it has become clear that our environment influences various metabolic pathways. Notably, it has been shown that one-carbon metabolism (OCM) is impacted by nutritional deficiencies, especially in vitamins B9 (or folates) and B12 [1]. OCM is a universal metabolic process which is essential to synthesize and/or recycle the molecules that provide the one-carbon group for various metabolic functions, such as methylation reactions [2], purine and thymidine synthesis [3], and antioxidant generation [4]. Some teams investigated the impact of OCM disorders on various physiological or pathological situations like pregnancy [5, 6], including metabolic complications during pregnancy [7], medically assisted reproduction (MAR) [8], cancer [9, 10], cardiovascular disease [11], or in health in general [12]. In the field of endocrine disease, no recent review has been published, and no review specifically on the interplay between OCM and steroid metabolism.

Aim and Methods

Our review aimed to provide an overview of the most recent data on the links between OCM and steroidogenesis, and their consequences for human health. It includes clinical data when available and, given that OCM is a highly conserved metabolic pathway between species, preclinical data. In vitro and preclinical data have been included to provide mechanistic explanations for clinical observations. Keywords such as “homocysteine,” “one carbon metabolism,” “folates,” “steroids,” “cortisol,” “testis,” etc. were used to carry out searches in PubMed and Google Scholar. Articles have been classified according to relevance and publication date, in order to reflect the most recent and relevant data on the subject.

Overview of the OCM

OCM comprises the folates cycle, the methionine cycle, and the trans-sulfuration pathway [13] (shown in Fig. 1). Folates, absorbed through the digestive tract, provide the one-carbon group after being reduced to dihydrofolate (DHF) and tetrahydrofolate (THF). THF then receives a methylene group, before engaging in one of the following three pathways: thymidine synthesis, purine synthesis or methylation process. A key step is the reduction of 5,10-methylene-THF to 5-methyl-THF by the methylene-THF-reductase (MTHFR). The 5-methyl-THF provides the methyl group that will integrate the methionine cycle. This methyl group is a substrate of the methionine synthase enzyme to methylate homocysteine (HCY), thus forming methionine. An important cofactor for this reaction is vitamin B12. Methionine is the precursor of S-adenosylmethionine (SAM). SAM gives his methyl group to a receiver via the methyltransferase activity and becomes S-adenosylhomocysteine (SAH). SAH is then hydrolyzed into HCY by the adenosyl-homocysteinase, to be recycled into methionine and reintegrate into the cycle. OCM is thus a key metabolic pathway for the methylation process, particularly of DNA and proteins. DNA methyltransferases (DNMTs) are responsible for the transfer of a methyl group from SAM to DNA on the 5-position of cytosine residues, while protein arginine methyltransferases (PRMT) are enzymes that catalyze the arginine methylation reaction. This is a major mechanism for regulating gene expression. There are 9 different PRMTs, and PRMT1 is responsible for 85% of the methylation reactions.

Fig. 1.

The one-carbon metabolism. Green cycle = folate cycle; blue cycle = methionine cycle; orange pathway = trans-sulfuration pathway. Enzymes (gray boxes): BHMT, betaine-homocysteine S-methyltransferase; CBS, cystathionine beta-synthase; CHDH, choline dehydrogenase; CTH, cystathionine gamma-lyase; DHFR, dihydrofolate reductase; MAT, methionine adenosyltransferase; MTHFD, methylenetetrahydrofolate dehydrogenase (type 1 or 2); MTHFR, methylenetetrahydrofolate reductase; MTR, methionine synthase; MTs, methyltransferases; SAHH, S-adenosylhomocysteine hydrolase; SHMT1, serine-hydroxymethyltransferase 1; TYMS, thymidylate synthase. Enzymes cofactors (red cycles): B2, vitamin B2; B6, vitamin B6; B9, vitamin B9; B12, vitamin B12. Substrates: −CH3, methyl group; 5-mTHF, 5-methyltetrahydrofolate; 5,10-CH-THF, 5,10-methenyltetrahydrofolate; 5,10-CH2-THF, 5,10-methylenetetrahydrofolate; 10-f-THF, 10-formyltetrahydrofolate; α-KB, α-ketobutyrate; Bet, Betaine; Chol, choline; Cth, cystathionine; Cys, cysteine; DHF, dihydrofolate; DMG, dimethylglycine; dTMP, thymidine monophosphate; dUMP, deoxyuridine monophosphate; Glu, glutathione; Hcy, homocysteine; Hse, Homoserine; Met, methionine; NH3, ammonia; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; Ser, serine; SO4, sulfate; Tau, taurine; THF, tetrahydrofolate.

HCY can also integrate the trans-sulfuration irreversible pathway: HCY is converted into cystathionine and cysteine by the action of cystathionine beta-synthase (CBS) and cystathionase, respectively. Cysteine is involved in the biosynthesis of sulfur compounds, taurine and glutathione, which is an important molecule for the oxidation-reduction reactions. In the case of alteration of the OCM, hyperhomocysteinemia is typically observed, which is why HCY is used as a biomarker in clinical practice.

The Central Role of Homocysteine

HCY is, therefore, a central player in the OCM. Hyperhomocysteinemia suggests a blockage of one or more stages of the cycle and is commonly observed in situations of nutritional deficiency (folates, vitamin B6, vitamin B12, or betaine), but other causes are possible. Some of them are acquired and most of the time induces mild hyperhomocysteinemia. They can be linked to lifestyle (smoking, alcohol consumption, sedentary lifestyle…) [14, 15] or not (chronic kidney disease [CKD] [16], hypothyroidism [17], anemia [18], malignant tumor [19], iatrogenia [20]). However, genetic variants remain the most common causes of major hyperhomocysteinemia. Genes involved are classically those encoding for OCM cycle enzymes, and the most concerned is MTHFR [21, 22]. Hyperhomocysteinemia, defined by a plasma concentration in humans above 15 µmol/L [23], is significantly associated with other clinical manifestations. HCY is a pro-inflammatory and pro-thrombogenic factor involved in endothelial dysfunction [24] and promotes thrombo-embolic events [22]. Thus, hyperhomocysteinemia is recognized as a cardiovascular risk factor. It is mainly associated with stroke [25] but also hypertension [26] and cardiovascular diseases [25, 27]. Conversely, folate supplementation, aimed at reducing hyperhomocysteinemia, has been shown to be effective in stroke prevention [28]. Some authors have also found a link between hyperhomocysteinemia and some neurological pathologies [29], such as Alzheimer’s disease [30], Parkinson’s disease, or epilepsy [31], or with other various pathologies including rheumatological autoimmune disorders [32], deafness [33], age-related macular degeneration [34], migraine [35], or psoriasis [36]. Other studies highlighted the effects of hyperhomocysteinemia on other metabolic pathways. More particularly, insulin secretion appears to be impaired in case of hyperhomocysteinemia, with an improvement in glycemic control with folate treatment [37]. Moreover, hyperhomocysteinemia seems to have an impact on the metabolism of steroids, with varying degrees of understanding of the underlying pathophysiological mechanisms.

Metabolic Links between OCM and Steroid Metabolism

The Wnt/β-Catenin Pathway

To date, the metabolic link between OCM and steroid metabolism has not been clearly established, but the data converge on a strong link with the Wnt/β-catenin pathway. The canonical Wnt signaling is a vital pathway for cell growth and tissue remodeling, especially in endocrine tissues [38]. This pathway is dysregulated when the OCM is disrupted, which could contribute to the pathophysiology of endocrine pathologies. Thus, an alteration in one or other intermediate of the Wnt/β-catenin pathway can be responsible for pathologies such as Cushing’s disease or Addison’s disease [38]. Wnt signaling seems to play a role in testis [39] and ovarian [40, 41] physiology too, but there are few data on steroidogenesis in these organs in humans and no data in pathological situations.

Among the metabolites involved in the Wnt pathway, methionine seems to have a privileged role since post-transcriptional modifications are central to the regulation of this pathway. Indeed, methionine restriction alters PRMT activity [42] and decrease in SAM levels by methionine restriction or methotrexate administration decreased Wnt signaling in cultured cell models of cancer [43]. More directly, the loss of PRMT1 decreased Wnt transcriptional activity in this study on HeLa cells [44], underlining the importance of the methylation process in regulating the Wnt pathway. Indirectly, a recently identified tumor suppressor called methylthioadenosine phosphorylase appeared to render cancer cells sensitive to methionine restriction by lowering SAM levels [45].

Since most studies on the Wnt pathway have focused on cancer, mechanistic data are mainly available in cancer cell models. However, recent studies have shown that similar alterations in this metabolic pathway can lead to non-cancerous endocrine pathologies. We can, therefore, suppose that steroidogenesis is indirectly impacted by OCM alteration through Wnt/β-catenin dysregulation, by lowering the quality or quantity of steroidogenic cells, but further investigations are needed to explore this hypothesis.

Methylation and Steroidogenic Enzymes

Furthermore, OCM could directly impact steroidogenesis by modifying the DNA methylation of enzymes involved in the synthesis of steroids and their receptors. Zhang and Ho published in 2011 a review on this topic [46]. At this time, only 5 have been studied: CYP11A1 [47], HSD3B1 [47], CYP17A1 [48, 49], CYP19A1 [49–53], and CYP27B1 (retracted article). Following this, several teams published work on the link between epigenetic modifications and steroidogenesis in testis [54, 55], ovarian [56], and adrenal gland (summarized in this review from a study by Baquedano and Belgorosky [57]), mainly due to a growing interest in endocrine disrupting chemicals (EDCs). Overall, these studies show that methylation changes in genes involved in steroidogenesis are associated with different levels of expression of steroidogenic enzymes.

In short, while epigenetic modifications seem clearly involved in the regulation of steroidogenesis, we still lack the data to understand how they are organized, which limits the possibilities of clinical trials. We can assume that OCM is at least partially involved, but studies on this subject most often pass directly from the OCM to steroids without exploring methylation modifications. We will now review the preclinical and clinical data on the various steroids and their link with OCM. Data regarding the metabolic connections between OCM and steroidogenesis are summarized in Figure 2.

Fig. 2.

Synthesis of metabolic connections between OCM and steroidogenesis. OCM (blue circle) regulates steroidogenesis (green circle) by acting on inflammation, mainly via the trans-sulfuration pathway, which modulates the activity of steroidogenic enzymes. Indirectly, OCM regulates the expression of steroidogenesis enzymes by applying epigenetic modifications to their DNA (orange circle), as well as to players in the Wnt/β-catenin pathway (purple circle). The Wnt/β-catenin pathway then influences steroidogenesis by regulating the proliferation of steroidogenic cells. Steroids provide parallel feedback to the OCM, maintaining a balance between these metabolic pathways. The arrows between the large circles symbolize the influence of one element on another. The solid arrows inside the steroidogenesis circle represent a conversion of one component into another, after conversion by the enzyme whose name is written in blue. The broken arrow represents a conversion with intermediates not shown in the figure. Cyp, cytochrome P450; DNMTs, DNA methyltransferases; Hcy, homocysteine; HSD3B1, 3 beta-hydroxysteroid dehydrogenase/delta(5)-delta(4)isomerase type I; PRMT1, protein arginine methyltransferases type 1; SAM, S-adenosylmethionine.

Sexual Steroids

Sexual steroids, particularly female steroids, have probably been the most extensively studied for their interactions with OCM. First, there is a clear sexual dimorphism in the OCM: women of childbearing age have lower SAM [58], lower HCY [59], and higher choline and betaine (two metabolites of the methionine cycle) [60] in comparison with men. Clare et al. [61] highlighted that several genes involved in OCM and related metabolic pathways are regulated by androgens, progesterone, and/or estrogen receptors (ERs), directly or indirectly. Therefore, clinical manifestations of OCM disorders vary according to gender and life stage. For instance, Fisher et al. showed, in a randomized control trial, that men and postmenopausal women require a higher intake of choline to reverse fatty liver disease and muscle damage induced by a choline-deficient diet, in comparison with premenopausal women [62]. Because of this dimorphism, we have chosen to present the data differently according to gender.

OCM and Sexual Steroids in Women

OCM, Sex Steroids, and Menstrual Cycle: Clinical Data

For women, a critical period for steroidogenesis is the preconception period. In healthy women of childbearing age, higher homocysteine levels were associated with lower total estradiol levels across the menstrual cycle and lower luteal phase progesterone in prospective longitudinal cohorts [63, 64]. Conversely, the increase in estrogen during pregnancy was associated with a 30% drop in plasma HCY concentration in a prospective observational study on 10 healthy women [65]. More generally, HCY is significantly lower in pregnant women, women of childbearing age, or postmenopausal women with hormone replacement therapy, compared with untreated postmenopausal women [66–69]. Some authors have also observed variations in HCY levels within the menstrual cycle, in conjunction with variations in estradiol levels in computerized models [70]. This suggests that the OCM is impacted by the woman’s estrogenic impregnation. A clinical open-label pilot study is currently being conducted to assess the effect of SAM treatment in women between 18 and 45 with premenstrual disorders [71]. This will probably yield interesting data that could be used for larger-scale studies.

Consequences for Ovulation and Procreation

Higher homocysteine at expected ovulation was associated with a 33% increased risk of sporadic anovulation and a higher folate/homocysteine ratio at ovulation was associated with a 10% decreased risk of anovulation. The protective effect of folate intake against anovulation has been confirmed by Gaskins et al. [63]. In their prospective study, women with a higher folate intake (3rd tertile) had on average 16% higher luteal progesterone levels compared to women in the 1st tertile, and 64% decreased odds of anovulation. The higher rate of anovulation associated with hyperhomocysteinemia could be the consequence of ovarian follicle-stimulating hormone (FSH) resistance. Indeed, women with hyperhomocysteinemia due to homozygous MTHFR 677C>T genotype require significantly more recombinant-FSH to achieve pregnancy and produce less estradiol and oocytes than heterozygous or homozygous 677T genotype. These results were consistent in retrospective [72] and prospective studies [73], but to date, no randomized controlled trial has been conducted to investigate the specific effect of folic acid supplementation on ovulation rate in the general population.

Preclinical Data on Menstrual Cycle and OCM

In vitro culture of granulosa cells from homozygous T/T women undergoing controlled ovarian hyperstimulation for in vitro fertilization (IVF) showed results consistent with observational studies: basal and post-stimulatory estradiol levels in follicular fluid were lower than in heterozygous C/T and homozygous C/C individuals [74], reinforcing the hypothesis of FSH resistance in case of alteration of the OCM.

Some teams have speculated that menstrual cycle disorders and reproductive difficulties are linked to accelerated degradation of the corpus luteum [63, 64]. Progesterone synthesis is dependent on the maintenance of the corpus luteum, and the regression of the corpus luteum after ovulation is influenced by certain inflammatory cytokines that accelerate the phenomenon of apoptosis [75, 76]. According to cellular models, hyperhomocysteinemia can stimulate the production of these cytokines [77], notably tumor necrosis factor alpha (TNFα) and nitric oxide [78–80]. We can, therefore, assume that hyperhomocysteinemia is indirectly linked to a drop in progesterone levels in the luteal phase, which could contribute to procreation difficulties due to abnormalities in embryo implantation.

Finaly, a recent study conducted on rats showed that methionine supplementation during the estrous cycle increases the level of SAM and GSH, increases the number of embryo implantation sites and enhances the expression of DNMTs [81]. So, methionine seems to promote follicular growth and estrogen synthesis in rats via an anti-inflammatory effect and by improving DNA methylation.

OCM, Sex Steroids, and Metabolic Concerns in Women

Given that OCM-related disorders are associated with cardiovascular disease and that women’s cardiovascular risk varies according to their life stage, other authors have investigated the association between OCM, cardiovascular risk, and sexual steroids’ metabolism. Prospective clinical data showed that declines in estradiol across stages of the menopause transition may lead to elevations of HCY and cysteine. This was associated with a lower brachial artery flow-mediated dilation, which is an indirect marker for endothelial dysfunction [82].

Preclinical models have confirmed these results and provided mechanistic hypotheses for this observation. Dimitrova et al. [83] treated Wistar rats with placebo, 1 mg, or 2 mg of 17beta-estradiol and added HCY (100 mg/kg/day) to the drinking water for half of them. They observed a protective effect of estrogens on the relaxation response of aortic ring segments to acetylcholine and against aortic endothelial denudation in hyperhomocysteinemic rats. Hyperhomocysteinemia without estrogen treatment was associated with lower glutathione and glucose-6-phosphate dehydrogenase (G6PDH) activity, suggesting that the trans-sulfuration pathway is probably involved. Another animal model in dogs showed that estrogens increase the myocardial glutathione concentration and G6PDH activity, with a protective effect against ischemia/reperfusion-induced myocardial systolic shortening [84]. Moreover, it has been shown in a rat model that the improved action of G6PDH by estrogens enhances the bioavailability of nitric oxide [85]. This reduces the synthesis of free radicals and offers protection against oxidative stress and, thus, against cardiovascular diseases. Finally, in vitro experiments showed that estrogen can activate CBS [86, 87], and activates phosphatidylethanolamine n-methyltransferase (PEMT), an enzyme that converts phosphatidylethanolamine to phosphatidylcholine, with a SAM-dependent reaction [88]. This reaction generates more betaine, which further activates CBS. The trans-sulfuration pathway is, therefore, globally activated by estrogen, thus explaining the drop in HCY level, and potentially the protective effect of estrogens against cardiovascular disease.

OCM in the Case of Polycystic Ovary Syndrome

Polycystic ovary syndrome (PCOS) is associated with cardiovascular diseases. Women with PCOS show a slightly elevated androgens level, with a decreased progesterone level, while estradiol levels are high and correspond to those of the early follicular phase of eumenorrheic women [89]. A number of teams identified hyperhomocysteinemia in women with PCOS (see review of Grodnitskaya and Kurtser [90]), and some have speculated that the increased cardiovascular risk of these patients is linked to hyperhomocysteinemia, partly via the associated insulin resistance [91, 92]. The precise role of steroids disturbances in cardiovascular events is poorly known, as well as the link between these disturbances and alteration of the OCM. Nevertheless, some observational studies have provided clues, suggesting an association between biological hyperandrogenism and hyperhomocysteinemia, like in this study on PCOS women and idiopathic hyperandrogenism [93]. In a prospective study conducted on 45 women with PCOS, testosteronemia was significantly higher than in control subjects, with a tendency toward hyperhomocysteinemia (although not statistically significant) [94]. However, in another interventional study comparing 23 lean women with PCOS treated with ethinyl estradiol/drospirenone (3 mg/30 µg) and spironolactone (100 mg/day) during 6 months and 23 age- and body mass index-matched healthy control women, HCY level was similar at baseline, despite a higher androgen level in PCOS women [95]. Nevertheless, the treatment induced a joint increase of androgens and HCY in this group. These conflicting data do not allow the conclusion that there is an association between hyperandrogenism and hyperhomocysteinemia in this population.

Other studies suggest that hyperhomocysteinemia is rather the consequence of insulin resistance. As an example, this is the conclusion of this study that compared patients with PCOS, i.e., with insulin resistance, to others with hyperandrogenism without insulin resistance (congenital adrenal hyperplasia). They found no difference in HCY levels between congenital adrenal hyperplasia and control group, whereas PCOS women had higher HCY levels than the control group [96]. This reinforces the idea of an indirect link via insulin resistance rather than a direct effect of hyperandrogenism. Interestingly, a recent mendelian randomization analysis that involved 8,741 women with PCOS and 415,735 control women showed no sufficient evidence to support the causal association of HCY with the risk of PCOS [97]. So, clinical data in women do not establish a cause-consequence relationship between hyperandrogenia and hyperhomocysteinemia.

Preclinical models have provided some interesting insights in this area. This study recently conducted on third-generation PCOS-like mice revealed in a large RNA-seq analysis that PCOS is associated with the negative regulation of insulin secretion, folliculogenesis and ovarian steroidogenesis [98]. Interestingly, other studies suggested that levels of androgens during pregnancy may be responsible for the fetal programing of PCOS [99].

OCM and Sexual Steroids in Men

OCM undoubtedly plays an important role in the reproduction process in men too since the homozygous MTHFR 677C>T genotype is 2 times more frequent in infertile men than in non-infertile men [100]. Finkelstein et al. [101] showed in 1971 on rat tissue that testosterone increases the activity of betaine-homocysteine methyltransferase and MTHFR [101], but the mechanistical link between testosterone and OCM has not been further investigated. To date, recent data are only available from preclinical models and clinical studies.

OCM and Sexual Steroids in Men: Preclinical Data

On the one hand, a preclinical model supports a link between testosterone synthesis and OCM with a positive correlation between HCY and testosterone. In the Sakamuri et al. [102] protocol in which Sprague Dawley rats were exposed to a fructose-rich diet in order to induce a metabolic syndrome (MS) [102], testosteronemia decreased by 47.9% after 4 weeks, compared to control rats, in line with a slight decrease in testis weight. Similarly, homocysteinemia was 35.4% lower at week 4 in this group. However, homocysteinemia and testosteronemia normalized at week 12 and 24 of the fructose-rich diet. So, the lack of persistence of the anomalies over time, even though the diet had not been modified, suggests that the observed differences are not diet-related.

Recently, a study conducted on a model of rats with oligoasthenozoospermia showed that treatment with betaine improves sperm parameters [103]. Other studies confirmed the association between OCM and spermatogenesis [104, 105] but none of them investigated the role of steroidogenesis. Interestingly, in Lin et al. protocol, rats with oligoasthenozoospermia had a lower level of testosterone with higher levels of luteinizing hormone, suggesting some kind of testicular insufficiency, and betaine treatment was associated with a restoration of testosterone secretion and a decrease in luteinizing hormone level, in a dose-dependent manner. As these two models explore the OCM in different ways, the results are not necessarily contradictory. However, clinical studies have provided additional data.

OCM and Sexual Steroids in Men: Clinical Evidence

Observational studies performed in humans identified no trend to hyperhomocysteinemia in men with MS and hypotestosteronemia. In particular, Sung et al. [106] found no difference in HCY levels between men with and without MS in a cohort of 8,606 men, while men with MS had a lower testosterone level (5.8 versus 6.6 ng/mL, p < 0.001). However, HCY and testosterone levels showed a significant, but weak correlation (+0.046, p < 0.001) that do not seem clinically significant.

Some interventional studies have been performed and the effect of testosterone supplementation on HCY level is controversial as well. In the Krysiak et al. [107] protocol, in which hypogonadal men with atherogenic dyslipidemia were treated with oral testosterone, homocysteinemia was not significantly affected. But in parallel groups, patients treated with fenofibrate increased homocysteinemia by 24%, while homocysteinemia in patients with fenofibrate + testosterone remained statistically stable. Notably, there was a negative correlation between HCY and testosterone level, but also with uric acid, high-sensitivity C-reactive protein and fibrinogen (r values between –0.34 [p < 0.01] and –0.51 [p < 0.01]). Furthermore, the same team, in another trial on 30 men with hypogonadism and MS, found that oral testosterone treatment combined with metformin is more effective in lowering homocysteinemia than metformin alone as HCY was 35% lower in the testosterone-treated group [108].

Since folic acid supplementation is the first-line treatment to reduce HCY, we could assume that folic acid could improve testosteronemia if it is the consequence of hyperhomocysteinemia. A meta-analysis including 7 studies on infertile men from various etiologies showed no beneficial effect of treatment with folic acid or folic acid and zinc on testosterone levels [109]. However, only 2 studies were included in the analysis regarding testosterone [110, 111]. In these studies, patients had fertility disorders related to testicular pathologies treated surgically (varicocelectomy [111]), or of unspecified cause [110]. It is, therefore, difficult to draw conclusions about the secretory reserve of the testes in these situations. Moreover, the authors acknowledged the considerable heterogeneity of the studies included (I² = 93%) but highlighted the positive effect of the supplementation on sperm characteristics. In summary, clinical data suggest that testosterone synthesis could be improved by intervention on the OCM, but preclinical data remain very limited, and we lack high-powered clinical trials to confirm this trend.

OCM and Neurosteroidogenesis

Some teams have studied the in situ synthesis of steroids in brain tissue independently of gender. This process, known as neurosteroidogenesis, appears to be impaired in the case of hyperhomocysteinemia. As explained above, HCY is frequently increased in neurodegenerative diseases, both in plasma and in cerebrospinal fluid (CSF). More particularly in Alzheimer’s disease, clinical data showed that the androgen dehydroepiandrostenedione level is higher compared to cognitively intact control subjects [112–114]. These observational results have led other teams to carry out experimental studies on animal models. Studies conducted on folate-deficient rats showed that neurosteroidogenesis is impaired in the event of folate deficiency, with defective expression of two key enzymes involved in steroidogenesis: steroid acute regulatory protein (StAR) and aromatase [115]. These abnormalities are also present in the offspring of methyl-donor deficient mothers, probably due to epigenetic modifications, resulting in developmental and olfactory disorders [116].

In addition, in this recent mendelian randomization study on 687 patients with brain atrophy, an association was established between estradiol level and brain atrophy in women but not in men [117]. There was little evidence of a causal link between elevated plasma HCY and brain atrophy, but genetic predisposition to elevated plasma HCY was associated with lower estradiol levels. We can, therefore, imagine that there is an indirect link between HCY and cerebral atrophy, but this requires further investigation. Clinical data for sexual steroids are shown in Figure 3.

Fig. 3.

Synthesis of clinical consequences of the interactions between one-carbon metabolism and sexual steroids in men, women and children. Upper part of the figure, with blue arrow: consequences in men. Lower part of the figure, with pink arrow: consequences in women. Glu, glutathione; Hcy, homocysteine; HRT, hormonal replacement therapy; PCOS, polycystic ovary syndrome.

Adrenal Steroids

While the literature is relatively rich on sex steroids, it is sparser on gluco- and mineralocorticoids. Nevertheless, the hypothalamic-pituitary-adrenal (HPA) axis physiology seems to be influenced by the OCM.

Glucocorticoids, Stress Response, and OCM

Cortisol is frequently referred to as the “stress hormone,” due to the activation of the HPA in acute situations. In the case of transient elevation of cortisol, i.e., under stress, a rise in homocysteinemia has been observed in some studies [118].

Conversely, in cases of chronic stress, cortisol secretion is lowered, typically in post-traumatic stress disorder (PTSD) [119]. This is associated with smaller volumes of the thalamus, right frontal pole, left occipital pole, and right superior occipital gyrus in comparison with healthy controls. In this study on 28 patients with PTSD, compared to 223 healthy controls, PTSD was positively associated with homocysteinemia, and duration of PTSD was found to predict serum homocysteine levels [120]. Jendricko et al. [121] found the same positive association in a cross-sectional study on 66 war veterans with PTSD, compared with 33 without PTSD and 42 healthy controls. Unfortunately, none of these studies investigated the cortisol levels of patients, and no other studies on chronic stress and OCM have been published.

The Key Role of the Glucocorticoid Receptor

Since the endocrine system, particularly the HPA axis, is responsible for the organization’s adaptation to external stimuli, and epigenetics gives responses to external and internal environmental stimuli, we can easily imagine HPA regulation via epigenetic modifications, designed to respond to stressful situations. In a study conducted on rats, the expression of glucocorticoid (GC) receptor (GR) mRNA was significantly affected by antenatal hypoxia [122]. In this study, prenatal hypoxia reduced the expressions of GR mRNA and protein in adult male offspring, but not in females. This sexual dimorphism seems to be related to the differential expression of exon1 mRNA of the GR gene between male and female offspring. In addition, the GR promoter appeared to be hypermethylated, contributing to its inactivation.

Clinical studies on this subject are rare, but some were published on early-life abused adult suicide victims. In this population, compared with non-abused people, studies showed a lower GR mRNA and increased cytosine methylation of the promoter of NR3C1, the gene coding for the GR [123]. Moreover, NR3C1 methylation was linked to neurodevelopmental development in children, suggesting that it may influence behavioral and biological aspects of the stress response [124].

GCs and OCM in Physiological and Pathological Conditions

HPA Axis, OCM, and Psychiatric Disorders

Since stress in childhood is associated with psychiatric pathologies in adulthood, some teams have studied the association between HPA, OCM and psychiatric pathologies. Some have found an association between lower folate concentration and higher cortisol levels in patients with schizophrenia. In this prospective observational study on 31 schizophrenic patients compared to healthy controls [125]. Concordant data have been found in patients followed for psychiatric pathologies, with a positive correlation between homocysteinemia, cortisolaemia, and progression to type 2 diabetes [126].

HPA Axis and OCM during Pregnancy

If disturbances of the OCM and HPA axis during childhood are at the root of psychiatric disorders in adults, other studies suggest that the same is true for disorders during pregnancy. Apparently, circulating cortisol is not directly affected by changes in OCM during pregnancy, as shown by Christian et al. [127]. In a randomized control trial comparing the effect of folate supplementation or not in 737 healthy women, folate supplementation induced no significant effect on blood cortisol levels. If the blood cortisol level is not affected, Pavlik et al. [128] showed an impact of OCM alteration on cortisol level in follicular fluid of women with the homozygous MTHFR 677C>T genotype. They found a lower cortisol concentration in the follicular fluid of women without folate supplementation, and a normalization of this level to 800 µg of folic acid per day. Even if the consequences of pregnancy have not been studied, it could be significant for the offspring. Krishnaveni et al. [129] showed that hyperhomocysteinemia of the mother during pregnancy induces a greater rise in cortisol in the offspring under stress, with a greater heart rate response and output. Similarly, maternal hyperhomocysteinemia seems to be associated with greater insulin resistance in offspring [130], which could be explained by a higher cortisol level since this steroid is known to induce glucose tolerance impairment. All these examples are in line with the fetal programming concept, according to which exposures during fetal life can contribute to the development of diseases in the offspring after birth.

HPA Axis and OCM in Neurological Disorders

Other studies focused on the respective roles of GCs and HCY in neurological pathologies. In normal-pressure hydrocephalus (NPH), CSF cortisol levels appear to increase in cases of hyperhomocysteinemia [131, 132]. Evacuation of excess CSF leads to a higher cortisone/cortisol ratio in CSF but a lower ratio in plasma [133]. Since cortisone is the inactive form of cortisol, this suggests an activation of GC metabolism in the central nervous system in patients with NPH, that is reversible after evacuation of excess CSF. In this study, HCY was stable in CSF but increased significantly in blood during the 2 years of the study. The blood-brain-barrier could explain the discordance between the metabolite levels, but its integrity has not been investigated in this condition. Moreover, in Alzheimer’s disease, which is associated with hyperhomocysteinemia as mentioned above, there is a certain form of hypercorticism and elevation of inflammatory cytokines such as interleukin-6, whose transcription is promoted by GCs [134]. Remember that hyperhomocysteinemia is associated with the synthesis of inflammatory cytokines too, as said above. Nevertheless, no study has been carried out specifically on the association between HPA and OCM in Alzheimer’s disease.

OCM and Cushing’s Syndrome

In Cushing’s syndrome (CS), an affection induced by chronic GCs excess, homocysteinemia is significantly higher according to some observational studies, and recovers after disease remission [135], what seems to hold true even for subclinical hypercortisolism [136]. This observation was supported by another study in which homocysteinemia was higher in patients with active disease, with higher cystine level too, lower taurine, and unmodified methionine [89]. It should be noted that folate and vitamin B12 levels were lower in patients with active disease and improved with disease control. The authors suggested that these changes were partly induced by GC activation of CBS. Conversely, another observational study conducted on 27 patients with CS, compared with control subjects, showed no statistical difference in HCY level between these groups [137], but this study does not provide any information about other factors that may impact homocysteinemia. Despite these discrepancies, some authors point out that CS causes excess cardiovascular mortality that is not fully explained by “classic” cardiovascular risk factors. Since hyperhomocysteinemia is an independent cardiovascular risk factor, we could assume that altered OCM contributes to the cardiovascular excess risk of these patients. However, there are no studies on the subject to provide answers to this hypothesis.

GCs and OCM: Metabolic Considerations

Nicolaides et al. [138] showed that GC sensitivity in healthy subjects is associated with lower levels of blood serine, glycine, and threonine. This could suggest that GCs are involved in the transfer and use of these amino-acids in cells. The authors hypothesize that this activation of OCM could be a physiological counter-regulatory mechanism to reduce oxidative stress, increase energy production, and provide methyl groups for epigenetic modifications. Berf et al. [139] showed that administration of adrenocorticotropic hormone or cortisol in healthy volunteers leads to a drop of folate (23% and 24%, respectively) and cobalamin (13% and 19%, respectively), and a decreased in homocysteinemia, but with no statistical difference. This reinforces the hypothesis of an activation of OCM by the HPA. However, the literature shows contradictory results for the consequences of administration of synthetic GCs. [140–142]. Unfortunately, no more preclinical data are available to better understand the physiological dialog between these 2 pathways.

Mineralocorticoids and OCM

Data on the renin-angiotensin-aldosterone system are even rarer. Only one preclinical study on an animal model was conducted and identified an in situ synthesis of HCY in the adrenal glands of aldosterone-treated rats [143], which could potentially contribute to the very high cardiovascular risks of patients with aldosteronism.

On the other hand, few clinical data are available. Observational studies in humans have shown an increase in homocysteinemia in case of hyperaldosteronism [144, 145]. This was associated with higher pulse wave velocity, in a linear relationship. In this cross-sectional study on 145 patients with essential hypertension, hyperhomocysteinemia was associated with resistance to angiotensin conversion enzyme inhibitors (ACEi), a higher inflammation level and a lower regression of hypertension mediated organ damages [144]. The reduced effectiveness of ACEi was confirmed in a clinical trial on 10,783 patients with untreated essential hypertension [146]: after a 3 week treatment period with enalapril, those with HCY between 10 and 15 µmol/L and those above 15 µmol/L showed a smaller reduction in systolic blood pressure of 1.39 and 3.25 mm Hg, respectively, in comparison with those with HCY below 10 µmol/L. However, in Carnagarin et al. [144] study, the use of an angiotensin II receptor inhibitor (valsartan) was effective, irrespectively of the level of HCY. This underlines the key role of the angiotensin II type 1 receptor. Conversely, Zacharieva et al. [147] investigated the evolution of HCY levels in patients with CS after 1 month of treatment with valsartan, resulting in hyperreninism and a drop in aldosteronemia. There was no significant difference in HCY level between baseline and reassessment at 1 month. This could suggest that aldosterone is not responsible for hyperhomocysteinemia, but patients with CS show a so-called “pseudo-aldosteronism.” We cannot, therefore, rule out the possibility that the improvement in HCY levels was masked by the effect of cortisol itself and this result cannot be transposed to primary aldosteronism.

In the population of patients with CKD, hyperaldosteronism is frequent, as is hyperhomocysteinemia. As CKD is a major cardiovascular risk factor, we can assume a link between hyperaldosteronism, hyperhomocysteinemia and cardiovascular events in this population. Nevertheless, there is no specific study investigating the association between HCY, aldosterone and cardiovascular events in patients with CKD to date. Data regarding adrenal steroids are shown in Figure 4.

Fig. 4.

Synthesis of clinical consequences of the interactions between one-carbon metabolism and adrenal steroids. Dark red: hypothalamus-pituitary-adrenal-related elements. Dark blue: renin-angiotensin-aldosterone-related elements. Light red: higher level of one-carbon metabolism substrates or cofactors. Light blue: lower level of one-carbon metabolism substrates or cofactors. Green: clinical consequences indirectly related to GCs and one-carbon metabolism. Arrow = cause-consequence relationship; line = association. ACTH, adrenocorticotropic hormone; ACEi resist, angiotensin conversion enzyme inhibitors resistance; B9, vitamin B9; B12, vitamin B12; Cys, cystein; GC, glucocorticoid; GC sensit, higher glucocorticoid sensitivity; Gly, glycine; Hcy, homocysteine; HTS-related dmgs, hypertension-related damages; primary aldo, primary aldosteronism; psy disorders, psychatric disorders; Ser, serine; Tau, taurine.

Vitamin D and OCM

A Link between Vitamin D and OCM: Clinical Evidence

Vitamin D shows the properties of a steroid hormone and is classically considered the sixth hormone of this family. Its metabolism is not fully understood, but it seems to interact with OCM. Vitamin D deficiency is associated with hyperhomocysteinemia, and some clinical studies showed an inverse linear correlation between HCY and vitamin D levels [148, 149]. In addition, some studies showed that vitamin D supplementation reduces homocysteinemia [150].

Further, indirect evidence of the connection between vitamin D metabolism and OCM is that exposure to ultraviolet light, which is responsible for vitamin D synthesis by skin tissue, induces a reduction of homocysteinemia [151]. Seasonality and ultraviolet exposure, which influence cutaneous vitamin D synthesis, appear to be particularly associated with folate status.

However, HCY was not lowered by vitamin D supplementation in this population of patients with CKD [152]. Since the main site of hydroxylation in position 1 for 25-hydroxyvitamin D are the kidneys, we can assume that it is the 1,25-dihydroxyvitamin D that interacts most with the OCM. However, there are no published studies to confirm this hypothesis to date.

A Link between Vitamin D and OCM: A Matter of Inflammation

Other studies have also demonstrated that treatment with 1,25-dihydroxyvitamin D can reduce HCY levels by regulating CBS activity, such as this one conducted on pre-osteoblastic MC3T3-E1 cells [153], suggesting an action on the trans-sulfuration pathway and on inflammation.

The association between vitamin D, OCM, and inflammatory process has been shown in other organs. As an example, vitamin D deficiency is correlated with increased oxidative stress in the placenta through reduced CBS according to the observational study on 119 women (50 with preeclampsia and 69 normotensive) [154]. In this study, Nandi et al. showed that women with preeclampsia had lower placental protein and mRNA levels of CBS, while CBS was positively associated with the vitamin D receptor (VDR) level in the placenta. Thus, the anti-inflammatory action of vitamin D could be by partly explained by the activation of the trans-sulfuration pathway.

Importance of Vitamin D for Folate Transport

A potential regulatory mechanism of OCM by vitamin D is its action on folate transporters. Their expression is in part regulated by the VDR, so that vitamin D increases the capacity of transport of folate carriers [155–157]. Digestive absorption of vitamin B9 could, therefore, be improved, which could explain why blood levels of vitamins B9 and D are linked, as suggested by this cross-sectional study on 1,416 healthy adolescents [158]. Interestingly, the presence of these transporters in blood-organ barriers like the blood-brain barrier [159] and the blood-testis barrier [160], could contribute to disturbances in steroidogenesis at neurological [161] and gonadal levels in case of OCM alteration.

The links between these metabolic pathways do not seem to be limited to folate carriers. Indeed, Lucock et al. [162] identified an impact of polymorphisms of genes encoding for enzymes involved in OCM (MTHFR, serine-hydroxymethyltransferase, and methylenetetrahydrofolate dehydrogenase) on plasma 25-hydroxyvitamin D concentration [162], but the clinical consequences of these polymorphisms remain unknown.

Vitamin D and OCM: Implications for Human Pathology

Reduction of homocysteinemia by vitamin D supplementation appears to have a beneficial effect since it is associated with a decrease of the risk of cardiovascular diseases such as stroke [163, 164] and coronary artery disease [165], and with an improvement of liver balance parameters [166, 167].

Neurological pathologies are also affected by the dialog between these two metabolic pathways. Neurodegenerative diseases such as multi-system atrophy are accentuated by hyperhomocysteinemia, and an association between vitamin D deficiency and hyperhomocysteinemia has been demonstrated in these patients in a cross-sectional study on 53 patients [168]. Interestingly, the level of Klotho, a key protein for the metabolism of vitamin D, was lower, like in another study on vascular dementia [169]. In psychiatric disorders like schizophrenia, some teams found a trend toward hyperhomocysteinemia with elevation of Klotho (statistically not significant) but with no difference in vitamin D level [170].

Since vitamin D is a major determinant of bone turnover, several teams have prospectively evaluated the link between hyperhomocysteinemia and fracture incidence. Their work identified an association between homocysteinemia and fracture risk [171, 172]. Others have evaluated the effect of folate supplementation on bone mass and fracture risk. Data from these studies are summarized in the recent review of Mariangela Rondanelli published in 2021 [173]. Data concerning vitamin D are shown in Figure 5.

Fig. 5.

Synthesis of clinical consequences of the interactions between one-carbon metabolism and vitamin D metabolism. Light blue: vitamin D-related elements. Dark blue: decreased element. Light red: increased element. Green: regulation without precision. Arrow = cause-consequence relationship; line = association. 1,25OH vitamin D, 1,25-dihydroxy-vitamin D; CBS activ, cystathionine beta-synthase activation; CV risk, cardiovascular risk; Hcy, homocysteine; MTHFD, methylenetetrahydrofolate dehydrogenase (type 1 or 2); MTHFR, methylenetetrahydrofolate reductase; neurodeg, neurodegenerative; OCM, one-carbon metabolism; SHMT1, serine-hydroxymethyltransferase 1; UV rays, ultraviolet rays.

Clinical Implications of OCM and Steroid Metabolism Crosstalk in the Spectrum of Endocrine-Related Diseases

To date, interventions targeting OCM have been considered to treat several non-endocrine pathologies and developmental abnormalities [174–176]. Moreover, several substrates of OCM, especially HCY, methionine, folate levels, and SAM/SAH ratio, have been proposed as biomarkers for monitoring general health status, disease progression, and methylation status of individuals [177, 178]. In addition, as we have shown, OCM and steroid metabolism are closely related, but clinical data remain limited, so that transposability into routine care for endocrine disorders remains difficult to consider. We have chosen to focus on situations where data are relatively solid, enabling us to consider adapting management in the short to medium term.

Implications for Anti-Cancer Therapies

Targeting OCM as a supportive treatment to conventional anti-cancer therapies seems a serious approach. Cancer immunotherapies are probably the treatments for which the OCM has been most studied since OCM plays a role in the function of immune cells. So, inhibitors of several key enzymes of OCM are being studied such as SHMT, MTHFD, TYMS, DHFR, ACHY (S‐adenosyl‐l‐homocysteine hydrolase), or 1 (DNA methyltransferase type 1) inhibitors. Preclinical and clinical preliminary data suggest that these inhibitors are promising, and that OCM assessment may be a predictive marker of treatment response. Ren et al. [174] published a review on the subject recently, so it will not be detailed further. It should be noted, however, that the only key organ of steroidogenesis studied was the ovary [179–181], while no data are available for adrenal cancer. However, Assié et al. [182] showed that methylation profile has a major impact on the prognosis of adrenocortical carcinoma [182], but to date, no study of a treatment targeting epigenetic modifications has yet shown convincing results [183].

Apart from cancers of steroidogenic organs, some teams have studied the association between steroid receptor methylation and cancer. For breast cancer as an example, estradiol receptor inactivation by estradiol deprivation induces epigenetic changes at enhancers, which may lead to resistance to hormone therapy [184]. Furthermore, a recent meta-analysis conducted on 49,707 women with breast cancer and 1,274,060 controls found that high intake folate, vitamin B6, and vitamin B2 might decrease the risk of breast cancer and that folate vitamin B6 might decrease the risk of ER-negative/progesterone receptor-negative breast cancer. Similarly, in prostate cancer, androgen receptors appeared to contribute to epigenetic regulation directly by controlling the expression and activity of DNA and histone methyltransferases, and indirectly by controlling the expression of enzymes of OCM [9]. In addition, androgen deprivation therapies used in adjuvant therapies of prostate cancer seem to have a negative effect on the OCM, which could contribute to treatment escape. These two examples illustrate that steroid hormone receptors play an important role in treatment sensitivity in hormone-dependent cancers, and that the OCM is involved in treatment sensitivity or resistance depending on whether it is functional or not.

Implications in Reproductive Medicine

Data on sex steroids and their link with OCM are the most comprehensive to date. It is clearly accepted that maintaining a healthy OCM during the preconceptional period in the mother is essential for the healthy development of the unborn child, and there is some evidence to suggest that the same is true for the father. Otherwise, developmental abnormalities can occur, with serious consequences for the child [185, 186]. The World Health Organization, therefore, recommends treating mothers with folic acid from the preconception period [187].

Regarding MAR, data seem to confirm the importance of the OCM and the value of maintaining its homeostasis to improve the efficiency and safety of procedures. This was developed in a review published by Sfakianoudis et al. [8] in 2024, so we will not go into detail here. In a few words, the endocrine system affects both OCM and fertility status, while IVF techniques, especially culture conditions, directly impact OCM activity in gametes and embryos. In addition, serum homocysteine levels seem to be a promising biomarker for predicting IVF outcomes. The availability of the various metabolites involved in OCM, such as folates, vitamin B12, choline, betaine, and zinc, appears to be crucial to improving MAR performance, but further studies are needed before modifying current care recommendations as most studies used preclinical models. But the consequences of OCM alterations during pregnancy are not limited to the chances of a successful IVF. Indeed, influencing the mother's OCM during pregnancy could have an impact on this metabolic pathway in the child.

Implications in Public Health and Environmental Exposure to Endocrine Disruptive Chemicals

EDCs have the capacity of interfering with the normal hormonal action, metabolism, and biosynthesis, causing notably infertility in women [188]. More specifically, EDC can influence the endocrine systems in different ways, such as by exerting the same action as the natural hormone, inhibiting their activity, or modifying their synthesis, transportation, and degradation. Moreover, they can interact with membrane receptors, nuclear receptors and various intracellular pathways. Their binding to hormone receptors largely explains the deleterious effects they induce. EDCs are able to bind, among others, with androgen receptors, ER, GR, constitutive androstane receptor, estrogen-related receptor, aryl hydrocarbon receptor, pregnane X receptor, thyroid hormone receptor, and retinoid X receptor [182, 189–192]. So, sex steroid receptors are particularly concerned and their metabolism is easily affected [193–196].

On the other hand, environmental pollutants and EDCs can also significantly impact OCM [197–201]. Recent studies showed that intervention on OCM can reverse some side effects of EDC, as in studies in which folic acid treatment was used to counteract the deleterious effects of EDCs induced by their binding to Aryl hydrocarbon receptor (see review from Karl Walter Bock [202]). All these metabolic disorders, most of the time resulting from interactions with the hormonal signaling pathways mediated by nuclear receptors [203], are responsible for epigenetic modifications that partly explain the deleterious effects of EDCs, particularly on reproduction. For more details on the impact of EDCs on epigenetic and endocrine disorders, read the review of Sibuh et al. [204]. To date, the data in the literature do not allow us to envisage specific treatments targeting the OCM to restore functions impaired by EDCs. However, given the growing problem represented by EDCs, the collective awareness of their existence and their harmful role on health, and the ever-finer understanding of their mode of action and their interactions with other metabolic pathways such as the OCM and steroid pathways, more and more studies are being initiated to find therapeutic options.

Conclusion

To our knowledge, our review is the first to summarize literature data on interactions between OCM and steroid metabolism, considering all steroids in different clinical situations. We have also analyzed data on associated epigenetic modifications and reviewed the main clinical implications, and their consequences in terms of short- and medium-term treatment. These data allow us to draw several conclusions.

First, as we have shown, OCM and steroid metabolism are closely linked. The literature is rich regarding sexual steroids in women, with convincing studies concerning the effects of OCM disruption on the menstrual cycle, fertility, and pregnancy. Data are more contradictory for men, but an association between MS, altered androgen synthesis, and hyperhomocysteinemia which may contribute to increased cardiovascular risk seems plausible. GCs and mineralocorticoids have been much less studied, but available data also suggest an association between these metabolic pathways and OCM, which may also be related to cardiovascular mortality in patients with hypercortisolism and primary aldosteronism. Finally, several studies have shown that folate and vitamin D metabolism are linked with significant consequences for the body.

Clinical implications of these observations are limited for the moment since the level of evidence of most studies remains low. Previously cited clinical studies are summarized in Table 1, along with their level of evidence. Some guidelines suggest exploring OCM by measuring HCY, or treating people preventively by giving them folic acid, like in pregnancy for prevention of neural tube defects [205], or to the slow progression of dementia in the elderly [206], but there are currently no recommendations concerning endocrine pathologies. We have discussed the most advanced fields to date, namely, reproductive medicine, cancer, and EDCs, which open promising avenues from a therapeutic point of view. Therapeutic modalities may differ depending on the case and the target population. For the most common situations, we can imagine nutritional recommendations based on adaptations of dietary intake (or the use of nutritional supplements) to increase the availability of certain metabolites (folates, vitamin B12, methionine, choline, etc.), as is already the case with pregnancy. In other cases, such as cancer or exposure to EDCs, it seemsf more sensible to consider targeted treatment, focusing on a specific enzyme or metabolic pathway, rather than an overall increase in OCM.

Table 1.

Summary of the clinical studies discussed in the review with their main findings, methodology, level of evidence, but also their limitations

First author	Year	Findings	Limitations	Design	Level of evidence
Sex steroids in women
Smolders et al. [58]	2005	Oral estradiol reduced plasma HCY, vitamin B6, and albumin in postmenopausal women but did not alter whole-body homocysteine flux	Small sample size; short duration; did not assess long-term metabolic, or cardiovascular outcomes	RCT
Fischer et al. [62]	2007	Men and postmenopausal women had higher dietary requirements for choline than premenopausal women; estrogen appears to influence choline metabolism	Limited by sample diversity and potential hormonal variation; small subgroup sizes	RCT
Gaskins et al. [63]	2012	Higher dietary folate intake associated with higher progesterone and lower risk of anovulation in premenopausal women	Observational design; potential confounding factors	Prospective cohort study
Michels et al. [64]	2017	Found that higher HCY is associated with lower estradiol and progesterone during the menstrual cycle in healthy women	Observational cohort; cannot infer causality	Prospective cohort study
Dasarathy et al. [65]	2010	Observed a 30% drop in plasma HCY during pregnancy in healthy women, suggesting estrogen's regulatory role	Small sample (n = 10); no control group	Observational cross-sectional study
Mijatovic et al. [66]	1998	HRT lowered HCY levels in postmenopausal women	Short-term trial; not powered for clinical endpoints	RCT
Giri et al. [67]	1998	Oral estrogen improved HCY and lipid profile in elderly men	Small cohort; no placebo group	Open-label clinical trial
Mijatovic et al. [68]	1998	Raloxifene and conjugated estrogen reduced HCY levels in a double-blind study	Effects on hard outcomes (e.g., cardiovascular events) not measured	RCT
van Baal et al. [69]	1999	HRT decreased HCY levels in postmenopausal women	No long-term cardiovascular outcome data	RCT
Stevenson et al. [71]	2024	Proposed that S-adenosylmethionine may alleviate premenstrual syndrome by supporting methylation and monoamine regulation	Open-label design (no placebo control); small sample size	Open-label pilot clinical trial
Marci et al. [72]	2012	MTHFR 677T variant associated with lower estradiol and fewer oocytes in IVF.	Retrospective analysis; no dietary data	Retrospective cohort study
Thaler et al. [73]	2006	Women with MTHFR variant needed more FSH and had poorer IVF outcomes	Small sample, observational design	Prospective cohort study
Keller et al. [82]	1985	Higher HCY and cysteine levels were associated with endothelial dysfunction in women across menopausal stages	Cross-sectional design, surrogate marker for cardiovascular risk	Observational cross-sectional study
Maleedhu et al. [91]	2014	PCOS patients had significantly elevated plasma HCY levels compared to healthy controls	Small sample; did not control for dietary or genetic factors influencing HCY.	Observational case-control study
Badawy et al. [92]	2007	HCY levels were elevated in women with PCOS and correlated with insulin resistance	No longitudinal follow-up; other metabolic factors not fully controlled	Observational case-control study
Carmina et al. [93]	2005	HCY was higher in PCOS women without ovulation, in comparison with women with idiopathic hyperandrogenism and women with PCOS and ovulation	Cross-sectional design; phenotypic categorization may introduce heterogeneity	Observational cross-sectional study
Gözüküçük et al. [94]	2021	PCOS patients had significantly higher HCY and CRP levels, indicating possible inflammation and cardiovascular risk	No hormonal intervention studied; limited control for confounding variables	Observational case-control study
Harmanci et al. [95]	2013	Combined oral contraceptive and spironolactone reduced hyperandrogenism and increased HCY level	Short follow-up; tested combination therapy	RCT
Bayraktar et al. [96]	2004	HCY levels were higher in women with PCOS and congenital adrenal hyperplasia than in controls	Did not explore causality or long-term clinical outcomes	Observational cross-sectional study
Sex steroids in men
Bezold et al. [100]	2001	Observation of an association between the homozygous MTHFR C677T variant and male infertility	Short observational study; speculative pathophysiological mechanism	Observational cross-sectional study
Sung et al. [106]	2019	Found no significant difference in HCY between men with and without MS; testosterone was lower in MS patients	Weak correlation; cross-sectional design	Observational cross-sectional study
Krysiak et al. [107]	2015	Testosterone and fenofibrate co-treatment prevented HCY increase seen with fenofibrate alone	Small sample; limited to men with dyslipidemia and hypogonadism	RCT
Krysiak et al. [108]	2015	Testosterone plus metformin lowered HCY more than metformin alone in hypogonadal men with MS	Small sample; short duration	RCT
Ebisch et al. [110]	2006	Folic acid and zinc sulfate supplementation increased sperm concentration but did not affect endocrine parameters	Small sample size, and heterogeneity in participant baseline characteristics	RCT
Nematollahi-Mahani [111]	2014	Supplementation post-varicocelectomy with folic acid and zinc did not affect significantly the level of testosterone	Did not assess long-term fertility outcomes; testosterone secretion possibly limited by secretory reserve of remaining testicle	RCT
Adrenal steroids
Agarwal et al. [118]	2016	Cortisol and HCY levels were positively associated in patients with central serous chorioretinopathy under chronic stress, compared to controls	Single-center study with a small sample size and no prospective follow-up	Observational cross-sectional study
Levine et al. [120]	2008	Male PTSD patients exhibited significantly elevated serum HCY levels compared to controls	Study restricted to males; no assessment of diet or lifestyle confounders	Observational cross-sectional study
Jendricko et al. [121]	2009	PTSD patients showed higher plasma HCY compared to controls	No cortisol measures; limited psychiatric profiling	Observational cross-sectional study
Kale et al. [125]	2010	Elevated cortisol and HCY in unmedicated patients with schizophrenia	Case-control design; psychiatric medications not considered	Observational cross-sectional study
Kontoangelos et al. [126]	2015	Found correlations between cortisol, HCY, psychopathology, and metabolic factors like diabetes in psychiatric patients	Correlational analysis only; heterogeneous sample with multiple comorbidities	Prospective cohort study
Christian et al. [127]	2014	No effect of folic acid supplementation on serum cortisol in pregnant women in a large RCT (n = 737)	Did not assess HCY changes; only cortisol measured	RCT
Pavlik et al. [128]	2011	Found lower cortisol in follicular fluid of women with MTHFR variant; normalized with folate supplementation (800 µg/day)	Small cohort; indirect clinical relevance	Observational cross-sectional study
Krishnaveni et al. [129]	2020	Maternal hyperhomocysteinemia associated with higher stress response (cortisol and heart rate) in offspring	Longitudinal design but no mechanistic data; confounding possible	Birth cohort study
Krishnaveni et al. [130]	2014	Maternal HCY during pregnancy predicted higher cortisol and insulin resistance in children	Single-center, observational; did not test interventions	Longitudinal birth cohort study
Sosvorova et al. [133]	2015	Changes of blood and CSF steroid hormones, and HCY levels, induced by shunt insertion in patients with normal pressure hydrocephalus	Limited sample size, single-center study, and non-randomized design	Prospective cohort study
Yeram et al. [134]	2021	Found a correlation between elevated HCY, IL-6, and cortisol levels in Alzheimer’s patients	Small sample size, correlational data only	Prospective cohort study
Terzolo et al. [135]	2004	Patients with Cushing’s syndrome had higher plasma HCY, potentially linking cortisol excess and cardiovascular risk	Small cohort, lack of longitudinal follow-up	Observational cross-sectional study
Świątkowska-Stodulska et al. [136]	2012	Subclinical hypercortisolemia was associated with elevated HCY and altered alpha-1 antitrypsin levels	Small sample size and absence of causal analysis	Observational cross-sectional study
Ozsurekci et al. [137]	2016	Patients with uncontrolled Cushing's syndrome showed a non-different level of HCY, in comparison with healthy subjects	Small sample size; lack of longitudinal follow-up	Observational cross-sectional study
Nicolaides et al. [138]	2021	Found that GC sensitivity in healthy individuals affects OCM-related amino acid levels	Mechanistic in nature; limited clinical translation	Observational cross-sectional study
Berg et al. [139]	2006	ACTH and cortisol infusions in humans increased HCY and decreased B6, B12, and folates, showing a direct metabolic link	Small sample size; acute effects studied, not long-term outcomes	RCT
Kim et al. [140]	1997	Elevated cortisol and estradiol levels were associated with higher HCY in healthy subjects	Cross-sectional; no control for dietary/lifestyle variables	Observational cross-sectional study
Martinez-Taboada et al. [141]	2003	Corticosteroid therapy reduced HCY levels in patients with polymyalgia rheumatica and giant cell arteritis, possibly by lowering inflammation	Observational; effect of corticosteroids not isolated from disease impact	Prospective cohort study
Lazzerini et al. [142]	2003	Pulsed GC therapy in rheumatoid arthritis patients led to a significant reduction in plasma HCY levels	Small sample; effect of corticosteroids not isolated from disease impact	Non-randomized Interventional study
Carnagarin et al. [144]	2021	Higher HCY in primary aldosteronism associated with increased arterial stiffness and inflammation	Cross-sectional design; causality not demonstrated	Observational cross-sectional study
Tzamou et al. [145]	2013	Essential hypertensive patients had elevated aldosterone, inflammatory markers and HCY, suggesting aldosterone's role in vascular inflammation	Limited to hypertensive patients; anti-hypertensive drugs not considered	Observational cross-sectional study
Qin et al. [146]	2017	Elevated HCY levels attenuated the efficacy of ACE inhibitors in hypertensive patients	Potential confounding factors; lack of long-term outcome data	RCT
Zacharieva et al. [147]	2008	Valsartan reduced aldosterone but had no effect on HCY in patients with pseudo-aldosteronism (Cushing)	Indirect assessment of aldosterone; not primary hyperaldosteronism	Non-randomized interventional study
Vitamin D
Mao et al. [148]	2016	HCY levels were inversely correlated with folates, vitamin D, and B12 in patients with T2DM, hypertension, or CVD.	Cross-sectional study; does not establish causality	Observational cross-sectional study
Glueck et al. [149]	2016	25-hydroxyvitamin D levels were inversely correlated with HCY and were associated with lipid profiles	Single-center study; confounding nutritional or lifestyle factors not controlled	Observational cross-sectional study
Amer Qayyum [150]	2014	Found inverse relationship between serum 25-hydroxyvitamin D and HCY in asymptomatic adults	Cross-sectional design; no interventional component	Observational cross-sectional study
Bouazza et al. [152]	2022	Oral vitamin D3 reduced cardiometabolic risk markers including HCY in CKD patients, with racial differences observed	Small number of participants; short duration of intervention; only CKD Stage 3	RCT
Nandi et al. [154]	2021	Maternal vitamin D deficiency impacted polyunsaturated fatty acids metabolism and pregnancy outcomes, linked to altered one-carbon metabolism	Study based on associations, limited to observational interpretation	Observational cross-sectional study
Toole et al. [163]	2004	HCY-lowering treatment with B vitamins reduced the risk of recurrent stroke in patients with high HCY and low dose of B vitamins supplementation	Low level of HCY at enrollment; possible impact of folate fortification of the US grain or B12 malabsorption	RCT
Spence et al. [164]	2005	Post hoc analysis on patients without B12 malabsorption or taking B12 supplementation. HCY lowering was associated with reductionin the risk of events in the high-dose group compared with the low-dose group	Post hoc analysis; underpowered for subgroup effects	Post hoc analysis of an RCT
Verdoia et al. [165]	2021	Vitamin D deficiency was associated with higher HCY and more severe coronary artery disease	Observational design; cannot prove causality	Observational cross-sectional study
Walentukiewicz et al. [166]	2018	Nordic walking + vitamin D3 supplementation reduced HCY and ferritin in elderly women	Limited to older women; small sample size; no blinding	RCT
Al-Bayyari et al. [167]	2021	Vitamin D3 supplementation significantly reduced HCY and improved cardiovascular/liver risk	Short-term trial duration; potential population bias	RCT
Guo et al. [168]	2017	Klotho, vitamin D, and HCY levels predicted prognosis in patients with multiple system atrophy	Observational, predictive model only; small sample	Observational cross-sectional study
Brombo et al. [169]	2018	Low plasma Klotho levels were associated with vascular dementia, and negatively correlated with HCY	Observational cross-sectional design, no assessment of nutritional habits	Observational cross-sectional study
Yazici et al. [170]	2019	In schizophrenia patients, acute episodes were associated with lower Klotho and vitamin D and higher HCY.	Observational, cross-sectional; psychiatric medication effects not fully controlled	Observational cross-sectional study
McLean et al. [171]	2004	Elevated HCY was predictive of hip fractures in older adults, independent of vitamin B levels	Observational; fracture risk is multifactorial	Prospective cohort study
van Meurs et al. [172]	2004	Higher HCY levels were independently associated with increased risk of osteoporotic fractures	Observational; genetic and dietary factors not controlled	Prospective cohort study

The light-blue circles in the “level of evidence” column correspond to low to moderate levels of evidence, and the dark-blue circles to high levels of evidence.

ACE, angiotensin converting enzyme; ACTH, adrenocorticotropic hormone; CKD, chronic kidney disease; CRP, C-reactive protein; CSF, cerebrospinal fluid; FSH, follicle-stimulating hormone; GC, glucocorticoid; HCY, homocysteine; HRT, hormonal replacement therapy; IVF, in vitro fertilization; MS, metabolic syndrome; MTHFR, methylene-THF-reductase gene; OCM, one-carbon metabolism; PCOS, polycystic ovary syndrome; PTSD, post-traumatic stress disorder; RCT, randomized controlled trial.

To date, the literature is based mainly on animal experimentation or retrospective studies and very few clinical trials have been conducted. Mechanistic studies provide partial clues to the underlying pathophysiology but deserve to be extended. Epigenetic modifications associated with OCM disorders undoubtedly play a major role in the pathophysiology of endocrine diseases associated with steroid metabolism. However, studies on the subject have only recently begun, and more work is needed to better understand the underlying mechanisms and identify the most promising therapeutic avenues. In this sense, preclinical and in silico models remain indispensable, while keeping a clinical eye on the situation. Translational research applies perfectly to this theme: a “from bench to bedside” approach will bring real benefits to the population in terms of morbidity and mortality, and quality of life.

OCM and steroid metabolism have a close relationship. Research efforts must continue to better understand their interactions and, thus, propose effective therapeutic options for the management of endocrine pathologies.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

This study was not supported by any sponsor or funder.

Author Contributions

N.S. carried out the literature review. N.S. wrote the initial manuscript. B.L.-M., R.-M.G.R., and M.A. reviewed the manuscript and made modifications as specialists in OCM (B.L.-M. and R.-M.G.R.) and in pathologies related to steroid metabolism (M.A.).

Funding Statement

This study was not supported by any sponsor or funder.