Aberrant gut microbiota composition in preterm neonates is linked to adverse health consequences. Little is known about the impact of perinatal factors or maternal gut microbiota on initial preterm gut colonization.Fecal samples were collected from 55 preterm neonates , 51 mothers, and 25 full-term neonates during the first 3–4 postpartum days. Gut microbiota composition was assessed using 16S ribosomal RNA gene sequencing.Preterm neonates exhibited significantly lower gut microbiota alpha diversity and distinct beta diversity clustering compared to term neonates. Spontaneous preterm birth was associated with distinct initial gut microbiota beta diversity as compared to iatrogenic delivery. Gestational age or delivery mode had no impact on the preterm gut microbiota composition. The cause of preterm delivery was also reflected in the maternal gut microbiota composition. The contribution of maternal gut microbiota to initial preterm gut colonization was more pronounced after spontaneous delivery than iatrogenic delivery and not dependent on delivery mode.The initial preterm gut microbiota is distinct from term microbiota. Spontaneous preterm birth is reflected in the early neonatal and maternal gut microbiota. Transmission of gut microbes from mother to neonate is determined by spontaneous preterm delivery, but not by mode of birth.The initial gut microbiota in preterm neonates is distinct from those born full term. Spontaneous preterm birth is associated with changes in the gut microbiota composition of both preterm neonates and their mothers. The contribution of the maternal gut microbiota to initial neonatal gut colonization was more pronounced after spontaneous preterm delivery as compared to iatrogenic preterm delivery and not dependent on delivery mode.Our study provides new evidence regarding the early gut colonization patterns in preterm infants.Altered preterm gut microbiota has been linked to adverse health consequences and may provide a target for early intervention. While the exact cause for spontaneous preterm delivery can often not be identified, bacterial infection and conditions related to disturbances in the maternal microbiota such as inflammatory bowel disease, poor dental health, or bacterial vaginosis have been associated with preterm delivery risk.4 Recent studies suggest that more subtle alterations in the maternal gut microbiota composition may also be associated with an increased risk of spontaneous preterm birth.6 In addition to its potential role in spontaneous preterm delivery, the maternal gut microbiota is one of the most important factors shaping the initial gut colonization of the newborn.8 The compositional development of the vaginally delivered full-term and breast-fed child’s gut microbiota has been relatively well characterized, while in the preterm counterpart, our understanding of the microbiota establishment remains incomplete.Preterm birth, a global challenge with significant health consequences,9 The immediate perinatal exposures in preterm neonates, including birth by cesarean section, frequent antibiotic exposure, lack of skin-to-skin contact, and breast milk, may disturb early gut colonization. Interestingly, initial gut colonization in preterm neonates appears to be distinct, as the microbiota composition of meconium, defined as the first stool passed and reflecting the life in utero, has been reported to be different in preterm as compared to term neonates.10 However, it is not known whether the microbiota alterations defined in the preterm neonate are associated with factors causing preterm birth, the preterm birth per se, immaturity, or whether they are mere consequences of the different treatment and feeding procedures.The preterm gut microbiota profile differs from full-term gut microbiota with decreased diversity and a higher abundance of proinflammatory bacteria.12 and poor postnatal growth.15 Understanding the early colonization process in the preterm gut may provide new means to prevent these complications. In the present study, we focused on the initial microbial pioneers in order to address the early differences in gut microbiota between preterm and full-term neonates and the factors influencing the gut microbiota establishment in preterm neonates. In particular, the purpose of this study was to investigate whether the cause of preterm birth and the maternal gut microbiota affect the microbial composition of preterm gut microbiota. In addition, the contribution of other factors potentially affecting the early preterm gut microbiota composition including gestational age, mode of delivery, intrapartum antibiotics, and intrauterine growth was investigated.Specific microbial features of the preterm gut microbiota have been linked to a higher risk for common preterm short-term morbidities including necrotizing enterocolitis (NEC)n = 65) and their newborns (n = 79) with a duration of gestation <35 weeks. The threshold of 35 gestational weeks was chosen to include a clearly preterm population, admitted in the neonatal intensive care unit (NICU) and receiving uniform treatment according to the hospital protocol. A substantial number of preterm neonates with a gestational age of 35–37 weeks are not admitted in the NICU, but treated in the maternity ward together with the mother, and therefore this population was excluded. Children with severe congenital anomalies were excluded. Also, the children (n = 10) and mothers (n = 14) without adequate samples as well as B-twins (n = 14) were excluded, resulting in a total of 55 preterm neonates and 51 mothers. The exclusion process as well as matched newborn–mother pairs are presented in Fig. The study was conducted prospectively in the Turku University Hospital in Turku, Finland. For the preterm group, we recruited mothers , whether they were small for gestational age , starting day for enteral feeding (mother’s own or donor milk), and starting day of mother’s own milk. Similar data regarding the term neonates were collected.For the preterm population, the cause of preterm birth  or iatrogenic) was collected from patient records. In this study, iatrogenic preterm delivery was defined as delivery due to a maternal or fetal reason, i.e., pre-eclampsia, placental insufficiency, or hepatogestosis without spontaneous rupture on membranes or onset of labor. In addition, data regarding the following clinical characteristics were obtained: gestational age, sex, mode of delivery, intrapartum and neonatal antibiotic use, birth weight . DNA was extracted following the commercial kit InviMag Stool DNA Kit  using the automated KingFisher DNA System . The KingFisher’s system protocol steps included nucleic acid binding on magnetic beads, five-step washing, and elution. Then, the total DNA concentration was measured using a Qubit® 2.0 Fluorometer  and normalized. A specific 16S rRNA gene region (V3–V4 region) was amplified following the 16S rDNA gene Metagenomic Sequencing Library Preparation Illumina protocol (Cod. 15044223 Rev. A). After 16S rDNA gene amplification, the multiplexing step was performed using Nextera XT Index Kit (FC-131-2001). One microliter of the PCR product was run on a Bioanalyzer DNA 1000 chip to verify the size; the expected size on a Bioanalyzer trace is ~550 bp. The libraries were sequenced using a 2 × 300 bp paired-end run (MiSeq Reagent Kit v3) on a MiSeq-Illumina platform according to the manufacturer’s instructions.Total DNA was extracted from the biological samples as previously described.17 was used to study differences in the taxonomical abundances between the groups. Group comparison was conducted with either analysis of variance or permutational multivariate analysis of variance statistical analysis. The statistical significance was determined as a corrected p value of <0.05.18 Calypso software version 8.24  was used, along with total sum normalization for the statistical analysis.The raw sequences were analyzed with QIIME2 (version 2019-7 and 2020-11) pipeline. The data were imported using the Phred33-importing tool for paired-end data and quality filtered using the DADA2 method. The taxonomy was formed by using the Greengenes v.13.8 database and 99% amplicon sequence variant taxonomic classifier creating a phylogenetic tree. Alpha diversity was assessed using Faith’s Phylogenic Diversity; in addition, alpha-diversity evenness and Shannon indexes were used. Beta diversity was conducted using Bray–Curtis and Unweighted Unifrac distance matrices. Analysis of composition of microbes (ANCOM)Z-score, maternal and neonatal alpha diversity as assessed by Shannon index) were examined with Spearman’s rank correlation coefficient because of non-normal distribution. Associations between the relative abundances of microbial taxa and categorical variables were analyzed with Wilcoxon’s rank-sum test. The cause of prematurity (iatrogenic/spontaneous), mode of delivery , intrapartum antibiotic exposure (yes/no), and neonatal antibiotic exposure (yes/no) were treated as categorical variables. The level of significance was set at p value <0.05. The analyses were performed with the SAS software, version 9.4 for Windows .The contribution of maternal gut microbiota composition on the preterm gut microbiota composition was selected as a response variable. Relationships between the percentage of microbes and continuous variables , whereas the neonates born by iatrogenic preterm delivery had lower birth weight (p = 0.046) and were more often small for gestational age (p = 0.002). All neonates in the study received human milk at the time of sample collection. None of the subjects had received the formula. There were three cases of chorioamnionitis in the preterm study population, with a premature delivery at 30, 31, and 34 weeks of gestation, and out of these cases, two newborns and one mother participated in the analyses.The clinical characteristics of the preterm study subjects born after spontaneous or iatrogenic delivery are presented in Table The characteristics of the term population are presented in Table n = 55) initial gut microbiota composition was compared to the initial gut microbiota composition of spontaneously born, term neonates (n = 25). The preterm gut microbiota was mainly composed of bacteria belonging to the phyla Firmicutes (60.4%), Proteobacteria (26.3%), and Bacteroidetes (8.8%)  Fig. . The thr8%) Fig. . Wide va8%) Fig. . The thr8%) Fig. .Fig. 2Prp = 0.02) and Shannon index (p = 0.03)  and Unweighted Unifrac (p = 0.001) beta-diversity matrices according to the two groups , Streptococcus (W = 178), and Bacteroidetes (W = 177) in the term microbiota compared to preterm.Significant differences were observed in both alpha and beta diversity as well as in specific relative abundancies when comparing the two groups. Significantly higher alpha diversity was observed in the term population compared to the preterm population as assessed by alpha-diversity evenness (03) Fig. . Signifiups Fig. . In the p = 0.047)   Fig.  and post45) Fig.  as asses45) Fig. , or Brayp = 0.007). It is of note that gestational age or mode of delivery did not affect the extent to which the maternal gut microbiota contributed to the preterm gut microbiota. Indeed, a manifest contribution from the maternal gut microbiota was also seen in the microbiota of neonates born by cesarean section.The contribution of the maternal gut microbiota composition to the preterm gut microbiota composition was assessed with Source Tracker using QIIME version 1.9  Fig. . The ratRoseburia (W = 118) and mothers without antibiotic treatment a higher abundance of Macellibacteroides (W = 63).In the maternal gut microbiota, Firmicutes (62.4%) was the dominant taxa at the phylum level, followed by Bacteroidetes (29.6%) and Actinobacteria (4.3%)   and evenness (p = 0.003), as well as Bray–Curtis (p = 0.004) and Unweighted Unifrac beta diversity (p = 0.002).Distinct and significant clustering of the gut microbiota was detected among mothers who delivered spontaneously, iatrogenically or after PPROM delivery by Bray–Curtis beta-diversity analysis (41) Fig. . There w19 The present study for the first time provides data indicating that spontaneous preterm birth, often triggered by infection or other maternal microbial perturbation, may also affect the initial gut colonization in preterm neonates. Two distinct clusters were seen according to the cause of prematurity. However, there was nonsignificant variation in the ANCOM analysis in these two groups, not forming a consistent colonization pattern. Interestingly, other perinatal exposures such as intrapartum antibiotic use, delivery mode, or gestational age were not associated with the initial preterm gut microbiota composition in the present study. Furthermore, our results suggest that the initial preterm gut microbiota might at least in part originate from the time in utero as differences were seen in both preterm neonatal and maternal samples according to cause of prematurity. Several possible maternal–fetal transmission routes have been suggested.20 Changes in the gut microbiota in mothers with spontaneous preterm delivery may therefore affect early preterm gut colonization.The initial gut microbiota in preterm neonates is distinct from that of those born full-term. The aberrant gut colonization in preterm neonates may at least in part be explained by detrimental perinatal and postnatal exposures, including cesarean section delivery, antibiotic use, and delayed or reduced exposure to breast milk, which often cluster in these fragile individuals. However, we have previously reported that prematurity per se affects the preterm gut microbiota composition.4 More recently, it has been shown that alterations in the maternal gut microbiota composition may also be associated with an increased risk of spontaneous preterm birth.6 Taken together, our study suggests that the events leading to spontaneous preterm birth are reflected not only in the gut microbiota of the mother but also of the newborn. This notion is corroborated by the higher contribution of the maternal gut microbiota to the neonatal gut microbiota after spontaneous preterm birth.Significant differences in the gut microbiota were detected between mothers after spontaneous preterm delivery as compared to iatrogenic preterm delivery. Previously, numerous studies have indicated that changes in maternal microecology resulting from bacterial vaginosis, inflammatory bowel disease, or poor dental health may be associated with an increased risk for preterm birth.21 In the present study, however, the preterm children born with cesarean section did not differ from vaginally delivered in terms of the gut microbiota composition. This observation is consistent with the studies by Dahl et al.,22 as well as Ardissone et al.,10 who have provided evidence suggesting that the impact of delivery mode is not substantial in the gut colonization process in preterm neonates. Notably, bacteria usually connected with the birth canal and vaginal birth (Lactobacillaceae family) were detected in the early gut microbiota of preterm neonates born both by vaginal delivery and by cesarean section in the present study. Furthermore, no differences in the contribution of the maternal gut microbiota to the preterm neonate gut microbiota were detected between subjects born by vaginal or cesarean section delivery in the Source Tracker analyses. This suggests that the early colonization of the preterm gut does not follow the same patterns as in term infants.It has previously been extensively shown that the mode of delivery is a strong modulator of the early gut microbiota in term infants.The relatively small sample size may affect the applicability of the results. However, the study subjects were characterized in detail, and a structured study protocol was followed while collecting the samples and treating the preterm neonates in the NICU. All the preterm neonates received breast milk, either from donors or their own mothers, from the first day of life as recommended by the European Society of Paediatric Gastroenterology, Hepatology and Nutrition. Antibiotics were used according to the unit protocol; initial empirical antibiotics consisted of a combination of penicillin G and gentamicin. The term population chosen for the comparison was a uniform group with no neonatal antibiotic treatment, and the term fecal samples were collected during the first days of life post partum. A limitation is that the preterm samples were collected 0 to 3 days postpartum, and therefore possibly promoting a higher microbial variation and a lower bacterial biomass. In addition, the maternal fecal samples were collected after birth and were therefore affected by intrapartum antibiotic exposure. Prenatal maternal samples might have been more informative but predicting preterm delivery is difficult. The differences in the rates of cesarean section, intrapartum antibiotic exposure and fetal growth restriction between the neonates born by spontaneous or iatrogenic preterm delivery is an obvious concern. Terminating the pregnancy for an iatrogenic cause with a cesarean section delivery is more common in the cases with placental insufficiency and poor intrauterine growth, which could be seen in our patient population. However, potential confounding is ruled out by the fact that the mode of delivery, intrapartum antibiotic exposure or being small for gestational age displayed no association with the preterm gut microbiota in our analyses as independent factors. We may therefore consider our results, where the cause of prematurity emerged as the sole significant factor, reliable.Enterobacter, Enterococcus and Lactobacillus genus, are reportedly typical for the early preterm gut microbiota.10 The most abundant bacterial families in the preterm neonates in our study were Planococcaceae, Lactobacillaceae and Enterobacteriaceae, which all include common pathogenic bacteria. A proinflammatory, low diversity gut microbiota composition has been described earlier as typical for preterm infants.9 When compared to the term gut microbiota composition in our study, the preterm gut microbiota composition exhibited significantly lower alpha diversity, significant beta diversity clustering, and higher levels of specific proinflammatory bacteria. As previously reported, preterm infants may have delayed colonization with Bifidobacteria,23 which are thought to reduce inflammation and promote a healthy gut composition.24 Our findings regarding the microbiota differences in these two groups are therefore consistent with the previously reported data.The preterm neonates in this study exhibited low microbial diversity and great variation in the gut microbiota composition. Uniform colonization patterns could not be seen, which is consistent with previously reported data. High abundance of specific bacteria, including 15 suggest that the initial gut microbiota composition in preterm infants is associated with growth in the neonatal period. This finding may be of clinical significance since poor postnatal growth, a highly common problem in preterm neonates, has been linked to neurodevelopmental problems later in life. Ensuring adequate growth is a top priority in treating these fragile patients. There are studies suggesting that the early gut microbiota composition of the preterm infant may affect the later gut microbiota composition, which in turn has been linked to several clinical outcomes, including obesity25 and allergic diseases.26 Our results provide intriguing new information about the establishment and exposures connected to the initial gut microbiota establishment in preterm neonates. Future research should focus on the connections between spontaneous preterm birth, early gut microbiota composition, and short- and long-term health outcomes.The connection between initial gut microbiota composition and health outcomes remains largely unknown. However, data from a study by Arboleya et al.Supplementary information


Extrauterine growth restriction (EUGR) in preterm birth infants could have long-term adverse impacts on health. Less is known about the gut microbiota regarding its establishment in early life and its role in long-term growth in preterm birth infants.A prospective, longitudinal observational study was conducted with 67 preterm infants in a level III neonatal intensive care unit. Clinical information was obtained from medical records, and fecal samples were collected weekly during hospitalization and processed for 16S rRNA gene sequencing.Staphylococcus, and lack of obligate anaerobes, including Clostridium and Veillonella. In the 2-year follow-up, infants with a high level of obligate anaerobes-to-facultative anaerobes ratio at stage 4 had a lower risk of long-term growth restriction at the margin of statistical significance.The bacterial profiles from the weekly sampling of preterm infants demonstrated that the early-life gut microbiota was clustered into the following four stages in chronological order: stage 1: 0–4 days, stage 2: 1–2 weeks, stage 3: 3–7 weeks, and stage 4: 8–10 weeks. The development of gut microbiota showed latency at stage 4 in EUGR infants compared with that in non-EUGR infants, which resulted from their consistently high level of facultative anaerobes, including Enterobacteriaceae and The results of this study indicate that the development of gut microbiota in the early life of EUGR infants is delayed compared with that of non-EUGR infants. The obligate-to-facultative anaerobes ratio could be an indicator of the maturity of gut microbiota development and associated with the risk of long-term growth restriction in preterm infants. Preterm birth is one of the most common adverse pregnancy outcomes and a leading cause of neonatal mortality . ExtrautBifidobacterium and Lactobacillus, remained in a relatively low abundance. However, with the development of the intestine, the Clostridiales were gradually elevated, meaning that obligate anaerobic bacteria increased  of Shenzhen BaoAn Maternal and Child Care Hospital from August 2018 to July 2019. Infants born under 37 gestational weeks and with parental written informed consent were prospectively included, and those with congenital malformations, necrotizing enterocolitis, or who withdrew from the study during hospitalization were excluded. The weight of infants was measured daily during hospitalization and at 24 months old after discharge by follow-up visits. EUGR was diagnosed when discharge weight ≤10th percentile derived from Fenton's postnatal growth charts (https://www.ebi.ac.uk/) with accession number PRJEB45532.We collected meconium and stool samples (0–1 day) from preterm infants' soiled diapers. After the meconium collection, another stool sample would be collected in the middle of the first postnatal week (day 3 or 4) and then collected weekly (week 1–10) during hospitalization. DNA was extracted using the Minka Gene Stool DNA Isolation Kit , and the V4 region of 16S ribosomal RNA (rRNA) gene was amplified as previously described  and addepheatmap to demonstrate the change of bacterial abundance and cluster of EUGR infant age. The cluster method of heatmap was calculated using complete-linkage clustering (setting “method='complete”') in R package hclust. BugBase was used to annotate potential oxygen phenotypes , taxonomic annotation (using RDP against GreenGenes Database v13_8), followed by the construction of an amplicon sequence variant (ASV) table, and α- and β-diversity calculation using QIIME2 according to its online tutorial . We totaenotypes . Then, wWe enrolled 67 preterm infants. By comparing the discharge weight of infants with Fenton's postnatal growth charts, we divided participants into EUGR and non-EUGR groups . Of note, 20 (29.9%) infants were born by vaginal delivery; 44 (65.7%) infants' weights were <1,500 g at birth; eight infants were diagnosed as small for gestational age; and all developed into EUGR. Early-onset infection occurred in 11 infants from the non-EUGR group (55.0%) and 30 infants from the EUGR group (63.8%). Detailed statistics along with gender, family, and feeding characteristics are reported in The rarefaction curves illustrated the adequacy of sample size for 16S rRNA sequencing . We obseEnterobacteriaceae-unassigned remained >25% from birth to discharge in the EUGR group  and positively correlated with chronological age , whereas the facultative anaerobes did just the opposite, decreasing from stage 2 to stage 4 (p < 0.001) and negatively correlated with chronological age   . Compare= 0.001) . To vali= 0.001) . The res= 0.001) , C.Enterobacteriaceae-unassigned and Staphylococcus were the dominant bacteria of facultative anaerobes in both groups. The relative abundance of Staphylococcus increased from stage 1 to stage 2 and then decreased at stages 3 and 4. The relative abundance of Enterobacteriaceae-unassigned increased from stage 1 to stage 3 in both groups. The divergence appeared at stage 4 as Enterobacteriaceae decreased in the non-EUGR group, whereas it increased in the EUGR group. In addition, the relative abundance of Streptococcus for all stages is stable in the EUGR group, whereas it decreased in the non-EUGR group. In contrast, Clostridium and Veillonella were the dominant bacteria of obligate anaerobes in both groups. The increase in the relative abundance of these two bacteria was slow in the EUGR group but rapid in the non-EUGR group  had a closer relationship with long-term growth than that during 38–40 weeks corrected gestational age , D.In this study, we characterized the bacterial dynamic profiles of preterm infants and suggested that the development of gut microbiota in EUGR infants was stunted during hospitalization. Key support to this finding was that facultative anaerobes remained at a constantly high level, and obligate anaerobes remained at a low level in the gut microbiota of EUGR preterm infants compared with that of non-EUGR preterm infants. This phenomenon could be observed regardless of antibiotics and ventilator use. Although our data could not indicate the causal role of bacterial oxygen utilization in EUGR development, the follow-up results showed the potential of gut microbiota oxygen utilization ratio being an alerting signal for infant catch-up growth.Recent studies have provided insights into the gut microbiota maturity during the early life and its association with healthy growth , 15, 21.Firmicutes-to-bacteroidetes ratio (F/B ratio), a widely reported indicator in studies of obesity, has a close link with energy harvest and nutritional utilization, which recently has been reported to increase with age . HoweverIn clinical practice, the postnatal course, nutritional management, and even medication were substantially differed by the gestational age of preterm birth infants. While a previous study suggested an association between gestational age and early life gut microbiota establishment, our study found that the delayed maturation of gut microbiota in EUGR infants was associated with both chronological age and corrected gestational age. Many studies have found the stunt of gut microbiota in preterm birth infants. Our data prompted that the profile of early life gut microbiota establishment is closely relative to the growth of preterm birth infants, which is consistent with a previous study . It is pVentilatory oxygen support is necessary and is the most commonly used therapy for preterm infants to prevent neonatal respiratory distress syndrome and meet the needs of oxygen required for growth . HoweverAlthough the criteria of EUGR by measuring anthropological indicators provide a convenient diagnosis for clinical practice, the lack of consensus on EUGR measurement still constrains research. Fenton et al. argued that the measurement and definition of growth charts could be arbitrary and unhelpful to medical practice ; thus, nOur study has some limitations. We only collected fecal samples from preterm infants without detecting the oxygen content in the infants' guts, which could provide direct evidence of the oxygen concentration and the relative consumption. However, despite the availability of the detection method, it would have been unethical to obtain these data by applying a cutting-edge oxygen sensor to NICU preterm infants. Another major limitation is that the sample size of our cohort was small, which limited us from examining the effect of innate factors and clinical practices in the development of preterm infants. The sample size also limited us from enabling a powerful analysis of antibiotic types and the relation between antibiotics and time of each sample. The oxygen utilization ratio was an observed risk factor for long-term growth on the boundary of significance, which suggests the requirement of future research on a larger population to examine this observation. It should be pointed out that our study is a monocentric study, and we used 16S rRNA gene sequencing for analysis, which limited our interpretation of species level and microbial function.Our findings indicate that the maturation of gut microbiota in the EUGR infants showed a significant delay compared with non-EUGR infants. This maturation delay is manifested in the consistently high level of facultative anaerobes and low level of obligate anaerobes. Furthermore, we suggest that the obligate-to-facultative anaerobes ratio during hospitalization may be correlated with long-term growth restriction.PRJEB45532.The original contributions presented in the study are publicly available. This data can be found here: EBI: The studies involving human participants were reviewed and approved by Medical Ethics Committee, Shenzhen Baoan Women's and Children's Hospital, Jinan University, Shenzhen, Guangdong Province, China. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. Written informed consent was obtained from the individual(s), and minor(s)' legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.EE-O, HZ, and PL designed the study, reviewed, and revised the manuscript. LM, YiZ, YS, YoZ, and ML collected the specimens. Y-EH, XS, and PL processed the samples. YH, XS, and PL searched the literature and drafted the manuscript. XS, SL, FH, JC, and PL analyzed the data. All authors approved the final version of the manuscript.This study was funded by the Research Foundation of Shenzhen Baoan Women's and Children's Hospital, Jinan University (BAFY 2021002).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


For the study group plasma blood samples at 9–13 weeks before diagnosis and at 22–24 weeks after start of therapy were selected. Using high-throughput sequencing technology we detected differences in the levels of 15 miRNAs  (log2(FC) ≥ 1.5; FDR ≤ 0.05) during the first trimester compared with the control (non-high-risk of preterm birth pregnant women). All downregulated miRNAs in the first trimester from the placenta-specific C14MC cluster. During the second trimester no differentially expressed miRNAs were found. Our results suggest that the miRNA profile in plasma during early pregnancy may predict a high risk of preterm birth, which is important in preventing gestational problems as early as possible.In recent years evidence has been accumulated showing that miRNAs can act as potential biomarkers or targets for therapy of preterm birth, one of the most important problems in modern obstetrics. We have performed a prospective study of the miRNA profile in the plasma during the first and second trimesters in pregnant women with high risk of preterm birth ( Preterm birth (PTB) is one of the most important problems in modern obstetrics. Relevance of this issue is due to predominance of preterm births among obstetric complications and significance of their consequences in the future of the newborns and their families .According to literature, PTBs account for 7–11% of all births. Thus, based on WHO data, about 15 million children are born prematurely every year and this figure does not tend to decrease. More than 1 million premature newborns die in their first year of life . A signiIn obstetric practice, it is customary to name the following main predictors of PTB: pathogenic infections, multiple pregnancy, a history of preterm birth, shortening of the cervix less than 25 mm in the second trimester of pregnancy (according to ultrasound) ,5. DespiStudies—the main purpose of which were to improve the ability to predict preterm birth, for which various biochemical markers in maternal blood were studied—have been conducted . Some ofIn recent years, ncRNAs have proven to be promising biomarkers or targets for treatment of various diseases, including pregnancy complications . There aMost researchers used patients with spontaneous preterm birth (sPTB) as the study group. We hypothesized that the miRNA profile in blood plasma may change in pregnant women at risk of preterm birth. To test this hypothesis, we have performed a prospective study of the miRNA profile in the blood plasma during the first trimester in pregnant women who were later found to be at high risk of preterm birth. Also, we evaluate the miRNA profile in these same women in the second trimester after the diagnosis. For miRNA profiling we used high-throughput sequencing, which allows de novo miRNA analysis with high resolution and accuracy .We used blood plasma samples from “The Bioresource Collection for the Detection of Early Biomarkers of Pregnancy Complications” biobank, created at the D.O. Ott Research Institute of Obstetrics, Gynecology, and Reproductology, which houses about 17,000 biosamples of various types of biomaterial from 355 pregnant women. Samples were taken prospectively in the first (9–13 weeks), second (22–24 weeks), and third (32–34 weeks) trimesters, and at birth, and then the outcomes of pregnancy and childbirth were analyzed . For then = 13). All pregnant women had a short cervix of less than 25 mm on the second ultrasound screening (19–21 weeks); had pain in the lower abdomen; and were hospitalized and received maintenance therapy  and magnesium sulfate for fetal neuroprotection for at least one week.The study group (high-risk of PTB) group consisted of pregnant women with a high risk of preterm birth in the second trimester (at the time of taking the second sample) (n = 11). For the control group, samples were taken at gestational ages corresponding to those of women at high risk of preterm birth.The control group had a cervical length > 25 mm on the second ultrasound screening (19–21 weeks), and did not have pain in the lower abdomen (2); and singleton pregnancy.Inclusion criteria for all participants were as follows: age (25–35 years); normal BMI , APS and others); gestation complications ; and the absence of late pregnancy complications .The study was approved by the Institutional Review Board of the D.O. Ott Research Institute of Obstetrics Gynecology and Reproductology , No. 97 from 27 June 2019. Informed consent was signed by all women prior to their inclusion in the study and to processing of their personal and medical data. The study was performed in accordance with the Declaration of Helsinki.g for 10 min at 4 °C. The supernatant was carefully transferred to a sterile tube and centrifuged again at 2500× g for 10 min at 4 °C in order to pellet any debris and insoluble components. After the centrifugation steps, plasma samples were immediately aliquoted in cryotubes, frozen and stored at −80 °C until further processing.Blood samples were collected in Improvacuter tubes with K2EDTA  (for plasma); tubes with blood were centrifuged at 1500× MiRNA was extracted from 200 μL plasma samples using miRNeasy Serum/Plasma Kit , according to the manufacturer’s instructions, and the RNA was resuspended in 12 μL of RNase-free water and then stored at −80 °C until library preparation.MiRNA libraries were prepared using the QIAseq miRNA Library Kit , according to the manufacturer’s protocol. Briefly, RNA samples were ligated with 3′ and 5′ adapters. After ligation, reverse transcription was done. The cDNA was purified using magnetic beads, then eluted with 17 µL nuclease-free water. The cDNA samples were used as templates for subsequent PCR. The samples were barcoded with unique indexes during the PCR amplification to allow pooling of libraries before sequencing. The PCR products were cleaned up using the magnetic beads and eluted with 25 µL nuclease-free water. For assessment of the yield, size distribution, and molar concentration of the amplified DNA libraries, the samples were run on 2200 Tapestation Instrument with High Sensitivity D1K ScreenTape and High Sensitivity D1K Reagents . The quantity of libraries required for sequencing was determined, according to the manufacturer’s protocol using the miRNeasy Serum/Plasma Kit .All libraries were sequenced together on a HiSeq 2500  with single-end 75 bp reads, according to the manufacturer’s protocol.Clinical data were analyzed using Statistica 10.0 . Continuous variables are presented as the mean ± standard error. Categorical variables are presented as number/total number.https://geneglobe.qiagen.com/us/analyze, accessed on 12 September 2022). The average of the number of high-throughput sequencing reads per RNA category in plasma of pregnant women was calculated per group (n = 13 and n = 11 samples per group). Differential expression analysis was performed on the resulting raw count matrix of 24 individual samples using DESeq2 R package [p-value < 0.05 and absolute value of log2(fold change) > 1.5. miRWalk v3.0 database used to predict miRNA targets [Small RNaseq data were processed, including adapter trimming and mapping to miRBase to yield raw count data, using the web-based tools of the GeneGlobe Data Analysis Center , 13 in the group with a high risk of preterm birth (hrPTB). The characteristics of the pregnant women included in the study, the gestational ages of sampling, and the pregnancy outcomes are presented in An analysis of the clinical characteristics of the groups showed that, in the group at high risk of preterm birth, 8/13 were primiparous with a singleton pregnancy, making it impossible to rely on the two most significant early predictors of preterm birth . However, it should be noted that in the study group, 8 out of 13 patients had a history of miscarriage in early pregnancy, in contrast to the control group, with 3 out of 11 patients having a history of miscarriage. Also, 4 of 13 high-risk group women had recurrent miscarriage , while the control group had none. In 6/13 high-risk pregnant women, early pregnancy was complicated by threatened miscarriage with retrochorial hematoma. The presence of a history of miscarriage and the presence of retrochorial hematoma in the early stages can be important predictors of preterm birth, which requires the attention of researchers . Spontan2(Fold Change) ≥ 1.5; FDR ≤ 0.05) in the group with a high risk of preterm birth compared to the control group .All 12 downregulated miRNAs in the first trimester are members of the C14MC cluster. The C14MC cluster is a pregnancy-associated cluster and is highly expressed in the placenta. Upregulated miRNAs are also associated with pregnancy and pregnancy complications but are not members of the C14MC cluster.According to miRWalk v3.0 database, a total of 291 genes can be potential targets of differentially expressed miRNAs. Gene enrichment analysis of the predicted target genes showed that there were 55 GO term gene sets and 42 KEGG pathways that were significantly overrepresented in the list of potential miRNA targets. Today, preterm birth prediction, risk assessment and risk management are the key pillars of preterm birth prevention. In this regard, the search—for the earliest and most reliable predictors allowing stratification of the risks of the development of the PTB as early as possible in addition to preventive measures—is of utmost importance .Despite the necessity for early markers of PTB, the search for them is complicated by a plethora of factors. First of all, it involves a complex design of clinical trials, including acceptance of a pregnant woman at an early stage and assessment over time in the second and third trimesters of pregnancy; this could be thwarted by withdrawal of study participants due to delivery and other obstetric complications, preterm pregnancy, and refusal to participate in further studies. Second, such studies require long running time due to the need to obtain outcomes and must include many research centers since low frequency of PTB leads to a small number of participants in the main group. Finally, the presence of serious confounders is inevitable due to the impossibility of leaving a pregnant woman with threatening preterm birth without therapeutic measures for ethical reasons. All of the aforementioned factors impede forming groups for reliable statistical analysis.The detection of miRNAs in early gestation in the maternal circulation makes them good candidate biomarkers for monitoring the course of normal pregnancy and the presence of gestational diseases , and forIn recent years evidence has been accumulated that miRNAs can play the key role in pathogenesis and act as potential biomarkers or targets for therapy of preterm birth. However, whether the miRNA profile is changed in the blood plasma of patients with high risk of preterm birth is yet to be known.In our prospective study, we chose an approach that allows us to determine the high risk of PTB implementation by the miRNA profile in blood plasma. The main objective of this approach was as follows: to assess the possibility of determining the risk of threatening preterm birth before the onset of its clinical manifestations, which allows taking therapeutic measures as early as possible, and to assess the dynamics of the miRNA profile after the start of therapy, which determines the possibility of additional control over the effectiveness of the measures taken. Only such an approach is suitable for an miRNA-based diagnostic test system for determining the risk of preterm birth .We compared the miRNA expression profile in plasma in the first and second trimesters of pregnancy in women with a high risk of preterm birth and pregnant women without complications (control group).In the first trimester of pregnancy, 15 differentially regulated miRNAs  were found in plasm. In the second trimester, no significant differences were found between miRNA profiles in pregnant women at high risk of preterm birth and in the control group.It is worth noting that for the second trimester, samples from women after the start of therapy were used, so the lack of differences in miRNA profiles in the second trimester may be related to the effectiveness of therapy in preventing preterm birth. In turn, Gray et al. found a difference in plasma miRNA expression in mid-term pregnancy in pregnant women with sPTB and term birth , but theInterestingly, all miRNAs with downregulated expression in the first trimester belong to the C14MC cluster. The C14MC cluster is a placenta-specific cluster associated with many obstetric pathologies . MiRNAs In our study, we found increased expression of hsa-miR-34a-5c and hsa-miR-34c-5p. The miR-34 is associated with pregnancy complications, including placental diseases (preeclampsia and intrauterine growth restriction) ,40. HassmiR-122 plays a role in metabolic diseases, in particular in the development of gestational diabetes mellitus (GDM) ,45. AlsoWe also analyzed the enrichment of differentially regulated miRNA target genes, and a total of 291 miRNA target genes were found.Enrichment analysis of possible targets of miRNAs showed the effect of differentially expressed miRNAs on the p53 signaling pathway. p53 is a tumor suppressor and it has been shown to be involved in the senescence of placental cells, decidual cells, and fetal membranes, which possibly leads to preterm birth . PreviouAn association was also found between differentially expressed miRNAs in patients at high risk of PTB and signaling pathways associated with cytokines . As is known, the maturation of the cervix is associated with an inflammatory signature, in particular with the regulation of cytokines . Tarca eThe limitations of our study are the lack of validation of RNA-seq data by RT-qPCR in a larger cohort and the exclusion of other complications of pregnancy. Despite this, our study corroborates previous findings about the biology of PTB and enables future large population-based studies to confirm the relevance of discovered potential miRNA markers.Our results highlight the unique expression profiles of early circulating miRNAs at high risk of preterm birth and identify potential candidate plasma miRNAs as predictive markers of preterm birth. The ability to identify women at high risk of preterm birth early in pregnancy would allow for earlier clinical interventions to prevent preterm birth. Our study showed that in the first trimester, the miRNA profile changes in the blood plasma of pregnant women in whom the risk of PTB was later identified. It is quite possible that the identified miRNAs can be used as a biomarker for PTB. In the second trimester, the miRNA profile will not change, which is likely due to the start of PTB therapy, or compensatory mechanisms. These assumptions need to be tested in future studies.


Birth at term comprises a period with heterogeneous neonatal outcomes that tend to be worse for infants born earlier. However, few studies have analyzed this period, in which each day can make a difference. Therefore, we aim to assess neonatal mortality (NM) according to gestational age (GA) at birth measured in days in term liveborn infants born in 2012–2017 in São Paulo, the largest city in Latin America. This population-based cohort study assessed term liveborn infants followed until the end of the neonatal period. We analyzed 7 models for NM according to GA in days: crude NM adjusted for maternal and prenatal variables, NM additionally adjusted for type of birth and type of hospital, and adjusted NM stratified by type of birth  and by type of hospital (public and private). We included 440,119 live infants born at 259–293 days of gestation. The median GA at birth was 274 days. In all models, NM was higher for infants born early term, decreasing in infants born full term and rising again in infants born late term. In the unadjusted model, hazard ratios of NM changed daily, decreasing from 3.34 to 1.00 on day 278 and increasing again thereafter. In the stratified analysis according to type of hospital, being born in a public hospital was associated with a reduced risk of NM for infants born at 278–283 days of pregnancy. There was a decrease in GA related to obstetric interventions, especially cesarean sections, which increased NM. The loss of days of pregnancy was larger in private hospitals. Increasing the granularity of GA to days is feasible and has the potential to drive public policies. To the best of our knowledge, this is the first Brazilian study on GA in days using a national live births database. Birth at term is defined as birth between 37–42 completed weeks of gestation (259 to 293 days). Babies born before 37 completed weeks (< 259 days) are classified as pre-term, while those born after 42 completed weeks (≥ 294 days) are post-term . HoweverIn a cohort study involving term births in the United States in 1995–2001, Zhang and Kramer  reportedSeveral Brazilian studies also found differences in outcomes of infants born at term. In a cohort study of live births (1982–2004) in a southern Brazilian city, Barros et al.  reportedSeveral researchers have recommended that term births be subdivided into early term (37–38 6/7 weeks), full term (39–40 6/7 weeks), and late term (41–41 6/7 weeks). Some have also recommended that the period between 34–36 6/7 weeks be called late pre-term and that births occurring after 41 weeks be called post-term . The selThe objective of this study was to assess neonatal mortality (0–27 days of life) according to GA in days in a cohort of liveborn infants born in 2012–2017 in the city of Sao Paulo, Brazil.This population-based cohort study analyzed live births between January 1, 2012, and December 31, 2017, in the city of São Paulo, Brazil, followed until the end of the neonatal period (28 days of life). According to the Brazilian Live Births Information System (SINASC), there were 1,202,843 live births during this period. In this study, we only included liveborn infants with information on GA in days who were born at term (between 259 and 293 days of pregnancy), resulting in 440,119 liveborn infants .This study is part of the "Potential pregnancy days lost (PPDL): An innovative gestational age measure for assessing maternal and child health interventions and outcomes” project , process number 443775/2018-4).The project that was approved by the research ethics committee of São Paulo Faculty of Public Health (protocol number 98163018.2.0000.5421). Individual records were assigned a unique number to anonymize names, and we analyzed the data confidentially.We obtained data from the Brazilian Live Births Information System (SINASC) and the Mortality Information System (SIM). Both databases are managed by the Epidemiological Coordination Unit (CEInfo) of the São Paulo Municipal Health Department. All variables in these databases are extracted from live birth certificates and death certificates.To link the records of the two databases, we used three Visual Basic macros in Microsoft Excel and Microsoft Access. The first macro created graphic phonemes for the fields in text format. We used phonemes to identify similar first names of the mothers in the SINASC and SIM databases. The second macro cleaned first name fields by excluding both words that were not first names and numbers. The third macro created a similarity score between the fields that were transformed into graphic phonemes. We performed the following linkage steps: exclusion of newborn infants with implausible GA between 18–45 weeks; cleaning first names of the mothers in both databases (SINASC and SIM); elimination of unusual words ; creation of “graphic phonetic phonemes” and “Soundex” phonetic algorithms for the first and last names of the mothers; generation of pairs through deterministic linkage between the number of the birth certificate and newborn sex in the two databases (SINASC and SIM); generation of the similarity score between the “graphic phonetic phonemes” and the “Soundex” algorithm pairs, plus exact match for date of birth and newborn sex; and verification of the pairs (cases with uncertainties were not considered pairs).The process was made in a deterministic step using Microsoft Access, a second step probabilistic using Miscrosift Access and finaly a probabilistic step using Linkplus 2.0 from CDC, in order to confirm the pairs found by steps 1 and 2 and add any pairs not found in the previous steps. All three steps were manually reviewed by the researchers to ensure that the paired information was correct. Thes process produced a single merged database with all the relevant information on live births from 2012–2017 and all deaths of children up to 5 years of age in 2012–2018. This resulted in 13,347 pairs from the SINASC (2012–2017) and SIM (2012–2018) databases. We included only the pairs that had information on NM and GA at birth in days in this study. We used this merged database (from the SINASC and SIM databases) for all analyses.The main study outcome was all-cause neonatal mortality (NM), defined as the death of liveborn infants in the first 27 days of life from any cause.The main independent (predictor) variable was GA at birth in days (between 259 and 293 days) estimated on the basis of the first day of the last menstrual period (LMP) reported by the mother. The following maternal covariates were included as additional independent variables in the adjusted models: age , self-reported skin color (white or nonwhite), education , living with a partner (yes or no), number of antenatal care visits , and parity . We also included type of birth  and type of hospital (public or private) as associated variables.We used Cox regression models to calculate hazard ratios (HR) and 95% confidence intervals (95% CIs) to assess the association between GA in days and NM, adjusted for several covariates. We censored children who did not die by the last observation date . We modeled NM by GA in days using restricted cubic splines with five knots ; 279 days was the reference knot because it was closer to 280 days, the central point of the full-term period. We first conducted a crude univariate analysis with GA in days (Model A). In Model B, we adjusted the results for maternal  and gestational  variables. Model C was identical to the previous model with additional adjustments for type of birth  and type of hospital (private or public). We also conducted stratified analyses according to type of birth (Models D and E) and type of hospital (F and G). We used Stata version 15  for all statistical analyses and to create the graphs.We performed a sensitivity analysis for the sample using all 1,202,843 live births during the study period as a parameter but using gestational age in weeks. Crude and adjusted  models were performed, with results similar to models made using only the sample with gestational age in days , women with GA between 294–321 days , and four women without information on the date of birth, we had a total of 440,119 eligible live births that were included in the study .In all GA categories, 70.4% of women were 20–34 years of age, 59.3% had white skin, 86.7% had a high school or university education, 60.2% lived with a partner, 52% were giving birth for the first time, 65.3% had a cesarean section and 59.4% gave birth in private hospitals. More detailed information is provided in Out of the 2160 offspring deaths that occurred in 2012–2018, 797 took place in the first 27 days of life, 84.4% had at least one potential pregnancy day lost, and most were born by cesarean section (64%). See All NM models had U-shaped curves . In the In stratified analyses according to type of birth , both haIn the analyses according to the type of hospital , we obseIn the city of São Paulo, babies born in the first days of the term period had a higher risk of NM in all models. This risk decreased for infants born closest to the median GA and increased again afterward. This U-shaped curve suggests that babies born closest to the probable due date (280 days) have the lowest risk of NM.In all the models, the curves were U-shaped, with higher NM risks at the extremes of the term period. The risk of NM was significantly higher for early-term infants in the three models. The risk of NM was significantly higher for late-term infants only in the crude model, losing its significance in the models that adjusted for maternal characteristics, number of antenatal care visits, and type of birth.Our results using adjusted models were similar to those reported by Wu et al.  in a stuThe decrease in the risk of NM in later-term infants in our adjusted models (B and C) showed the effect of covariates, especially type of birth and type of hospital, on the risk of NM in infants born after their due date. This effect was more evident in the stratified models. For early-term infants, vaginal birth (Model D) seemed to be safer than birth by CS (Model E). For example, for babies born at 259 days, the risk of NM for infants born by cesarean section was 3.75 (95% CI 2.87 to 4.90) versus 2.69 (95% CI 1.72 to 4.22) for those born vaginally. The lower risk of NM for babies born vaginally was constant until near the estimated date of birth; this should alert clinicians to avoid CSs before this date. To reduce perinatal risks, several specialist associations, including the American College of Obstetrics and Gynecology, recommend delaying elective CS until 39 weeks of pregnancy . In BrazOur results indicated a progressive, albeit nonsignificant, increase in the risk of NM in babies born vaginally after 284 days of gestation. In Brazil, there is a high incidence of interventions in women who have a vaginal birth, including artificial rupture of membranes, oxytocin to induce or augment labor, and fundal pressure in the second stage, which are often associated with iatrogenic adverse events . AccordiThe type of hospital had a differential effect on NM in different term periods. While the risk of NM was higher for early-term infants in both types of hospitals (Models F and G), the risk of NM for babies born full term (278–283 days) was lower in public hospitals. This protective effect was not found for babies born in private hospitals.The median GA at birth in the São Paulo city cohort was 274 days, which was lower than the average GA at birth of 280 days in most international studies . HoweverThe high number of neonatal deaths in infants born at term signals the need to improve the quality of care offered to pregnant women in the antenatal period and during birth, since many of these deaths are potentially preventable . One of We acknowledge several limitations, such as the lack of data on maternal morbidity that could affect NM, information that is not available in the SINASC database. The lack of data on sonographic GA is another study limitation because of the possible discrepancies in GA estimated by LMP. The lack of information on GA in days in over half of the original sample is an additional limitation. However, a sensitivity analysis did not find significant differences between GA in days between the study population and a sample of the complete original dataset with GA in weeks. This lack of information on GA in days in a large proportion of birth certificates, the main source of information for the SINASC database, indicates the need to improve and standardize how these forms are filled in São Paulo city hospitals.A strong point of the study was that we used a cohort of live births in the largest city in Brazil, which also has one of the best datasets on births in the country. Additionally, to the best of our knowledge, this is the first Brazilian study to use granular data for GA in days, available in the SINASC database, to analyze the complex problem of NM in a setting where all women who give birth have access to health care.In all models, the risk of NM was significantly higher for early-term babies, decreasing until approximately 280 days of gestation, with daily differences. However, contrary to some results of studies conducted in other countries, we found in some models an increased risk of NM for late-term babies. The main hypothesis for this finding is that pregnancies that progress to the late-term period in São Paulo receive more potentially iatrogenic interventions , which could increase the risk of NM in these cases.The results of this study, from the largest city in South America, can help to understand the role of “potential pregnancy days lost”  in the causes of neonatal mortality, better informing public health policies on maternal and child health. To the best of our knowledge, this is the first Brazilian study on GA in days using a national live births database. Analyses using GA in days and other factors associated with NM at term can help to generate an important discussion on changes in the way data are collected in birth and mortality information systems, showing more clearly the effect of each day on main neonatal outcomes. In this way, this study has the potential to impact health policies across Brazil as well as the health care of women and babies.S1 Table*Adjusted for maternal variables , number of antenatal care visits, type of birth and type of hospital.(DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.


Glaucoma is the first major category of irreversible blinding eye illnesses worldwide. Its leading cause is the death of retinal ganglion cells and their axons, which results in the loss of vision. Research indicates that glaucoma affects the optic nerve and the whole visual pathway. It also reveals that degenerative lesions caused by glaucoma can be found outside the visual pathway. Diffusion tensor imaging (DTI) is a magnetic resonance imaging (MRI) technique that can investigate the complete visual system, including alterations in the optic nerve, optic chiasm, optic tract, lateral geniculate nuclear, and optic radiation. In order to provide a more solid foundation for the degenerative characteristics of glaucoma, this paper will discuss the standard diagnostic techniques for glaucoma through a review of the literature, describe the use of DTI technology in glaucoma in humans and animal models, and introduce these techniques. With the advancement of DTI technology and its coupling with artificial intelligence, DTI represents a potential future for MRI technology in glaucoma research. Glaucoma is a diverse group of diseases characterized by damage to the retinal ganglion cells (RGC) and their axons, the retinal nerve fiber layer (RNFL), which leads to progressive vision loss . If leftDiffusion tensor imaging (DTI) is now the most prevalent method for investigating WM degeneration in glaucoma patients . In addiRoutinely used methods for glaucoma evaluation include gonioscopy, IOP measurement, corneal thickness measurement, fundus photography, VF inspection, and optical coherence tomography (OCT) . MoreoveThe term “visual field” refers to the area the human eye can perceive, and the “VF examination” primarily refers to the perimeter measuring this region. VF examination is a subjective test used to diagnose glaucomatous damage and quantify the degree and severity of visual impairment in glaucoma patients . The autOptical coherence tomography is a non-invasive imaging technology that permits the quantitative measurement of changes in the ON, RGC axon, and RGC body layer, RNFL . Since iOptical coherence tomography angiography is a non-invasive, dye-free imaging technique that can measure the retina, optic papilla, and choroidal blood vessels in qualitative and quantitative ways. OCTA can provide information on the extent of perfusion damage at various depths in the retina and choroid. Because advanced glaucoma has a lower vascular density , OCTA caConventional glaucoma diagnostic technologies, including fundus photography, OCT, OCTA, and VF, focus exclusively on the retinal region and disregard information regarding the WM of the brain, which transmits and systematically processes vision. The visual pathway consists mainly of the WM of the brain, and DTI is the advanced imaging technology currently available for evaluating the WM of the brain ; consequDiffusion-weighted imaging (DWI) is a magnetic resonance imaging (MRI) technique used to assess water diffusion . Due to Changes in DTI parameters of the glaucomatous ON and beyond the ON are hot areas of current research . The folin vivo DTI was demonstrated to detect RGC axonal damage earlier than immunohistochemistry in the early stages of the disease in a mouse model of optic nerve crush-induced glaucoma (In vivo evaluations of axonal, myelin, and trans-synaptic degeneration in canine PACG can be done using DTI. Research on DTI is currently being done primarily in rat eyes, and future research on glaucoma in large animals, including dogs, pigs, and monkeys, will also be done.Diffusion tensor imaging has been examined extensively and thoroughly in glaucoma-affected animal models with altered visual pathways in experimental animal models. Numerous investigations on rat glaucoma models have documented an increase in MD and a decrease in FA of the ON and OT . Moreoveglaucoma . DTI canglaucoma . The evaGlaucoma-related modifications to DTI parameters in the visual pathway constitute a significant research focus. Multiple studies have found that glaucoma patients had lower FA and higher MD on DTI . MoreoveResearchers are particularly interested in determining whether abnormalities occur outside the visual pathway in glaucoma patients. In contrast to nature controls, NTG patients show decreased FA of OR and forceps major in the occipital lobe, according to a DTI study by As a result, we can now consider glaucoma a neurodegenerative disorder affecting the visual and extra-visual pathways owing to analyzing various types of glaucoma utilizing DTI. Additionally, DTI offers objective and quantifiable changes in the visual pathway and improves understanding of the central visual pathway’s degenerative process in human and animal glaucoma.Multiple investigations have demonstrated that as the disease progresses, FA and MD values in the ON and OR decrease and increase, respectively . In addiApplication DTI has some limitations. The human ON is small, only 3–4 mm in diameter. There may be specific areas throughout the inspection when artifacts lead to examination errors . This reMoreover, the DTI signal acquisition time is extended, which could cause motion distortions if the patient moves a lot during the examination. DTI should be enhanced and modified prior to its widespread usage in clinical glaucoma testing, which is now employed primarily for research. The accuracy and reproducibility of DTI results can be impacted by oculomotor interference, large magnetic sensitivity differences in the orbit caused by gas and bone in the sinus combined with the thin diameter of the optic nerve, encapsulation by cerebrospinal fluid in the sphincter cavity, and surrounding orbital fat . The finIn this review, we only discuss the alterations of DTI in the visual and extra-visual pathways because not only WM alterations but also gray matter alterations are present in the central visual pathways of glaucoma patients, and intracranial gray matter alterations are typically detected by functional MRI , which iThe area of glaucoma and ophthalmology, in general, is primarily image-based, and AI is situated to solve many of these problems . Deep leIn summary, DTI often showed a rise in MD and a decrease in FA, strongly linked with increasing disease severity in glaucoma patients or animal models. DTI is a promising non-invasive technique for assessing the severity and prognosis of glaucoma. As clinical and scientific uses of DTI continue to develop, practitioners in the area will engage with ophthalmologists to overcome its limits and enhance patient care.All authors contributed to draft and revise the manuscript, gave final approval of the version to be published, and agreed to be accountable for all aspects of the work.


The infant's anthropometric characteristics  were compared at birth, at 32, 34, and 36 weeks of gestational age, at the time of full oral feeding, and at discharge from the unit.A total of 66 infants were included, gestational age at birth range from 24 to 30. Full oral intake occurred at 37.1 ± 2.1 weeks postmenstrual age (PMA). We found an inverse correlation between gestational age at birth and birth weight with PMA at which full oral feeding (FOF) is achieved. PMA at discharge was 38.6 ± 2.5 weeks. Age of full oral intake and discharge occurred later in infants who had patent ductus arteriosus, retinopathy of prematurity, and sepsis or received a blood transfusion. A positive correlation was found between days of oxygen and both parameters. However, we found no relationship between necrotizing enterocolitis or intraventricular hemorrhage with age at full oral feeding or age at discharge.The transition from gastric tube to oral intake did not affect growth. We found a close relationship between preterm infants birth, earlier younger than 30 weeks of gestational age, and low birth weight, with a delay in full oral feeding achievement that correlated with age at discharge. Preterm infants have higher nutritional requirements per kilogram than term infants and are less tolerant of high fluid volumes . PhysiolSuccess in oral feeding depends on several factors: the infant's neurological and physiological maturity, the ability to concentrate on feeding, to organize oral-motor functioning, to coordinate swallowing with breathing, and to maintain physiological stability; as well as the ability of the caregiver to regulate the baby during feeding and to recognize and respond in a timely manner to the baby's behavioral and physiological signals, with the aim of preventing physiological decompensation and repeated stress. It is especially important to have a unit's consensus on nutrition/feeding and the nutritional protocol . Illnessz-score for weight between birth and 35 weeks of 0.52, and another decline of 0.48 between 35 weeks and discharge , from January 1, 2019 to December 31, 2019.The study sample included all infants younger than 30 weeks of gestational age. Infants were excluded if they had congenital anomalies, genetic diseases, were admitted after 48 h of life, died in the first 7 days of life, were not receiving enteral nutrition, had incomplete medical records, or if at the time of discharge they were receiving gastric tube feeding.Of the 98 preterm infants younger than 30 weeks of gestational age admitted during the study period, 7 died prior to discharge  and 25 met the exclusion criteria.The study, therefore, included 66 infants, with a range of gestational age from 24 to 30, mean ± standard deviation (SD) gestational age, and weight at birth of 28.3 ± 2 weeks and 1.1 ± 0.3 kg, respectively see .International recommendations on early nutrition were followed, starting with parenteral nutrition immediately at birth. Enteral nutrition was introduced in the first 24 h after birth. Advances in enteral feeding volumes were made based on the signs of tolerance: vomiting, abdominal distension, and gastric residuals. The goal of enteral nutrition was to reach 150 ml/kg/day using OMM or donor human milk (DHM) when OMM was unavailable.Donor human milk was available for infants born at <32 weeks or <1,500 g, at least during the first 3 or 4 weeks of postnatal age or until they reached 1,500 grams in weight, whichever occurred last. Once gestational age or the indicated weight was reached, if there was not enough OMM, the premature formula was administered for premature newborns. Standard fortification of human milk started when enteral feeding reached 100 ml/kg/day and continued until discharge.Exclusive oral feeding is defined as the time of achieving full milk volume intake at the breast or by the bottle, for 24 h.Persistent ductus arteriosus (PDA) was only considered if treatment  was reported as all hemodynamically significant PDA are considered for treatment in our protocol . Moderatz-score for each of these measurements was calculated based on the 2013 Fenton anthropometric neonatal charts . First, a description of the sample included in the study was made.The data were processed using a database in Microsoft Excel format, which was later imported for statistical treatment in the SAS program, version 9.4 . Statistically significant differences were those that presented a probability of error <5% (z-scores) were compared at birth, at 32, 34, and 36 weeks of gestational age, at the time of full oral feeding, and at discharge from the unit.As a pilot study, no power calculation analysis was performed. The infant's anthropometric characteristics  when the analysis involved 3 or more groups. When necessary, non-parametric tests were used, Kruskal–Wallis or Mann–Whitney U non-parametric tests. The frequency analysis between qualitative variables was performed using the chi-square test or Fisher's exact test when necessary.Comparisons between continuous quantitative variables between independent groups were assessed mainly by parametric tests, using the Student's z-Score) were analyzed with repeated measures ANOVA, together with the Greenhouse–Geisser test. When the result was significant, the Bonferroni post-hoc test was used to explore all possible differences between two-to-two time points.The correlation analysis between the continuous quantitative variables was performed using Pearson's R correlation coefficient. The variables measured longitudinally . At 34 weeks' PMA, further decreases in weight and length z-scores were observed compared with 32 weeks (p = 0.005). No differences in head circumference z-scores were observed between these 2 periods (p = 1.000).At 32 weeks' PMA, we observed a decrease in weight, length, head circumference, and p = 1.000). A significant increase in the head circumference z-score was observed at 36 weeks' PMA and at discharge compared with 34 weeks' PMA (p < 0.001).At 36 weeks' PMA and at discharge, no differences in weight or length z-scores were found compared with 34 weeks' PMA  was also related to full oral feeding age. HL (days) = 1.041 ×  + 7.528 .The OMM consumption during hospitalization decreased over time. The percentage of OMM intake relative to total enteral intake was 63, 64, 57.5, and 24% during the first 14 days and at 32 weeks' PMA, 34 weeks' PMA, and 36 weeks' PMA, respectively. Before 32 weeks' PMA, OMM intakes are completed with DHM, depending on the patient's weight  and the availability of DHM in the milk bank. From 32 weeks' PMA, feedings were completed with premature formula. At the time of discharge, only 33.8% had managed to establish breastfeeding directly at the breast .We found a correlation between days of oxygen, the presence of PDA, retinopathy of prematurity, sepsis, and transfusion, and age at full oral feeding and age at discharge. However, we found no relationship between necrotizing enterocolitis or intraventricular hemorrhage with age at full oral feeding and age at discharge .z-scores from 34 weeks' gestation to discharge, when full oral feeding occurred. The weight and length z-scores decreased from birth to 34 weeks' PMA. The head circumference decreased from birth to 32 weeks' PMA. The head circumference z-score increased from 32 weeks to discharge, and the weight and length z-scores remained stable from 34 weeks' PMA to discharge.This study aimed to evaluate the effect of the transition to oral feedings on growth and age at discharge. We did not find a further decrease in weight or length z-score from 34 weeks' PMA to discharge, weight, and length gain continue during the transition phase, from gavage to oral, without slowing down, and showed an increase in the head circumference z-score, in head circumference growth. A possible explanation of our findings is that full oral feeding was achieved by bottle, not at the breast, in 66% of the infants. The milk volume administered did not change between before and after achieving this goal. In our study infants were followed for 5 weeks.The weight gain is maybe influenced by oral feeding. Full oral feeding was achieved after 34 weeks' PMA and before discharge. The present study results showed no changes in the weight or length z-scores at 35 weeks of gestational age and at discharge, but not in head circumference, with a population similar to the one reported here, with a percentage of breastfeeding at the discharge of 78.1%.These results are consistent with those reported by Lehnart Vargas et al. , with a p < 0.001) Kangaroo mother care was promoted in our study,  weeks. These results are similar to those reported by Khan et al.  in whichr study,  but other study, .Our findings confirmed that gestational age at birth influenced the development of full oral feeding, preterm neonates who were less mature at birth were more mature at attainment of independent suckle-feeding. It is acknowledged that extremely preterm infants have immature motor skills and lack coordinated sucking ability. This result is in accordance with the results of Khan et al.  and JackPreterm infants must achieve independent oral feeding before discharge. Achievement of independent oral feeding remains the most common barrier to discharge in preterm infants. The timing at which full oral feeding is attained is associated with the age at hospital discharge and may reduce associated hospital costs. Our results can thus help predict when patients will be discharged: once they have achieved full oral feeding, it will be 1 week after achieving this milestone. The next step is to evaluate prospectively whether a practice change to earlier attainment of full oral feeding enables earlier hospital discharge. Finally, we found several morbidities related to prematurity that delay the goal of achieving full oral feeding, especially those related to the use of oxygen- and PDA-treated infants. However, we found no association between full oral feeding and necrotizing enterocolitis or intraventricular hemorrhage, probably because a larger sample size would be necessary . Few stuThe main limitation of this study was its retrospective design. Other limitations: Information on mechanical ventilation was not collected; we did not have into account when the transition from oral tube to oral intake begins and the duration, only when it was achieved; no measurement tool was used for the evaluation of readiness for feeding and it was conducted only in one Center.In our study, in which only one third of the infants established breastfeeding at discharge, two thirds were bottle fed, transition from gastric tube to full oral feeding does not affect growth. We found a relation between preterm birth, earlier than 30 weeks gestation and low weight at birth with a delay in full oral feeding achievement that correlated with age at discharge.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by Comité de Ética de la Investigación con Medicamentos del Hospital Universitario La Paz. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.The planning, conception, and design of the study were proposed by BC together with MS. Data collection was carried out by SF and BC. The statistical analysis was performed between JD, MS, and BC. And finally, the conclusions and writing of the article were carried out by BC and MS, with the critical review of SF and JD. All authors contributed to the article and approved the submitted version.This work was funded by the Fellowship 2021-2023 ESPGHAN Networking Grant 2021 to MS (PI).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The main risk factors associated with PTSD after birth were obstetric interventions and obstetric violence such as emergency caesarean section or a non-compliant birth plan, a previous mental illness, having suffered from of a traumatic event or depression and/or anxiety, and having poor social support throughout pregnancy and/or during birth. Conclusions: Obstetric interventions, obstetric violence, experiencing a traumatic event or depression and/or anxiety, and a previous mental illness are factors associated with the diagnosis of PTSD after birth. Protective factors are multiparity, adherence to the mother’s birth plan and skin-to-skin contact.Background: Post-traumatic stress disorder (PTSD) after birth has generated a growing interest in recent years. Although some risk factors associated with PTSD have been studied, information is still scarce to date on risk factors associated with PTSD. This systematic review aims to identify risk factors associated with the diagnosis of PTSD after childbirth. Methods: We searched on PubMed, Web of Science and SCOPUS databases, from inception to May 2022. Quality assessment of the articles was performed using the Newcastle-Ottawa Quality Assessment (“NOQAS”) scale. This systematic review was performed according to the PRISMA guidelines. Inclusion criteria were women with age ≥18 years; articles in English or Spanish; articles focused on physical, social, psychological, medical-obstetric, and environmental risk factors. Results: A total of  Post-Traumatic Stress Disorder (hereafter PTSD) is a disorder that occurs after suffering a stressful and extremely traumatic situation . During PTSD, the person experience a real danger to his or her life . PTSD afPTSD after childbirth is considered the third most common psychiatric disorder among pregnant women, after depression and nicotine dependence . Ten perThe symptomatology of PTSD after birth can increase during the first four to six weeks after childbirth. Five symptoms have been revised by psychologist Cheryl Beck, (1) persistent thoughts and memories of childbirth, through flashbacks and/or nightmares; (2) disconnection from their babies, which can appear immediately after the birth; (3) repetitive expression of what happened during childbirth; (4) feelings of anger towards professionals, family and self and (5) negative experience of motherhood . By idenThe aim of this systematic review was to identify risk factors associated with post-traumatic stress disorder after childbirth. The importance of conducting a systematic review on this topic lies in the scarcity of risk factors associated with PTSD after childbirth.This systematic review was performed according to the PRISMA guidelines  11]. Th. Th11]. Searches were carried out on Web of Science, Scopus and PubMed databases, from inception to May 2022. The following queries and MesH  terms were used: ((PTSD OR PTSD after childbirth) AND (mothers OR newborn)) OR ((PTSD OR PTSD after childbirth) AND (mothers OR newborn) OR ((PTSD OR PTSD after childbirth) AND (risk factors)) OR (PTSD OR PTSD after childbirth) AND (risk factors)) OR ((PTSD OR PTSD after childbirth) AND (traumatic birth) OR ((PTSD OR PTSD after childbirth) AND (traumatic birth)) OR ((PTSD OR PTSD after childbirth) AND (pregnancy and childbirth)) OR ((PTSD OR PTSD after childbirth) AND (pregnancy and childbirth)). In order to obtain a more precise search, additional terms were included in the search strategy: . Studies were independently screened for eligibility and selected by two researchers (MMR y de IFK), as a result of a multi-step approach .The article selection process was carried out by a first reviewer (M.M.R), who checked whether the study met the planned selection criteria, and discarded by title and/or abstract, and a second independent reviewer (I.F.K) Discrepancies between the two reviewers were resolved by discussion.Similarly, data were extracted through independent work by two reviewers  and disagreements were cross-checked by a third reviewer (B.R.G).Inclusion criteria were (a) women with an age ≥18 years who may suffer or have suffered from PTSD after childbirth; (b) articles in English or Spanish language; (c) articles focusing on physical, social, psychological, medical-obstetric, and environmental risk factors that may be related to the occurrence of PTSD after childbirth. Post-traumatic stress disorder is a persistent memory in which the traumatic experience is relieved. The person engages in avoidance behaviour and may show “hypervigilance”, to ensure that the traumatic event is not repeated. Symptoms persist for more than a month after the event and affect the person’s ability to function .Exclusion criteria were (a) articles in languages other than English and Spanish; (b) articles whose results were not relevant; (c) papers whose full text was not available; (d) articles not available free of charge; (e) experimental animal studies were excluded.Assessing the quality of such studies is essential for a proper understanding of non-randomised studies. As tabulation and description of the included reviews and articles addressing postpartum PTSD, authors of the study, year, country of study, research design, average age of participants, gestational age at delivery, risk factors associated with post-traumatic stress following childbirth and study quality Newcastle-Ottawa Quality Assessment Scale (NOQAS) . This scOur search provided 8415 citations from inception to May 2022. We excluded 3561 duplicates. We rejected 3959 references because the titles or abstracts of the articles were unrelated to the objective to be investigated, leaving 57 full-text studies. Of these, 35 were excluded due to lack of relevant results, unavailability of full text or because they were in a language other than English or Spanish. Finally, 22 studies were selected for data extraction .n = 101 [n = 2990 [n = 800 women, shows a positive association between fear of childbirth  or lack of exercise during pregnancy  with PTSD after birth [Obstetric interventions and obstetric violence were the main risk factors for developing PTSD after birth. Women with previous mental illness is also considered an important risk factor. The main social factor influencing the development of PTSD after childbirth is the low social support that women may have, both throughout the pregnancy process, and in the birth experience itself ,17,18,19er birth . A cohorer birth . Finallyer birth .Experiencing a caesarean delivery, or an emergency delivery are the most influential risk factors   in triggChildbirth complications, such as lower likelihood of co-homing, lack of skin-to-skin contact, and an absent of breastfeeding, have been associated with PTSD after birth . These bWomen who experience depression and/or anxiety during pregnancy  , as wellLack of maternal social support during pregnancy and/or during birth positively influence the development of PTSD after birth ,25,28,32This systematic review examined the main risk factors influencing the occurrence and development of PTSD after birth. PTSD after birth is a mental illness related to the birth experience, such as having had a caesarean section, emergency caesarean section and an obstetric violence experience ,18,19,22In turn, the most common psychological factors that has been found to cause PTSD after birth are depression and/or anxiety throughout pregnancy ,20,21,29Obstetric violence has been considered the main risk factors for the development of PTSD after birth. According to López et al., these different forms of obstetric violence can be a risk for both the mother and the baby, such as anaesthesia complications at delivery . Women wTraumatic events seem to be a factor strongly associated with PTSD after birth ,25. PTSDIn relation to demographic and social risk factors, Milosavljevic et al.  found noIt is important to highlight domestic/partner violence and its relevance in the occurrence of PTSD after birth. This factor remains understudied, but Oliveira et al.  have shoWe have established some of the most statistically significant ORs to determine the variables most strongly associated with the development of PTSD, such as severe expected intensity of pain (OR = 4.65) , anteparThis systematic review tackles risk factors that may cause PTSD after birth. It has been carried out based on the PRISMA guidelines, establishing a PICO research question. A clear inclusion and exclusion selection criteria were defined, in order to be accurate. In turn, studies that were selected for analysis range from medium to high quality. Of these, 11 were of average quality ,30,31,43n = 11) and cohort studies (n = 2), including nine cross-sectional studies. Heterogeneity regarding the methodology of the included studies, and consequently from their findings, is somewhat evident. Heterogeneity is also true regarding different validity of the diagnoses [Limitations of the review were the lack of information on the risk factors that can cause PTSD after birth. Studies included in the systematic review were mostly case-control , we still do not have a registration number.Studies focused on the risk factors involved in PTSD after birth were from moderate to high quality. This systematic review emphasises the importance of knowing these risk factors for mental illness. Moreover, measures and actions can be taken throughout pregnancy to reduce factors involved in the development of PTSD after birth. The systematic review shows that obstetric violence or emergency caesarean section, are well known as a risk factors but other such as demographic situations, maternal morbidity, or insomnia, are lesser known. The inclusion of a short screening in a preconception consultation could greatly avoid PTSD after birth. In this screening variables that should be addressed are, among others, mother’s thoughts, women mental health (depression and/or anxiety), social support, fears about pregnancy and/or birth process.Although there is not much information to date on what aspects are effective in preventing a traumatic birth experience, more direct support and care for women giving birth and their birth experience could be one of them .Future research could include high-risk samples with the aim of understanding whether various predictive pathways can be applied, as well as considering different time points for collecting predictive data. Predictive data can identify risk before trauma occurs, this informing possible primary preventive measures. However, predictive data that is stored immediately after birth can be helpful, and more specifically, important for the creation of secondary preventive measures that can be administered after delivery .On the other hand, it would be interesting for future research to perform a meta-analysis of the pooled effect sizes/odds ratios for the most important risk factors. This is a topic that could be addressed in future reviews when more information is available in this area.Obstetric interventions, obstetric violence, depression and/or anxiety and previous mental illness are factors associated with the diagnosis of PTSD after birth. Protective factors are multiparity, adherence to the mother’s birth plan and skin-to-skin contact. Maternal physical and psychological factors are risk factors of PTSD after birth. Maternal social health, and social support, is even more important in the association with PTSD after birth occurrence. Further research and information will help to better understand and possibly prevent to reduce factors involved in its incidence.