Periodontal disease and its bacteria can be responsible for pregnancy complications and transmission of periodontal bacteria from mother to newborn.A salivary swab to 60 healthy, full-term newborns and their mothers was taken immediately after birth. The test was performed with Real Time PCR method to evaluate the expression of the gene through DNA amplification. The species considered were: Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum ssp.The newborn oral microbiome was composed primarily by saprophytes (98.38 + 4.88%), just like the mothers (98.8 + 3.69%). There was a statistically significant difference of the total microbiological density in newborns and mothers (p = 0.0001). Maternal and neonatal oral microbiome had a correlated total microbiological density only in 33.3% (N = 20/60) of cases. The analysis of the oral microbiome showed a pathological composition only in 12/60 babies (20%). The most frequent detected specie in newborns was Fusobacterium nucleatum , as well as for the mothers (53.3%). However, the pathogen was present both in baby and his mother only in 3 dyads. Porphyromonas gingivalis showed the highest association mother-baby . Porphyromonas gingivalis was the pathogen with the highest bacterial load in the 12 mothers. We found a statistically significant difference in the total load of Porphyromonas gingivalis in mothers and babies (p = 0.02).There was a statistically significant difference in the richness of the microbiome from newborns and mothers. Even comparing the microbiological density in the oral cavity of the individual mother-child pairs, we did not find a significant concordance. These results seem to suggest a low influence of maternal oral microbiome on the richness of the oral neonatal one. We didn’t find mother-child concordance (p = 0.0001) in the presence of pathogenic periodontal micro-organisms. Fusobacterium nucleatum was the most frequent specie detected. Porphyromonas gingivalis instead was the bacteria with the higher possibility of transmission. In conclusion in our study maternal oral health doesn’t affect healthy, full-term newborns’ oral microbiome. Further studies are needed to understand the maternal influence on newborn’s oral microbiome and its effects on babies long-term health. Even if the origin of the neonatal oral microbiome is poorly understood, it is clearly that richness and change in composition of neonatal oral microbiome impact both short and long term outcomes , 2. It iThe influence of maternal oral microbiome to early oral newborn colonization isn’t well understood and there is a need for more in-depth research to understand if cariogenic bacteria colonize the fetus’ mouth in healthy pregnancies too.Oral diseases are progressive, cumulative and grow in complexity over time, which is why it is very important for good oral health to be established early in life.In order to motivate the oral hygiene of women of childbearing age and to prevent future diseases in the systemic and oral health of children, we analized the neonatal oral microbiome to estabilsh if there is an influence of the maternal oral microbiome in healthy pregnancies on the neonatal mouth colonization at birth.This Retropective observational cohort study was performed in the Neonatal Unit of the IRCCS Ospedale Policlinico San Martino Genoa, Italy in collaboration with the Academy Unit of Obstetrics and Gynecology and the Orthodontics Department of Genoa University between 2017 and 2018.A total of 60 newborns  and their mothers were enrolled to study their oral microbiome. The average birth weight was 3205.9 g, ranged 2600–3850 g. Women ages was ranging from 20 to 42 years old (mean 32.08 y.o.).Inclusion criteria were: newborns must have to be born by vaginal delivery, a term , in health and adeguate for gestional age (AGA). Babies just after birth must stay skin to skin with their mothers. Mothers must not have received antibiotics during delivery and must not have taken antibiotics during the last 6 mounths of pregnancy. Furthermore, the women in this study must not have drunk or eaten or brushed their teeth half an hour before the test. None of the mothers had received dental treatment during pregnancy.Exclusion criteria were: newborns by caesarean section, prematurity , newborns with difficult birth or any health problem who don’t permitte them to stay immediatly after birth skin to skin with the mother because they necessity hospitalization in Neonatal Intensive Care Unit.The outcome of this study was to analize oral neonatal microbiome and to evaluate the trasmission in healthy pregnancies of the major pathogenic bacteries in oral diseases comparing it with the maternal oral cavity microbiome.The study provides for the execution of a salivary swab to the neonate and his mother; it was a multi-site microbiological test. The samples were taken from the baby’s immediately after birth, before start breastfeeding, during skin to skin to accurately reflect colonization prior to and during birth. Samplings were obtained using steril paper cones swiped on the oral mucose of the babies and inserted into the periodontal sulcus of the mothers for at least 1 min. The test was performed with Real Time PCR method, to evaluate the expression of the gene through DNA amplification.The microbial density in the test was expressed in number of bacteries (copies per µl).The species considered for this research were: Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum ssp.Written informed consent was obtained from parents or caregiver of patients after full explanation of the study, in according to the declaration of Helsinki.Data are described as mean and standard deviation (SD) or median and range for continuous variables, and as absolute and relative frequencies for categorical variables. Non-parametric analysis  for continuous variables and the Chi square or Fisher’s exact test for categorical variables were used to measure differences between groups. P values ≤ 0.05 were considered statistically significant, and all P values were based on two-tailed tests. Statistical analysis was performed using SPSS for Windows .Ethical clearance for the publication of this study was obtained from the Regional Ethical Commitee .This study analyzed 60 babies’oral microbiome at birth. All infants were born at full term by vaginal, eutocic delivery. Newborns were adeguate for gestational age (AGA) and had not health problems.We analyzed and compared newborn’s oral microbiome at birth with their mothers’oral microbiome to understand if it could be influenced by their mothers during pregnancy.3 copies per µl in 10/60 newborns (16.7%); 17/60 babies (28.3%) were between 103 and 104 copies per µl; 14/60 babies (23.3%) had microbiological density higher than 104 but lower than 105 copies per µl. Richer microbiome (> 105 copies per µl) was found in 19/60 babies (31.7%), only 4 of them had a microbial community density > 106 copies per µl.We found a microbial community density lower than 105 copies per µl. In 2 cases both mother and neonate had microbilogical density < 105 copies per µl. In all the other couples, mothers had a richer microbiome > 105 copies per µl, but their children weren’t.There was a statistically significant difference of the total microbiological density in newborns and mothers (p = 0.0001). Maternal and neonatal oral microbiome had a correlated total microbiological density only in 33.3% (N = 20/60) of cases. 18 mothers and babies had both a microbiome > 105 copies per µl). In comparison, the oral microbiome of mothers showed the presence of pathogenetic bacteria in oral diseases in 80% (N = 48/60), so in 36/60 cases we didn’t find mother-child concordance (p = 0.0001) in the presence of pathogenic periodontal micro-organisms.The oral cavity microbiome of the 60 full term babies was composed primarily by saprophytes (98.38 ± 4.88%), just like the mothers (98.8 ± 3.69%). The analysis of the oral microbiome showed a pathological composition only in 12/60 babies (20%). Only 4/12 of these newborns had a richer microbiological density too . In addition, comparing the percentage of saprofite and pathogens in the microbiome, there was also a significative difference in the microbiome composition of the 12 babies and their mothers (p = 0.03).5 copies per µl.About investigation of the periodontal micro-organisms in the 12 babies, in 4/12 babies (33.3%) there was a polimicrobial contamination: in 1 case of them we found contamination by 3 different species, the other 3 cases was contaminated by 2 different species, they had higher total microbial community > 10We found Fusobacterium nucleatum in 9/12 babies (75%): it was the most frequent specie present in these 12 newborns. Even in mothers Fusobacterium nucleatum was the most frequent oral bacteria detected: the frequence in the 60 mothers was high 53.3%. In 6 mothers of these 9 babies, Fusobacterium nucleatum wasn’t found, so only in three of the dyads the presence of Fusobacterium nucleatum spp was associated.The frequence of the species Prevotella intermedia and Porphyromonas gingivalis in the babies’ oral microbiome was the same , lower than Fusobacterium nucleatum. The frequence of Prevotella intermedia in all 60 mothers was 31.7% (N = 19/60). Prevotella intermedia was present in 4/12 mothers, but only in 2 of the 12 dyads (16.6%) Prevotella intermedia was observed in both mother and newborn. Porphyromonas gingivalis was present in 29/60 mothers (48.3%) and in 7 of the 12 mothers (58.3%). It showed an higher association mother-baby than all the others specie examinated because we observed trasmission of Porphyromonas gingivalis in 4 of the 12 dyads (33%).Despite the specie with the highest bacterial load in newborns was Fusobacterium nucleatum (1266 ± 2634 copies per µl), Porphyromonas gingivalis was the oral pathogen with the highest bacterial load in the 12 mothers. There was a statistically difference in the total load of Porphyromonas gingivalis in mothers and babies . This difference wasn’t observed for the other species examinated, in fact the bacterial count of Fusobacterium nucleatum, Prevotella intermedia was similar in the 12 dyads mother-newborn (p > 0.05).None of the 12 babies showed the presence of Aggregatibacter actinomycetemcomitans in their oral microbiome. Aggregatibacter actinomycetemcomitans was the less frequent in the mothers’ oral microbiome too: it was present in only 1 mother of the 12 dyads and in 7/60 mothers (11.7%).Several studies confirm that the microbiome in the first years of life is fundamental for health in future. Even if the most of oral microbial colonization is traditionally considered to take place after birth, the beginning of the development of the microbiome seems to takes place just at the fetal level with the passage of bacteria through the placenta and then becomes more complex from birth onward . The hum6 copies per µl. There was a statistically significant difference in the richness of the microbiome from the 60 newborns and mothers (p = 0.0001). Even comparing the microbiological density in the oral cavity of the individual mother-child pairs, we did not find a significant concordance (33.3%). It is supposed that the presence of caries in mothers don’t influence microbiome density and composition in infants in the first year of life [In our study, the microbiological test executed at full term birth by a salivary swab, shows us the density of microbiological population in the mouth of the neonates and their mothers. We examinated the richness of oral microbial community of the babies immediately after birth, before skin to skin contact with the mother and before start breastfeeding, to reduce the influence of them on the density of the microbial community in the mouth at birth, so to accurately reflect colonization prior to and during birth. It is discussed if the delivery mode influence oral microbiome, some recent studies conclude that newborn microflora isn’t related to the kind of delivery but instead primarily driven by body habitats , 21. In  of life , 23, res of life . Microbi of life  and in p of life , 24. So, of life .Oral microbiome is a complex community and its composition and persistence is strictly influenced by food, oral hygiene practices and salivary flow . SeveralOral diseases are progressive, cumulative and grow in complexity over time, which is why it is very important for good oral health to be established early in life. The etiology of both dental caries and periodontal disease is polymicrobial and occurs when there is a shift in the overall ecological balance of microbes in the oral cavity .It is known that the oral health and oral microbiome of a woman may directly affect her pregnancy and her developing fetus: if the mother has periodontal disease, she has higher risk for giving preterm birth, for delivering a low-birth-weight infant, for preeclampsia, and 3.4 times higher risk for preterm birth plus delivery of an infant of low birth weight . So, eveStudies have found that in the case of oral diseases (gingivitis or periodontitis), bacteria in the oral cavity may reach amniotic fluid through transient bacteremia, indicating that maternal microbes may be transmitted to the amniotic fluid with blood.The available studies have focused their attention on the immediate complications of fetal and neonatal colonization by these pathogenic germs of the oral cavity, but it isn’t yet understood whether transmission of these pathogens is possible even in uncomplicated pregnancies, in healthy full-term infants and it is not possible to exclude that a colonization already at birth can determine a worse prognosis of oral health in the child.The genera considered in this study, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, are all Gram negative oral pathogens responsible for periodontal disease. They are characterized by the production of various metabolites and virulence factors that lead to the destruction of periodontal tissue or inactivation of the local defenses of the immune system.We found that the newborns’ oral cavity microbiome was composed primarily by saprophytes. The analysis of the oral microbiome showed in fact a pathological composition only in 20% of babies, this data doesn’t correlate with the incidence of periodontal pathogens in the mothers . So in our samples there weren’t significative trasmission of periodontal micro-organisms and we could conclude that the oral health and oral microbiome of the mothers don’t affect a full term pregnancy. So the hypothesis of colonization in utero is still controversial and these datas would make us suspect that in some pregnancies, but not in all, there may be a bacteremia which may be followed by a complication of gestation. However, from the analysis of these data it would not be clear what is the mechanism that leads to the unleashing of bacteremia, since the presence of pathogens of the oral cavity in our 12 children did not correlate with the highest maternal bacterial charges.Fusobacterium nucleatum was the most frequent specie detected in both mothers and babies, although again there was no correlation in the dyads. Fusobacterium nucleatum is an opportunistic periodontal pathogen of the oral cavity, but the dysbiosis can determine its pathogenicity with local and systemic consequences. We can’t determinate what influence will have for the future the presence of Fusobacterium nucleatum in the oral cavity immediately at birth but it is interesting that, even if in few babies of our sample, this bacteria was in some way able to trough the placenta also in healthy gestations.Analysis of the other pathogenic genera of the oral cavity of our study showed a lower incidence of presence than Fusobacterium nucleatum in the oral mouth at birth. Porphyromonas gingivalis showed an higher correlation mother-babies  than all the other genera examinated, so it could be the bacteria with the higher possibility of trasmission, but it is at the moment a too low evidence to demostrate it. None of the babies showed the presence of Aggregatibacter actinomycetemcomitans in their oral microbioma. Even in mothers it was the bacterium with the lowest incidence in the oral cavity.In 4 babies there was a polimicrobial contamination too. Sometimes the association between two or more pathogens can be even more aggressive and harmful, polymicrobial infections in fact can be more serious than those caused by a single pathogen, when the interactions between individual species turn a mixed infection into a synergistic infection.We cannot still know exactly what influence the presence of Fusobacterium nucleatum and other periodontal microorganisms in the oral cavity just at birth will have for the future infant oral health, but we could assume for these newborns a greater future risk of pathology if the balance between the different species of the microbiome under the influence of environmental factors will be broken.In conclusion, in our study maternal oral microbiome doesn’t influence healthy, full-term newborn’s oral microbiome. There are currently not enough studies that analyze the oral microbiome at full-term birth and compare it with the maternal one. Research into oral pathogenic microorganisms in newborns has also focused on the effects on gestation, but the possible transmission to healthy full-term infants and its possible effects on long-term health has not yet been investigated. Further studies are needed.


Trichilemmal cyst (TC), also known as trichodermal cyst, trichodermal isthmus-degenerative cyst. It is a benign skin lesion originating from the outer hair root sheath, with low incidence and few reports.A 41-year-old patient had found a scalp lump for more than 10 years. A 2.0 cm × 1.0 cm × 1.0 cm lump on the right occipital region was touched more than 10 years ago without special treatment. In the past 2 years, the lump has gradually increased. Physical examination: 4 protruding lumps can be reached in the scalp. One lump in the right occipital region is about 3.0 cm × 2.0 cm × 2.0 cm, with 1 lump immediately below and 2 lumps in the left temporal region. All lumps can be pushed.The lesion is located in dermis, The lesion is solid, and the contents of the cyst were cheese-like white material, and the inner and outer walls of the cyst were smooth and shiny. Pathological results showed that the lesion was TC. The cyst wall is epidermal tissue, the spinous layer and basal layer are intact, there is no granular layer, and the protein in the cyst is dense.All lumps were completely surgically removed.The wound healed well after TC resection. There was no recurrence of TC after 1 year follow-up.The clinical manifestations of scalp TC are not specific, and the diagnosis needs pathological examination, and the prognosis of total excision is good. It is a benign skin lesion originating from the outer hair root sheath, with low incidence and few reports.A 41-year-old patient was admitted to our hospital because he had found a scalp lump for more than 10 years. A 2.0 cm × 1.0 cm × 1.0 cm lump on the right occipital region was touched more than 10 years ago without special treatment. In the past 2 years, the lump has gradually increased. Physical examination: 4 protruding lumps can be reached in the scalp. One lump in the right occipital region is about 3.0 cm × 2.0 cm × 2.0 cm, with 1 lump immediately below and 2 lumps in the left temporal region. All lumps can be pushed, with no tenderness, no wave motion, normal surface hair, no redness and ulceration. Local anesthesia with lidocaine was given during the operation; The lesion is located in dermis, and the boundary with epidermis is clear; The lesion is solid, the cyst wall is smooth and complete, there is no obvious blood supply vessel, and the boundary with surrounding tissues is clear. All lumps were completely surgically removed, and the contents of the cyst were cheese-like white material, and the inner and outer walls of the cyst were smooth and shiny  listed them as lumps with hair follicle differentiation, and the pathological morphology was divided into a continuous morphological pedigree from benign to malignant. There are rare cases of malignant transformation in TC, and if malignant transformation occurs, it can lead to distant metastasis. TC lesions are often multiple, and there are a few single lesions.–7 The lesions are located in the parts with dense hair follicles and vigorous hair growth. More than 90% of them are found in the scalp, and a few are located in vulva and bulbar conjunctiva. TC is more common in middle-aged people, especially in women. Clinically, the course of the disease develops slowly, and most of them are round, hard and protruding masses, with no lesions on the surface skin and hair. CT or ultrasound is feasible for auxiliary examination, and postoperative pathological examination is needed for diagnosis, and the prognosis is good.In 1969, Pinkus confirmed that TC originated from the outer hair root sheath of the isthmus of hair follicle, and the cells of the outer hair root sheath of hair follicle proliferated due to gene changes, but what kind of gene changes are still unclear. Seidenari et al; The epithelial cells of the cyst wall keratose rapidly and there is no granular layer; The cyst has clear boundary with poor blood supply and solid nature without cystic degeneration under the naked eye; The patient has a family history of TC.TC has the following characteristics: The contents of the cyst tend to be eosinophilic hair keratin and its destruction products, and the form of keratosis is hairy keratosis and non-epidermal keratosis; That content of the cyst may bind to human hair-derived antibodies; 25% of the lesions showed calcification in the cyst. The lesions always have sheet or arenaceous calcification The cyst wall was composed of 2 layers of tissue, with the outer layer being fibrous connective tissue and the inner layer being stratified squamous epithelium with a granular layer. The basal lay is arranged on that out layer of the cyst wall, and the stratum corneum is arranged on the inner lay of the cyst wall; Dermoid cyst, which is often found in children, has cyst wall with skin adnexa such as sebaceous gland, hair follicle, hair and other germ layer structures in addition to squamous epithelium and fibrous tissue, belonging to the category of mature teratoma; The sebaceous cyst, commonly known as “pink tumor,” is caused by obstructed sebum excretion and is more common in young people.,14 The cyst wall is composed of stratified squamous epithelium with focal sebaceous gland cells. TC progresses slowly, and the masses are small and small in number, which may justify conservative management, but further treatment is required in the event of ulceration and infection. In general, the treatment is based on complete surgical resection, and recurrence can be caused by residual cyst wall. In our patient, all the lumps were completely excised during the operation, and the wound healed well without recurrence after operation.This disease needs to be distinguished from other cysts: Epidermoid cyst: it occurs mainly in young people and children, but rarely in the scalp. The contents of the cyst can bind to antibodies derived from human corpus callosum.Conceptualization: Zipeng Zhu.Investigation: Zipeng Zhu, Xiaoqian Yang.Methodology: Zipeng Zhu, Xiaoqian Yang.Project administration: Zipeng Zhu.Resources: Zipeng Zhu, Jinyue Fu.Supervision: Zipeng Zhu, Shubao Zhang.Validation: Zipeng Zhu, Shubao Zhang.Writing – original draft: Zonglei Chong.Writing – review & editing: Zonglei Chong.


Early neonatal death, which occurs during the prenatal period, is defined as the death of a newborn between 0 and 7 days after birth. It is one of the major public health problems in several developing countries. This study aimed to determine the early neonatal mortality rate and identify determinants of early neonatal mortality in Somalia region of Ethiopia.The data used for this study were drawn from the 2019 Ethiopia Mini Demographic and Health Survey (2019 EMDHS) data. A multivariable logistic regression model was used to identify the determinants of early neonatal mortality. Adjusted odds ratio (AOR) with 95% CI was used to examine the association of factors with early neonatal mortality.A total of 637 live births were included in this study. The early neonatal mortality rate in the study was 44  deaths per 1000 live births. Male babies , babies delivered at home , and babies born from uneducated mothers  were at increased risk of death in the first 7 days of life after birth. Contrarily, decreased risk of death of babies in the first 7 days of life after birth was associated with urban residence  and singleton .The early neonatal mortality rate in the region was high. The study revealed that sex of child, place of residence, birth type, mother’s educational level, and place of delivery were the determinants of the death of babies in the first 7 days of life after birth. Hence, providing health education to uneducated mothers and enhancing institutional delivery is recommended to minimize the early neonatal mortality rate in the region. HighlightsThis study was an attempt to determine the early neonatal mortality rate and identify determinants of early neonatal mortality in Somalia region of Ethiopia using 2019 Ethiopia Mini Demographic and Health Survey data (2019 EMDHS).The early neonatal mortality rate in the study area was 44  deaths per 1000 live births.Sex of the child, residence, birth type, mother’s educational level, and place of delivery were determinants of the death of babies in the first 7 days of life after birth.Providing health education to uneducated mothers and enhancing institutional delivery is recommended to minimize the early neonatal mortality rate in the region.Early neonatal death, which occurs during the prenatal period, is defined as the death of a baby between 0 and 7 days after birthNeonatal death is significantly influenced by perinatal mortality, which comprises stillbirths and early neonatal deaths. Worldwide, perinatal mortality accounts for about three-fourths of neonatal deaths, and the day of birth is the most perilous moment for newbornsIn Ethiopia, neonatal mortality, death in the first month after birth, increased from 29 deaths per 1000 live births in 2016 to 33 deaths per 1000 births in 2019Place of residence, sex of the child, antenatal care, birth type, having a skilled attendant at birth, previous history of perinatal mortality, obstetric complication during labor, birth weight, maternal educational level, father’s educational level, history of previous abortion, preceding birth interval, gestational age, wealth index of family, birth order, maternal anemia, maternal HIV, maternal employment, marital status, parity, age of mother, family size, place of delivery, mode of delivery, maternal death at birth, and late breastfeeding initiation were factors reported to be significant determinants of early neonatal mortality by several studiesIn spite of the efforts that have been made in Ethiopia to reduce the death rate of babies in the perinatal period, it is still high in some regions of EthiopiaThe data used for this study were drawn from the 2019 Ethiopia Mini Demographic and Health Survey data (2019 EMDHS). The 2019 EMDHS is the second EMDHS to be implemented in Ethiopia, and the data were collected from 21 March to 28 June 2019The outcome variable was the death of a baby within the first 7 days of life after birth. It is coded as 1 if the baby died in the first 7 days of life after birth and 0 otherwise. Independent variables were sex of child, place of residence, birth type, birth order, mode of delivery, place of delivery, age of mother, maternal educational level, marital status, wealth index, and family size.P value less than 0.05. Adjusted odds ratio (AOR) with 95% CI was used to examine the association between the independent variables and early neonatal mortality. The Hosmer and Lemeshow test was used to check the goodness of fit of the model. The model, with a Hosmer and Lemeshow test significance value of 0.319, was found to be a good fit.The data analysis was done using SPSS version 26. Frequencies and percentages were used to describe the background characteristics of the participants of the study. A bivariate analysis was done to select the independent variables to be included in the multivariable logistic regression analysis. The multivariable logistic regression analysis was done to identify the determinants of early neonatal mortality. The statistical significances of the independent variables were declared at A total of 637 live births, of which 28 died in the first 7 days of life after birth, were included in this study. Of the total live births, 356 (55.9%) were males, 527 (82.7%) were from urban, 615 (96.5%) were singletons, 393 (61.7%) had birth order of fourth and above, 632 (99.2%) were born vaginally, and 517 (81.2%) were born at home. With regard to mother’s level of education, age of mother, family size, marital status, and wealth index of the household, 538 (84.5%) were uneducated, 543 (85.2%) were in the age range of less than 25 years, 529 (83.0%) were from families of size 5 and above, 608 (95.4%) were married/living together with partners, and 545 (85.6%) were from poor families.The early neonatal mortality rate was 44 95% CI: 31, 65) deaths per 1000 births. The early neonatal mortality rate was higher among male babies (51 deaths per 1000 live births), rural babies (46 deaths per 1000 live births), multiple births (182 deaths per 1000 live births), first-born babies (90 deaths per 1000 live births), home deliveries (46 deaths per 1000 live births), babies born to mothers aged 35 years and above (66 deaths per 1000 live births), and babies born in poor families (46 deaths per 1000 live births). In the bivariate analysis, sex of child, place of residence, birth type, place of delivery, maternal educational level, age of mother and family size had an association with early neonatal mortality at 5% level of significance. These variables were included in the multivariable analysis  times higher compared to female babies. The odds of the death of babies in the first 7 days of life after birth among urban residences was 0.669  times lower compared to rural residences. The odds of the death of babies in the first 7 days of life after birth among single births was 0.345  times lower compared to multiple births. The odds of the death of babies in the first 7 days of life after birth among those born at home was 2.288  times higher compared to those born at a health institution. Moreover, the odds of the death of babies in the first 7 days of life after birth among babies born from uneducated mothers was 2.130  times higher compared to babies born from mothers with secondary and higher education  died within the first week of life. The early neonatal mortality rate was 44 deaths per 1000 births .This study found that babies born from mothers living in urban areas were less likely to die within the first 7 days of life after birth compared to babies born from mothers living in rural areas. This agrees with studiesThis study also found that the babies born at home were more likely to die within the first 7 days of life after birth compared to babies born at health institutions. This result is in line with the studiesThe strength of the study was that it used national representative data. The main limitation of this study was that some important variables such as weight at birth, ANC follow-up, gestational age, breastfeeding, preceding birth interval, HIV status of the mother, and anemia status of mother, which could have an association with early neonatal mortality, were not included in the study because of unavailability or high missing values in the dataset we used for the analysis.The aim of this study was to determine the early neonatal mortality rate and identify the determinants of early neonatal mortality in Somalia region of Ethiopia. The study revealed that the early neonatal mortality rate in Somalia region of Ethiopia was 44  deaths per 1000 births. The study revealed that factors such as sex of child, place of residence, birth type, mother’s educational level, and place of delivery determine the death of babies in the first 7 days of life after birth. Hence, providing health education to uneducated mothers and enhancing institutional delivery is recommended to minimize the early neonatal mortality rate in the region.The data used for this were secondary data and are publicly available and have no personal identifiers.Not applicable.This study was not funded.G.G.M.: conceptualization, data curation, formal analysis, investigation, methodology, software, validation, and writing – original draft; G.A., K.B.G., B.H.R., B.W., T.S.W., G.T., A.G., K.L.A., and H.K.: formal analysis, methodology, software, and writing – review and editing.The authors declare that there are no conflicts of interest.Name of the registry: not applicable.Unique identifying number or registration ID: not applicable.Hyperlink to your specific registration (must be publicly accessible and will be checked): not applicable.gizmake@gmail.comGizachew Gobebo Mekebo, E-mail: All data used for this study are available upon reasonable request from the corresponding author.Not commissioned, externally peer-reviewed.


Importantly, they were more likely to have vaginal birth (p = 0.03). Pre-pregnancy BMI influenced the week of delivery, i.e., inadequate, too-high BMI contributed to an increase in the percentage of premature births [OR (95% CI) = 1.19 ]. The findings indicate that promoting physical activity and weight management remains a priority in public health policy, and women of childbearing age should be encouraged to adopt or maintain an active and healthy lifestyle during pregnancy in order to avoid sedentary- and obesity-associated risks affecting birth and newborns’ health.There has been a dramatic worldwide increase in the prevalence of obesity or overweight and physical inactivity in women of reproductive age. Growing evidence suggests that pre-pregnancy maternal abnormal body mass index (BMI) and lower physical activity level are associated with poor maternal health and perinatal outcomes. The aim of this study was to assess how self-perceived exercise and pre-pregnancy BMI are associated with preterm birth, low birth weight, and type of birth. We conducted a retrospective cross-sectional study of 394 Polish women in the postpartum period. We used a questionnaire with the structure of the medical interview. To analyze factors related to birth outcomes, we used the Pearson’s Chi-squared test of independence and odds ratio (OR), with a corresponding 95% confidence interval (CI), followed by a multiple logistic regression. Women who reported being physically active before pregnancy ( The recent data have shown that regular physical activity during pregnancy and before pregnancy has a positive effect on the physical and psychological condition of the future mother, fetal development, parturition, and functioning during the postpartum period ,3,4,5,6.The presence of abnormal body weight before pregnancy and in the first trimester of pregnancy is a crucial risk factor of preterm birth (PTB) and low birth weight (LBW) ,10. The In many studies, researchers have mentioned that abnormal pre-pregnancy BMI is a very important independent risk factor of PTB. This value also determines the recommended maternity weight gain during pregnancy. The data obtained in this way are considered the most important indicators of the nutritional status of a pregnant woman . AbnormaThe aim of this study was to assess how pre-pregnancy BMI and self -perceived maternal exercise are associated with preterm birth, low birth weight, and other newborn and birth outcomes.The present study was a multi-center, retrospective, cross-sectional study among a group of 394 Polish women  in the postpartum period and after singleton pregnancy; of which, 153 women delivered preterm 22–37 weeks) (38.8%) and 241 had a full-term birth (38–42 weeks) (61.2%). We used a questionnaire with the structure of the medical interview. Also, medical history, especially maternal and neonatal outcomes, were analyzed see the . We foll–37 weeksThe study was conducted in 2 hospitals of tertiary referral centers ; level II of perinatal care: the care of intermediate-level pathological pregnancies; and level III of perinatal care: the care of the most severe pregnancy pathology) in the northern region of Poland. The period of data collection and patient eligibility was from 1 January 2016 to 1 January 2018.All participants were informed of the study objectives and provided their voluntary consent to participate, by marking consent to participate in the survey in the questionnaire and consent for access to medical records (it was one of the inclusion criteria). The parents were informed that taking part in the research would not affect their child’s diagnostic and therapeutic process and that the child would not be exposed to additional procedures. The children’s mothers, as their legal carers, also consented to access to the documentation concerning the newborn child.The principal investigator had a personal conversation with each mother of the children, explaining all concerns about the conduct of the study and the recruitment process. Mothers were invited to the study during their stay in the maternity ward after delivery or during a visit to the newborn in the neonatal intensive care unit . The children’s parents had the right to ask questions. Each child and mother included in the study were given an identification number, based on data from the medical records  that were completed, to minimize information bias.The study included 394 mothers aged 19 to 44 years  and their children born, alive, between 22 and 42 weeks’ gestation, who met the inclusion criteria .Approximately 5000 mothers received an invitation to take part in the study. Only 10% responded, and 136 participants were subsequently excluded from the study due to not meeting the eligibility criteria (as a result the rate of preterm birth was 38.8%). We conducted the study at the level III reference medical centers, where the rate of premature births was assumed to be higher than in other hospitals, due to the access to specialized equipment. Nevertheless, for our study, this increased rate of preterm birth compared to the general population appeared to be beneficial, providing access to a larger group of newborns born prematurely. This allowed for more accurate statistical analysis and more accurate inferences. The participants’ flow through the study is presented in The inclusion criteria were mothers having a single, alive birth after 22 weeks of pregnancy, those that gave birth at the public hospitals, and those understanding the Polish language.We excluded multiple gestations , lack of consent to access medical records, obtaining an incomplete interview questionnaire, death of a newborn in the perinatal period, stillbirth, and gestational age of the newborn below 22 weeks .Understanding the importance of pre-pregnancy exercise on the pregnancy and newborn outcomes, we included two subcategories of the study women in terms of their exercise habits:Self-perceived exercise before pregnancy—exercise performed by the mother 6 months before becoming pregnant, excluding normal home- and work-related activity, but including active transportation, and autonomous and supervised physical exercise. As “exercise” we considered all forms of physical activity that were planned, structured, repetitive, and performed with the goal of improving health or fitness . We noteSelf-perceived exercise during pregnancy —exercise started after becoming pregnant and performed by the mother during pregnancy, excluding normal home and work-related activity, but including active transportation, and autonomous and supervised physical exercise. We noted these data from survey addressed to mothers. This variable was categorized as YES or NO.2 (in m2). The number generated from this equation was marked as the individual’s pp-BMI number. These classifications for BMI are in use by the National Institute of Health (NIH) and the World Health Organization (WHO) for White, Hispanic, and Black individuals [2;Overweight and obesity—BMI greater than or equal to 25.0 and greater than 30 kg/m2;Normal weight—BMI greater than or equal to 18.5 to 24.9 kg/m2;Underweight—BMI under >18.5 kg/mPre-pregnancy BMI category—this value was calculated by taking a mother’s weight (before becoming pregnant or in the first weeks of pregnancy-data obtained from pregnancy chart), in kilograms, divided by their height, in meters squared, or BMI = weight (in kg)/heightividuals . The BMIWe connected these two categories (overweight and obesity) because of the small group of obese mothers.Overweight and obesity (25.0–29.9 and 30.0 or higher)—7.0–11.5 and 5.0–9.0 kgNormal weight (18.5–24.9)—11.5–16.0 kgUnderweight (less than 18.5)—12.5–18.0 kgMaternal weight gain (WG) (kg)—this was calculated by recommendations for total and rate of Weight Gain During Pregnancy, by Pre-pregnancy Body Mass Index (pp-BMI), data from the Institute of Medicine/National Research Council :OverweigMethods of labor—the method of pregnancy completion was recorded from the participants’ medical records. Vaginal delivery, caesarean section, or operative delivery  were considered. Perineal episiotomy and degrees of perineal tears were not analyzed in cases of vaginal delivery.Gestational age at delivery (number of weeks category)—the newborn’s age at birth, determined by the week of gestation according to Neagele’s rule or, if there was a discrepancy between the gestation date and the first-trimester ultrasound, according to the first-trimester ultrasound. The information on the week of pregnancy completion was recorded from the medical records and verified by the medical staff.Extremely preterm birth (less than 28 weeks);Very preterm birth (between 28 and 32 weeks);Moderate and late preterm birth (32 to 37 weeks).Particular consideration was given to completion of the pregnancy prematurely , i.e., between 22 and 27 weeks’ gestation. The subcategories of preterm births, according to the WHO, by gestational age were used ,27:ExtreThe proposed subdivision was taken into account due to the different proportion of preterm births in each category, with the largest group being late preterm births, which also has an impact on the prognosis and further development of the newborn. It is also important to underline here that the gestational age at delivery categories were related to the referral of the patient to the appropriate reference center , which is crucial issue in planning the care of pregnant women.Newborn’s birth weight (in grams)—the birth weight of the neonate is an important clinical criterion. Many times, the birth weight of a neonate born prematurely corresponds to that of a full-term neonate, i.e., between 2500 g and 4000 g. It also happens that a neonate born on time is born with a low birth weight, which can be indicative of hypotrophy. We noted these data from medical records, and qualified newborns for subcategories of newborn weight .Hypertrophic neonate, too large for gestational age , defined as macrosomia-neonatal weight above 4000 g;Eutrophic neonate, weight appropriate for gestational age —body weight between 2500 g and 4000 g;Hypotrophic neonate, too small for gestational age (SGA)—body weight below the 10th centile for gestational age or less than 2500 g;Low birth weight (LBW) neonate—weight between 2500 g and 1500 g;Very-low birth weight newborn (VLBW)—weight between 1499 g and 1000 g;Extremely low birth weight (ELBW)—weight between 1000 g and 750 g;Incredibly low birth weight newborn —weight less than 749 g.Division of neonates by birth weight:Preterm birth is the leading cause of low birth weight in both developing and highly developed countries. These pathologies (LBW and PTL) most often occur together. In our work we described normal weight and compared the results with LGA, LBW, VLBW, ELBW, and ILBW because of the potential negative influence of this factor on the babies; according to previous studies, they were more likely to die during their first month of life and those who survived face lifelong consequences, including a higher risk of stunted growth, lower IQ, and adult-onset chronic conditions such as obesity, diabetes, and cardiovascular disease (CVD) .Apgar score in 1st minute (0–10 points)—As an indicator of the general condition immediately after birth, the Apgar scale was chosen as a universal and easily obtainable parameter  from thep = 0.05; power = 0.95; allocation ratio N2/N1 = 2; result: sample size group 1 = 68; sample size group 2 = 135; total sample size = 203, using variable: maternal exercise before pregnancy). The calculated sample size was smaller than our final study group; however, due to access to more women after childbirth, we decided to analyze larger data, which in turn allowed for subgroup analysis.The data were analyzed using statistical software: the Statistica 12.0 version (advanced package) and PQStat v 1.8.0.476. Pearson’s Chi-squared test of independence was used to present the differences in the rates of full term vs. preterm deliveries and the differences in the birth weight categories in subgroups of active and inactive women. With the same test, we analyzed the rates of various modes of delivery in subgroups of women with the different pre-pregnancy BMI. Logistic regression analysis was performed . Multiple regression analysis was performed in order to analyze the strength of the relationship between maternal BMI and newborn variables . The level of significance adopted was 0.05. Sample size was computed using G-power 3.1.  and 241 who had a full-term birth (38–42 weeks-control group). Demographic characteristics, maternal data, and pregnancy outcomes are shown in We found that 25.1% women with normal weight (BMI 18.5 to 24.9) had EWG, excessive gestational weight gain; 40.0% had AWG, appropriate weight gain; and 34.9% LWG, too little weight gain (recommended WG 11.5–16.0 kg). Women who were overweight and obesity (BMI 25.0 to <30) had EWG in 62.0%, AWG in 20.6%, and LWG in 17.4% (recommended WG 7.0–11.5 kg). Women who were underweight (>18.5) had EWG in 5.5%, AWG in 66.7%, and LWG in 27.8% (recommended WG 12.5–18.0 kg).p = 0.05). In the group of women who were active, 69.9% (n = 93) delivered full term, 19.5% (n = 26) had moderate to late preterm birth, 9.0% (n = 12) had very preterm birth, and 1.4% (n = 1) had extremely preterm birth. In the group of women who were not active, 56.7% (n = 148) delivered full term, 24.1% (n = 63) had moderate to late preterm birth, 14.2% (n = 37) had very preterm birth, and 5.0% (n = 13) had extremely preterm birth. The results are shown in In our research, we assessed self-perceived exercise before pregnancy and we compared the results with the week of pregnancy category. Chi-squared Pearson’s test showed statistically significant differences between the groups (p < 0.001). In the group of women who started to be active during pregnancy, 83.0% (n = 50) delivered full term, 11.9% (n = 7) had moderate to late preterm birth, 5.1% (n = 3) had very preterm birth, and 0.0% (n = 0) had extremely preterm birth. In the group of women who were not active, 57.6% (n = 192) delivered full term, 24.5% (n = 82) had moderate to late preterm birth, 13.4% (n = 46) had very preterm birth, and 4.5% (n = 15) had extremely preterm birth. The results are shown in We also asked women about self-perceived maternal exercise during pregnancy  and we compared the results with that of the gestational age at delivery (weeks) category. Pearson’s chi-squared test showed statistically significant differences between the groups (p = 0.03). In the group of women who were active, 10.2% (n = 6) newborns had LGA, 79.6% (n = 47) newborns had a normal birth weight, 5.1% (n = 3) had low birth weight (LBW), 5.1% (n = 3) had very-low birth weight (VLBW), 0.0% (n = 0) had extremely (ELBW), and 0.0% (n = 0) had incredible low birth weight (ILBW). In the group of women who were not active, 9.2% (n = 31) newborns had LGA, 61.8% (n = 207) newborns had a normal birth weight, 15.5% (n = 52) had low birth weight (LBW), 7.5% (n = 25) had very-low birth weight (VLBW), 4.5% (n = 15) had extremely (ELBW), and 1.5% (n = 5) had incredible low birth weight (ILBW). The results are shown in In the next step, we assessed self-perceived maternal exercise during pregnancy and we compared results with the newborn birth weight category. Pearson’s chi-squared test showed statistically significant differences between the groups (p = 0.03). Of the women who had normal weight before pregnancy, 56.5% (n = 144) had a VB, 42.3% (n = 108) had a CS, and 1.2% (n = 3) had forceps or vacuum delivery. Of the women who were obese or overweight, 41.3% (n = 50) had VB, 57.0% (n = 69) had CS. and 1.7% (n = 2) had an assisted vaginal birth. Of the mothers who were underweight, 72.2% (n = 13) had VB and 27.8% (n = 5) had a CS or had an instrumental delivery. The data are shown in We also wanted to know how pre-pregnancy BMI affected the mode of birth. Pearson’s chi-squared test showed statistically significant differences between the groups  with the specified variables during this simulation. Based on the interpretation of the obtained results, we can assume that some of the variables do not have a significant impact on the gain and may be redundant.We built a multiple linear regression model by selecting the following variables: pp-BMI (pp-BMI), newborn’s birth weight (NBW), and APGAR (1st minute). As a result, the coefficients of the regression equation and measures were calculated to assess the quality of the model. Based on the estimated value of the b-factor, the relationship between week of pregnancy completion (WPC) and all the independent variables can be described with the equation:2 = 0.780825; corrected coefficient multiple determination R2adj = 0.78; and the result of the F test of the analysis of variance: p < 0.000.The model fits well, as evidenced by a small standard error estimate SE = 1.96; high value of the coefficient multiple determination R2, the time to complete pregnancy will be shortened by an average of 4 days, assuming ceteris paribus. If the pregnancy is lengthened by 3.5 days, the Apgar index increases by 1 point, assuming ceteris paribus. If the time to complete the pregnancy increases by 3 weeks, the weight of the child at birth increases by 1000 g, assuming ceteris paribus. Additionally, 78.08% of the total variability in the time of completion of pregnancy was explained by the model . Note that the multivariate regression equation only applies to the study population under unchanged conditions (ceteris paribus).Interpretation of the model: if the mother’s pp-BMI increases by 10 kg/m2Pseudo = 0.03, R2Nagelkerke = 0.05, and R2Coxa − Snella = 0.04). At the same time, the model was statistically significant (p = 0.001 of the likelihood-ratio test) and, therefore, the independent variables in the model were statistically significant. The Hosmer–Lemeshow test result indicates no statistical significance (p = 0.09). However, in the case of the Hosmer–Lemeshow test, the lack of significance is desirable because it indicates the similarity of the size of the observed groups and the predicted probability.The fit quality of the model was not high  is associated with poor maternal and perinatal outcomes ,30. PropWith the increasing amount of scientific evidence on the positive effects of prenatal physical activity, authors from different countries observe its insufficient level in pregnant women . The gesp < 0.001) [Maternal exercise is associated with reduced odds of macrosomia and is not associated with neonatal complications or adverse childhood outcomes ,39,40,41< 0.001) .Importantly, the literature data indicate that prematurity is not directly related to high BMI , but results from medical complications that usually accompany high BMI . Our anaOne of the most important findings of our study is that pre-pregnancy BMI affected the mode of the childbirth. Women who had normal pre-pregnancy BMI or were underweight had a lower risk of having a cesarean section (CS) compared to women with overweight or obesity. The last two decades have seen an increase in CS worldwide. In the middle and highly developed countries, almost half of the women gave birth by CS. Poland has one of the highest percentages of cesarean sections (42%) in Europe, where the average is 27% . We are There were changes in behavioral patterns during the COVID-19 pandemic that were associated with lockdowns and restrictions of access to certain resources. During the lockdown, trends of unfavorable changes were observed: decreased physical activity, increased sedentary time, increased snacking, decreased consumption of fresh food , and increased consumption of sweets, cookies, and cakes. Yet, the opposite trends were also observed: increased home cooking and increased physical activity. All of these trends displayed associations with various individual characteristics ,37,38. AThe study findings align with recommendations from the American College of Obstetricians and Gynecologists (ACOG), which emphasize the positive impact of physical activity on pregnancy outcomes. Women who reported being physically active before and during pregnancy exhibited a significantly higher likelihood of achieving full-term births, accompanied by a noteworthy reduction in the incidence of very-premature and extremely premature births .Additionally, the Chief Medical Officers’ Physical Activity Guidelines in the UK support the notion that promoting physical activity is integral to overall health, with potential benefits extending to maternal and perinatal outcomes . ImportaThe main strength of the study was that the data were collected in multi-centers in Poland, allowing for generalization of our conclusions to the Polish population. The relatively large number of study participants allowed for the subgroups analysis. Medical records used for the data collection were objective and reliable and could not be affected by the subjective assessment of mothers.From this study we excluded women with multiple pregnancies, recognizing that this condition could limit their physical activity. On the one hand, this assumption provided us with greater group homogeneity and avoided misinterpretation of the data. Multiple pregnancies statistically lead to preterm birth more often and, in consequence, babies with lower birth weight due to their prematurity. These are also variables that can indirectly influence Apgar scores. In addition, multiple pregnancies are mostly terminated by CS . TherefoIn the context of our study, it is noteworthy to address the upper limit for recommended exercise levels during pregnancy. While our findings demonstrate the positive impact of self-perceived exercise before and during pregnancy on various birth outcomes, it is crucial to acknowledge that there exists a need for clear guidelines regarding the upper threshold of physical activity for expectant mothers. The current literature, including recommendations from reputable sources such as the American College of Obstetricians and Gynaecologists (ACOG), underscores the importance of regular physical activity during pregnancy . HoweverOne of the limitations of our study was that analysis did not include indications for caesarean section. However, it seems that the data obtained may be useful in the future to distinguish the analysis in only CSs performed due to lack of progress of labor, prolonged second period of labor, or threatened asphyxia. Moreover, for the determination of neonatal weight, centile charts for girls and boys were not used to determine percentile values; instead, only birth weight was used, which was then classified into specific birth weight categories. In future analyses, it would be extremely valuable to use a reference to centile charts for this variable.Another limitation of the study was the lack of information on the type, intensity, duration, and frequency of physical activity undertaken and the length of time for which it was carried out. The data collected was based only on the subjective assessment of the exercise undertaken by the mothers. In future studies it would be valuable to use standardized tools, e.g., Pregnancy Physical Activity Questionnaire  or accelAccording to the literature, an important issue in assessing the nutritional status of a child’s mother is taking into account the quality of consumed products and identifying deficiencies in the diet. Particularly important is the deficit of elements such as zinc or iron, an inadequate concentration of which in the body may disturb the functioning of the immune system, thus increasing the risk of preterm labor . In our Since national minorities constitute a marginal part of Poland’s population (around 2%), we did not ask the study participants about their ethnicity. However, due to the increasingly intense migration process in Europe, this issue is worth addressing in future research.Despite the indicated weaknesses of our work, the obtained results may be useful in planning future, more complex studies aimed at improving a healthy lifestyle before and during pregnancy.The study revealed that women who reported being physically active before and during pregnancy were more likely to have full-term birth and had a lower incidence of very premature and extremely premature births. Women who exercised during pregnancy more often gave birth to children with a normal body weight and were less likely to have children with low, very-low, or extremely low body weight. Pre-pregnancy BMI affects the method of pregnancy termination—in the group of women with a higher BMI there were more cesarean sections. Pre-pregnancy BMI also influenced the week of pregnancy termination, i.e., incorrect BMI contributed to an increase in the percentage of premature births.Our findings indicate that promoting physical activity and weight management remains a priority in public health policy, and women of childbearing age should be encouraged to adopt or maintain an active and healthy lifestyle during pregnancy in order to avoid sedentary- and obesity-associated risks affecting birth and newborns’ health.


Empagliflozin also enhanced cardiac robustness by maintaining intracellular ATP levels and the recovery capacity in the infarcted area during ischemic-reperfusion. Our findings suggest that empagliflozin enters cardiac mitochondria and directly causes these effects by increasing mitochondrial ATP via inhibition of NHE1 and Nav1.5 or their common downstream sites. These cardioprotective effects may be involved in the beneficial effects on heart failure seen in clinical trials.Empagliflozin, a sodium-glucose co-transporter 2 inhibitor developed, has been shown to reduce cardiovascular events in patients with type 2 diabetes and established cardiovascular disease. Several studies have suggested that empagliflozin improves the cardiac energy state which is a partial cause of its potency. However, the detailed mechanism remains unclear. To address this issue, we used a mouse model that enabled direct measurement of cytosolic and mitochondrial ATP levels. Empagliflozin treatment significantly increased cytosolic and mitochondrial ATP levels in the hearts of  The SGLT2 inhibitor, Empagliflozin, is shown to exert cardioprotective effects by increasing mitochondrial ATP in a mouse model of diabetes. In the EMPA-REG OUTCOME trial, a large randomized controlled clinical trial, the highly selective SGLT2 inhibitor empagliflozin significantly reduced the risk of three-point major adverse CV events, cardiovascular death, heart failure hospitalization and composite renal outcomes in patients with type 2 diabetes (T2D) with established cardiovascular disease2.Sodium-glucose cotransporter 2 (SGLT2) inhibitors are a unique class of oral antidiabetic medications that reduce glucose reabsorption in the renal proximal tubules, thereby enhancing urinary glucose excretion4, which can be multifactorial and acute/chronic5; the most common one is via an effect on diuresis/natriuresis, but the mechanisms involved in these impressive cardiac benefits are incompletely understood. Several research groups have suggested the potential efficacy of empagliflozin on cardiac energetics. Changes in cardiac energy production play a critical role in the pathophysiology of heart failure. The failing heart faces an energy deficit primarily because of a decrease in mitochondrial oxidative capacity, which is partly compensated for by an increase in ATP production from glycolysis occurring in the cytoplasm6. Several studies have shown that empagliflozin improved cardiac energy production in animal experiments8. Indeed, a single dose of empagliflozin in fasting diabetic db/db mice was associated with an improvement in cardiac energetics, which is also associated with an increase in ketone levels7. Similarly, Verma et al. reported that chronic administration of empagliflozin enhanced ATP production in the heart of db/db mice, although they argued that this was due to an increase in glucose and fatty acid oxidation rather than the utilization of ketone bodies8. To investigate the mechanism underlying the improvement in cardiac energy status with empagliflozin, real-time and accurate monitoring of the pathways and amount of energy production in vivo is needed.Multiple hypotheses have been proposed to explain the beneficial effects of SGLT2 inhibitorsAteam (GO-ATeam9), which can directly quantify intracellular ATP levels via Fluorescence Resonance Energy Transfer (FRET) in cytosol and mitochondria, we generated transgenic mice to monitor subcellular ATP levels in the whole body, organ and cells, as well as in the beating heart . These mice enabled us to visualize ATP levels in real time and in a living state by measuring the FRET/GFP ratio and proved ideal for studying drugs that affect energy metabolism.Recently, using a modified 11, prompting us to investigate the real-time ATP change in cardiac energy production in an ischemic-reperfusion model of MI. Furthermore, to gain insight into the direct effects of empagliflozin on the myocardium and its underlying molecular pathway, we also conducted experiments using isolated cardiomyocytes.In the present study, we crossed cytoATP-Tg or mitoATP-Tg mice with a mouse model of T2D and assessed the effects of empagliflozin on the cardiac energy status of those mice. In addition, several studies have demonstrated the beneficial effects of empagliflozin on myocardial infarction (MI) in diabetic animal modelsdb/db mice were significantly decreased compared to controls  in wild-type mice  in the heart was significantly reduced to 1.44 ± 0.01 (about 1.03 mM) in ice Fig. . This AT10, which is a severely stressed condition, might be mediated by the maintenance of the ATP levels in the heart. Thus, we investigated the effect of empagliflozin on the temporal changes in ATP levels in the infarcted myocardium, particularly during ischemic-reperfusion.The increase in ATP levels induced by empagliflozin implies an improvement in energy maintenance in the heart. We therefore hypothesized that the beneficial effect of empagliflozin on an ischemia/reperfusion modeldb/db; cytoATP-Tg mice were treated with control or empagliflozin for 10 weeks, starting at 7 weeks old, and at 17 weeks old, they underwent the ischemic/reperfusion procedure, and the ATP levels at the infarct sites of the heart were measured over time  was induced in the infarct region compared to before ligation after 20 min of ligation , which is almost the same as that observed in the wild-type mice   . At this time, the ATP level in the cytoplasm was not changed . The results showed that the mitochondrial membrane potential was significantly increased in the cardiomyocytes treated with empagliflozin .The mitochondrial membrane potential was measured after adding vehicle or empagliflozin to mature cardiomyocytes isolated from 8-week-old wild-type mice, and 1 h later, JC-1 was applied to the cells (0.30 ± 0.004: +/H+ exchanger 1 (NHE1)13 and Nav1.514. To investigate the possibility that these ion channels influence mitochondrial ATP levels in mature cardiomyocytes, we next performed experiments in which we added each inhibitor to mature cardiomyocytes isolated from 8-week-old +/+; mitoATP-Tg mice and incubated them with either vehicle or empagliflozin. The ATP levels in the mitochondria were measured 1 h after the addition of 300 nM empagliflozin. The FRET ratio was 1.37 ± 0.005 in control and was significantly increased to 1.39 ± 0.007 in MA-5-treated cardiomyocytes, which facilitates mitochondrial ATP production via ATP synthase oligomerization . In empagliflozin-treated cardiomyocytes, the FRET/GFP ratio was significantly increased to 1.42 ± 0.006 . To investigate the effect of cariporide and ranolazine, inhibitors of NHE1 and Nav1.5, respectively, we measured the ATP level in mitochondria of cardiomyocytes 1 h after the addition of cariporide. The FRET/GFP ratio was significantly increased to 1.42 ± 0.006 after the addition of cariporide and to 1.42 ± 0.006 after the addition of ranolazine . We then examined whether or not the addition of empagliflozin to these inhibitors had an add-on effect. Consequently, the FRET/GFP ratio was 1.42 ± 0.004 when cariporide plus empagliflozin was added, showing no significant difference from cariporide alone (1.42 ± 0.006) or empagliflozin alone (1.42 ± 0.006) . The addition of ranolazine plus empagliflozin resulted in a FRET ratio of 1.42 ± 0.006, showing no significant difference from the addition of ranolazine alone (1.42 ± 0.006) or empagliflozin alone (1.42) . These results, combined with the previous findings that empagliflozin directly inhibits NHE1 and Nav1.5, suggest that empagliflozin may enhance ATP levels in mitochondria via the inhibition of these ion channels or common downstream sites of these channels.Empagliflozin reportedly inhibits not only SGLT2 but may also directly inhibit Nadb/db, 17 weeks old). Empagliflozin attenuated the decrease in and enhanced recovery of ATP levels at the ischemic site in an ischemia-reperfusion model of MI. Furthermore, the mitochondrial ATP level was increased 3 h after a single administration of empagliflozin. These results prompted us to examine cardiomyocytes derived from the model mice, and the direct effects of empagliflozin on the mitochondria of cardiomyocytes were demonstrated.The current study was performed to evaluate the effect of empagliflozin on cardiac energy metabolism in T2D mice in order to directly quantify cytosolic and mitochondrial ATP levels. The hearts of empagliflozin-treated mice had significantly higher ATP levels in the cytoplasm and mitochondria after 10 weeks of treatment than control mice  in a variety of organs/cells.Empagliflozin has been shown to increase ATP in the heart of db/db mice, which suggested the robustness of its effect on the cardiac energy state, we monitored the changes in cytosolic ATP concentrations in the ischemic region of the ischemia-reperfusion model after 10 weeks of empagliflozin administration. The changes in the cytoplasmic ATP levels during ischemia and reperfusion were clearly ameliorated in the empagliflozin-treated group, thus demonstrating that the energy production had become robust in vivo. In vitro studies using isolated cells, by contrast, revealed that 1-h treatment with empagliflozin significantly increased mitochondrial ATP but not cytoplasmic ATP. Furthermore, it also significantly increased mitochondrial ATP in wild-type-derived cardiomyocytes. Furthermore, we showed that empagliflozin alleviated the changes in mitochondrial ATP levels in cardiomyocytes during low-oxygen exposure, suggesting that the energy production had become robust in vitro. These in vivo/in vitro results suggest that empagliflozin not only quickly increases ATP production in mitochondria, but also increases ATP production in the entire cell in the long term. Such qualitative changes in myocardial energy metabolism, which we call “remodeling”, may underlie the cardiovascular benefits of empagliflozin seen in clinical trials and support the ongoing clinical trial evaluating the effect of empagliflozin on patients after acute MI (EMPACT-MI)15.Encouraged by the finding that empagliflozin increased mitochondrial ATP in 17, the primary target tissue and target molecule for its cardioprotection has long been discussed. In the present study, we showed that empagliflozin rapidly reached the cardiac mitochondria in vivo. Furthermore, in vitro studies revealed that empagliflozin immediately increased mitochondrial ATP in isolated cardiomyocytes, suggesting that empagliflozin has a direct effect on the myocardium. In addition, our findings also showed that the cytosolic ATP levels did not increase in vitro, while the mitochondrial ATP levels and membrane potential both increased. These in vivo/in vitro results suggest that the target in cardiomyocytes may reside in the mitochondria.Since SGLT2, the molecular target of empagliflozin, is expressed specifically in the proximal tubules of the kidney but not in the heart14. In our study, we found that both cariporide and ranolazine, inhibitors of NHE1 and Nav1.5, respectively, increased mitochondrial ATP production. Empagliflozin, however, did not induce any additional benefit, suggesting that empagliflozin may increase mitochondrial ATP by inhibiting both NHE1 and Nav1.5. This conclusion is consistent with reports that NHE1 is expressed in mitochondrial membranes, with its inhibition maintaining the membrane potential18, and that Nav1.5 is functionally linked to mitochondria in cardiomyocytes19. However, this should be interpreted with caution: first, a report was recently published arguing that empagliflozin does not inhibit NHE120. If this is the case, to reconcile these findings with our own, the mechanism underlying the effects of empagliflozin may involve the common pathway of both inhibitors, i.e., reduction in Ca2+ overload by decreasing the intracellular Na+ concentration. Second, cariporide and ranolazine used as tool compounds in this study may not have been specific to each target23, and the concentrations used are in the micromolar range; therefore, we cannot exclude the possibility that ATP is induced by their off-target effects. Further studies are needed to identify bona-fide molecular targets of empagliflozin in the heart.Some researchers have proposed the possibility of NHE1 and Nav1.5 as off-targets of empagliflozin in the heartdb/db mice, which recapitulate the features of cardiomyopathy and heart failure25. However, given that there are many etiologies of heart failure and the clinical evidence indicating that empagliflozin is effective against heart failure with or without diabetes27, to what extent our findings can be applied remains to be seen. Third, our in vitro experiments were conducted with cardiomyocytes isolated from the heart, so the molecular mechanism underlying ATP enhancement by empagliflozin that we postulated here is not necessarily the same as that seen in the in vivo native environment.Several strengths and limitations associated with the present study warrant mention. First, our ATeam mouse is a very useful tool for visualizing energy metabolism in vivo, even at organellar levels, which is not limited to the heart. It should be pointed out, however, that this assay only measured cardiac surface cells, so the results need to be validated using two-photon microscopy or fiber microscopy, which can measure deeper areas. Second, we clearly showed the energetic effects of empagliflozin in the failing heart of diabetic db/db; cytoATP-Tg/mitoATP-Tg or control  mice were fed a diet with/without 30 mg/kg empagliflozin for 10 weeks. The dose of empagliflozin was chosen based on the data from previously published pre-clinical studies10.Seven-week-old Prior to starting the experiments, baseline urine and blood samples were collected. The body weight and systolic blood pressure in awake mice were measured. The body weight was measured 8 weeks after the start of drug treatment, and the systolic blood pressure was measured 8 weeks after the start of drug treatment. Blood samples were collected from the tail vein of mice for measurement of non-fasting blood glucose and ketone levels at eight weeks after the start of drug treatment, and the blood pressure was measured at 8 weeks after the start of drug treatment. After 8 weeks of drug treatment, mice were housed in metabolic cages to collect 24-h urine. At the end of the 10-week treatment, 17-week-old mice were anaesthetized with isoflurane, and the chest was opened surgically. The beating heart was observed under a fluorescent microscope for ATP imaging in vivo.Regarding the observation method, in brief, the object was exposed to an excitation light (ET470/40) using a fluorescence microscope , and the absorbed light was separated into GFP  and RFP  by a Dual-View , and the image was captured by a cMOS camera  to obtain dual-images simultaneously.Analyses were performed using the MetaMorph software program  and displayed as IMD images. For cytosolic ATP, since we were able to make a calibration curve showing the relationship between the FRET/GFP ratio and ATP level, we were able to estimate the ATP level from the FRET/GFP ratio (paper submission). However, for mitochondrial ATP, only the FRET/GFP ratio was described, as it is technically difficult to make a calibration curve.All male mice used were 8 weeks old at the start of each experiment and were housed individually or in groups of 2–3 per cage at a temperature of 22 ± 1 °C with a 12-h light–dark cycle and ad libitum access to food and water. All procedures were performed in accordance with the guidelines of the Laboratory Animals Care and Use Committee . Efforts were made to minimize the number of animals used and to limit their suffering.db/db; cytoATP-Tg mice were intubated and artificially ventilated. After opening the chest using electrocautery, ischemia was induced by ligating the left anterior descending artery with a suture needle and surgical thread  under a microscope while monitoring the electrocardiogram (ECG). Reperfusion was performed by removing the suture 30 min after the creation of the infarction while monitoring the ECG. In vivo ATP imaging was performed using a fluorescence microscope (Leica M165FC) at six time points: before ligation (0 min), after ligation (20 and 30 min), just after reperfusion, and 15 and 30 min  after reperfusion.After anesthesia with isoflurane, overnight-fasted 17-week-old db/db; mitoATP-Tg or control mice received a single dose of empagliflozin (30 mg/kg body weight) via oral gavage after 4 h of fasting. After another 3 h of fasting, the mitochondrial ATP level of the heart was monitored in vivo under fluorescent microscopy.Eight-week-old We isolated mature cardiomyocytes from the indicated genotype mice using a Langendorff perfusion system . To investigate the effect of empagliflozin, cariporide, and ranolazine on cardiomyocyte energetics in mitochondria or cytosol, we studied the mitochondrial or cytosolic ATP levels of cardiomyocytes with/without empagliflozin , cariporide (10 μM), ranolazine (30 μM) or MA-5 (10 μM). To investigate the effect of empagliflozin on the mitochondrial membrane potential of cardiomyocyte, we used the JC-1 with empagliflozin (300 nM). Two-photon microscopy (Leica SP8MP) was used to observe fluorescence.2PO4, 50 μM MgCl2, 0.2% BSA, pH 7.5) and lysed by being passed through a 1-mL syringe with a 27-G needle 10 times. Homogenized lysates were next centrifuged at 800 × g for 5 min at 4 °C, and supernatants were further centrifuged at 6000 × g for 15 min at 4 °C. The resulting pellets containing mitochondria were resuspended in a mitochondria buffer. Protein concentrations were determined using the BCA protein assay (Thermo Fisher Scientific) according to the manufacturer’s instructions.A single oral dose of empagliflozin was administered to 8-week-old wild-type mice, and 3 h later, they were sacrificed, with the heart, kidney, liver, small intestine and brain tissue collected. The tissues were homogenized using a homogenizer (Nippi Inc.) containing mitochondria buffer . Subsequently, hypoxia treatment was performed by switching from 95% O2, 5% CO2 state to 10% O2, 5% CO2, 85% N2 state, and the mitochondrial ATP level was imaged by two-photon microscopy after 20 and 30 min. Further imaging was performed 15 and 30 min after switching again to 95% O2 and 5% CO2 .Cardiomyocytes were isolated from 18-week-old db/db; mitoATP-Tg, treated with empagliflozin (1000 nM) or DMSO in Tyrode solution for 1 h, and then transferred to Tyrode saturated with 95% Ot-test to compare two groups and an analysis of variance (ANOVA) . All data were normally distributed, and variance was similar between groups. Biological replicates were derived using different samples derived from different mice. The results represent data from at least three independent experiments. We estimated the required sample size considering the variation and mean of the samples. We used the fewest animals required to draw statistically valid conclusions. Our protocol required excluding mice if we observed an abnormal size, weight, or both or disease symptoms before performing experiments. However, this was unnecessary, as all mice were phenotypically normal and healthy.Data are expressed as the mean ± SEM. Statistical analyses were performed using the unpaired two-tailed Student’s Further information on research design is available in the Peer Review FileSupplementary FiguresReporting Summary


Very low birth weight infants (VLBW) are at risk for adverse growth and neurodevelopmental outcomes. We aimed to evaluate the association between growth during Neonatal Intensive Care Unit (NICU) stay and long-term neurodevelopmental outcomes in a cohort of preterm VLBW newborns.We conducted a longitudinal observational study in the Follow-up Service of our Clinic from January 2014 to April 2017. All preterm VLBW infants born at our hospital and enrolled in our follow-up program were considered eligible for the study. The neurodevelopmental assessment was performed using the Griffiths Mental Development Scales at 12 and 24 months corrected age.Study population included 172 subjects  with a mean gestational age of 29 weeks and a mean birth weight of 1,117 g. A unitarian Δz-score increase in head circumference from birth to discharge was associated with a 1.6-point increase in General Quotient at 24 months corrected age. An association with subscales C and D was also found. Likewise, an increase in length Δz-score was associated with better 24-month subscale C scores although not reaching statistical significance. No relationship with the outcome at 24 months was found for weight gain.Growth during NICU stay appears to be related to a more favorable neurodevelopmental outcome at 24 months corrected age, especially in the hearing and language domain . The longitudinal evaluation of auxological parameters during hospitalization can contribute to the identification of subjects at risk for adverse neurodevelopmental outcomes in the first years of life. Our hospital is a tertiary referral center for neonatal care, covering around 6,000 births per year. Our center offers an outpatient Follow-up service for prematurely born newborns, with low birth weight or relevant comorbidities, who have been discharged from the NICU.This study is part of a larger project that investigated the neurological development of preterm VLBW infants admitted to the NICU of our centre from January 2014 to April 2017.The study was approved by the Ethics Committee of the Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico. Informed consent was obtained from both parents. All procedures were carried out according to the Declaration of Helsinki.2.2.All preterm infants  with a birth weight <1,500 g consecutively born at our hospital from January 2014 to April 2017 and subsequently enrolled after discharge in the follow-up program routinely provided at our Institution were considered eligible for the study.Infants affected by genetic syndromes and/or major congenital malformations, infants who died during NICU stay or after discharge, who were transferred to other hospitals before discharge, who abandoned the follow-up before reaching 24 months CA (drop-outs), whose data could not be retrieved or whose parents did not provide informed consent were excluded from the study.2.3.The baseline characteristics, perinatal history and follow-up information of the first 24 months CA of the infants enrolled were collected from the patients' computerized medical charts .Basic socio-demographic characteristics were collected .Perinatal information collected included: gender, gestational age, mode of delivery, birth weight, length and head circumference, length of hospital stay, mode of feeding at discharge.The mode of feeding was classified according to the WHO definitions .The occurrence of common prematurity-associated pathologies was also collected.During follow-up visits anthropometric measurements and neurodevelopmental assessment scores according to the Griffiths Mental Development Scale – Extended Revised (GMDS-ER) were recorded.CA was calculated adjusting chronological age for gestational age.Data were collected and analyzed anonymously.2.4.During hospitalization and at follow-up visits infants' anthropometric parameters  were measured by registered pediatric nurses using standard techniques.The weight was assessed with electronic scales, placing the naked infant on a light sheet. The measurement was approximated to the nearest gram.The vertex-heel length was assessed with the Harpenden neonatometer. To guarantee the accuracy of the measurements, two health professionals were needed: one to hold the infant's head firmly against the headpiece, while keeping the shoulders fixed to the instrument, and the other one to fully extend the infant's right leg and hold the foot at a 90° angle to the sliding carriage. The measurements were approximated to the last completed millimeter.The head circumference was measured by detecting the maximum occipito-frontal circumference using a non-extendable measuring tape placed on the frontal bones, above the supraorbital arches, and then passed around the head at the same height on both sides and posteriorly, passing through the most protruding point of the occiput. The measurements were approximated to the last completed millimeter.During hospitalization the newborns' weight was assessed daily, whereas length and head circumference were assessed weekly, according to the hospital internal protocol.2.5.During follow-up visits the completion of the main stages of neuro-cognitive development was assessed by three trained and qualified examiners using the GMDS-ER. The Italian back-translation of the GMDS-ER was validated by Battaglia and Savoini in 2007 . The sca2.6.Demographic characteristics of the study population, growth parameters and neurodevelopmental outcomes at 12 and 24 months CA are presented using descriptive statistics. Continuous variables are expressed as mean and standard deviation or median and interquartile range; categorical variables are expressed as absolute and relative frequencies.The z-scores for weight, length and head circumference at birth, during hospitalization and at discharge were assessed according to the Intergrowth-21st charts for Newborn Size for Very Preterm Infants and the longitudinal Charts for Post-Natal Growth of Preterm Infants , 19, resTo better evaluate the longitudinal growth of the subjects, the difference between the z-scores (Δz-score) of the anthropometric parameters  measured at discharge and those measured at birth was calculated. With regards to the weight, the Δz-score between its values from the time of weight recovery to discharge was also calculated.A multivariate linear regression model was used to evaluate the association between auxological growth during hospitalization and neurodevelopmental outcome at 12 and 24 months CA as measured by the GMDS-ER scales .The model was corrected for the mode of feeding at discharge  and the Comorbidity Score. The Comorbidity Score is a severity score calculated for each newborn on the basis of their comorbidities, owing to their known impact on neurodevelopmental outcomes in VLBW infants . For eacBrain lesions were identified on both cranial ultrasound (cUS) and brain magnetic resonance imaging (MRI) performed according to our Institution's internal clinical imaging protocol that includes sequential cUS scans from birth to term equivalent age (TEA) and conventional brain MRI at TEA.SBL were defined as: grade III intraventricular hemorrhage and/or parenchymal hemorrhagic venous infarction and/or posthemorrhagic ventricular dilation and/or cystic periventricular leukomalacia and/or >6 punctate white matter lesions and/or focal cerebellar hemorrhage and/or brain malformations.In a subgroup analysis, the effect of anthropometric growth during hospitalization on neurodevelopmental outcomes was evaluated excluding the subjects who had developed SBL. A subgroup analysis was also performed considering small for gestational age  patientp-value <0.05 was considered statistically significant. The statistical analyses were performed using R software .All tests were two-tailed. A 3.3.1.Patients' flow through the study is represented in The basic socio-demographic characteristics of the study population are summarized in The study population included 172  subjects with an average gestational age of 29 weeks and an average birth weight of 1,117 g. Among subjects included in the analysis, 108 were VLBW , corresponding to 62.8% of the population studied, and 64 were Extremely Low Birth weight . Mean length and head circumference at birth were 37 cm and 26 cm, respectively. The 23.8% of the subjects included in the study were SGA.Mean hospitalization in the NICU lasted 61 days, and 22.7% of infants were exclusively breastfed at discharge.3.2.The auxological parameters of the study population, expressed as z-scores, are summarized in According to extra-uterine growth restriction (EUGR) longitudinal definitions, we found that 45 (26.2%) patients had a loss of weight z-score >1 SDS from birth to discharge, 33 (19.2%) patients had a loss of weight z-score >1 SDS from weight recovery to discharge, 77 (44.8%) patients had a loss of length z-score >1 SDS from birth to discharge, 68 (39.5%) patients had a loss of head circumference z-score >1 SDS from birth to discharge.3.3.All infants included in the analysis underwent GMDS-ER. Only one infant resulted unable to perform the GMDS-ER, due to severe cerebral palsy, and was therefore scored <49 in General Quotient and all subscales. However, because of missing data, said patient was not included in the final analysis.At 12 months CA mean General Quotient was 91.4, with a mean score of 92.4 in subscale A—locomotor, 91.1 in subscale B—personal-social, 92.4 in subscale C—hearing and language, 91.5 in subscale D—eye-hand coordination, and 92.3 in subscale E—performance.At 24 months CA mean General Quotient was 89.0, with a mean score of 93.4, 88.3, 87.5, 91.4 and 90.7 in subscales A—E, respectively.3.4.p = 0.08 and p = 0.07, respectively).In the studied population a significant association was found at 12 months CA between weight and length Δz-score from birth to discharge and GMDS-ER subscale C scores  . Conversp = 0.06) and E (p = 0.08). As for the increase in length, the greatest effect was observed on subscale C . On the other hand, no relationship with the neurodevelopmental outcome at 24 months could be found with regards to weight gain.At 24 months CA an increase in head circumference of 1 unit of Δz-score was found to be associated with an average increase in the General Quotient of 1.6 points. A statistically significant association with subscales C and D was also found, with a gain of 2 and 1.4 points, respectively, as well as a non-statistically significant association with subscales B , and no relationship was found between the weight increase from recovery of birth weight (data not shown).The increase in weight from birth to discharge showed a non-statistically significant association with subscale C at 24 months CA  newborns were born SGA. The association between auxological parameters of SGA patients and neurodevelopmental outcome at 12 and 24 months CA is shown in p = 0.07). No association was found at 24 months CA.The increase in weight from birth to discharge showed an association with subscale C at 12 months CA, although not reaching statistical significance  . HoweverMore in line with our findings, Maiocco et al.  showed iper se, deeming the heterogeneity of existing definitions a limit to the interpretation of results and their comparability between studies. Indeed, EUGR is variously defined in the Literature and can be measured either at a given time  or between two time points   , 38, 39.al EUGR) . We thusOur results could depend on the fact that weight, albeit easier to measure than other auxological parameters, is a rather crude indicator of growth , especiaIn clinical practice the assessment of body composition is often complicated by the clinical instability of the hospitalized newborns and the limited availability of adequate methods, such as air plethysmography or dual-energy x-ray absorptiometry (DEXA). The longitudinal evaluation of more readily assessable anthropometric parameters, like length and head circumference, can therefore represent a valid proxy of the quality of growth.Further studies are needed to clarify the relative importance of weight gain during NICU stay in terms of long-term neurodevelopmental outcomes of preterm VLBW infants.Major strengths of our study are the inclusion of a relatively large cohort of premature VLBW infants, who received standardized nutritional care and were followed longitudinally up to 24 months CA with a low drop-out rate (0.9% at 12 months CA and 13% at 24 months CA) . The useIn our cohort, the increase in length and head circumference during NICU stay was related to a more favorable neurodevelopmental outcome at 24 months CA, especially in the hearing and language domain. Conversely, weight gain was not associated with neurodevelopmental outcomes at 24 months CA in our cohort. We suggest to longitudinally evaluate auxological parameters  throughout hospitalization, using suitable tools and appropriate curves for the study population. This can contribute to the identification of subjects at risk for adverse neurodevelopmental outcome in the first years of life, allowing for timely nutritional interventions that favor growth and neurodevelopment.