Necrotizing enterocolitis (NEC) is a common and potentially fatal disease that typically affects preterm infants (PIs) and very low birth weight infants (VLBWIs). NEC is an inflammatory disease of the intestines, which ranges from mucosal injury to bowel necrosis and perforation. Although NEC is extensively studied, the current therapeutic approaches are unsatisfactory and mortality and long-term morbidity remain high (1). Breast milk (BM) feeding has a protective role on the development of NEC and is associated with reduced NEC incidence when compared to formula feeding (2). Since factors that are present in BM are also found in amniotic fluid (AF), it has been hypothesized that AF has similar protective effects (3). AF is widely used for its diagnostic purposes and is often considered as “biological waste”. However, researchers have started to elucidate the therapeutic potential of AF in in vitro and in vivo studies in animal models (4, 5). Our intent was to identify relevant research on AF, its impact on the gastrointestinal tract (GIT) and to investigate the potential therapeutic application of AF on the development of NEC. We aim to define a theoretical framework to guide future research on clinical application of AF-based therapy against NEC.

The pathogenesis of NEC is understood as a complex and multifactorial process, in which the immature intestines and microbial dysbiosis play a pivotal role (1). PIs are particularly susceptible to developing NEC by facilitating an exaggerated inflammatory response to colonizing bacteria in the premature gut. The abnormal gut microbiota of PIs has been linked to NEC pathogenesis (6). Moreover, Toll-like receptor 4 (TLR4) signaling is proposed to play a role. TLR4 is expressed at higher levels on the intestinal epithelium in PIs and leads to increased apoptosis of enterocytes, hampered intestinal mucosal healing and the production of pro-inflammatory cytokines (6, 7). BM, and potentially AF, provides important protective mechanisms to diminish the inflammatory response in the neonatal intestines and reduce the risk of NEC.

The fetus develops in the amniotic sac. Initially, AF is synthesized from maternal plasma and absorbed through the fetal skin. Subsequently, the fetus contributes to the production of AF by urination, through the trans-membranous pathway and other pathways of secretion. After keratinization of the skin, the fetus takes up AF through the GIT. Until the 20th week of gestation, the content of AF is similar to that of fetal plasma. Following skin keratinization, the content changes (8). Water generally accounts for 98%, while the remainder consists of soluble components: minerals, proteins, carbohydrates, lipids, steroids and hormones (9). AF also contains exfoliated skin, respiratory tract, urinary tract and GIT cells as well as immune cells. Furthermore, AF consists of a heterogenous pool of AF-specific cells, a small percentage of which are AF stem cells (AFSCs) (10). AF functions as a protective fluid for the fetus by protecting against physical trauma, supporting the umbilical cord and lubricating the fetal skin. AF also helps to protect against fetal infection through immune cells, microbial peptides and enzymes. Lastly, the majority of nutrition is provided by the placenta but AF is also a key source of nutrition (8, 9).

The formation of the primitive gut initiates during organogenesis. Following this, the rudimentary gut tube forms and the fetus starts contributing to AF. During late gestation, the crypts and villi mature, resulting in a gut that is ready for extra-uterine life (11). Exposure to AF plays an important role in the development of the fetal intestines. Studies in pig fetuses that had undergone esophageal ligation, which limits the ingestion of AF, demonstrated that ligated pig fetuses have diminished GIT development (12). In line with this, multiple trophic factors (TFs) in AF benefit fetal intestinal development (13). Exposure to growth factors (GFs), including fibroblast GF (FGF), epidermal GF (EGF), hepatocyte GF (HGF), insulin-like GF (IGF)-1, IGF-2 and transforming GF (TGF)-α, promoted fetal intestinal growth, similar to BM, in an in vitro model (14). After birth, exposure to AF comes to a halt and development of the GIT continues in the neonatal phase, when the GIT is exposed to microbes. Feeding with BM contributes to mucosal differentiation and further intestinal development (15).

Evaluation of AF has taken a prominent place in prenatal diagnostics. AF can be obtained during pregnancy through amniocentesis or chorionic villus sampling. Cytogenic assessment of isolated AF is routinely used to diagnose chromosomal disorders (16). More recently developed AF-based tests aim to optimize monitoring of fetal and maternal health, for example through analysis of cell-free DNA and whole exome sequencing (17). AF volume is also critical for fetal growth and development. Abnormalities in its volume are associated with pathologies. The assessment of AF volume can thus improve pregnancy outcomes (18).

In addition to using AF for diagnostic purposes, studies on its therapeutic use are increasing. Considering that AF promotes the development of a healthy GIT and has a similar composition to BM, including the presence of EGF, TGF-β, IGF-1, IGF-2, interleukins (ILs), lactoferrin and immunoglobulins (Figure 1), it has been hypothesized that AF and its derivatives may limit the development of NEC (13, 19). Here, we discuss key studies on this topic. To assess the risk of bias in the animal studies that were employed the SYRCLE's risk of bias tool was used (Supplementary Table 1) (20).

Here, we have assessed the state of the art regarding the use of AF in the prevention and treatment of NEC in neonates (Figure 2). Although current literature lacks relevant clinical human data on this topic, we closely examined in vitro and in vivo studies as well as human studies on feeding tolerance, which have taken the first steps toward realizing the clinical application of AF-based therapy.

Each approach is accompanied by its own challenges. To be able to mimic AF swallowing by enteral PAF administration, high volumes of PAF must be supplemented. Fortunately, lyophilization is able to preserve TFs in HAF as well as improve cellular proliferation and reduce IL-8 production, supporting the notion that PAF administration may benefit vulnerable infants (47). Moreover, the PAF- and AFSCs-approach utilizes the body's own fluid in contrast to SAF. The PAF approach provides the benefits of all soluble components. Noteworthy, AF also contains components with antimicrobial and prebiotic properties that can potentially protect against NEC (13). The contribution of many of these molecules have yet to be identified. Since the cellular components of AF are removed during centrifugation, treatment with PAF excludes the protective effect of AFSCs. Vice versa, the administration of merely AFSCs lacks the beneficial effect of the soluble AF content.

Although SAF represents a carefully selected content of AF, it is likely that, unintentionally, equally important molecules are neglected in the formulation and the manufactured SAF might not represent AF accurately. A vast number of biologically important molecules have not been studied yet in the context of SAF. Therefore, our notion to explore large numbers of molecules expressed in AF and craft more complex SAF formulations is supported. Similar to PAF, SAF does not contain AFSCs and fails to incorporate their beneficial effect. While creating a personalized solution has advantages, it also makes resembling the in utero composition of AF more difficult. Significant research efforts are needed to characterize the functions of the single components in order to craft meaningful SAF. Noteworthy, SAF administration does not raise ethical issues compared to the use of HAF. In addition, issues how to effectively sterilize and store HAF for long-term use do not play a role in SAF production. Finally, careful consideration of the donor AF is required when choosing the PAF- or AFSC-approach. For the latter, specific AFSC characteristics should be taken into account. AFSCs derived during early second trimester are believed to have better potential than AFSCs derived from late second trimester. The more potent AFSCs have a smaller cell size, a more convenient cell density and a shorter dividing time; these characteristics are associated with more vigorous stem cell potential. AFSCs collected at an earlier stage of pregnancy may therefore have more therapeutic value than AFSCs derived from full-term pregnancies (48).

Of particular interest would be to investigate the potential effects of AF, AFSCs, and SAF on the intestinal microbiome. The preterm gut microbiome differs from the term microbiome, among other reasons, due to early initiation of enteral feeding. PIs often have higher numbers of facultative anaerobic bacteria, reduced levels of Bifidobacterium and Bacteroides and increased numbers of Escherichia coli, Staphylococcus, and Klebsiella, which can potentially be pathogenic. In addition to the role of TLR4 signaling, previous studies have demonstrated an association between the preterm gut microbiome and NEC (49). Various pre- and postnatal factors contribute to the development of this microbiome. The knowledge that the fetus ingests large volumes of AF during pregnancy and the finding that microbial DNA is present in meconium suggests that the fetal gut might be exposed to AF microbes in utero, although it is not yet known whether the fetal GIT is significantly affected by this (50). In addition, studies on BM, which has similar components to AF, have demonstrated that BM also modulates the neonatal microbiome (51). Future research should point out whether AF-based therapy results in alternation of the neonatal microbiome and what the subsequent effect would be on NEC development.

Although the research field of AF-based therapy has vastly expanded, key knowledge is still lacking to design and conduct a definitive randomized controlled trial. We argue that various steps need to be taken beforehand to fill in the gaps of knowledge in current literature. At this point research has focused on a variety of animal models, making it complicated to directly compare findings and translate the results to a human setting. Relevant preclinical human models that more closely resemble the immature intestine susceptible to NEC need to be studied to gain more knowledge on the effect of AF-based therapy in a clinical setting. The use of organoids from fetal intestines or induced pluripotent stem cells can provide insights on the effect of AF compounds on intestinal epithelial maturation, barrier function and innate immune response (52, 53). Moreover, further studies are needed to characterize the ideal dose and route of administration for the various approaches. Finally, research should focus on comparing the various AF-based approaches and determine which approach has most clinical potential in the context of NEC.

In conclusion, we have taken a close look at the current body of knowledge on the potential of AF-based approaches in the prevention and/or treatment of NEC. Future research is necessary to investigate whether these proposed approaches will benefit neonates susceptible to develop NEC and/or neonates suffering from NEC. We hope that this framework will help toward clinical application of AF for the treatment and/or prevention of NEC.

MvW, TdB, SS, and RdK were involved in the conception and design of the study. RdK drafted the manuscript. All authors revised the manuscript critically and read and approved the final manuscript.

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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