Prenatal balanced protein energy supplementation consumed by undernourished women improves mid-upper arm circumference in early infancy. This study aimed to identify whether locally produced maternal food-based supplementation improved anthropometric measures at birth and early infancy.n = 8; control: n = 4) in Pirganj sub-district, Rangpur District, northern Bangladesh. Daily supplements were provided.A village-matched evaluation, applying principles of a cluster randomised controlled trial, of a locally produced supplemental food to 87 undernourished pregnant women. 12 villages (intervention: p < 0.05). The mean birth weight in babies of supplemented mothers  was higher than in babies of mothers in the control group , and these changes persisted until 6 months. Also, the proportion of low birth weight babies in the intervention group was much lower (event rate = 0.04) than in the control group (event rate = 0.16). However, none of these differences were statistically significant . The intervention reduced the risk of wasting at 6 months by 63.38% (RRR = 0.6338), and of low birth weight by 88·58% (RRR = 0.8858), with NNT of 2.22 and 6.32, respectively. Only three pregnant women require this intervention in order to prevent wasting at 6 months in one child, and seven need the intervention to prevent low birth weight of one child.Anthropometric data at birth were available for 77 mother-infant dyads and longer-term infant growth data for 75 infants. Mid-upper arm circumference (MUAC) was significantly larger in infants of mothers in the intervention group compared with the control group at 6 months . This project had human research ethical approval from the James Cook University  Ethics committee (H4498) and the Bangladesh Medical Research Council (BMRC/NREC/2010–2013/58).The online version of this article (10.1186/s12884-018-1915-x) contains supplementary material, which is available to authorized users. Size at birth and growth in early infancy are important indicators of early childhood survival and health. Maternal undernutrition, an indicator of poor fetal growth, is associated with lowered birth weight . Low bird = 0·203, 95% CI, 0·03–0·38, p = 0·021) [In a recent systematic review and meta-analysis, balanced protein energy supplementation among undernourished pregnant women was found to significantly improve birth weight in low and middle-income countries (= 0·021) –12.Although a number of studies have included food-based supplements using locally available and preferred foods –16, the 2) [Bangladesh has among the highest rates of maternal and child undernutrition worldwide . Materna2)  and low 2) . Among c2) . In rura2) , 23.The purpose of this study was to identify whether a locally developed balanced protein energy supplementation for undernourished pregnant women in rural areas of northern Bangladesh, affected anthropometric measures at birth and during early infancy.2, consists of 332 villages, and has 385,499 inhabitants [The study was conducted in the Pirganj sub-district of Rangpur District, northern Bangladesh. The Pirganj sub-district covers an area of 411·34 kmabitants . As in oabitants . Rangpurabitants . We prevabitants , 28.The study reported here was the third phase of a multiphase study design. All three phases were committed to local level applied research. The previous two phases were published accordingly , 27–30. A village-matched evaluation, using principles of a cluster randomised controlled trial, was conducted. Recruitment occurred from February 2013 to February 2015. Rangpur district was selected based on its rural location. Pirganj sub-district was purposively selected for this study from eight sub-districts in Rangpur. Two of 15 unions,In the selected villages, the study recruited women of reproductive age who were (1) confirmed to be pregnant ; (2) undernourished, as defined by a mid-upper-arm circumference (MUAC) ≤22·1 cm3Information on the study recruitment and community nutrition volunteers has been published elsewhere . In summEight female community nutrition volunteers and two  supervisors from the selected villages were trained on the basics of nutrition, and the study purpose and design. The community nutrition volunteers had at least a primary school education, spoke the local dialect, and were aged between 21 and 49 years.Women in the intervention group received the intervention by visiting community nutrition volunteers at a designated community site on a daily basis. If the women were unable to visit the community nutrition volunteer, the volunteer would visit the household. All enrolled women in the intervention villages received one serving of the food-based supplement per day, within seven days of enrolment to term. The community nutrition volunteers closely monitored compliance to the food-based supplement at every visit through observation and discussion. Women from the control villages were unable to access the intervention supplement from the intervention villages due to the careful enrolment and follow-up process conducted by the community nutrition volunteers.Details on how the supplement was developed are published elsewhere . In summWomen in intervention and control groups received identical services and support except for provision of the supplement. All pregnant women (regardless of study enrolment) in the intervention and control groups were screened for undernutrition through village-level monthly campaigns conducted by the community nutrition volunteers. Referral pathways with existing government- and NGO-supported ante-natal care (ANC) and post-natal care (PNC) services were established at the onset of the study.Regardless of study group, or nutrition status, all pregnant women living in the villages selected to participate in this trial received iron-folic acid supplementation through access to routine ANC and PNC services. In addition, all women were tested for anaemia at a mobile clinic and were provided treatment if identified as anaemic. The partnership with the mobile clinic was established for the purpose of this study.Upon enrolment, community nutrition volunteers collected the following data from participants: 1) background demographics; 2) household food security; 3) dietary diversity and 4) anthropometry. Household food insecurity was identified using the validated Food and Nutrition Technical Assistance (FANTA) Household Food Insecurity Access Scale (HFIAS) questionnaire (Version 3) , 33. WomThe community nutrition volunteers measured height, weight and MUAC at participant enrolment and each month of the data collection period, using the standardised procedures recommended by the World Health Organisation , 37. HeiAt birth, skilled health professionalsTo identify whether a locally produced food-based supplement improved birth and infant anthropometric measures, the primary outcomes included birth weight and longer-term infant growth (weight and MUAC) at 1, 3, and 6 months. The secondary outcomes were preterm birth (defined as birth at < 37 weeks of gestation), miscarriage (delivery of infant ≤20 weeks), stillbirth (delivery of an infant showing no signs of life ≥20 weeks), perinatal death (stillbirth or death of a live-born infant in the first 7 days after birth), and neonatal death (death within the first 28 days of life). We also measured acute malnutrition (wasting) in infants at 6 months. Wasting in infants was defined by a MUAC < 12·5 cm .Compliance was calculated by dividing the total number of days the supplement was taken under direct observation, by the total number of days possible, i.e., the number of days between enrolment and delivery. We defined loss to follow-up as a participant leaving the study area for a period longer than 2 consecutive weeks or delivering their infant in a place outside the study area.The Consolidated Standards of Reporting Trials (CONSORT) statement was used to ensure comprehensive reporting of this study. The data quality was ensured by quality checks associated with the data entry process, double entry, and data cleaning. Data management was conducted using Microsoft Excel. Statistical analyses were performed using the statistical software package IBM SPSS software, Version 23© .p-value of < 0·05 was considered statistically significant. To estimate the effect of the intervention, absolute measures of risk were also calculated. Specifically, Relative Risk Reduction (RRR), Absolute Risk Reduction (ARR), and Number Needed to Treat (NNT) were calculated in relation to the proportion of participants categorised as low birth weight (< 2·5 kg), and the proportion of participants with MUAC< 12·5 at 6 months.For categorical variables, between group differences were assessed as suggested by Donner and Klar (1994) . CategorThis research was registered with the ISRCTN registry (ISRCTN97447076). This project had human research ethical approval from the James Cook University  Ethics committee (H4498) and the Bangladesh Medical Research Council (BMRC/NREC/2010–2013/58).n = 49, control: n = 28) and infant anthropometric outcome data were available for 75 children . All births were singleton. No caesarean sections were recorded for the mothers in either group. The main reason for missing infant anthropometric outcome data was that the duration of the data collection phase did not adequately allow for 6 months of follow up data to be collected from infants enrolled later in the study. Other reasons for missing longer-term growth data included mothers being unavailable to bring their child to the assessments due to household work or caring for their other children. The number of women lost to follow-up differed between groups . The reason that one woman was lost to follow-up was that she returned to her father’s village to give birth; the other was unknown. Due to low numbers of preterm birth, miscarriage, stillbirth, perinatal death, and neonatal death, analyses on secondary outcomes were not conducted. Instead, these data were excluded from analyses . Two cases were excluded from longer-term analyses; one due to death (intervention), and one missing for unknown reasons (control). All infants identified with moderate or severe acute malnutrition were immediately referred to community health centres for treatmentThe participation and recruitment data are presented in Fig. p > 0·05), except that women in the intervention group were more religiously diverse . The mean age of the women was 22·6 years (SD: 5·3). Most women attended school, with the majority reaching either a primary or lower secondary education level . The mean height of the women was 148·5 cm (SD: 5·5), with more than half below 148 cm (52·6%), indicating chronic malnutrition. Most pregnancies were identified in the first or early second trimester .The background characteristics of the 87 enrolled women are presented in Table n = 57). One woman did not consume the supplement for a one-month duration during the third trimester, as she was not reachable and therefore did not receive the supplement. No women refused the supplement.Compliance was high among the intervention group; almost all women consumed the full supplement on a daily basis (p > 0·05). We grouped birth weight into two categories: healthy birth weight (≥2·50 kg) and low birth weight (< 2·50 kg). Low birth weight occurred much less frequently in the intervention group  compared to the control group (event rate = 0·16 ± 0·18), but this was not significant (p > 0·05).The primary outcome data are presented in Table p > 0·05).Although mean weight in the intervention group was higher than the control group at 1, 3, and 6 months, these differences were not statistically significant (p < 0·05).The mean MUAC was significantly larger in the intervention group compared to the control group at 6 months .We conducted analyses to explore the event rate of wasting at 6 months in the intervention and control groups. The event rate was significantly lower in the intervention group compared to the control group , and MUAC < 12·5 at 6 months by 63·38% (RRR = 0·6338). The absolute risk reduction was 15·82% for LBW, and 45% for MUAC< 12·5. The Number Needed to Treat (NNT) was 6·32 (low birthweight) and 2.22 (MUAC< 12·5). That is, 7 pregnant women need to experience this intervention in order to prevent low birth weight in one child, and 3 pregnant women need to experience the intervention in order to prevent wasting at 6 months in one child.In northern Bangladesh, we found that daily supplementation with a locally produced food-based balanced protein energy supplement during pregnancy among undernourished women resulted in infants with significantly larger MUAC measurements at 6 months and subsequently a lower event rate of wasting.p > 0·05).Although a clear trend was observed of heavier babies at birth to 6 months in the intervention group compared to the control group, the difference was not significant , and that only seven pregnant women require this intervention in order to prevent low birth weight of one child. This finding is supported by a recent systematic literature review on balanced protein supplementation among undernourished pregnant women in low- and middle- income countries . In addiFinally, our study highlighted that locally produced food-based balanced protein energy supplementation for undernourished pregnant women can be produced at a local level. While our finding is limited to the context of northern Bangladesh, similar studies have demonstrated that RUSF and Ready to Use Therapeutic Food (RUTF) can be locally produced and sustainable , 50.Our study had a number of limitations. First, we calculated our sample size based on the prevalence and estimated incidence using the most recent Demographic Health Survey data . Our calIn Rangpur district in northern Bangladesh, locally produced balanced protein energy supplementation, using a community-based small business enterprise, reduced wasting in children at 6 months when targeted to undernourished pregnant women. Further, this research illustrates how communities can be empowered to identify and address maternal undernutrition. The intervention reduced the risk of wasting at 6 months by 63.38%, and of low birth weight by 88·58%. Only three pregnant women require this intervention in order to prevent wasting at 6 months in one child, and seven need the intervention to prevent low birth weight of one child. This supplementation thus contributed to better development outcomes for the mother and child. Our study findings highlight that programme decision-makers have alternatives to the commonly used Ready to Use Foods (RUFs) to treat acute maternal malnutrition, which may not be sustainable, cost effective or acceptable to some populations. While our study identified that the supplementation improved infant lean mass, we recommend that studies with larger sample sizes further explore this association. This study can inform future studies, with larger sample sizes, that aim to investigate the effect of maternal supplementation on longer-term infant growth .Effective balanced protein energy supplementation for undernourished pregnant women can be locally produced.Communities can be empowered to develop effective balanced protein energy supplementation, identify maternal acute malnutrition, and provide treatment.Daily supplementation with a locally produced food-based balanced protein energy supplementation during pregnancy may contribute to reducing acute malnutrition in infants at 6 months of age. Further studies with larger sample sizes are required to explore this association.Further research using larger sample sizes is required to explore the effect of daily supplementation using locally produced food-based balanced protein energy supplementation during pregnancy among undernourished women on a baby’s weight at birth ad early infancy.Findings from this study can inform, guide and motivate policies by providing evidence on a sustainable nutrition intervention that improves birth weight.Additional file 1:Table S1. Composition of the food-based balanced protein energy supplement per serving. (DOCX 8 kb)Additional file 2:Table S2. Village means for birth weight, weight at 1, 3, and 6, months, and MUAC at 6 months. (DOCX 9 kb)


Objective: To anticipate the type of childbirth according to the health belief model. Materials and methods: The present cross-sectional research was conducted on 222 primiparous women visiting the healthcare center in Khorram Abad. A combination of simple randomization and clustering was used to do the sampling. The data collection instrument was a validated four-part questionnaire the first part of which contained demographic information. The second part was comprised of awareness questions while the third dealt with the constructs of the health belief model. The final part consisted of the behavioral intention derived from the logical action theory. SPSS 16 was used to statistically analyze the data and the significance level was set at p ˂ 0.05.Results: The average age of the participants was 27.40 ± 6.07 years. Intention to go for a vaginal birth showed to be significantly correlated with awareness, perceived sensitivity, intensity, barriers and benefits (p ˂ 0.001) as well as self-efficacy (p = 0.025). The best predictor of the type of childbirth turned out to be the perceived barriers  and only then awareness .Conclusion: Strategies to remove the barriers of preferring vaginal childbirth, raising women’s awareness of the side effects of C-section and the benefits of vaginal birth, strategies to enhance women’s beliefs in their capability of natural childbirth can be used to reduce the prevalence of unnecessary C-sections. Childbirth as a science which terminates pregnancy aims to maximize the chances of giving birth to a healthy infant without threatening mother’s health . In the According to the existing statistics, C-section accounts for 25.7% of all deliveries and this rate is reported to be 2.3% in Angola and 46.2% in China . This moA vast majority of the related literature so far has only dealt with the prevalence of C-section and its correlates. Little work has focused on theory-driven and psychological factors. Due to the contrary results obtained based on the health belief model about the choice of childbirth type -12, TherThe present descriptive, analytical study was cross-sectional in design. The target population was all primiparous women in their third month of pregnancy who visited the healthcare centers of Khorramabad. The sample size was determined to be 220 through a formula considering a correlation coefficient of 0.25 and CI of 0.95. The sampling method was a combination of simple randomization and clustering method. Initially, from among the existing 20 healthcare centers in Khorramabad, 12 were randomly selected. Then, pregnant visitors’ files were analyzed to extract qualified cases to enter the study. The inclusion criteria were: age range of 18-35 years, height above 145 centimeter, weight range of 50-90 kilos, primiparity, pregnancy age of at least 28 weeks and willingness to take part in the research. The exclusion criteria were: placenta problems, multiple pregnancy, narrow-shaped pelvis, pregnancy diabetes, history of hypertension, pregnancy preeclampsia and previous abortion. The data collection instrument was a reliable and valid questionnaire by Sharifi rad et al. (2009) . The tarThe mean age of the participants was 27.40 ± 6.07 years; their mean age of marriage was 23.40 ± 5.20 years. 17.7% of the participants were below 20 years of age; 79.5% had a moderate socioeconomic level’ 74.1% of them were housewives and finally 49.1% held a diploma .As the findings revealed, statistically significant correlations were found between birth type and: awareness (p ˂ 0.001), perceived sensitivity (p ˂ 0.001), perceived intensity (p ˂ 0.001), perceived barriers (p ˂ 0.001), perceived benefits (p ˂ 0.001) and self-efficacy (p = 0.025).Across all constructs the significant findings belonged to those who opted for a vaginal birth . MoreoveLogistic regression analysis was conducted for the predictors of fertility-related factors and the type of childbirth. The independent variables awareness, perceived sensitivity, barriers, benefits, intensity and self-efficacy entered the model. The most determining predictor of the type of birth was perceived barriers (OR=1.153) (p˂.001) and only then awareness (OR = 1.108) (p ˂ 0.001) .The present study sought to determine the predictors of the birth type among primiparous women in Khorramabad according to the health belief model. According to the present findings, the number of those who intended to have a vaginal delivery was twice as many as those who went for a C-section. These findings were consistent with those reported by Yuen Loke et al.  and YousIn the present research, about one-third of the participants tended to have a C-section which was higher than that in Yuen Loke et al. . This fiThe key predictor of the type of childbirth in the present research was found to be awareness and perceived barriers. Sharifi rad et al. indicated that perceived barriers formed the primary construct of the health belief model in suggesting a healthy behavior . The feaThe results revealed a statistically significant correlation between perceived sensitivity and the intention of natural childbirth. In other words, the mean score of perceived sensitivity was higher in women who intended to have vaginal delivery. Fuglenes et al.  drew attAccording to the present findings, the perceived intensity and one’s intention of natural delivery showed to be significantly correlated. As pinpointed by Disney et al. , the perMoreover, as the present findings revealed, perceived benefits showed to be significantly correlated with the birth type. In fact, the mean score of perceived benefits was high in women who intended to have a natural delivery. These findings were consistent with those by Yuen Loke et al. , and HasThe present findings further revealed that self-efficacy and birth-type were significantly correlated. In other words, the mean self-efficacy score was higher in women who intended to have a natural birth. Kan’ani et al. indicated that self-efficacy was effective in the choice of the birth type . Dilk etOne limitation of the present research was that all women visitors were urban residents and there was no access to rural residents. The limited sample size, the mere incorporation of personal factors involved in selecting the birth type and neglecting social and contextual factors are among the other limitations.Considering the predictors of the birth type, there seems to be a great need for strategies to remove the barriers to natural delivery. These can include a correct way of raising mothers’ awareness and changing their wrong perceptions of natural delivery and its pain. These can be accompanied by efforts to correct women’s belief in their capability of handling a natural birth. All these can help to cut down on the rate of unnecessary surgical deliveries.


Lymphatic cyst is a collection of lymphatic fluid with an epithelial lining which can either represent lymphatic malformations or may occur after trauma or operation. Idiopathic lymphatic cyst of the thigh in adulthood is extremely rare. We report a case of a 27-year-old Thai female presented with a rapidly growing mass over her right thigh. The magnetic resonance imaging scan demonstrated a well-defined, thin rim, enhancing simple cyst. The cyst disappeared for a few days after aspiration. Completed surgical resection was performed for definitive diagnosis and treatment. Immunohistochemical studies revealed the cell lining was positive for CD34, CD31, and D2-40 and negative for calretinin. To our best knowledge, idiopathic lymphatic cyst of the thigh in young adult has never been reported and should be kept in mind in the patient who presents with abnormal thigh mass. Lymphatic cyst is a collection of lymphatic fluid with an epithelial lining which can either represent lymphatic malformations or may occur after trauma or operation . IdiopatWe present an unusual case of a young adult female who presented with symptomatic mass at the thigh which has not previously been reported in English literature as the origin of a lymphatic cyst.A 27-year-old Thai female presented with a rapidly growing mass over her right thigh for 3 months. She had no underlying or previous surgery. She had pain when she walked. She had slightly limited her right hip function on flexion and abduction. Physical examination revealed a firm nonpulsatile mass over the right thigh measuring ~8 × 6 cm. in diameter . No notaThe magnetic resonance imaging (MRI) scan demonstrated a 7.8 × 5.8 × 5.7 cm, in vertical × transverse × AP diameter of well-defined, thin rim, enhancing simple cyst with vascularity at the right anterior intermuscular fascia of the upper thigh, just inferior to the right inguinal region. The cyst was located between the pectineus muscle and sartorius muscle, accompanied with anterior bulging to subcutaneous fat. The right common femoral artery and vein and superficial artery and vein were displaced posteriorly .Percutaneous aspiration of cyst revealed clear yellowish fluid about 60 ml., and the cytological examination showed hypocellularity consisting of few small lymphocytes and foamy macrophages in background of few erythrocytes and concluded cystic fluid. The cyst disappeared for a few days and presented in the same size within one week.The operation was performed on supine position under general anesthesia. Vertical incision was performed; the cyst was identified and found dense adherance to femoral artery. Carefully, dissection was done. Complete cystic removal with femoral artery preservation was successful with minimal leakage of cyst wall . Cystic Microscopic examination was demonstrated as Spontaneous thigh mass in adolescent or young adult is extremely rare. The accurate preoperative diagnosis is difficult. The differential diagnosis of thigh mass includes lipoma, liposarcoma, aneurysm, and inguinal hernia. Although, the cyst is unilocular and filled with clear fluid, the malignant cyst cannot be excluded. The cytological examination cannot give the detail and exclude the malignancy. The absolute guideline for lymphatic cyst is not established. Total removal of the cyst is the definitive diagnosis and treatment.Clinically, primary cysts occur predominantly in children and young adolescents; often asymptomatic until large sizes, they may then present with local or referred pain. However, rapidly swelling of thigh cyst without history of small cyst, trauma, or surgery of the thigh is rare.Lymphatic cyst is rarely seen than lymphocele and lymphangioma; it is different from a lymphocele that is a collection of lymphatic fluid without an epithelial lining that develops in anatomical compartments, often after surgery or trauma .Lymphangioma is a congenital lymphatic malformation that usually presents as a congenital mass in infancy and classified into microcystic (<2 cm) and macrocystic (>2 cm) . These mAn acquired lymphangioma (lymphangiectasia) can occur after surgery or radiotherapy because of damage to the draining lymphatic channels .Tagge and Baron reported the etiology of lymphatic cysts may be attributed to ectasia of the lymphatic vessels or to cystic degeneration of a hamartoma .Large lymphatic cysts have been reported in adrenal gland . HoweverThe definitive diagnosis of lymphatic cyst is confirmed by immunohistochemistry. Percutaneous aspiration of the cyst under ultrasound guidance is the initial treatment modality used. However, recurrence rates of up to 80% have been reported, with repeated aspiration being a risk factor for the development of infection . InstillSurgical excision is the mainstay and definitive therapeutic option and can achieve total excision and prevention of recurrence, as the cyst wall is removed.Lymphatic cyst is considered benign with excellent prognosis . MalignaTo the best of our knowledge, idiopathic lymphatic cyst of the thigh in young adult has never been reported and should be kept in mind in the patient who presents with abnormal thigh mass.Lymphatic cyst of the thigh remains a rare condition and should be among the top in differential diagnosis of unilocular cyst in adolescent or young adult. Completed surgical resection, keeping the cyst intact, is recommended for definitive diagnosis and treatment. Long-term follow-up to check for recurrence and/or neoplastic transformation is recommended.


Predicting language performances after preterm birth is challenging. It is described in the literature that early exposure to the extrauterine environment can be either detrimental or advantageous for neurodevelopment. However, the emphasis mostly lies on the fact that preterm birth may have an unfavorable effect on numerous aspects of development such as cognition, language, and behavior. Various studies reported atypical language development in preterm born children in the preschool years but also in school-aged children and adolescents. This review gives an overview of the course of language development and examines how prematurity can lead to atypical linguistic performances. In this paper, we mainly focus on environmental and neurophysiological factors influencing preterm infant neuroplasticity with potential short- and long-term effects on language development. Further research, however, should focus on examining the possible benefits that early exposure might entail. Preterm (PT) birth is a phenomenon that affects a large and variable group of newborns due to its many underlying causes. According to a 2010 estimation by the World Health Organization, approximately 15 million babies are born PT, worldwide, each year ). In parPrior to the development of language comprehension and speech production, the auditory system has to develop. Already very early in gestation, the fetus's auditory system is formed. Between the 23rd and 25th weeks of pregnancy, important structures of the auditory system such as the cochlea are already in place. After 26 weeks of gestation, hair cells in the cochlea become fine-tuned for specific frequency bands, converting acoustic signals into electrical stimuli and forwarding them through the auditory nerve to the auditory cortex in the brain. Therefore, between the 26th and 30th weeks, the fetus is able to detect and react to sound stimuli . This fiBecause hearing is functional during the last trimester of gestation, it is of interest to know whether and how these immature cortical circuits process speech. In utero, information about prosody and rhythm of the mother's speech is led to the fetus' inner ear by means of bone conduction through which they have the opportunity to learn about properties of their native language. Perceiving this speech signal is sufficient enough to shape an infant's phoneme perception prior to birth . In addiLanguage acquisition after birth is made possible through the interaction between structural characteristics of the mother language and language offerings in the environment (child-directed speech). The overall process can be seen as a set of language skills that continuously grows. An infant learns to interact with the environment by producing sounds, actions, and behaviors. Different phases can be distinguished such as the prelingual phase (from birth to 12 months of age) when an infant starts vocalizing and babbling. Second is the early-lingual phase (from 1 to 2.6 years of age), during which a child shows signs of word comprehension and starts producing isolated words and short, telegraphic sentences. Next, in the differentiation phase (from 2.6 to 5 years of age), grammar starts to develop and sentences become more complete. Finally, during the completion phase (starting from 5 years of age), bases acquired in the preceding phases are further elaborated, for example, by developing reading and writing skills through education  Figure .In the first year of life, development of receptive language is crucial. During this period, the infant will learn to understand the mother language and how to respond accordingly. A prerequisite for this development are language offerings in the infant's environment ) and a correct-functioning hearing organ. In order to understand and produce speech, the infant needs to listen to a caregiver. Prelingual skills such as vocalizations, eye movements/gazes, gestures, and shared attention with a parent or caregiver are an important part of that process. During the first months of life, an infant establishes an infant-caregiver relationship by using eye contact, smiling, producing sounds, etc. . Thus, sBy listening to speech, an infant becomes more sensitive to characteristics of their native language, while during the second half of the first year of life, those of other languages disappear . In typiAfter the first year of life, when the foundation of language comprehension has been laid, the infant will start to experiment more with spoken language. Expressive language involves learning to pronounce speech sounds and engage in communication. The expressive aspect of language can be subdivided into different skills, (1) lexicon/semantics: vocabulary, learning the meaning of words and (2) morphosyntax: grammatical development. A child learns to understand changes of word forms in different syntactic contexts and learns how to form correct sentences. A child will develop his or her vocabulary around the second year of life. On average, a child has acquired about 200-400 words at this age. Possessing an early lexicon has a highly predictive value for later language skills. In PT children, a linear relationship was found between gestational age at birth and later language outcomes. The lower one's gestational age at birth, the smaller the vocabulary size and quality of word use . Stolt eAs the child grows older, he or she will master the language better and start forming longer sentences. The meaning of words becomes better understood, and they can be formulated more accurately. This development is stimulated by education and acquiring reading skills. Reading requires good cognitive and intellectual development and is an essential skill for later academic purposes. Several prerequisites have to be established in order to learn how to read, i.e., phonological awareness, speech perception, and verbal skills. Each of these abilities contributes to the acquisition of two processes that are essential for becoming literate: firstly, decoding (single word reading) in which words are extracted from the mental lexicon, and secondly, word comprehension (semantics). In a meta-analysis performed by Kovachy et al. , fourteeLanguage development is not solely dependent on language processes but also depends on basic cognitive processes  . As abovSeveral recent studies have shown that cerebral abnormalities associated with PT birth may be a substantial determinant of cognitive and language development. Therefore, identifying predictors of development disorders through neuroimaging studies should help improve our knowledge.Variations in cognitive, language, and speech development are likely to be the result of underlying abnormalities in the brain associated with functional organization outcomes. During normal development, language organization is extensive and bilateral in the infant's brain. Later, with increasing age, it becomes more lateralized in the left hemisphere. In healthy 3-month-old infants, mature cortical areas are active when processing language. By this time, speech perception is already left-lateralized with activity in the superior temporal gyrus and the angular gyrus . In PT nApproximately 50-70% of VPT born infants are affected by diffuse white matter abnormalities such as loss of white matter volume, corpus callosum thinning, and delayed myelination . The preThe corpus callosum plays a crucial role in the exchange of interhemispheric information. Thus, a deviation may be associated with weaker cognitive performances. A study, in which the relation between the corpus callosum regions and preverbal skills was assessed in 14-15-year-old born VPT using structural MRI and neuropsychological tests, found a negative effect of VPT birth on the development of the corpus callosum. More specifically, a decreased volume in the corpus callosum posterior areas was positively associated with lower verbal IQ and reduced verbal fluency scores. Overall, this study demonstrates the involvement of the corpus callosum in speech and language processes and describes an interhemispheric asymmetry . AdditioConsidering brain development is mostly shaped by early sensory experiences, exposure to language in the infant's environment is of utmost importance . Since PWhen born prematurely, infants spend the first weeks or even months of their life in the NICU. During this critical period for development, they are deprived of the sounds they would otherwise be hearing in utero. As discussed earlier, the intrauterine environment allows the fetus to perceive low-frequency sounds in an attenuated fashion, ensuring the development of the auditory system . HoweverA study by Caskey et al.  showed tFor infants residing in the NICU, full-time parental presence is not the case. Dysfunctions in early social communication, due to long periods of separation in the NICU, can have negative consequences on an infant's behavior and emotional and physiological well-being. Moreover, an important aspect affecting language development in PT born children is the quality of the infant-caregiver relationship. Multiple studies show that when a child and their caregiver participate in quality interactions, language development will improve. Increased psychological stress experienced by mothers of PT infants has been linked to differences in the mother-infant interactions in this population , 74. On Although most abovementioned findings suggest a negative impact of PT birth, some studies suggest that PT infants can also benefit from early natural auditory exposure. Already at 29 weeks of gestational age, PT infants possess the ability to process subtle changes in phonemes and voices . Hence, An early intervention found to be effective is interaction through maternal speech and singing, showing favorable effects on an infant's physiological state such as heart rate, oxygen saturation levels, and respiration rate . FurtherThe aim of this paper was to provide a detailed review of the literature on language development in PT-born children and to examine how prematurity can lead to atypical linguistic performances. According to the research discussed, it can be concluded that during the first years of life, crucial for gaining adequate social and adaptive skills, language development can be affected by PT birth. In VPT, altered brain maturation, leading to atypical functional organization and structural changes, was associated with abiding language impairments. In addition, environmental factors such as a long stay in a NICU with underexposure to significant auditory stimuli and nonoptimal infant-caregiver interactions have been associated with weaker language outcomes. Several intervention methods were proven useful in promoting the parent-child relationship, resulting in better interactions which have positive effects on cognitive and language development of children born PT. Moreover, we described some evidence of beneficial effects from early exposure to language, voices, and music in PT children. Further research is needed to assess the influence of this exposure on language development more thoroughly.


We demonstrated that the lowest intensity of 108 V/m, although not inducing structural changes, can produce electrostatic modifications on the reaction centre of the enzyme, as apparent from the dipolar response and the electric field distribution of the protein active site. Electric pulses above 5x108 V/m produced a fast transition between the folded and a partially denatured state, as inferred by the secondary structures analysis. Finally, for the highest field intensity used (7x108 V/m), a not reversible transition toward an unfolded state was observed.Electric fields can be a powerful tool to interact with enzymes or proteins, with an intriguing perspective to allow protein manipulation. Recently, researchers have focused the interest on intracellular enzyme modifications triggered by the application of nanosecond pulsed electric fields. These findings were also supported by theoretical predictions from molecular dynamics simulations focussing on significant variations in protein secondary structures. In this work, a theoretical study utilizing molecular dynamics simulations is proposed to explore effects of electric fields of high intensity and very short nanosecond duration applied to the superoxide dismutase (Cu/Zn-SOD or SOD-1), an important enzyme involved in the cellular antioxidant defence mechanism. The effects of 100-nanosecond pulsed electric fields, with intensities ranging from 10 The Superoxide is generated by many life processes, which include aerobic metabolism, oxidative phosphorylation, and photosynthesis, respiratory burst in the immune response of stimulated macrophages and neutrophils . Superox2- , but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.b)  Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc. http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and  there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” . The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The authors try to explore the effect of electric field on enzyme active sitesby exploring the response to field of SOD.Unfortunately, the paper does not present a comparison between calculated and observed values. This includes the argument that the rate changes.Unless I missed something the paper is compared to some experimentsI cannot see advantage in publicationReviewer #2: The authors present a molecular dynamics simulation study exploring the effect of electric field pulses on the superoxide dismutase protein. There are two main motivations for this study as presented by the authors: (i) to understand the structural stability of the protein in respect to an external electric field and (ii) to explore the possibility that the external electric field modifies the electrostatic guidance, i.e., the local electric potential that drives the superoxide to Cu2+ for the catalytic electron-transfer reaction. The goal (i) was already studied in a number of previous papers and the authors confirm the basic conclusion that sufficiently strong fields lead to partial protein denaturation. Question (ii) is potentially more novel and I believe here the authors' analysis was insufficient.The authors explore the distribution of the electric field, and its modification by an external pulse, in the vicinity of the active site. The first question to ask here: why should one care about the electric field? The ability of the superoxide to reach the active site should be driven by the potential of mean force, a free energy, and not by the electric field, which provides the force at a local point. I believe the authors are not calculating the property which is critical for the question they have posed. I also have significant questions regarding how the electric field was calculated. The calculation is based on eq 1, which is the Coulomb law in vacuum. The resulting numbers are based on the magnitudes of partial charges and the distances to them. This is clearly not the entire picture. Electrostatic interactions are screened by water. Near the protein surface one cannot simply used the bulk dielectric constant of 78 and one has to specifically calculate the electric field by the water dipoles. However, the crudest estimate suggests that the numbers presented by the authors are overestimated by a factor of ~78. This is clearly not acceptable. In addition, I assume simulations were done in the standard Ewald protocol. Therefore, Ewald corrections have to be used in the calculation of the electric field as well. The calculations presented by the authors have no physical meaning unless these problems are addressed.It would be useful to have a consistency check for the electrostatic calculations. Can pKa be calculated to make sure the results are solid? None of the plots presented in the paper are testable by observations. The authors should make some minimum effort to connect to the observable reality.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  11 Aug 2019Reviewer #1: The authors try to explore the effect of electric field on enzyme active sites by exploring the response to field of SOD.Unfortunately, the paper does not present a comparison between calculated and observed values. This includes the argument that the rate changes. Unless I missed something the paper is compared to some experiments. I cannot see advantage in publicationIt seems that our initial statement was not sufficiently clear, and we apologize for this.We tried to improve clarity changing the introduction, materials and methods and discussion as evidenced in the text of the new version of the manuscript.The manuscript is a fully computational study aimed to investigate possible response of the SOD1 enzyme to the application of intense electric field pulses. We do not address specific experimental data to compare with, but the references to experimental works are aimed to demonstrate that a response is feasible and worthwhile to be deepen, due also to the biomedical and biotechnological potentialities as evidenced in .Computational models are becoming increasingly affordable . The high performances of computing resources, accompanied by refined protein design methodologies, has allowed for the design of increasingly sophisticated proteins with diverse topologies and functions. Protein dynamics, ranging from local fluctuations around equilibrium conformations to large-scale conformational changes upon binding can be captured by molecular dynamics (MD) simulations, which uses a physics-based potential energy function to simulate protein dynamics as a function of time according to classical Newtonian mechanics. While biophysical techniques, such as NMR spectroscopy, can yield insights into protein dynamics, MD has the power not only to identify functionally relevant conformations, which may be ‘hidden’ to experimental techniques, but it can also provide the details of transitions between these conformations .The hypothesis we tested using MD simulations, was the possibility to modulate the SOD1 active site electric environment by external intense electric fields as the ones produced in nsPEFs applications. For these reasons, we observed the response of SOD1 to external electric fields, studying whether this enzyme results sensitive to the application of a 100 nsPEF with different characteristics.Fully atomistic MD studies are acquiring and increasing importance and are finding increasing acceptance in top level journal as PlOS ONE. For example only in the year 2019 there are 8 fully computational (MD) papers puplished in PlOS ONE (see below). Therefore, we believe that even in absence of experimental data our manuscript can be of interest of this journal.https://doi.org/10.1371/journal.pone.0211381• Daghestani M, Purohit R, Daghestani M, Daghistani M, Warsy A (2019) Molecular dynamic (MD) studies on Gln233Arg (rs1137101) polymorphism of leptin receptor gene and associated variations in the anthropometric and metabolic profiles of Saudi women. PLoS ONE 14(2): e0211381. https://doi.org/10.1371/journal.pone.0212065• Kandeel M, Kitade Y, Al-Taher A, Al-Nazawi M (2019) The structural basis of unique substrate recognition by Plasmodium thymidylate kinase: Molecular dynamics simulation and inhibitory studies. PLoS ONE 14(2): e0212065. https://doi.org/10.1371/journal.pone.0213393• Silva SG, da Costa RA, de Oliveira MS, da Cruz JN, Figueiredo PLB, Brasil DdSB, et al. (2019) Chemical profile of Lippia thymoides, evaluation of the acetylcholinesterase inhibitory activity of its essential oil, and molecular docking and molecular dynamics simulations. PLoS ONE 14(3): e0213393. https://doi.org/10.1371/journal.pone.0213646• Angladon M-A, Fossépré M, Leherte L, Vercauteren DP (2019) Interaction of POPC, DPPC, and POPE with the μ opioid receptor: A coarse-grained molecular dynamics study. PLoS ONE 14(3): e0213646. https://doi.org/10.1371/journal.pone.0214554• Schuster KD, Mohammadi M, Cahill KB, Matte SL, Maillet AD, Vashisth H, et al. (2019) Pharmacological and molecular dynamics analyses of differences in inhibitor binding to human and nematode PDE4: Implications for management of parasitic nematodes. PLoS ONE 14(3): e0214554. https://doi.org/10.1371/journal.pone.0217992• Turner M, Mutter ST, Kennedy-Britten OD, Platts JA (2019) Molecular dynamics simulation of aluminium binding to amyloid-β and its effect on peptide structure. PLoS ONE 14(6): e0217992. https://doi.org/10.1371/journal.pone.0218042• Miguel V, Villarreal MA, García DA (2019) Effects of gabergic phenols on the dynamic and structure of lipid bilayers: A molecular dynamic simulation approach. PLoS ONE 14(6): e0218042. https://doi.org/10.1371/journal.pone.0213262• Woerner P, Nair AG, Taira K, Oates WS (2019) Sparsification of long range force networks for molecular dynamics simulations. PLoS ONE 14(4): e0213262. Reviewer #2: The authors present a molecular dynamics simulation study exploring the effect of electric field pulses on the superoxide dismutase protein. There are two main motivations for this study as presented by the authors: (i) to understand the structural stability of the protein in respect to an external electric field and (ii) to explore the possibility that the external electric field modifies the electrostatic guidance, i.e., the local electric potential that drives the superoxide to Cu2+ for the catalytic electron-transfer reaction. The goal (i) was already studied in a number of previous papers and the authors confirm the basic conclusion that sufficiently strong fields lead to partial protein denaturation. Question (ii) is potentially more novel and I believe here the authors’ analysis was insufficient.The authors explore the distribution of the electric field, and its modification by an external pulse, in the vicinity of the active site. The first question to ask here: why should one care about the electric field? The ability of the superoxide to reach the active site should be driven by the potential of mean force, a free energy, and not by the electric field, which provides the force at a local point. I believe the authors are not calculating the property which is critical for the question they have posed. The referee asked a very interesting question that we do not properly addressed in the previous version of the manuscript. Our analysis was driven by previous works by D’Alessandro et al. and Amadei et al.  where authors combined mixed QM/MM methods with basic statistical mechanical relations to study the chemical events and the atomic motions of the complex environment of the SOD1 reaction center. Their results clearly showed that the protein-solvent environment fluctuations are essential to understand the reaction mechanism which is based on the concerted rupture of the copper-histidine coordination bond and the copper-superoxide bond in the active site. Such environmental fluctuations have been conceived as a perturbation electric potential exerted by the environment on the quantum center, hence the sum of each elementary electric potential produced by i) water molecules solvating the protein; ii) counterions; iii) all protein atoms not belonging to the reactive center (treated via QM methods).Amadei et al. concluded that such perturbing environmental electric field is essential for the modelling of the reaction process, pointing out dramatic effect of the protein and solvent interactions on free energy surface at the quantum center.In the present work, we wanted to ask the question if an exogenous electric perturbation (in addition to the endogenous one) can significantly modify the electric environment at the quantum center, tackling the problem with a classical approach, i.e. established that the reaction free energy is affected by the local electric field at the active site.I also have significant questions regarding how the electric field was calculated. The calculation is based on eq 1, which is the Coulomb law in vacuum. The resulting numbers are based on the magnitudes of partial charges and the distances to them. This is clearly not the entire picture. Electrostatic interactions are screened by water. Near the protein surface one cannot simply used the bulk dielectric constant of 78 and one has to specifically calculate the electric field by the water dipoles. However, the crudest estimate suggests that the numbers presented by the authors are overestimated by a factor of ~78. This is clearly not acceptable. In addition, I assume simulations were done in the standard Ewald protocol. Therefore, Ewald corrections have to be used in the calculation of the electric field as well. The calculations presented by the authors have no physical meaning unless these problems are addressed.The referee is right; we just presented an oversimplified version of the perturbing field calculation. What we actually did is now presented in more details in the new version of the manuscript. Such perturbing field is an atomic electric field (on the order of GV/m), made by three terms: the first one representing the perturbation due to each aminoacidic residue of the protein, the second which considers the perturbation due to local electric field generated by water molecules, the last one due the presence of couterions. The new Eq. 1 now explicitly takes into account the contribution of all the constituents of the simulation box, as also explained in  for a different protein, with all simulation data already taking into account Ewald corrections. Also note that MD systems correspond to needle-like ellipsoidal systems with the applied field along the major axis, hence no depolarizing field is present in the simulations.We hope the issue raised by the referee is now clarified, the electric field perturbation at the active site was explicitly calculated at full atomistic scale.It would be useful to have a consistency check for the electrostatic calculations. Can pKa be calculated to make sure the results are solid? None of the plots presented in the paper are testable by observations. The authors should make some minimum effort to connect to the observable reality.The point raised by the referee drove us to calculate pKa values for some specific residues at the active site known to be fundamental for the reaction process. D’Alessandro et al.  already investigated the endogenous electric perburbation effects on such residues, evidencing the relevance of each residue for the catalytic process. In general, residues producing a positive average electric field projected along the Cu-O2- bond favour the electron transfer process, while negative average electric fields are associated to inhibitory effects. Then, we analysed via PROPKA algorithm  the specific residues indicated in  to affect the electronic transfer process.We added in the new version of the manuscript a new Figure , where we present pKa data of four fundamental residues: i) His 63 and His 120, directly involved in the reaction process; ii) Asp 83, which is known to favour the electron transfer; iii) His 71, which is known to inhibit the electron transfer. Data are presented together with their standard deviations, obtained collecting data of the simulation equilibrium states .Results indicate a significant effect on pKa values for all the considered residues , suggesting that a single high intensity (10^8 V/m) bipolar pulse is able to affect the local environment at SOD1 reactive site. In particular, negative variations of pKa values are associated to the build-up of a negative charge on residue side chain. Interestingly, such effects are consistent with the ones presented in Fig. 7, where the electric field shifts  due to the applied Bipolar pulse was supposed to be a consequence of possible rotation of specific protein residues, and hence a charge distribution alteration.All the above discussion and clarifications have been added in the main text.AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 14 Aug 2019Nanosecond pulsed electric signals can affect electrostatic environment of proteins below the threshold of conformational effects: the case study of SOD1 with a molecular simulation studyPONE-D-19-16769R1Dear Dr. Apollonio,I do not like purely theoretical works, conclusion of which are not checked experimentally but the rules of PLoS One allow publishing such studies. For this reason:We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.Shortly after the formal acceptance letter is sent, an invoice for payment will follow. 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Permyakov, Ph.D., Dr.Sci.Academic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 20 Aug 2019PONE-D-19-16769R1 Nanosecond pulsed electric signals can affect electrostatic environment of proteins below the threshold of conformational effects: the case study of SOD1 with a molecular simulation study Dear Dr. Apollonio:I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. 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Infants with respiratory distress syndrome, bronchopulmonary dysplasia, patent ductus arteriosus ligation, periventricular leukomalacia, and retinopathy of prematurity were more common in the compliance group. Follow-up rates showed significant differences according to NICU volume. Using multivariate logistic regression, high birth weight, low NICU volume, siblings, foreign maternal nationality and high 5 min APGAR scores were significant independent factors associated with the non-compliance of VLBW infants for follow-up at 18–24 months of age. This is the first nation-wide analysis of follow-up for VLBW infants in Korea. Understanding factors associated with failure of compliance could help improve the long-term follow-up rates and neurodevelopmental outcomes through early intervention.The critical need to emphasize preterm infant follow-up after neonatal intensive care unit (NICU) discharge assures early identification of and intervention for neurodevelopmental disability. The aims of this study were to observe the follow-up rates in high-risk follow-up clinics, and analyze factors associated with non-compliance to follow-up among very low birth weight (VLBW) infants. The data was prospectively collected for 3063 VLBW infants between January 2013 and December 2014 from 57 Korean neonatal network (KNN) centers at a corrected age of 18–24 months. Correlations among demographic data, clinical variables, and neonatal intensive care unit (NICU) volume (divided into 4 quartiles) with the occurrence of non-compliance were analyzed. The overall follow-up rate at the corrected age of 18–24 month was 65.4%. The follow-up rates were inversely related to birth weight and gestational age. Apgar score, hospital stay, maternal age, and maternal education were significantly different between the compliance and non-compliance groups. The follow-up rate was higher for mothers with chorioamnionitis, abnormal amniotic fluid, multiple pregnancy, and  Improvement of survival rates for very low birth weight (VLBW) and extremely preterm infants resulted in advances in perinatal and neonatal care –3. HowevHigh-risk infants who missed follow-up were more likely to present with severe impairment, such as cognitive impairment, or sensory adverse events. Prior research has found many factors to be associated with NICU follow-up appointment compliance ,12–15. TThe Korean Neonatal Network (KNN) was established in 2013 to improve data collection systems in participating facilities and study various factors associated with mortality and morbidity of VLBW infants in Korea. Long-term data were also collected at the corrected age of 18–24 months and postnatal age of 3 years for VLBW infants. A better understanding of long-term outcomes in a national cohort will lead to improved approaches to neonatal care for VLBW infants in Korea. However, successful compliance with appropriate care and presentation to follow-up clinics must be guaranteed first and foremost.The aims of this study were to observe the follow-up rates to high-risk follow-up clinics at the corrected age of 18–24 month, and to identify predictors of non-compliance to follow-up among VLBW infants in the KNN.Data were prospectively collected for 3522 VLBW infants born between January 2013 and December 2014 from 57 KNN centers by local staff using a standardized electronic case report form ,17. The This study was approved by the Samsung medical center institutional review board (2013-03-002). The data registry was approved by each institutional review boards of all 57 hospitals participating in the KNN. Written consent was obtained from the parents of infants during enrollment in KNN.Several factors potentially associated with non-compliance to follow-up clinics for VLBW infants were evaluated. Maternal, demographic, and NICU-related data were obtained through KNN records. Gestational age was determined by obstetric examination with ultrasonography early during the pregnancy or, via obstetric history based on the mother's last menstrual period. Birth weight was recorded for each baby as soon as they arrived at the NICU for admission. Bronchopulmonary dysplasia was defined by NIH classification . NecrotiUnadjusted comparisons of maternal demographics and neonatal characteristics between the compliant and non-compliant groups with high-risk follow-up were performed using a chi-squared or Fisher’s exact test for categorical data and Student’s t-test for continuous data. The correlations between compliance to follow-up clinics, gestational age, and birth weight were evaluated using Pearson’s correlation coefficients. Receiver operating characteristic (ROC) curve analysis with associated area under the curve (AUC) was conducted to explore the discriminate ability of NICU volume, gestational age, and birth weight in predicting compliance to follow-up clinics with the selection of the most suitable cut-off point of each parameter with the best sensitivity, specificity, and overall accuracy. Multivariate analyses of variables found to be significant on univariate analysis were also performed to identify independent predictors of non-compliance to follow-up clinics. All statistical analyses were performed using SPSS software version 25.0  and P values of < 0.05 were considered significant.in vitro fertilization, maternal age, chorioamnionitis, maternal education level, maternal nationality, Apgar score, and siblings.Among 3063 infants survived to discharge, 2003 infants (65.4%) were followed-up at the corrected age of 18–24 months. Significant differences were found for the following factors between the compliant and non-compliant groups, as shown in Follow-up rates were significantly and inversely correlated with gestation and birth weight (P < 0.001) .Neonatal morbidity such as respiratory distress syndrome (RDS), air leak, patent ductus arteriosus (PDA) ligation, periventricular leukomalacia (PVL), retinopathy of prematurity (ROP), and duration of supplement oxygenation showed significant differences between the compliant and not-compliant groups .rd quartile, and the largest NICU volume group showed significant differences in the follow-up rate compared to others  showed an AUC of 0.567 (sensitivity of 55.9% and specificity of 54.4% for a cutoff of 1162.5 g). For gestational age and follow-up (P < 0.001), the AUC was 0.560, with a sensitivity of 54.2% and specificity of 53.5% for a cutoff of 29.2 weeks. For NICU volume and follow-up (P < 0.001), the AUC was 0.612, with a sensitivity 53.0% and specificity of 59.1% for a cutoff of 3In multiple logistic regression, foreign maternal nationality, siblings, 5-min APGAR score, birth weight, and NICU volume were confirmed as significant predictors of non-compliance to follow-up .Preterm infant follow-up clinics after NICU discharge play important roles in monitoring growth and development; provides continuity of medical care; and provides assurance of appropriate therapeutic interventions . High foLack of developmental follow-up has been associated with higher rates of neurodevelopmental impairment. The American Academy of Pediatricians recommends that preterm infants be followed-up by physicians affiliated with a NICU ,23. HowePrior research has identified many factors associated with NICU follow-up appointment compliance. Maternal drug use (odds ratio [OR] = 0.049), multiple gestation pregnancy (OR = 0.163), male sex (OR = 0.308), and greater distance from the hospital (OR = 0.987) ,14,24–28In this study, the compliant group had higher rates of multiple pregnancies. Previous reports showed that non-compliance after a multiple pregnancy might be due to the fact that caring for two sick newborns puts even more stress on the resources and time of a family than caring for one newborn . This diHigher NICU volume was associated with greater odds for high-risk infant follow-up referrals . In thisrd quartile, gestational age ≤ 29 weeks, and birth weight ≤ 1160 g were found to be significantly associated with compliance to follow-up. The parents of infants with a large gestational age and a high birth weight reflects the feeling of decreased need for follow-up.The ranked order of variables for predicting compliance to follow-up, as assessed by AUC analysis is as follows: NICU volume (0.612), birth weight (0.567), and gestational age (0.560) with poor prediction. It may be caused by the fact that compliance to follow-up is related with more than a single birth variable. Multiple variables, such as comorbidities and maternal factors, may exist. NICU volume ≥ 3In order to improve the follow-up rate, it is essential to make an appropriate follow-up appointment at the time of NICU discharge . HoweverVarious interventions could potentially be used to increase the follow-up rate after NICU discharge. To provide patient education through printouts, conversations with primary health care physicians, and videos can help families recognize the importance of follow-up clinics. Reminder letters and phone calls have also been used with success . In partThe limitation of this study was that it narrowly defined compliance to follow-up as collecting the data at the corrected age of 18–24 months. Families ultimately attending a follow-up clinic during a different age window were considered non-compliant. Furthermore, the non-compliant group may also include cases of death after discharge. NICU volume assumed as total number of annual enrollments in KNN during study period, it may have variation within the year and sites. Additionally, regional aspects such as distance, and resources for the hospital were not analyzed.In conclusion, through this first nationwide study, we detected the factors associated with non-compliance to follow-up at the corrected age of 18–24 month. Strategies, such as patient education, reminder phone calls, and an organized system of storing and remembering appointments, should be used particularly targeted to families who may be at risk of non-compliant to follow-up based on the factors we have identified. This could contribute to improve patient care accordingly and long-term outcome through early detection and intervention.


Poor maternal nutrition during pregnancy is a leading modifiable risk factor associated with risks of adverse pregnancy outcomes (APO). Nevertheless, there is paucity of evidence if consumption of some food groups is associated with lower risk of APO, particularly in low-income settings. We aimed to determine whether consumption of some food groups is associated with lower risk of APOs such as: preterm birth (PTB), low-birth weight (LBW), and stillbirth in rural Central Ethiopia.A multi-center  prospective cohort study, enrolling 432 pregnant women during their initial antenatal care visit, was employed. All mothers were then followed monthly  from enrollment to delivery. Midwives in respective health centers assessed dietary diversity using the Women’s individual dietary diversity score and evaluated birth outcomes following standard procedures. Logistic regression models were run to predict association of food groups with the APO.Out of the 374 pregnant women who completed the study, one in five [74 (19.8%)] experienced at least one of the APO: 34 (9.1%) gave birth to LBW babies, 51(13.6%) had PTB and 17 (4.5%) experienced stillbirth. Poor or inconsistent consumption (<¾ assessments) of dark green leafy vegetables (adjusted odds ratio (AOR) = 2.01; 95% confidence interval (CI): 1.04–3.87), dairy products , and fruits and vegetables  were independently associated with higher APO risks. Whereas, being nonanemic at term  was independently associated with lower APO risks.Poor or inconsistent consumption of dairy, dark green leafy vegetables and fruits were associated with higher risk of APOs. While community-based trials and mechanistic studies are needed to substantiate these findings, efforts to promote dietary diversity through increased consumption of fruits, vegetables and dairy may be beneficial in this and similar settings. Some of the world’s poorest countries have managed to cut child mortality rates by >50% in just a couple of decade’s time2. The mortality rate of under-five children has also declined substantially from 12.6 (12.4–12.8) in 1990 to 5.6 (5.4–6.0) million deaths in 20162. Despite these achievements, 5.6 million under five children continue to die every year mainly from preventable causes. Majority of child deaths occur during the neonatal period (first one month). About one million of neonates die on the first day and another close to one million die in the first week3.In the last couple of decades, a substantial progress in child mortality was made worldwide5. In 2015 alone, close to 15 million premature (<37 completed weeks of gestation) babies were born; of which, more than a million died during the neonatal period due to complications, and those surviving often experienced lifetime of impairments6.Adverse outcomes of pregnancy, mainly: preterm birth (PTB), low-birth weight (LBW), and stillbirth continue to be major causes of neonatal and under five child deaths, worldwideIn Ethiopia, recent estimates suggest that prenatal death happens in 46 out of 1000 pregnancies and about 11% of newborns have LBW (7 and 8). These estimates rank Ethiopia among the ten top countries with the highest global burden of maternal (fifth) and neonatal (sixth) deaths, as well as stillbirth (seventh) incidence rates.15. Evidence is accumulating that maternal dietary patterns during pregnancy is associated with some APO risks. Studies from Europe19, New Zealand20, and Brazil21 showed that maternal adherence to certain dietary patterns reffered as “western”, “prudent”, or “traditional” diets were positively associated with birth weight, offspring bone size, bone mineral density, and forearm fractures; with additional protective effect against risks of having infants of small for gestational age. However, the associations between dietary diversity on APO risks remains unclear as some studies show heightened or null-effects23. Besides, many of the studies investigating such associations are mainly from high-income settings and hence studies from resource-poor settings are urgently needed. Therefore, in this study, we aimed to determine the association between consumption of some food groups with APO risks.Poor nutrition of women during pregancy is among the leading modifiable risk factors associated with elevated APO risks24. Briefly, the study employed a prospective cohort follow-up study design enrolling 374 pregnant women from their initial antenatal care (ANC). All mothers were then followed monthly  from enrollment to delivery.This is part of a larger study that aimed to investigate dietary patterns during pregnancy and several outcomes. Detailed information about the larger study and the methods applied are reported elsewhereThe study was conducted in eight health centers randomly selected from four rural districts of Oromia Regional State, Ethiopia. The four rural districts were selected purposively to represent all agro climatic areas and socio-cultural aspects of the zone. Pregnant women were enrolled to the study according to their exposure to diversify (exposed) and nondiversified (unexposed) diets.We used the following criteria to include mothers to the study: permanent residence (weather she lived at least for 6 months in the area or not), apparently healthy , and mother willing to attend the monthly ANC visits.25 and World Health Organization26. Portable devices equipped with calibrated and standardized height gauges (SECA 206 Body meter) were used to measure the height of mothers to the nearest mm during each visit. Using a nonstretch measuring, midwives also measured the Mid-upper arm circumference (MUAC).We collected socio-economic data including: age, educational status, land ownership and others using questionnaire-based and face-to-face interviews. Midwives in each health center collected anthropometric data during enrollment and follow-up ANC visit. Weight was measured to the nearest 100 g using electronic scales  with a weighing capacity of 10–140 kg following the standardized procedures recommended by World Medical AssociationWomen dietary diversity scores (WDDS) was collected monthly using a four 24 h dietary recall from enrollment to delivery. According to FAO, the WDDS has nine food groups that include: (i) cereals, (ii) roots and tubers; (iii) vitamin A rich fruits and vegetables; (iv) other fruit and vegetables; (v) legumes and nuts; (vi) meat, poultry, and fish; (vii) fats and oils; (viii) dairy; and (ix) eggs.A woman was considered to be either in the diversified (“consumers”) or a nondiversified (“nonconsumers”) group, based on data computed from the WDDS. Accordingly, if a woman completed the four monthly visits and remained in same group for at least three of the four visits, she was considered to be part of the group. Women who missed a visit or changed dietary diversity group more than once, were excluded from the analysis as shown in Fig. 20, and women whose altitude adjusted hemoglobin values below 11.0 g/dl were considered to be anemic26. They also recorded birth weight to the nearest 100 g, and assessed stillbirth (a baby born with no signs of life at birth after 24 completed weeks of gestation) as well as PTB (delivery < 37 weeks of gestation), immediately after delivery. The midwives have also estimated gestational age of pregnancy, by combing data from the women’s report of last menstrual period and performing fundal palpation27.The midwives were trained on how to take hemoglobin levels using a portable HemoCue . Accordingly, hemoglobin levels were taken twice at enrollment and at term for every women. The readings were adjusted for altitudeData entry and cleaning was conducted using Epi-data and SPSS (version 20.0) statistical software (3.1). Kolmogorov–Smirnov test was used to test normality of continuous variables. A log-binomial model was run to adjust baseline group differences of key variables including MUAC, educational level, hemoglobin, age, height, and gestational age.p values < 0.05 were considered statistically significant. A bivariate and multivariate logistic regression analyses were to test the association of food groups consumed and APOs.We reported Adjusted ORs with 95% CI values for regression models run. Differences with p value:Furthermore, false discovery rate adjustment was performed using the Benjamini–Hochberg (B–H) procedure to reduce the inflation of type 1 error due to multiple comparisons. The following formula was used to calculate B–H critical values for each i/m)Q; where,Benjamini–Hochberg (B–H) = .p values to the critical B–H values, and all p values were < 0.05, with the highest value obtained for dark green leafy vegetables (p = 0.043).We then compared our original n = 28), shift from study group (n = 12), and delivery at home or away from index health facility (n = 18)  eligible pregnant women who fulfilled the inclusion criteria. Amongst of these, 374 completed the study. This yielded an overall dropout rate of 13.4%, which was balanced across groups. The main reasons for dropout were: discontinuation of ANC visits (Close to 1 in every 5 (19.8%) women, of those who remained in the study, experienced at least 1 of the APOs. As such, 51 (13.6%) gave birth to premature baby, 34 (9.1%) had a LBW baby, and 17 (4.5%) had a stillbirth babies who died at, or immediately after birth, Fig. n = 159; 42.5%) of the women completed primary education, and majority  were in the early 20s24 by age category, and 241 (65.1%) had ≥4 ANC visits. A sizable proportion 166(44.9%) of women owned two or more hectares of land. Hematologic investigations showed that more than two-third (71.4 % at enrollment and 67.9 % at term) were nonanemic.Table Bivariate analyses results showed that pregnant women who had poor consumption of dark green leafy vegetables, vitamin A-rich foods, other fruits and vegetables, milk and milk products, experienced a higher risk of APO, compared to those women who reported better consumption. Nevertheless, dark green leafy vegetables and dairy products remained to be independent predictors of APO risks in the final multivariate logistic regression analysis, Table Among maternal nutritional characteristics run the multivariate logistic regression analysis, only anemia at term was found to be independently associated with higher APO risks. Accordingly, nonanemic women whose altitude adjusted hemoglobin level was higher than 11 mg/dl at term had a 77% reduced risk  of experiencing APO compared to those who were anemic  were associated with higher risk of experiencing at least one of the APOs: LBW, preterm, and stillbirth.6, 98% of stillbirths5 and 96.5% of LBW infants28 occur in low-income settings, mainly in Africa and South Asia, there is a critical shortage of evidence investigating the association of APO with dietary patterns during pregnancy. Most of the existing studies in Ethiopia are cross-sectional9; and hence, ill-fitted to relate outcomes with dietary patterns. However, another study in Ethiopia showed that dietary diversity ≥4 (out of 9 food groups) has been correlated with lower risk of maternal anemia, preterm birth, and low birth weight babies24, but which food groups are associated with reduced risk remained unknown.Although 60% of preterm births29, Spanish30, and Indian31 prospective studies that enrolled and followed pregnant women all showing reduced risk of APOs with increasing consumption of dairy, green leafy vegetables, and fruits.In the present analysis, our multivariate analysis suggests that poor intake of dairy and dark green leafy vegetables during pregnancy were found to be independent predictors of higher APO risks. Pregnant women who had poor consumption of dairy, fruits and vegetables had nearly two to four fold increased risks of APO, compared to those who consumed either of these food items. This is in line with the Iranian32. Second, proteins and growth hormones provided by dairy can support fetal growth and thus may increase birth weight. Beyond its nutritional contribution, milk can also promote anabolism and serve as an endocrine signaling system for postnatal growth by activating the nutrient-sensitive kinase mTORC134, thus increasing gestational age, placental, and fetal weight. On the other hand, fruits and vegetables have been found to have a protective role against APO. Perhaps, this role can be conferred by micronutrients and antioxidants found in these foods, which contribute to optimal immune and placental functions, and besides contribute to fetal growth36Many factors can explain the role of dairy consumption in reducing the risk of APO. First, dairy is a good source of essential nutrients, including calcium. The role of calcium in preventing preeclampsia, a leading contributor to APO, is well-documented7. Secondly, in spite of our efforts to adjust for baseline differences computing APOs risks, the effect of hidden factors in the model may not be completely controlled. Our study was not powered enough to evaluate associations of consumption of each food groups with individual APOs. Besides, given that this is a health-facility based study, our sampling might be subject to favoring women with better access to health facilities.The study had limitations that require curiosity and consideration when interpreting the findings. Firstly, unlike preconception nutrition is associated with pregnancy outcomes, we were only able to follow the women starting from their second trimester, mainly related to maternal habit of late ANC onset in the countryNotwithstanding the above limitations, our study highlights that consistent consumption of dairy products, fruits, and vegetables (dark green leafy vegetables) were found to be independently associated with APO risks. While community-based trials and mechanistic studies are needed to establish causal relationships, promotions of the consumption of these food groups in Ethiopia and other similar settings may be beneficial.