Forty-eight haemocultures were performed in this study, of which 18.75% were premature and 81.25% at-term. Gender, temperature, heart rate, respiratory rate, glycaemia and caesarean vs. vaginal delivery characteristics were not statistically different between the control and the positive haemoculture groups. However, neonates’ weight in both sepsis types was significantly different to C-reactive protein (CRP), which significantly differed in the EOS neonates. The clinical information of the 48 patients is shown in Table 1.
Changes in the miRNA‑23b expression levels in early onset sepsis
The miR-23b expression levels in the neonatal sepsis samples were analysed by the quantitative real-time PCR method. Our results showed that, compared to the control group, the miR-23b expression levels significantly differed in the neonatal sepsis samples either in the at-term or premature neonates (p < 0.001 KW) (Fig. 1). The miR-23b expression significantly lowered in the neonates who died of sepsis (p < 0.0001, p < 0.05 at-term and premature infants, respectively), and significantly increased in the neonates who survived with a positive haemoculture (p < 0.005, p < 0.001). These results reveal that miR-23b expression correlates with sepsis progression.
Changes in the miR‑23b expression levels in late onset sepsis
Figure 2 shows how the miR-23b expression in LOS significantly lowers in both the dead and surviving newborns with a positive haemoculture, with p < 0.005 and p < 0.05, respectively, compared to the controls and for all comparisons (p < 0.05 KW). Two cases presenting the clinical signs of sepsis died, but the haemoculture was negative. In this case, we recorded a significant drop in the miR-23b level with a negative haemoculture (p < 0.05). This case was considered a false-negative haemoculture, probably due to the sampling time  or another limitation, like the presence of unculturable or fastidious microorganisms that could decrease its sensitivity .
Change in the miRNA-23b expression levels in newborns in two different stages
The differences between our results when comparing EOS and LOS led us to think back to the starting point before sepsis appeared. Figure 3 shows the miR-23b expression level after the first 72 h of life and beyond that time in the control patients. The results revealed a significant increase in the miR-23b level after 72 h of live performance p < 0.05 compared to that before the first 72 h of life.
The correlation between miR-23b, sepsis and death during the neonatal period
Figure 4 shows a very strong negative correlation between miR-23b and death in early sepsis patients (r = -0.96, p = 0.002), the same for premature, with a negative correlation with miR-23b and death sepsis (r = -0.89, p = 0.0001), but the miR-23b level correlated negatively with sepsis (r = -0.81, p = 0.39). With late sepsis, a low negative non-significant correlation is observed between miR-23b and the appearance of sepsis ( r = -0.26, p = 0.32). Nevertheless, we show a positive correlation between miRNA-23b levels in both the control and non-survivor patients ( r = 0.70, p = 0.506).
Infant and late foetal deaths are key factors when assessing a country’s level of social protection [27, 28]. In neonatal sepsis, the leading treatment is antibiotics, which target the infecting pathogen, but not the inflammatory process that continues to increase. Therefore, an ideal treatment approach should include antimicrobial and anti-inflammatory drugs to neutralise the rising inflammatory cascade and the resulting "cytokine storm" in neonatal sepsis . Newborns immune status during the perinatal period differs from that of adults. Neonatal immune responses are generally directed against the generation of T helper type 1 (Th1)-related proinflammatory immune responses, while favouring the Th2-related anti-inflammatory /immunosuppressive response . This process represents an efficient strategy to address the unique challenges of the neonatal period, including maintaining tolerance to maternal antigens in utero and balancing the transition from the sterile intrauterine environment to the antigen-rich outside world .
miRNAs are endogenous, non-coding, single-stranded RNAs (~22 nucleotides long) with the ability to degrade mRNA or inhibit translation, which then regulates gene expression at the post-transcriptional level . We know that the expression of ≥ 30% of human genes is controlled by miRNAs . miRNAs also regulate molecular signalling pathways and immune activities . The invasion of pathogenic microorganisms, followed by rapid miRNAs production, promote the release of inflammatory factors that cause immune hyperactivity, and induce apoptosis or degrading inflammatory factors that can provoke immunosuppression [33, 34].
The biomarkers frequently used in neonatal sepsis are still not completely conclusive. but have shown some potential for in vitro diagnoses . Since their discovery, circulating miRNAs in human peripheral serum are used as biomarkers of various cancer types. The use of miRNAs as diagnostic and prognostic markers has extended to other diseases, including sepsis, but their role in infectious diseases has rarely been studied [36, 37]. One of the main obstacles to establish a well-defined link between miRNAs and sepsis lies in the fact that sepsis can be caused by very different factors that cause similarities and differences, which influence the patient’s situation itself and make sepsis so very complicated . This is why the association of miRNAs with sepsis diagnosis remains controversial .
miRNAs from different biological fluids can be used for the early prediction and evaluation of neonatal sepsis, where various miRNAs are down-regulated, and contribute to the initiation of the immune response to infection . To date, no studies are available on miRNAs in both neonatal sepsis types, i.e., EOS and LOS. To the best of our knowledge, no study has investigated miRNAs expression levels in haemocultures from septic patients and their change according to neonatal sepsis types.
The neonatal immune response to sepsis depends on the timing of onset, relative pathogens and developmental age , and is markedly different from the immune response in adults because of specific neonatal microbial susceptibility and atopic properties. Differences have been reported in the regulation of target gene expression by miRNAs in innate immunity . A study of ten immune-regulating miRNAs, whose expression significantly altered more than 2-fold in neonates with sepsis compared to uninfected neonates, showed that miRNA expression levels were altered, and that this alteration in miRNAs modulated the immune response during neonatal sepsis so as to represses inflammatory response . In another study , low miRNA-26a levels have been correlated with the up-regulation of IL-6 expression in blood mononuclear cells and serum. Nevertheless, neither newborns age nor sepsis type has been specified. There are also reports indicating that miR-15a/16 can be used as a potential biomarker for the diagnosis and prognosis of neonatal sepsis, and that miRNA15a/16 regulation may limit the inflammatory response to LPS .
Although the discovery of miR-23b is recent [11, 16], intense research efforts have been made to show that it is involved in various physiological and pathophysiological processes . So, it has been revealed as an essential moderator of several physiological pathways that regulate the differentiation of many cell lines, such as keratinocytes, chondrocytes and skeletal muscle. miR-23b also regulates inflammatory response in several autoimmune diseases through suppressing proinflammatory signalling pathways in resident cells, such as human fibroblast-like
synoviocytes, and in primary kidney cells and astrocytes from mice . miR-23b also plays a critical role in certain pathologies, including acute myocardial infarction, inflammatory heart diseases and sepsis-induced cardiac dysfunction [45, 46], diabetic nephropathy  and prostate cancer . We herein demonstrate for the first time the presence of miR-23b in haemocultures from neonatal sepsis and their interest for diagnosis and prognosis in early and late sepsis.
In sepsis, miR-23b has been reported to be down-regulated in peripheral blood mononuclear cells (PBMCs) from adult patients and in the LPS-induced THP-1 human monocytic cell line, and has been negatively correlated with the production of proinflammatory cytokines. Increased miR-23b expression has been shown to induce the down-regulation of proinflammatory cytokines production and LPS-stimulated apoptosis .
In the present study, we revealed that at-term birth, the neonates with negative haemocultures (the control group) presented low miR-23b levels during the first 72 h of life, which started to increase after 72 h. Conversely, the neonates with a positive haemoculture who did not survive infection always showed the lowest miR-23b levels, regardless of whether they were premature or born at term. In addition, the neonates with a positive haemoculture who survived infection had high miR-23b levels during the first 72 h of life, irrespectively of whether they were born at term or premature. Thus the increase in miR-23b levels during the first 72 h of life in septic neonates may be a potent prognostic factor for survival and a sensitive clinical marker in both preterm and at-term neonates.
It has been recently shown with an animal model of sepsis that miRNA profiles in CD8+ T cells from adult and neonatal mice were surprisingly similar during infection, but infection miRNA levels differed when it was absent. In particular, marked differences were observed in the miR-29 and miR-130 expression levels between adult and neonatal cells before infection. Likewise, changes in the expression of messenger RNA targets have been noted for both miR-29 and miR-130 .
Our study indicated a difference in the miR-23b expression levels in both EOS and LOS. The miR-23b expression levels increased in the EOS patients with a positive haemoculture and lowered in the LOS patients with either premature or full-term newborns. Nevertheless, we also observed a difference in expression over time in the control group before and after 72 h of birth. This could be due to differences in the genome expression patterns in newborns between EOS and LOS. Exclusively to newborns, uninfected status and host response to sepsis are significantly affected by time of birth [49, 50], in which immune system development is a continuous process throughout embryogenesis and into childhood. Hence the different miRNAs expression in neonatal sepsis could be considered a developmental characteristic of the immune response . Early and late sepsis responses considerably differ depending on the postnatal age at the time of sepsis . By controlling postnatal age in studies on epigenetic changes during neonatal sepsis, we were able to better understand the immune mechanism in newborns and to identify therapeutic targets. From these results, we suggest the possibility of using miR-23b levels as an in vitro diagnosis marker, which can be used to differentiate between EOS and LOS. miR-23b levels are up-regulated during the first 72 h of life and down-regulated over time during this period.