All nutrients come from the diet, and diet is one of the most important aspects impacting and modulating health and the gut microbiota. This ‘microbial’ organ within our guts, and the set of genes it contains, called the microbiome (El Hage et al., 2017) have been extensively studied over the last decade. Several of these studies, now published in prestigious journals, have uncovered that dysbiosis, or an imbalance of the intestinal microbial communities (microbiota) and the decrease in ecological diversity within the gut, are related to gastrointestinal, metabolic, and autoimmune diseases, mental disorders, and even some types of cancer (Derrien and Veiga, 2017; Deng et al., 2021; Zhao et al., 2021; Christovich and Luo, 2022; Horn et al., 2022). Since the gut microbiome has a crucial role in the absorption and metabolism of nutrients, both macro and micro, aiming for a balanced microbial community in the gut, helps maintain the host homeostasis, and builds the intestinal barrier (DAS & Nair, 2019). The presence or absence of specific microbial genera or species has been associated with multiple diseases, most of them, non-communicable ones, such as inflammatory bowel disease, diabetes, obesity, some types of cancer, Parkinson’s, and Alzheimer’s, among others (Novakovic et al., 2020; Zhang et al., 2020; Bardenhorst et al., 2023). Regarding micronutrient absorption, Hadadi and collaborators (2021) addressed the importance of the gut microbiome for maintaining the balance of the host vitamins and minerals. They also address the micronutrient-microbiome axis as a bidirectional entity, and according to other studies, several micronutrient deficiencies could be positively or negatively associated with the gut microbiota (Hadadi et al., 2021). Another study carried out by Maynard and Weinkove has revealed that certain host microbes, such as C. elegans and E.coli, play a role in the effective supplementation of micronutrients by the secretion of siderophores (iron and B12), or the uptake and conversion into more readily absorbable derivatives or micronutrients, such is the case of folic acid (Maynard and Weinkove, 2020). Moreover, Bielik and Kolisek (2021) reported the positive effect of probiotics on mineral absorption, stating they are promising due to their ability to modulate the composition and metabolism of the gut microbiota (Bielik and Kolisek, 2021).

On the other hand, the perinatal period is marked by hormonal, immunological, and—especially during the late stages of healthy pregnancies without complications—by inflammatory changes that alter the function and bacterial composition of the mother’s gut (Mandal et al., 2016). Estrogen and progesterone also impact this composition through their effect on bacterial metabolism and the increase in abundance of pathogenic bacteria (Edwards et al., 2017). It is also known that the gut microbiota contributes to the regulation of glucose metabolism in pregnancy (Brantsæter et al., 2011). For example, the abundance of the genus Collinsella sp. Is positively correlated with circulating insulin, and low dietary fiber intake was associated with a gut microbiota favoring lactate fermentation, while high fiber intake promotes short-chain fatty acid-producing bacteria (Fu et al., 2022). Related to this, low dietary fiber may enable the overgrowth of Collinsella sp. and alter the overall fermentation pattern in gut microbiota (Gomez-Arango et al., 2018). This suggests that dietary choices during pregnancy can modify the nutritional ecology of the gut microbiota. Besides, in a study conducted on pregnant women, it was shown that there are significant differences in the relative abundance of several genera in women on a vegetarian diet, specifically a reduction in Collinsella sp., Holdemania sp., and an increase in the relative abundances of Roseburia sp. and Lachnospiraceae sp. (Barrett et al., 2018). The most recent research on gut microbiome during the perinatal period in mice shows that the characteristic microbiota of the third trimester of pregnancy, increases weight gain, insulin resistance, and a greater inflammatory response when transferred to germ-free mice (Koren et al., 2012). Studies in other populations different from the American and European ones, such as those from Latin America, the Caribbean, Asia and African, or from women and children’s cohorts, are urgently needed as well as their underlying data (Magne et al., 2016), in order to properly acknowledge the gut microbiome in world-population scale, and be able to develop solutions to improve the health status of the groups belonging to these communities, in need of tools for this purpose.

In the quest for the characterization of generalizable traits of the gut microbiome, metabolomics has appeared as one of the most useful techniques to study it, being defined as a comprehensive analysis of all metabolites in a biological system with their proper identification and quantification (Fiehn, 2002), and is recognized as a powerful top-down systems biology approach, for understanding the genetics-environment-health paradigm and identifying clinically relevant biomarkers (Moco et al., 2013). Metabolomics studies within the gut, which we name here the gut metabolome, have been increasing in the last years due to the strong relationship found between some gut microbiome metabolic pathways and diseases, especially non-communicable ones, and due to the involvement of the gut microbiota in several biochemical functions directly associated with perturbations that can lead to the development of diseases (De Preter et al., 2015). Moreover, the identification and relative quantification of metabolites in these environments can point out lifestyle and dietary habits, and nutrient balance in the gut, which in turn, allows the highlighting of specific disease predispositions (Vernocchi et al., 2012), such as a mineral or vitamin deficiency of (Lai et al., 2022; Wan et al., 2022), an excess of an inflammatory molecule (Zhang et al., 2021), among others. Metabolomics is a technique that can be performed over different biological matrices such as cells, tissues, stool samples, and biofluids such as plasma, saliva, urine, and blood. The sample selection will always depend on the research or clinical question, but biofluids are typically used to identify biomarkers, whereas tissues and cells are used to investigate mechanisms associated with the pathophysiological process (Chetwynd et al., 2017). Regarding human stool samples, which reflect the gut metabolome, most of the published research has focused on characterizing its complex bacterial composition using next-generation microbial DNA sequencing and sophisticated metagenomic techniques. However, a growing number of microbiome researchers are recognizing that considerable information could be gained by using a more integrative approach that also includes comprehensive fecal metabolite analysis (Karu et al., 2018; Haffner et al., 2022).

One of the techniques widely used to study the gut (fecal) metabolome, is liquid chromatography coupled to mass spectrometry (LC-MS), which does not usually include derivatization steps. The technique can be performed in a targeted or untargeted mode, depending on the experimental design, and multiple approaches can be taken to analyze the raw data, thus allowing the recognition of multiple chemical families and the greater elucidation of the chemical space, phenotype, and nutrients composition of the gut. In this research, we used classical molecular networking and untargeted metabolomics to make a pilot and first approach toward the characterization of the chemical space of the gut microbiota (gut metabolome) of women from Antioquia, Colombia. These women conform to a pilot cohort (n = 23) of pregnant, 7) lactating 9), and reproductive-age women 7) acting as controls. By using LC-QTOF-MS/MS metabolomic techniques and data analysis, we aimed at the identification and quantification of several compounds of nutritional importance for the baby’s appropriate development, which are supplied by the mother during these fundamental stages of pregnancy and lactation. As stated above, macronutrients, micronutrients, and derived metabolites play a key role in the balanced nutrition of both mother and baby, and both are intrinsically related to the gut microbiome. Thus, with this pilot study, we wish to contribute to the maternal nutritional body of knowledge in our area of the world since to date, there are no published studies that explore the chemical diversity of the Colombian female population during the mentioned stages, despite these being key interventional periods for nutrition. It is our wish that the knowledge derived from this pilot study and its validation in larger cohorts can help avoid future developmental complexities in an individual during later stages of their lives also avoiding future health complications. Thus, the relevance of this kind of pilot study and as mentioned earlier, the further validation of its preliminary results in larger cohorts is evident, to broaden our knowledge of the gut microbiome chemical space and phenotype in the populations in Colombia, Latin America, and the Caribbean.

Frozen Stool samples were weighted, lyophilized at −80°C for 72 h, and weighed again to determine the removed water content percentage. Then, nitrogen gas was injected for 10 min into each sample to guarantee an inert environment. For extraction, 60 mg of each lyophilized sample was mixed with 300 µL of MeOH and vortex-mixed for 5 min. Subsequently, samples were taken to an ultrasound for 30 min and vortex-mixed again for 5 min. Finally, samples were centrifuged at 180,00x g, 4°C for 15 min and 100 µL of the extract was used for the analysis by LC-QTOF-MS (Cheng et al., 2020).

The gut microbiome of humans is estimated to comprise around 45 million non-redundant genes (Sender et al., 2016; Tierney et al., 2019). When compared to the human genome and its approximately 20.000 genes, the microbiota exceeds this capacity more than 1,000 times, evidencing its profound potential to influence the biochemical environment of the host (Lee-Sarwar et al., 2020). More surprisingly, the Human Microbiome Project discovery of metabolic pathway abundances in the gut is relatively consistent across populations, while taxonomic composition varies between individuals. This denotes that a core set of conserved pathways is associated with microbial genes, but their abundance varies depending on the taxonomic composition of this environment (Huttenhower et al., 2012). Besides, microbial functions are closely reflected by the composition of the metabolome, or better said, the collection of small molecules present in a sample. Although the human body houses many discrete microbiomes and metabolomes, the gut is taxonomically the most diverse and largest site (Thursby and Juge, 2017). Gathering the ideas exposed before, gut metabolome studies are now being considered the next Frontier to unveil the gut microbiome and are becoming prevalent in studies concerning this so-called organ, since they reflect the phenotype of the individual and thus, provide a more accurate perspective of the biochemical and metabolic processes taking place in this environment.

In this respect, our study offers various conclusions, some of them new and others reinforcing previous evidence from the scientific community on the gut microbiome and its associated metabolome. First, the gut metabolome or chemical space associated with the gut is a rich environment where important biomarkers of health can be detected (Figures 3–7). This is important in the way that the same metabolites are not always detected in serum metabolomics (Dhakan et al., 2019; Wen et al., 2020). Second, although our cohort is small (n = 23), the gut metabolome of pregnant, lactating, and women of reproductive age from our region of Antioquia (Colombia), evidences structural differences between groups both in its composition and relative abundance, denoting a plausible different core composition of microbial and host metabolism (Figures 2–6). In the case of microbial metabolism, these differences can be attributed to the differential taxonomic communities associated with each group of women according to their physiological state, as stated before (Koren et al., 2012). Specifically in this study, our findings suggest that, for the physiological stages of pregnancy and lactation, metabolites related to fats mobilization and membrane formation such as glycerophosphocholines, glycerophosphoserines, and fatty acids; hormones (corticosteroids); bile acids; carbohydrates and sterols are increased, being significantly overexpressed both in lactating and pregnant women’s gut metabolome (Figures 3–7). This result is expected since, specifically for lipids metabolism, previously published findings reporting multiple physiological changes that occur in healthy, gestating women, which contribute to the alterations in lipid profiles, mainly to support the developing fetus to whom cholesterol and essential fatty acids are essential for normal development (Wild and Feingold, 2000). Also, larger doses of foods rich in healthy fats are needed to meet the metabolic demand, especially for the nutrient choline, which is highly available in fats (Zeisel, 2013). Our findings, also correlate with a transformation of the gut microbiota into a proinflammatory immune state as pregnancy progresses (Koren et al., 2012; Trevisanuto et al., 2013), since prostaglandins, a main biomarker of both the promotion and resolution of inflammation (Ricciotti and Fitzgerald, 2011), are increased in this last group (Figure 3A). Also, corticosteroids such as hydroxypregnene are decreased in the lactating stage but increased in pregnancy, which could be an indication of prescription of corticosteroids to treat symptoms of autoimmune conditions or of inflammation, as well as being one of the most important antenatal therapies available to improve newborn outcomes before anticipated preterm birth (El-Sayed et al., 2017). Also, interestingly, glucocorticoid compounds with anti-inflammatory and immunosuppressive effects are commonly used to treat inflammatory bowel disease, asthma, allergies, and rheumatic diseases and are upregulated among the pregnant group as well, denoting a normal behavior in pregnancy where maternal glucocorticoids critically rise reaching up to a 20-fold increase of mid-pregnancy concentrations (Solano and Arck, 2020). Also, in lactating women, we found an increased ratio of palmitoyl dopamine, which is an endogenous, long-chain, linear fatty acid dopamide with entourage effects in the endocannabinoid system (Matsumoto et al., 2016). This is of high interest since its biological significance in lactation is understudied and it is then an interesting metabolite to further analyze as a biomarker.

Continuing with deeper insights into the unique metabolic traits of each group of the cohort, it can be observed in Figures 3A,C, that sphingolipids and ceramides are upregulated among the lactating group. These metabolites are involved in the regulation of insulin resistance during the perinatal period (Rico et al., 2017). It is also abundant in human breast milk and has a positive impact on cognitive functions and brain development of the infant (Dei Cas et al., 2020). In addition, prostaglandins are known to affect uterine contractility and cervical ripening and are important in the initiation of labor (Wood et al., 2021). These we found as being upregulated in pregnant women, which denotes the correct reflection of the gut chemical environment with the state of the individual. Also, these findings are in accordance with what Liang and collaborators found in 2022. They found nine metabolites differentially expressed in stool samples from pregnant women in the third trimester and full term. These included levels of lipids and lipid-like molecules, such as long-chain fatty acids and 21-hydroxysteroids, being upregulated in pregnant women compared to full-term, whereas the levels of amino acids and dipeptides showed a downregulation. On the other hand, 20-hydroxyarachidonic acid and palmitic acid were enriched at the time of full-term pregnancy. Other metabolites like cyclohexylsulfamate, 3,3-dimethylacrylic acid, hydroxyisocaproic acid, and phenylalanylphenylalanine (Phe-Phe) were also identified in fecal samples from Chinese pregnant women (Liang et al., 2022).

In summary, for the chemical space composition of the gut or as called by us in this research, the gut metabolome of our cohort, we mainly observe metabolites that are either produced by the gut microbiome bacterial metabolism or modified by it. Examples of these compounds are bile acids, bilirubin (van Best et al., 2020; Garcia et al., 2022), tryptophan (Stoll et al., 2016; Gao et al., 2020), hormones (Jiang et al., 2021; Marć et al., 2022), glycerophosphocholines. Thus, we can suggest that the gut metabolome can be seen as a reflection of an appropriate gut microbiome profile, understating appropriate as the balance of the communities according to what has been reported for a healthy state in a certain condition. Also, we prove that fecal samples, which contain small and large molecules from the gut microbiome, can indeed reflect the net result of nutrient ingestion, digestion, and absorption by both gut bacteria and the gastrointestinal tract (Ulaszewska et al., 2019.)

Another interesting finding in this study, regarding a more general behavior of the population, is the reinforcement that gut metabolomics reflects diet, drug consumption, and pharmacokinetics, even if the person does not declare it or the initial data collected in the enrolling questionnaires, such as the one used in this study. For example, metabolites such as coumarin, omega-3 and omega-6 fatty acids, and vitamin E were detected, which are associated mostly with a plant-based diet (Pistollato et al., 2015; Sebastiani et al., 2019); drugs such as antihistamines and anticoagulants were found in the volunteers that declared its consumption. Central nervous system stimulants like caffeine were found significantly higher in the control group, and it is coherent with behavior during pregnancy and lactation, a time when women avoid high doses of this metabolite.

These findings are in accordance with the ones by (Pires et al., 2019), where the authors found significant metabolic changes in the chemical ecology of the gut environment between populations of individuals living in the Amazon, and those from an urban, industrialized setting, which was mainly attributed to dietary differences as well as diverse patterns of environmental exposure. Furthermore, organosilicons and other toxins coming from the heating of food, plastics, and agrochemicals, which can be harmful as they accumulate over time only when they have small particle sizes, are significantly abundant among the group of pregnant women from our region (Antioquia) which is not an encouraging finding from the public health perspective. Considering these compounds, specifically, those with a low silicon particle size, can overcome biological membranes and skin barriers, being possibly transferred to the baby (Dixon and Williamson, 2016), and can be endocrine disruptors. Organosilicon compounds are widely encountered in commercial products such as sealants, adhesives, coatings, medical products, and cosmetics (Mojsiewicz-Pienkowska et al., 2016).

Additionally, other, natural, compounds were found at toxic levels suggesting the ability of the methodology followed in the study to detect abnormal levels of naturally present molecules. Such is the case of volunteer 14, part of the pregnant women group, who showed significantly higher levels of primary bile acids which could be related to cholestasis and cause irreversible toxicity to the fetus (Mazzella et al., 2001).

We would like to highlight as well, that studies in other populations different from the American and European ones, such as Latin peoples, Asian people, African people, or women and children, are urgently needed as well as obataining the underlying data to properly acknowledge the gut microbiome and its associated chemical space on a world-populationscale. This will allow an appropriate and significant characterization of the gut microbiome of other countries and regions, as well as of different conditions such as healthy pregnancies and lactation in women. Furthermore, gut microbial communities change with age and sex; with one study showing a strong positive association between age and alpha diversity in young adults (less than 40 years old), and women were found to have more diversity than men (De la Cuesta-Zuluaga et al., 2019), thus groups like women, which have been previously excluded from study cohort in microbiome studies, should be included. Finally, there is a need to convert findings like the ones in this study into affordable and accessible strategies to measure gut health in every population. This reinforces the need for more studies on the gut metabolome in larger, and the use of ordinated (e.g., PCAs), clustering approaches, supervised models, or the employment of unsupervised models like NMF which have the added advantage that pre-calculated signatures of bacterial assemblages can be reapplied to even a single metagenome, removing the need for large cohort sizes capturing microbiome variation (Frioux et al., 2023).

Overall, this exploratory study serves as a starting point to describe the gut metabolome of healthy pregnant and lactating women from Antioquia, Colombia, a special population regarding the requirements of these physiological states and the profound impact that maternity can have on child development in terms of healthy growth, but also in its adequate cognitive development, as well as its regional nature. The two approaches to analyzing metabolomics data were complementary in the study, we could say that molecular networking serves as the starting point to have a broad panoramic view of the metabolites present in the chemical space. These can be later found in a more stringent and quantitative way by the untargeted metabolomic analysis.

Gut metabolomics studies can shed light on the phenotype differences of a population with a specific condition, such as healthy pregnancies and lactation, from others. In this study, within a cohort of women from Antioquia, Colombia, we found that lactating women can be differentiated from other pregnant and reproductive-age non-pregnant nor lactating women by a gut metabolomic profile enriched in carnitine derivatives, glycerophosphocholines, bile acids, ceramides, glycerolipids, and glycerophosphoserines. Pregnant and lactating women, when compared to reproductive age controls, are enriched in glycerophosphocholines and glycerophosphoserines, corticosteroid hormones, bile acids, fatty acids, carbohydrates, and sterols. These metabolites can be further studied in a larger population, to scale their occurrence, and plausibly develop preventive biomarkers for healthy pregnancies. Metabolites such as toxins, xenobiotics, and environmental contaminants, which can be missed by other techniques, and are ubiquitous harmful foreign chemicals present in the environment, were detected in this study in fertile age, non-pregnant nor lactating women denoting a presence in the diet and lifestyle of women that can become pregnant in the future, posing a risk to the infant’s health. The metabolite Palmitoyl domamine was found as upregulated in lactating women, being reported for the first time in a gut metabolomics study, and in this specific population. Fibers and phytonutrients such as lignans and neolignans, glycosides, and lipid-derivatives from plant food sources, plant polyphenols, raffinose trisaccharides, glucosinolates, and lipopolysaccharides which are proven prebiotic compounds, were also found by molecular networking analysis in this cohort, denoting the capacity of this method to detect dietary compounds. Although our cohort is still limited for scaling these conclusions to a population level, this research sets an initial basis in our country and region, Latin America, for future population level measurements of a normal gut metabolome composition during the important perinatal period, which can provide valuable information to enhance public health nutrition strategies in middle-income countries.