Anemia is a serious public health problem affecting people all over the world, and is one of the most frequent complications involved in pregnancy, imposing a tremendous toll on well-being of approximately 40% of pregnant women in China (Li et al., 2018). Gestational anemia, according to the World Health Organization (WHO), is defined as a hemoglobin concentration (Hb) < 110 g L^-1. Gestational anemia mostly occurred in the second and third trimester (Zhao et al., 2018). Multiple factors account for gestational anemia, nutritional iron deficiency anemia (IDA) (approximately 75%) and folate megaloblastic deficiency anemia being the commonest (Sifakis and Pharmakides, 2000; Goonewardene et al., 2012).

Anemia in pregnancy has adverse effects on maternal and neonatal health. Weakness, fatigue, being vulnerable to infection, reduced work capacity, and productivity are typical symptoms during pregnancy (Prema et al., 1982; Hunt, 2002). Current evidence suggests severe gestational anemia could be associated with an increased risk of preterm birth, low birth weight, and even neonatal and maternal mortality (Khan et al., 2006; Rahman et al., 2016; Figueiredo et al., 2018; Guignard et al., 2021). Furthermore, it has been reported that babies born to anemic mothers are prone to exhibit future poor cognitive performance and delayed mental and motor development in adolescence and adulthood (Anker et al., 2009; Camaschella, 2015).

The current clinical method is generally applied to cross-sectional diagnosis rather than prediction in the long term. Although Hb concentration serves as a golden standard for the diagnosis of anemia in pregnancy, it has limitations in precise prediction of potential impending anemia in normal pregnant women. Additionally, it remains controversial to use Hb concentration to distinguish true or absolute anemia from relative anemia, ascribed to a normal physiologic increase of plasma volume (Sifakis and Pharmakides, 2000). Thus, an accurate determination of gestational anemia is essential and of enormous clinical importance for prevention and management of anemia in pregnancy.

The human gut hosts an immense number of resident microorganisms, collectively termed as the microbiota (Structure, Function and Diversity of the Healthy Human Microbiome, 2012; Bäckhed et al., 2004). There has been accumulating evidence that the gut microbiota is implicated in enteric, metabolic, and psychiatric diseases (Imhann et al., 2018; Vich Vila et al., 2018; Thomas et al., 2019; Wirbel et al., 2019). Previous studies have documented that gut microbial dysfunction (e.g., deficiency in lactobacillus) is related to IDA and gut microbiota could promote hematopoiesis, underlying the close relationship between gut microbiota and anemia (Balamurugan et al., 2010; Khosravi et al., 2014). During pregnancy, host profoundly remodeling of the gut microbiota could cause symptoms of metabolic syndrome, which might be related to transportation and storage of host Fe, and ultimately lead to the occurrence of gestational anemia (Koren et al., 2012). Microbial signatures have been elucidated to function as novel biomarkers to discriminate patients suffering from illness and healthy individuals (Liu et al., 2019; Wei et al., 2020). To date, an anemia classifier in pregnancy has been constructed (Long et al., 2021); however, no efforts have been made to predict the subsequent incidence of anemia in normal gestational women.

To reach a better understanding of how gestational anemia is developing and regulated, herein, we conducted a prospective study to rigorously evaluate dynamic landscape of gut microbiota varying from healthy status to identified anemic status in pregnant women. Leveraging the discriminative microbial signatures in the early stage of pregnancy when anemia did not occur yet, we prospectively built an accurate prediction model for the subsequent incidence of gestational anemia. Our findings help identify pregnant women with high risk of anemia in general population and ultimately improve quality of life and well-being of women and children.

In the current study, we delineated that GA microbial dysbiosis was characterized by several bacterial genera and structured CAG. A cross-sectional anemia classifier in the first trimester and second trimester has been constructed (Long et al., 2021); however, limited efforts have been made to prospectively predict future anemic women. The greatest advantage was that for the first time, a prediction model for upcoming anemia in normal pregnant women with relatively high discriminatory power was established based on novel gut microbial signatures.

It was challenging to determine the etiology of anemia and confirm IDA in pregnancy. Apart from Hb, more laboratory examinations (e.g., serum iron, transferrin receptor, transferrin saturation, and bone marrow iron) should be taken into consideration. The most common true anemia in pregnancy was IDA. In addition, there were no other types of pathological anemia (e.g., aplastic anemia and hemolytic anemia) remaining in our study. Thus, anemic women were basically postulated to suffer from IDA.

Clinical indices serve as generally accepted diagnostic criteria for GA cross-sectionally; however, they were confirmed to have very limited predictive value for potential impending anemia in normal pregnant women according to our study. Gut microbial signatures exhibited impressive performance in the prediction model. Bacteria were significantly differently abundant in GA and healthy control in the second trimester. Of note, 93.03% of healthy control and 97.44% of anemic women took folic acid and iron supplement from conception to the second trimester, implying the sufficient iron storage. From the perspective of potential mechanism, the altered gut microbiota in the early stage was conjectured to be subsequently associated with the altered health condition (i.e., from iron sufficiency to iron deficiency or malnutrition) and further accounted for the upcoming anemia.

Megamonas, Veillonella, and Haemophilus were enriched in GA. Megamonas could act as a beneficial bacterium, and it has been reported that compared to healthy microbiota, canine anal furunculosis (CAF) microbiota showed a decreased abundance of Megamonas (Maldonado-Contreras et al., 2020). Nevertheless, another study illustrated infant vitamin D supplementation was associated with a lower abundance of Megamonas in gut microbiota, implying the potential competitive relationship between vitamin D and Megamonas (Drall et al., 2020). Veillonella species, documented as the Fe (III)-reducing genera, were capable of supplying Fe (II) to combine with oxygen in Hb (Jin et al., 2019). We hypothesized there might be a negative feedback regulation, host generating more Veillonella species when detecting less Hb combined with Fe (II). Haemophilus is a genus of Gram-negative, containing several markedly pathogenic bacteria, such as Haemophilus influenzae causing septicemia. Indigenous bacteria might inhibit host Fe transport and storage via producing metabolites that suppress hypoxia-inducible factor 2α (HIF-2α), assumed as a master transcription factor of intestinal Fe absorption and increasing the Fe-storage protein ferritin (Das et al., 2020). Decreased incidence of Blautia has been detected in the gut microbiota of obese children and Blautia genera might help to reduce inflammation causally linked to obesity-related complications (Benítez-Páez et al., 2020).

Microbial network has been an increasingly popular tool to explore microbial community structure (Röttjers and Faust, 2018). Ecologically, gut microbiota exists in functional groups named “guilds” rather than isolation and thrives in communities with large numbers and develops close interactions, which are critical evolutionary pressures for natural selection in microbial evolution (Faust and Raes, 2012; Ma et al., 2020). We sought to reduce the dimensionality of microbial datasets to identify GA more effectively based on CAG, and interestingly, the prediction model exhibited a much higher discriminatory power.

It is noticeable that normal physiologic changes in pregnancy lead to a relative or absolute reduction in Hb concentration. However, it is still an open question that this “anemia” is physiologic or pathologic. Given gut microbial signatures were free from influence of hydremia, a better diagnostic or predictive performance using gut microbial signatures could be achieved when it comes to either true anemia or physiologic anemia.

There were some advantages of our study. Firstly, samples were collected prior to treatment initiation in a large and well-characterized cohort. Secondly, antibiotics use was controlled in analysis. On the other hand, most of pregnant women enrolled in the study claimed antibiotics were used very early in pregnancy at a low dose and once they were discovered to be pregnant, antibiotics use was avoided as much as possible, which meant a possible lesser impact of antibiotics on gut microbiota. Thirdly, we constituted the first exploration to prospectively predict the risk of anemia in healthy subjects based on gut microbiota. Lastly, a much more accurate prediction model was built based on CAG network.

There were several limitations. Firstly, as we have discussed above, diagnostic evidence of IDA was not sufficient enough. Secondly, we did not construct a GA classifier using gut microbiota in the third trimester since that Hb was supposed to be a better alternative, quicker and costing lower. There was no information on vitamin D levels or supplementation in women, which was supposed to be a factor associated to gut dysbiosis and consequently GA. In addition, lack of metagenomics sequencing limited data interpretation from the angle of species level and bacterial function. Finally, there was not an independent cohort to verify the prediction model; our study merely provided evidence of association rather than causality and further studies are supposed to be conducted to validate the association.

Our results showed that alterations in the gut microbial community were associated with anemia in pregnancy. Moreover, microbial signatures relatively accurately predicted the subsequent incidence of anemia in normal pregnant women. Our findings could provide new insights into understanding the role of gut microbiota in GA, identifying high-risk individuals, and modulating gut microbiota as a therapeutic target, thus improving quality of life and well-being of women and children.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

The studies involving human participants were reviewed and approved by the Ethics Committee of Nanjing Medical University [FWA00001501 No. (2017) 003]. The patients/participants provided their written informed consent to participate in this study.

Conceptualization: XW and YX. Formal analysis: HW and SD. Methodology: HW and YQ. Writing original draft: HW and SD. Verifying the underlying data: YQ and XY. Review and editing: YQ, XY, TC, XW, and YX. All authors contributed to the article and approved the submitted version.

This work was supported by China-U.S. Program for Biomedical Collaborative Research (NSFC-NIH) (81961128022), the fifth phase of “333 High-level Talent Training Project” of the Jiangsu Province (BRA2020070), Postgraduate Research & Practice Innovation Program of Jiangsu Province (JX10313741), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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