The objective of the study was to explore patterns of white matter (WM) alteration in preclinical stage familial Creutzfeldt–Jakob disease (fCJD) using diffusion tensor imaging (DTI).PRNP G114V mutation and six non-carriers were recruited from the same fCJD kindred and follow-up obtained from all asymptomatic carriers and two non-carriers 2 years later. Overlapping WM patterns were also explored in asymptomatic carriers and symptomatic CJD patients. All participants underwent clinical and neuropsychological assessments and DTI at baseline and follow-up. DTI data were subjected to whole-brain voxel-wise analysis of fractional anisotropy (FA) and mean diffusivity (MD) in WM using tract-based spatial statistics. Three comparisons were conducted: baseline carriers against non-carriers , changes after 2 years in carriers , and differences between patients with symptomatic CJD and healthy controls .Seven asymptomatic carriers of the Neither carriers nor non-carriers developed any neurological symptoms during 2 years of follow-up. Baseline analysis showed no differences between the carrier and non-carrier groups in MD and FA. Follow-up analysis showed significantly increased MD in multiple WM tracts, among which increased MD in the bilateral superior longitudinal fasciculus, bilateral anterior thalamic radiation, bilateral cingulate gyrus, and left uncinate fasciculus overlapped the patterns observed in patients with symptomatic CJD.Changes in integrity within multiple WM tracts can be detected during the preclinical stage of fCJD. PRNP, and sporadic CJD (sCJD) are two major forms of CJD that share similar pathophysiological features  is a rare, fatal neurodegenerative disorder characterized by rapidly progressive dementia, motion disturbances, and akinetic mutism. Familial CJD (fCJD), which is caused by mutations in the prion protein gene, features . Given tfeatures , asymptoin vivo and has been widely used to show WM changes in the early stages of CJD. Previous DTI studies in CJD cases have demonstrated mean diffusivity (MD) changes in multiple WM tracts (Diffusion tensor imaging (DTI) is a non-invasive MRI technique that can determine the orientation and integrity of white matter (WM) fibers M tracts . A pathoM tracts ; howeverM tracts . An FDG-M tracts , suggestPRNP G114V mutation from the same fCJD kindred, which were the same individuals as those included in our previous FDG-PET study  testing, and DTI at baseline. All evaluations were double blind to the genotypes .Data used in this study were from a Chinese Han fCJD kindred with a G114V mutation in s of CJD . Detailss of CJD . Thirtees of CJD . ParticiFollow-up evaluation was carried out a mean of 2 years after the baseline interview in seven carriers and two non-carriers, and consisted of in-depth clinical examination, neuropsychological assessment, EEG testing, and DTI. Follow-up was not obtained for five non-carriers due to refusal to participate further in the study. All carriers and non-carriers for whom follow-up was obtained had received no treatment for cognitive impairment or neurological symptoms in the previous 2 years.PRNP G114V fCJD kindred were enrolled from the clinic at Xuanwu Hospital, along with age-, and sex-matched healthy controls enrolled from the community. All patients with symptomatic CJD were diagnosed according to the European probable CJD criteria , Montreal Cognitive Assessment (MoCA), Clinical Dementia Rating (CDR), and neuropsychiatric inventory questionnaire assessments (NPI-Q). Clinicians who performed the assessments were unaware of the mutation status of participants.Venous blood samples (5 ml) were collected from 10 patients with symptomatic CJD in EDTA anticoagulant vessels and stored at -20°C. A blood extraction kit was used to extract blood DNA. DNA concentration and purity were determined using a NanoDrop2000. Specific primers were used to amplify DNA fragments in the region of the mutation site. Reaction system: 2 × Phanta Max Buffer 12.5 μl, Phanta Max Super-Fidelity DNA Polymerase (1 U/μl) 0.5 μl, dNTP Mix (10 mM each) 0.5 μl, Primer-F (10 μM) 1 μl, Primer-R (10 μM) 1 μl, DNA 1 μl, and water to 25 μl. Reaction procedure: 95°C denaturation 5 min; 95°C denaturation for 30 s, 65°C annealing for 30 s, 72°C extension for 30 s, 25 cycles, each cycle reduced by 0.6; 95°C denaturation for 30 s, 50°C annealing for 30 s, 72°C extension for 1 min, 20 cycles; 72°C extension 10 min. After PCR, 3 μl aliquots of products were analyzed by 2.5% agarose gel electrophoresis, and PCR products were also sequenced using an ABI 3730XL DNA Analyzer.All participants received a 2-h EEG at baseline and follow-up using 18-lead electroencephalographic transducers . Electrodes were placed according to the international standard 10–20 system. Conventional single lead, double lead, and sphenoid lead were traced. Eyes closed and deep breathing experiments were performed.b-value of 1,000 s/mm2, applying diffusion gradients along 30 directions. Seventy axial slices, with no slice gap, were acquired .Magnetic resonance (MR) scanning was performed on a GE Signa PET/MR 3.0 Tesla scanner  in Xuanwu Hospital, Capital Medical University. All 13 asymptomatic family members received their first PET/MR scans in March 2017. Nine of them received follow-up scans in March 2019. All 10 patients with symptomatic CJD received their scans on the same scanner during their hospital stays from 2018 to 2020. DTI data were acquired using a spin echo-echo planar imaging sequence  with a http://www.nitrc.org/projects/panda/), which was independently developed by the state key laboratory of cognitive neuroscience and learning of Beijing Normal University. Briefly, preprocessing involved correction of eddy current and head movement, creating a brain mask, and fitting the diffusion tensor model. Outputs were voxel-wise maps of fractional anisotropy (FA) and mean diffusivity (MD). The FA index of DTI is a sensitive neuroimaging measure of degeneration and describes overall WM health, maturation, and organization. MD is another index representing the average dispersion level and dispersion resistance of water molecules overall, which can reflect changes in brain tissue. Higher MD values indicate more free water molecules in the tissue, which influence the information transmission speed to some extent.Diffusion tensor imaging data were preprocessed using the PANDA software package  and covariates (age and sex), which were stored in the columns of a “design matrix”; significance thresholds were set at family-wise error (FWE) corrected P < 0.05 using the threshold-free cluster enhancement option.Tract-based spatial statistics (TBSS) analysis was performed to explore the influence of CJD pathology on WM integrity. In TBSS, all participant FA and MD data were projected onto a mean FA tract skeleton before applying voxel-wise cross-participant statistics. Voxel-wise statistical analyses were conducted using a non-parametric permutation-based inference tool  with the general linear model for statistical modeling. Paired t-test. Sex differences were assessed using the Chi-square test. Results were considered statistically significant at p < 0.05.In this study, SPSS 23.0 software was used to evaluate statistical significance. Differences in age, education, and cognitive scores were assessed using the Student’s Estimated years from expected symptom onset (EYO) values were calculated as the age of the participant at the time of assessment in this study minus the age of the parent at symptom onset; if the parent of the participant had not developed CJD symptoms, the age of the grandparent was used to calculate EYO. For example, if the participant was 35 years old, and the parent’s age at onset was 45 years, then the EYO value would be 10. Parental age at onset was determined using a semi-structured interview in which family members were asked about the age of first progressive cognitive decline.n = 13) were recruited from the same kindred [the same participants were reported in our previous FDG-PET study . After 2 years, TBSS analysis showed that asymptomatic carriers had increased MD across multiple WM tracts, including the left inferior fronto-occipital fasciculus, left uncinate fasciculus, bilateral anterior thalamic radiation, bilateral cingulate gyrus, bilateral superior longitudinal fasciculus, and bilateral corticospinal tracts (FWE correction, p < 0.05; At baseline, TBSS analysis showed that there was no significant difference in MD and FA between asymptomatic carriers of the p < 0.05; Furthermore, significant differences were detected between patients with symptomatic CJD and matched controls (FWE correction, Among WM tracts with increased MD in patients with symptomatic CJD, asymptomatic carriers showed overlapping patterns in the bilateral superior longitudinal fasciculus, bilateral anterior thalamic radiation, cingulate gyrus, and left uncinate fasciculus . DetailsPRNP G114V mutation carriers. We found that changes in WM were characterized by increased MD, accompanied by FA within the normal range. The observed alterations were mainly focused in several WM tracts in asymptomatic carriers, which included patterns overlapping those of patients with symptomatic CJD. Notably, the changes in WM tracts were also correlated with the gray matter metabolic alterations identified in our previous FDG-PET study. To our knowledge, our findings provide the first description of WM abnormalities in the preclinical stage of fCJD, which, together with the gray matter changes revealed by the FDG-PET study, describe pathological imaging changes occurring during the asymptomatic stage of CJD.In this prospective study, we applied DTI to detect global WM involvement in asymptomatic In our study, changes were found at follow-up in multiple WM tracts in asymptomatic carriers. Among the detected changes, those in the bilateral superior longitudinal fasciculus, bilateral anterior thalamic radiation, bilateral cingulate gyrus, and left uncinate fasciculus overlapped the patterns detected in patients with CJD. Previous investigations have also detected WM alterations in similar areas, consistent with our data. A DTI study of 26 patients with CJD found MD changes in WM, including in the uncinate fasciculus, superior longitudinal fasciculus, and anterior thalamic radiation . AnotherAn FDG-PET study was previously conducted in the same participants included in this investigation. The FDG-PET study focused on gray matter alterations, and the results suggested hypometabolic changes in the parietal and temporal lobes in the preclinical stage of CJD . AlthougIn our study, increased MD and normal FA were detected in the WM of asymptomatic carriers, while the same alterations were also detected in patients with CJD, and represent pathological imaging changes in the clinical stage of the disease. It may be assumed that the imaging patterns detected in asymptomatic carriers are preclinical pathological changes rather than being secondary to aging; however, the DTI alterations detected differ from those usually reported in patients with CJD , althougA strength of this study is that it shows the earliest known brain changes detected by DTI in the asymptomatic stage of fCJD, which were correlated with the gray matter alterations determined by FDG-PET; however, these results require further confirmation, as the study was limited by the small sample size, since fCJD is a very rare disease. Additionally, the symptomatic patients included in this study were not from the same kindred as the asymptomatic carriers and non-carriers. As our data were collected as part of an ongoing research project, we anticipate that further analyses will be conducted, to investigate the dynamic changes of indices between the asymptomatic and clinical disease stages.In conclusion, the preliminary results from our TBSS analysis indicate increased MD in several WM tracts and suggest the involvement of WM integrity in the preclinical stage of fCJD. Overall, this study may provide new insights into CJD pathogenesis.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Ethics Committee and local Institutional Review Board of Xuanwu Hospital, Capital Medical University, Beijing. The patients/participants provided their written informed consent to participate in this study.DJ, YCh, ZZ, and LWa were responsible for the study concept and design. CC, HL, LL, JY, ZL, RG, and LWa were responsible for clinical data collection. YCh and ZZ analyzed the image results. DJ, KX, and YCu were the major contributors in writing the manuscript. ZZ and LWu were responsible for critical revision of the manuscript for important intellectual content. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Estimating total body fat in public hospitals using gold-standard measurements such as air displacement plethysmography (ADP), deuterium oxide dilution, or dual-energy X-ray absorptiometry (DXA) is unaffordable, and it is challenging to use skinfold thickness. We aimed to identify the appropriate substitute marker for skinfold thickness to estimate total body fat in pregnant women and infants.Kappa statistics to assess agreement between these anthropometric markers with skinfold thickness.The study is part of a prospective cohort study titled MAASTHI in Bengaluru, from 2016 to 19. Anthropometric measurements such as body weight, head circumference, mid-upper arm circumference (MUAC), and skinfold thickness were measured in pregnant women between 14 and 36 weeks of gestational age; while measurements such as birth weight, head, chest, waist, hip, mid-upper arm circumference, and skinfold thickness were recorded for newborns. We calculated Kappa statistic, 0.42; 95 % CI 0.38–0.46) in pregnant women. For newborns, the highest agreement with total skinfold thickness was with birth weight . Our results indicate that MUAC higher than 29.2 cm can serve as a suitable alternative to total skinfolds-based assessments for obesity screening in pregnancy in public facilities. Similarly, a birth weight cut-off of 3.45 kg can be considered for classifying obesity among newborns.We found the highest amount of agreement between total skinfold thickness and MUAC (Mid-upper arm circumference and birth weight can be used as markers of skinfold thickness, reflecting total body fat in pregnant women and the infant, respectively. These two anthropometric measurements could substitute for skinfold thickness in low- and middle-income urban India settings.The online version contains supplementary material available at 10.1186/s12884-021-03915-1. The increasing prevalence of overweight and obesity among children is a significant public health issue attributing to immediate and long-term health problems . Howevern = 12)  followed by the head and chest circumferences, respectively. The cut-offs for circumferences at the waist, hip, and mid-upper arm and BMI showed fair agreement with total skinfold thickness. Therefore, on the basis of the extent of misclassification and agreement, the birth weight cut-off emerged as the best substitute .The extent of misclassification that would result from the use of the newly defined cut-offs instead of the accepted standard, i.e. 85th percentile of total skinfold in newborns, along with the amount of agreement  when they are not so. Further validation studies in India can establish the reliability and validity to steer policy-level actions to prioritise screening obesity in pregnancy. The third limitation is that this study mostly represents the source population comprising low-middle-income women who attend public facilities in Bengaluru. This needs to be validated in an even larger population to prove its wider applicability. However, we were able to capture the measurements among a large sample size of mother-child dyads and thus have shown the use at public facilities for urban populations that can be applied across the country.Mid-upper arm circumference and birth weight can be used as markers of skinfold thickness, reflecting total body fat in pregnant women and infants, respectively. These two anthropometric measurements could substitute for skinfold thickness in low- and middle-income urban India settings. Our results suggest that the MUAC and birth weight be used in pregnant women and infants, respectively, as markers for effective screening tools for detection of obesity in Indian states and other similar settings. The simple technique and low costs associated with measurements can enable the implementation by frontline health workers including in rural areas.Additional file 1Additional file 2Additional file 3Additional file 4


To investigate the impact of a 5-year follow-up on the incidence of identified birth defects in children conceived using assisted reproductive technologies (ART).A 5-year cohort study was performed in three ART centers from January 2013 to October 2018. 1,543 women with 1,985 infants who delivered successfully or underwent termination of pregnancy due to malformations were recruited in this study. Follow-up was conducted by phone interview, 7 days, 1 year, 3 years, and 5 years after birth. Collected data included whether one or more birth defects were diagnosed, the category of birth defects, and when the malformation was diagnosed. Cumulative incidence of birth defects and the loss to follow-up rate of each follow-up was compared.According to the diagnostic criterion of birth defects, 111 cases of one or more birth defects were recorded, with a total of 117 birth defects after the 5-year follow-up. 0.2%  of birth defects were diagnosed before delivery; 2.7%  at 7 days; 5.0%  after 1 year; 5.5%  after 3 years; and 5.6%  after 5 years. 3.4% (4/117) of defects were diagnosed prenatally, 45.3% (53/117) of defects diagnosed within the first 7 days after delivery, 40.2% (47/117) diagnosed during 7 days to 1 year, and 9.4% (11/117) of defects diagnosed in 1–3 years after birth. The remaining 1.7% (2/117) of defects were diagnosed between the ages of 3 and 5 years. Among the 1,543 patients, 99.9% patients  responded to the telephone interview at 7 days after delivery; the response rate was 89.0%  at 1 year, 81%  at 3 years, and 64.5%  after 5 years.We suggest that in ART, 1-year follow-up should be the minimum requirement and 3-year follow up the optimal length of follow-up that balances resource requirements with ascertainment completeness. It was estimated that ART has resulted in hundreds of thousands of infants born globally every year  that occur during intrauterine life and are identified prenatally, at birth, or sometimes are only detected later in infancy, such as hearing defects .The prevalence of birth defects after ART varies from 1% to 6.2% according to different studies –8. The iIn theory, the incidence of birth defects increases with longer follow-up. However, there is no consensus regarding follow-up duration with regard to capturing all, or nearly all, defects. For example, birth defects such as septate uterus, the most common uterine anomaly, with an estimated prevalence of 2%–3% of women, cannot be diagnosed until reproductive age . SeveralHowever, extending the follow-up duration means more resources would be required to maintain a registry or complete a study. In addition, as follow-up intervals lengthen, the loss to follow-up (LTFU) rate increases which may affect the accuracy of estimates. Therefore, it may be useful to identify where the balance lies between identifying more birth defects and the resources required to conduct research.The aim of this study was to gain a better understanding of which defects are diagnosed and when defects are diagnosed after ART treatment, and to document the trajectory of LTFU rates. We conducted a multicenter cohort study with 5-year follow-up of all infants conceived using ART in Guangdong Province, China, to investigate the prevalence of all-cause and specific cause of birth defects.This was an observational, longitudinal, multicenter, study. Participants were recruited from January 2013 to December 2015 from three reproductive centers in Guangdong, China. The study was approved by the Reproductive Medical Ethics Committee of Guangzhou Women and Children’s Medical center.in vitro fertilization (IVF) and/or intracytoplasmic sperm injection (ICSI) and had reached 28 weeks of pregnancy were invited to participate in this study. These couples were informed and signed a follow-up consent. Telephone interviews were conducted four times: 7 days, 1 year, 3 years, and 5 years after delivery. At the first interview (7 days after delivery), the maternal complications, neonatal complications, category and quantity of birth defects, and loss to follow-up information were recorded; meanwhile, terminations for birth defects and stillbirths of over 28 weeks of gestation were recorded as well. At the following three times, only birth defects and loss to follow-up data were included  according to the International Classification of Diseases and Health Related Problems, 10th Revision  . Birth dp value was less than 0.05. All data analyses were performed using SPSS for windows 20.0 .Categorical variables were expressed as frequency and percentage and were compared between groups by means of the Pearson chi-square test or Fisher exact test. A result was considered statistically significant if a two-tailed A total of 1,543 women and 1,985 fetuses were enrolled in this study. 99.9% of patients  responded to a request for telephone interview at 7 days after delivery. The response rates were 89.0%  at 1 year; 81%  after 3 years; and 64.5%  after 5 years  before delivery; 2.7%  7 days after delivery; 5.0%  after 1 year; 5.5%  after 3 years; and 5.6% after  after 5 years (3.4% (4/117) of defects were diagnosed prenatally, and 45.3% (53/117) of defects were diagnosed within the first 7 days after delivery. 40.2% (47/117) were first recorded at the 1-year follow-up, and 9.4% (11/117) of defects were first recorded at the 3-year follow-up. The remaining 1.7% (2/117) of defects were first recorded at the 5-year follow-up.Of the 4 fetuses that were diagnosed with congenital malformations before delivery, two fetuses underwent termination of pregnancy  and the other two fetuses  received treatment after delivery.50 babies (with 53 defects) were diagnosed at the 7-day follow-up. The main categories of congenital malformations included cardiovascular and circulatory malformations (8 cases); genitourinary malformations (9 cases), musculoskeletal malformations (14 cases), and other malformations (10 cases). Details of birth defects are listed in Forty percent of the cases  were diagnosed at the 1-year follow-up. The categories of new congenital malformations at this stage included cardiovascular and circulation malformations (6 cases), genitourinary malformations (17 cases), and musculoskeletal malformations (15 cases). Only 11 cases with defects were diagnosed after 1 year. The categories included cardiovascular and circulation malformations and musculoskeletal malformations  of defects were diagnosed prenatally in our study; however, in a previous literature from South Australia, the corresponding rate was up to 18.7% 7 of defe, which iSimilar with previous studies , 14, mosIn our study, 40% of all birth defects (47/117) were not recognized until after the 7-day follow-up. Examples of these defects include abdominal wall defects and cryptorchidism. As more than 40% birth defects were identified between birth to 1 year, we believe that the data presented suggest that 1-year follow-up should be the minimum requirement for future studies of birth defects after ART. If registries only collected data to 7 days after delivery, a range of cardiovascular, genitourinary system and musculoskeletal system defects would potentially be missed.After the 1-year assessment, only 11 birth defects were noted, accounting for 11% of the overall birth defects. Among them, half were cardiovascular defects; at 3 years after delivery, the cumulative prevalence of birth defects was 5.5% and 98.2% of all the defects eventually detected were documented.We found a paucity of articles that reported on their LTFU rate. In our study, the LTFU at 7 days after delivery was 0.1% and the LTFU was 11%, and 19% by 1 and 3 years after delivery, respectively. By 5 years after delivery, the LTFU rate increases to 35.5% which is almost three times compared to 3 years after delivery. Therefore, in order to better understand birth defects after ART treatment, we believe that 1-year follow-up after birth is the minimum requirement in ART treatment and 3-year follow-up is the optimal duration that allows for >95% ascertainment of all birth defects eventually diagnosed.There are some limitations of our study. Although we collected data from three hospitals over 5 years, a total of 1,985 babies may be not enough to detect some birth defects. Secondly, the fact that we obtained the information of birth defects from parents not from a professional pediatrician might affect the accuracy of the description of the specific diagnosis. Thirdly, accompanied by those loss-to-follow-up cases, some birth defects might be missed in data collection. The actual birth defect rate may be a little higher. In conclusion, we suggest that in ART, 1-year follow-up pulse should be the minimum requirement and 3-year follow-up the optimal follow-up interval for ascertaining the risk of birth defects.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Reproductive Medical Ethics Committee of Guangzhou Women and Children’s Medical Center. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.LS was responsible for the conception and design of the study as well as analysis and interpretation of data; prepared, drafted, and revised the article critically for important intellectual content; and approved the final draft for publication. C-LL, PL, and G-FC contributed to collection of the data, drafting and revision of the entire article, and analysis and interpretation of data. AM contributed to revision of the article for important intellectual content and editing of the English grammar. Z-HC, JL, and XZ contributed to collection of the data, as well as drafting and revision of the article for important intellectual content. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Via the UNC-NAMIC DTI Fiber Analysis Framework, our pediatric atlases provide the computational templates necessary for the fully automatic analysis of infant DTI data. To the best of our knowledge, these atlases are the first comprehensive population diffusion fiber atlases in early pediatric ages that are publicly available.Diffusion Tensor Imaging (DTI) is a non-invasive neuroimaging method that has become the most widely employed MRI modality for investigations of white matter fiber pathways. DTI has proven especially valuable for improving our understanding of normative white matter maturation across the life span and has also been used to index clinical pathology and cognitive function. Despite its increasing popularity, especially in pediatric research, the majority of existing studies examining infant white matter maturation depend on regional or white matter skeleton-based approaches. These methods generally lack the sensitivity and spatial specificity of more advanced functional analysis options that provide information about microstructural properties of white matter along fiber bundles. DTI studies of early postnatal brain development show that profound microstructural and maturational changes take place during the first two years of life. The pattern and rate of these changes vary greatly throughout the brain during this time compared to the rest of the life span. For this reason, appropriate image processing of infant MR imaging requires the use of age-specific reference atlases. This article provides an overview of the pre-processing, atlas building, and the fiber tractography procedures used to generate two atlas resources, one for neonates and one for 1- to 2-year-old populations.  Diffusion-Weighted Imaging (DWI) is a magnetic resonance imaging (MRI) technique that measures the diffusion of water molecules to characterize microstructural aspects of the brain. Diffusion rates of water molecules differ by brain tissue type, and thus, DWI allows researchers to examine the structural properties and connectivity of white matter fiber tracts. Along with the DWI technique, Diffusion Tensor Imaging (DTI) provides a means to analyze the diffusion tensor, which is the three-dimensional shape of the water diffusion, enabling the 3D visualization of white matter fiber pathways.Researchers utilize DWI and DTI techniques to build a 3D atlas that can provide a standardized reference space for anatomical guidance. Such reference spaces are considered crucial for consistency, reliability, and reproducibility of neuroimaging studies. A DTI atlas can serve as a tool for identifying changes in white matter tracts and for assessing the neurodevelopment of both healthy and at-risk children using, for example, tract-oriented statistics. Moreover, the use of an atlas can also enhance the diagnostic utility of neuroimaging and can quantitatively identify aberrant developmental patterns related to clinical conditions .To date, however, most atlases have been developed for the adult human brain , and manThe application of DTI analysis methods in infants also has the potential to enhance our understanding of the typical variation in brain development patterns and the structural correlation of rapidly developing cognitive capabilities. This potential is especially meaningful as it may enable the development of a model for monitoring white matter maturation as it relates to the observable behavioral and structural changes predictive of neurodevelopmental or psychiatric disorders.Some research groups have endeavored to build age-specific brain atlases for studies of neonates to overcome the aforementioned issues, although they are few in number and most are not publicly available. One exception however comes from Via tractography of white matter fiber bundles to examine neonatal and pediatric DTI data, such as in the UNC-NAMIC DTI Fiber Analysis Framework  database is a unique and innovative longitudinal dataset that has followed children, enrolled prenatally, with imaging and cognitive/behavioral assessments throughout their postnatal brain development. From the EBDS database, we selected participant scans collected within a few weeks of birth, and at 1- and 2-years of age from two ongoing longitudinal studies: a study of normative development that also includes children at genetic risk for psychiatric disorders ; and a tThe first atlas, the EBDS Neonatal DTI Atlas, was constructed from 144 participants, born between 30 and 42 weeks gestation (37.11 ± 2.6), with DWI scans acquired within the first few weeks of life, or between 38 and 48 weeks gestational age (41.88 ± 1.83). Participant demographic information for scans included in the neonatal atlas can be found in n = 170) and 2-year-old participants (n = 171), born between 27 and 42 weeks gestation (37.20 ± 2.7), with DWI scans acquired at an average age of 13.03 (±0.95) months for the 1-year-olds and 25.05 (±1.07) months for the 2-year-olds. Participant demographic information for scans included in the pediatric atlas can be found in The second atlas, the EBDS Pediatric DTI Atlas, was constructed from 341 scans acquired from 295 unique typically developing 1-year , BRIC, Chapel Hill, NC, United States using one of two different scan sequences: a 6-direction or a 42-direction sequence on the Siemens Allegra scanner and the same 42-direction protocol on Siemens Tim Trio scanner.All participants were scanned without sedation during natural sleep. Before scanning, neonate participants were fed and swaddled. Scans for 1- and 2-year-old participants were scheduled to take place just prior to nap/sleep time. Once asleep, participants were fitted with earplugs and earphones and placed in the MRI scanner with their heads in a vacuum-fixation device. We performed neonate scans with a neonatal nurse present, and a pulse oximeter to monitor heart rate and oxygen saturation. Scans for the older children were performed with a research team member present in the scanner room throughout the scan to monitor the child.b value of 1,000 s/mm2, were acquired. The acquisition was repeated five times to improve signal-to-noise ratio.A single-shot, twice refocused echo-planar spin echo DTI sequence was used with the following variables: TR 5,200 ms, TE = 73 ms, slice thickness = 2 mm, in-plane resolution = 2 mm × 2 mm, and 45 slices. One image without diffusion gradients  and DWIs along 6 gradient directions, with a 2 in unique directional diffusion gradients.A single-shot, twice refocused echo-planar spin echo DTI sequence was used with the following variables: TR = 7680 ms, TE = 82 ms, slice thickness = 2 mm, in-plane resolution = 2mm × 2mm, a total of 49 DWI volumes were acquired, 7 without diffusion gradients  and 42 with b = 1000 s/mmOur framework for the fiber tract-based analysis of DTI data closely follows the UNC Utah NAMIC pipeline , 2013, wUsing DTIPrep , we desiVia the DTIProcess software suite  with the average b0 image as the input. Manual editing of these initial masks using itk-SNAP , instead of the average b0 image, as input to BET2. This choice provided more accurate initial masks than those generated from the baseline images for this age group. This also significantly reduced the amount of time required for manually editing the 1 and 2-year-old data.For both the neonate and the pediatric data, the creation of brain-masked tensor images by applying the brain masks during the tensor estimation process served as the final pre-processing stage prior to atlas building.Via DTIAtlasBuilder, which is an in-house developed, publicly available tool. In the first step of the atlas building, a prior FA template is employed as an initial reference frame for affine alignment. We employed a rescaled  version of the JHU Eve atlas FA image  algorithm . It is noteworthy that a larger number of datasets were visually flagged during this quality control steps and then corrected  prior to rerunning the atlas building procedure. This level of quality control ensured optimal SNR and contrast in the final DTI atlas.White matter fiber tractography was applied to the DTI atlases built in the prior step. We employed 3D Slicer’s “Tractography Label Map Seeding” Module . A totalFor fiber bundles reconstructed in the neonatal atlas, tractography stopped where the FA value for the single tensor was less than 0.08 or the radius of curvature fell below 0.5 mm. In contrast, for the pediatric atlas, tractography stopped where the FA value was less than 0.1.Via FiberViewerLight  tractography, it was necessary to compute an arcuate-specific DTI atlas due the large inter-subject variance in arcuate morphology with a significant number of subjects missing larger sections of the fronto-temporal arcuate fiber . For thiThis tractography necessitates considerable interactive, manual efforts, both for the tractography ROIs as well as for the fiber cleaning/editing. While no exact book keeping was performed, we estimate that the full procedure took more than 12 months for the comprehensive set of fiber tracts, leading to several versions for each fiber bundle until a final, acceptable version was achieved. This significant effort highlights the need for the public dissemination of our DTI fiber atlases to facilitate efficient infant DTI studies. For the list of white matter structures, software terminology, and abbreviations, see link in supplementary materials for Appendix from Our infant DTI atlases are intended to be used in two ways as described below. In our own studies we favor the second/study-specific option  as a study-specific DTI atlas provides the optimal analysis reference space, though at significant effort cost:1.Via our DTI-Reg Slicer module, and diffusion properties are extracted along the atlas fiber tracts for statistical analysis.As a reference analysis space for infant studies, where subject DTI data is mapped directly into this atlas space, for example 2.Via an automated tractography approach, such as our AutoTract tool  tools in the DTI-reg processing procedure which allows for registration based on tensors.A limitation of our DTI atlases is that they are based on the registration of intensity-normalized FA images and thus the registration did not incorporate the local fiber orientation in the atlas generation process. Future atlases could improve upon our method by using a tensor-based registration algorithm which is superior to using scalar images for cross-subject image alignment. This can be done for example Furthermore, as some of the studies introduced in the previous section have shown, future research could utilize our neonatal and pediatric atlases as well as the library of white matter tracts specific to these atlases for the study of both typical development and investigations of clinical pathology. Providing resources for clinical implementation is essential as it enhances the diagnostic utility of neuroimaging and quantitatively identifies aberrant developmental patterns . Thus, oIn summary, this paper presents the generation of a new public resource for neuroimaging studies of early brain development. We have constructed two DTI atlases, one for neonates and another for pediatric populations. These atlases consist of both reference tensor templates, as well as a library of major white matter tracts delineated on these atlases. We encourage the field to use these high-quality standardized DTI brain templates and the associated white matter fiber bundles for studies of both typical and atypical brain development. Understanding of early brain maturation is still in its infancy and much remains to be discovered about normative brain development. The utility of these atlases also extends to clinical scenarios, where the use of such templates can enhance the diagnostic utility of neuroimaging and quantitatively identify aberrant developmental patterns. Furthermore, the white matter tract library and the 3D printed visualization of these tracts can also serve as interactive educational tools in multidisciplinary settings.https://www.nitrc.org/projects/uncebds_neodti NAMIC DTI Analysis Framework available at the following link: https://www.nitrc.org/projects/namicdtifiber/DTIAtlasBuilder: https://www.nitrc.org/projects/dtiatlasbuilder.The EBDS pediatric DTI atlases are available at the following link: john_gilmore@med.unc.edu.The datasets presented in this article are not readily available because NIH holds the rights to these data. Requests to access the datasets should be directed to JHG, The studies involving human participants were reviewed and approved by Institutional Review Board at the University of North Carolina at Chapel Hill. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.SJS, DJ, RJS, and MS wrote the manuscript. RLS, JBG, and JHG provided insightful comments and reviewed content. All authors contributed to the article and approved the submitted version.The views expressed in this manuscript are those of the authors and do not necessarily represent the opinions or position of listed funding agencies.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Findings from previous studies about the association of preterm birth as well as birth weight with the risk of T1DM were still inconsistent. We aimed to further clarify these associations based on Chinese children and explore the role of gender therein.A nationwide multicenter and population-based large cross-sectional study was conducted in China from 2017 to 2019. Children aged between 3 and 18 years old with complete information were included in this analysis. Multiple Poisson regression models were used for evaluating the associations of birth weight as well as preterm birth with T1DM in children.p for interaction=0.065). In addition, children with low birth weight were not associated with T1DM . The findings from matched data had the similar trend.Out of 181,786 children, 82 childhood T1DM cases were identified from questionnaire survey. Children with preterm birth (<37 weeks) had higher risk of type 1 diabetes . Children born with high birth weight  had no statistically significant risk of T1DM . However, children’s gender might modify the effect of high birth weight on T1DM (girls: OR: 3.15, 95%CI: 1.33-7.47; boys: OR: 0.99, 95%CI: 0.38-2.55, In China mainland, preterm birth increased the risk of childhood T1DM, but high birth weight only affected girls. Therefore, early prevention of T1DM may start with prenatal care to avoid adverse birth outcomes and more attention should be paid to children with preterm birth and girls with high birth weight after birth. What is already known about this subject?Some previous studies, including meta-analyses, have indicated that preterm birth and high birth weight had higher risk for childhood type 1 diabetes, but inconsistent results are still emerging.What is the key question?Whether the associations between preterm birth and birth weight and type 1 diabetes based on Chinese children are different for other countries and whether the gender plays a role therein?What are the new findings?Preterm birth was associated with higher risk of childhood T1DM in China.High birth weight was only associated with higher risk for T1DM in girls, which had not been reported before.How might this impact on clinical practice in the foreseeable future?Prevention of T1DM may start with prenatal care to avoid adverse birth outcomes.More attention should be paid to children with preterm birth and girls with high birth weight after birth to improve the adverse effects.Type 1 diabetes mellitus (T1DM) is an immune-mediated disease characterized by destruction of pancreatic β-cells, resulting in absolute insulin deficiency . AlthougThis study was a nationwide large population-based cross-sectional study, conducted at kindergarten, primary, secondary and high schools in 13 medical centers of China  from 2017 to 2019, but Jiangxi was not included in this analysis due to missing the information of T1DM. Schools were selected using stratified cluster random sampling. The study protocol was approved by the ethics board of Zhejiang University. All participants’ informed consents were acquired from children or their parents. 231,937 children aged between 3 and 18 years old were included, and children with serious disease, logic errors in the questionnaire, type 2 diabetes or missing information of key variables were excluded. Finally, 181,786 children were included in this final analysis Figure 1Birth weight (g), whether preterm birth or not  were self-reported by parents and children who were born prematurely were further asked about their delivery gestational age. According to the World Health Organization , we cateChildhood T1DM cases were identified by questionnaire surveys with the question: “Has your child been diagnosed with type 1 diabetes? ① Yes ② No.” The questionnaire was completed by their guardians. In addition, to minimize errors, we again asked those who answered yes to confirm the diagnosis of type 1 diabetes.The height was measured to the nearest 0.1 cm, whereas weight was measured with a precision of 0.1 kg. The children were weighted without shoes and wearing light clothing. Body mass index (BMI) was calculated as weight (in kg) divided by height (in m) squared. We categorized children’s BMI into four status according to the BMI Z-score , as defined by WHO 2007 standards and classifications . Other vP<0.05 was considered as statistically significant and all statistical analyses were performed using R software (version 3.6.0).We used mean and standard deviations (Mean ± SD) to describe continuous variables, and frequency and percentage  were reported for categorical variables. Student’s t test and Chi square test were used for continuous variables and categorical variables, respectively, to compare the characteristic difference between groups. Associations of preterm birth as well as birth weight with T1DM were analyzed by multiple Poisson regression, respectively. To explore independent effects, we adjusted potential confounders, including maternal age at delivery, maternal education, annual family income, diabetes of mother, father and siblings, maternal gestational diabetes, children’s characteristics including age, gender and breast feeding. To control the effect of gestational age when analyzed the effects of birth weight on T1DM, whether preterm birth or not was further adjusted. The crossover analysis was conducted to further elucidate the role of preterm birth and birth weight in increasing the risk of T1DM in children. In addition, we also explored the interaction between birth outcomes and gender on childhood T1DM. Interaction analyses with multiplicative interaction terms were conducted. The In sensitivity analyses, we repeated the analyses but used the matched data. We identified children with T1DM (n=82) as cases and randomly selected children without T1DM (n=328) as controls on a 1:4 ratio through Propensity Score Matching based on maternal age, maternal education, annual family income, diabetes of mother, father and siblings, maternal GDM, children’s characteristics including age, gender and breast feeding. And conditional logistic regression was used to test the association between preterm birth as well as birth weight and T1DM.A total of 181,786 children aged between 3 and 18 years old were included in the analysis Figure 1As showed in To further elucidate the independent effect of preterm birth, birth weight on the risk of T1DM in children, we took children with normal birth weight and term birth as reference and found that children with preterm birth but normal birth weight had higher risk of childhood T1DM  than children with high birth weight but term birth , using the matched sample size. Similar trends were shown when we used original data Table 3.P for interaction = 0.065 and 0.069 for adjusted model and matched data, respectively.). However, there was no interaction between gender and preterm birth .Considering the possible modification by gender, we conducted analyses stratified by gender. It turned out that findings were different between girls and boys. As shown in In present national study, both preterm birth and high birth weight were associated with increased risk of T1DM and the effect of preterm birth was stronger than that of high birth weight. However, children’s gender modified the effect of high birth weight on T1DM, with high birth weight only increasing the risk of T1DM in girls.The adverse effect of preterm birth on T1DM demonstrated in the current study was in line with the findings from previous meta-analysis with 18 studies and 22,073 cases published in 2014  After 20When further subdivided gestational age, we found that both children born before 32 and born between 32 and 36 gestational weeks were associated with increased T1DM, furthermore, the dose-response effect was observed. However, results from other studies were different. A population-based register study from Swedish with 14,949 cases found that compared to full-term infants, birth between 32 and 36 gestational weeks had a higher risk , while birth before 32 weeks of gestation had a lower risk of childhood-onset T1DM  . AnotherWe found that high birth weight was associated with increased risk of T1DM but low birth weight had no statistically significant effect on T1DM, using the matched sample. It was consistent with one meta-analysis published in 2010 . HoweverTo our knowledge, only one prior cohort study has reported there was no potential sex-specific difference between preterm birth and T1DM, which was consistent with present study . HoweverThere were several strengths in our study. Firstly, to our knowledge, this was the largest study to show the association between birth outcomes and childhood T1DM in China Mainland. Secondly, we considered a wide range of confounders, including demographic characteristics, family history of diabetes, intrauterine exposure and feeding patterns. Thirdly, we also explored the gender differences, which had been less taken into account in previous studies. However, some limitations also should not be ignored. Firstly, although present study was based on large sample size but the number of cases was still relatively small. The power of high birth weight was only 0.36, which might prevent us from finding that the effect of high birth weight was statistically significant. After using propensity score matching, the power increased to 0.68. However, due to the limited number of cases, we were still unable to further divide the gestational weeks into more subgroups. Secondly, in this study, we only investigated the gestational age of children with preterm birth, so it was not possible to assess whether all children had intrauterine growth restriction. However, we adjusted whether preterm birth when exploring the role of birth weight to avoid the effect of preterm birth on birth weight to some extent. Thirdly, cases in this study were from self-report, which might lead to recall bias. But generally, those without disease are not likely to be filled in as sick, so the prevalence rate is likely to be underestimated, and the impact of exposure on the outcome may be underestimated. However, recall bias may also be overestimated, because parents of children with diabetes may be more likely to associate possible factors and remember exposure factors. Finally, we still could not exclude potential confounders such as pregnancy history, pre-pregnancy obesity and pregnancy complications due to the limitations of the investigation and the cross-sectional study failed to consider the effects of recent life styles, which might be influenced by interventions after diagnosis. But previous studies with the adjustment only led to a little alteration, which would have probably made no difference.In conclusion, children with preterm birth had independently higher risk of childhood T1DM, and girls with high birth weight were more susceptible to have T1DM. Therefore, early prevention should start with prenatal care to avoid adverse birth outcomes and more attention should be paid to children with preterm birth and girls with high birth weight after birth for timely detection and treatment of type 1diabetes.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The study protocol was approved by the Institutional Review Board of The Children’s Hospital of Zhejiang University School of Medicine . Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.JF is the chief of the study and is responsible for the study concept and design, funding acquisition and critical revision of the manuscript. KH and SS contributed to analysis and interpreting of the data and writing of the draft. RC, CW, SC, YL, HY, RZ, FL, BC, ZS, MM, FHL, PL, HD, MZ, YY, and LC contributed to study design, investigation and data curation. YXY contributed to the study concept and design, analysis and interpreting of the data and critical revision of the manuscript. All authors contributed to the article and approved the submitted version.This study was funded by Chinese National Natural Science Foundation (81973055), the national key research and development program of China (2016YFC1305301) and the Fundamental Research Funds for the Central Universities. The funder/sponsor did not participate in the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The COVID-19 pandemic led to regional or nationwide lockdowns as part of risk mitigation measurements in many countries worldwide. Recent studies suggest an unexpected and unprecedented decrease in preterm births during the initial COVID-19 lockdowns in the first half of 2020. The objective of the current study was to assess the effects of the two months of the initial national COVID-19 lockdown period on the incidence of very and extremely preterm birth in the Netherlands, stratified by either spontaneous or iatrogenic onset of delivery, in both singleton and multiple pregnancies.Retrospective cohort study using data from all 10 perinatal centers in the Netherlands on very and extremely preterm births during the initial COVID-19 lockdown from March 15 to May 15, 2020. Incidences of very and extremely preterm birth were calculated using an estimate of the total number of births in the Netherlands in this period. As reference, we used data from the corresponding calendar period in 2015–2018 from the national perinatal registry (Perined). We differentiated between spontaneous versus iatrogenic onset of delivery and between singleton versus multiple pregnancies.The incidence of total preterm birth < 32 weeks in singleton pregnancies was 6.1‰ in the study period in 2020 versus 6.5‰ in the corresponding period in 2015–2018. The decrease in preterm births in singletons was solely due to a significant decrease in iatrogenic preterm births, both < 32 weeks  and < 28 weeks . For multiple pregnancies, an increase in preterm births < 28 weeks was observed .This study shows a decrease in iatrogenic preterm births during the initial COVID-19-related lockdown in the Netherlands in singletons. Future studies should focus on the mechanism of action of lockdown measures and reduction of preterm birth and the effects of perinatal outcome. The outbreak of the coronavirus disease 2019 (COVID-19) resulting from SARS CoV-2 has a major impact on healthcare worldwide, and was declared an official pandemic by the World Health Organization (WHO) on March 11, 2020 [In pregnant women with severe COVID-19, the preterm birth risk appears to be increased , 4. HoweWhile the COVID-19 pandemic rages on, threatening the lives of many adults, the finding that preterm birth decreased during the first lockdown warrants our full attention, since preterm birth affects millions of families worldwide each year.Preterm birth, defined as birth before 37 + 0 weeks of gestation, is a major contributor to perinatal mortality and morbidity, yearly complicating over 15 million pregnancies worldwide . PreventThe previously mentioned studies showed a decrease in preterm birth in COVID-19 lockdown used a relatively small sample size , 7, exclIn this study, we collected data on preterm births < 32 weeks of gestation from all ten perinatal centers with level-III Neonatal Intensive Care Facilities in the Netherlands. Our aim was to assess the association between the initial COVID-19 lockdown in the Netherlands and the incidence of preterm birth < 32 weeks of gestation, taking onset of delivery and composition of the pregnancy (singleton versus multiple) into account.The first national COVID-19 mitigation measures in the Netherlands were introduced on March 9, 2020. These measures were intensified on March 15, 2020: childcare facilities and non-essential services involving physical contact were closed and social distancing was introduced (1.5 m rule). Most mitigation measures were gradually phased out in May and June 2020. With the opening of restaurants and public facilities  on May 15, 2020, the first episode of most rigorous COVID-19 mitigation measures ended. For this study, we selected the period from March 15 up to and including May 15, 2020.+ 0 weeks to 31+ 6 weeks – i.e. very preterm and extremely preterm - take place in one of these centers. Dutch national guidelines advise against active management of neonates born at gestational ages less than 24+ 0 weeks [In the Netherlands, pregnant women with signs of preterm labor < 32 weeks of gestation or with a high risk of requiring iatrogenic preterm birth < 32 weeks are transferred to one of the ten perinatal centers with a specialized neonatal intensive care unit (NICU) if possible . Therefo 0 weeks .+ 0 weeks to 31+ 6 weeks, were provided by each perinatal centers as derived from local medical files. Collected items were: date of birth, gestational age at birth, type of onset of labor (spontaneous or iatrogenic), pregnancy composition (singleton or multiple), mode of delivery , child sex and birth weight. Data on maternal age, parity, previous obstetric history, BMI, pre-existing chronic disease, socio-economic status, ethnicity or other risk factors for preterm birth were not collected given the exploratory nature of the study.Data on all births from March 15 to May 15, 2020 with a gestational age between 24The total number of births in the study period in 2020 was obtained through the National Institute for Public Health and the Environment (RIVM) as extracted from Praeventis. In the Netherlands, all neonates are screened for a number of diseases after 72 h of birth using a dried blood spot card as part of our national screening program. Data about this screening and the provided limited maternal and neonatal characteristics such as gestational birth are collected in a national database called Praeventis , 13. Pre+ 0 weeks to 31+ 6 weeks in the corresponding calendar period (March 15 to May 15) in 2015 to 2018 from The Netherlands national perinatal registry, Perined [Since Praeventis contains no information on start of delivery (spontaneous vs iatrogenic), we were unable to use this database for our reference group. We therefore obtained data on preterm births between 24Since we were only able to collect data about live births during the lockdown period in 2020, and the Praeventis database only collects data on neonates having undergone neonatal screening, we excluded all cases with ante-partum and intra-partum fetal deaths, both in 2020 and in the reference years 2015–2018 .The Perined registry from 2015 to 2018 covers around 96–98% of all births . By subtOur primary outcome was preterm birth < 32 weeks (very preterm) and < 28 weeks (extremely preterm) in singleton and multiple pregnancies. Gestational age was predominantly based on crown rump length measurement in early pregnancy. In addition, we stratified the analysis by singletons and multiples and by type of onset of preterm birth (spontaneous vs iatrogenic). Preterm births were considered ‘spontaneous’ if labor started with spontaneous preterm contractions and/or preterm prelabor rupture of membranes (PPROM). Preterm births after elective cesarean section or after induction of labor were considered to have an ‘iatrogenic’ onset.Baseline characteristics of the 2020 preterm cohort and the 2015–2018 preterm cohort were compared using Student’s t-test and chi-squared test or Fisher’s exact tests where appropriate. Proportions of preterm birth for the subgroups were calculated and compared with chi-square test. Logistic regression was used to express an increased or decreased risk of preterm birth in the cohort of 2020 using unadjusted Odds Ratios (OR) with 95% confidence intervals (95%CI). All analyses were performed using SAS Statistics, version 9.4.The Medical Research Ethics Committee at the Amsterdam University Medical Centers, location VUmc, approved this study .Second, The National Institute for Public Health and the Environment approved the study protocol for using Praeventis data. Third, Perined approved the use of their data  for the years 2015–2018.None.From March 15 to May 15, 2020, Praeventis registered 27,351 live born children, of which 847 were part of multiple pregnancies. Accordingly, we conclude that there were 26,504 singleton births, and 420 multiple births.+ 0 weeks to 31+ 6 weeks). Of these births, 163 were singleton and 37 were multiple births. Two twin pregnancies had undergone fetal foeticide because of congenital anomalies before 24 weeks, they were still categorized as twins.In this cohort, we observed 200 live births with a gestational age < 32 weeks  was observed in the study period, solely due to an increase in births with spontaneous onset.We present a study assessing the association between COVID-19 lockdown and preterm births which was able to stratify between spontaneous or iatrogenic onset of preterm birth. As both types have a different etiology, distinction between these types is essential for correct interpretation of the effects, especially since we showed that the observed decrease is solely attributable to iatrogenic preterm births. Second, we were able to stratify between singleton and multiple pregnancies, although the total number of multiples were low.We used manually collected data from 10 perinatal centers, and used two different databases to compare the incidence of preterm during the COVID-19 lockdown period with previous years. The national databases used well-defined definitions for spontaneous or iatrogenic preterm birth, however due to the manual approach, differences in interpretation and registration might have occurred. Furthermore, the Praeventis database covers a higher percentage of births compared to the Perined database (99% versus 96%). We attempted to account for this issue by adding these births to the denominator in the reference group.+ 0 and 31+ 6 weeks of gestation. If lockdown measures indeed resulted in delay of delivery, it is possible that less women delivered with a gestational age less than 24 weeks, thereby influencing the incidence of PTB, especially < 28 weeks of gestation or more women between 32 and 37 weeks. The 2020 Perined data will shed light on this issue.We were only able to assess births between 24No data from 2020 on perinatal deaths could be collected , as many of these deliveries occur outside perinatal centers and the national database Perined is not yet available for 2020. In addition, early neonatal deaths were not collected since Praeventis screening is after 72 h. In addition, we could not collect data on maternal characteristics and neonatal outcomes.This study collected data from a limited time period (March–May 2020). We were therefore only able to compare the number of preterm births to the number of women who delivered in that period. A more precise comparison would have been if we were able to take into account all pregnant women in that period, including the women that remained pregnant / whom did not deliver preterm, and construct a time-to-event analysis. Unfortunately, due to the nature of this study this was not possible.We acknowledge the following limitations:A growing number of studies have revealed a decrease in preterm births during national COVID-19 lockdowns. This study supports evidence from previous studies around the globe showing a decrease in preterm births –7, 17–19Several theories have been put forward to explain the decrease in preterm birth during the COVID-19 lockdowns observed in various settings. Possible affected risk factors include a reduction in asymptomatic maternal infections due to hygiene measures, less physical activity and minimal direct social interaction due to home confinement, less work-related stress and reductions in air pollution. Alteration in these factors mainly influence the risk of spontaneous preterm birth . HoweverThere are several possible explanations for a decrease in iatrogenic preterm births.First, a decrease in the incidence of preeclampsia, fetal growth restriction or placental abruption would probably lead to a reduction in iatrogenic preterm births. During the initial COVID-19 lockdown pregnant women experienced less physical activity due to home confinement, and could take more rest during the day. Lockdown measures may have influenced the severity of placental obstetrics syndromes such as preeclampsia and fetal growth restriction, thereby reducing the need for early intervention by induction of labor or planned cesarean section. However, there is currently not enough evidence affirming rest is helpful in preventing pre-eclampsia and its complications . Based oSecond, a more likely explanation could be found in the patterns of hospital visits and admission for pregnant women at risk of iatrogenic preterm birth. Women might have been reluctant to visit the hospital for fear of contracting infection in the hospital , or not Third, during the COVID-19 lockdown, consultations with the midwives and antenatal outpatient clinic visits were reduced. Physical appointments were replaced by telephone appointments, without blood pressure measurement or ultrasound examination. Overall increases in telemedicine during COVID-19 quarantine up to 68% have been reported, which could have impacted quality of care . PossiblUnfortunately, we have no data on maternal morbidity and adverse perinatal outcome during the studied period. Stowe et al. found no evidence of an increase in stillbirths regionally or nationally during the COVID-19 pandemic in England when compared with the same months in the previous year . We cannAs Perined data will become available for 2020, we plan to validate our findings by undertaking a methodologically much more robust analysis of national data from a single data source, which will allow taking into account underlying temporal trends, appropriate adjustment for confounding, and exploration of subgroup-specific effects. Besides smaller initiatives, an international network has been set up to further investigate lockdown effects on perinatal outcome: the iPOP study . HopefulOur study confirms findings from a previous quasi-experimental study that the incidence of preterm birth was reduced following lockdown measures in the Netherlands. We provide essential novel information by indicating that the observed reduction was mainly due to iatrogenic and not spontaneous preterm births. Future studies should direct their focus on why the number of iatrogenic births were decreased and at what possible costs or benefits .


Empagliflozin, an established treatment for type 2 diabetes (T2DM), has shown beneficial effects on liver steatosis and fibrosis in animals and in humans with T2DM, non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH). However, little is known about the effects of empagliflozin on liver function in advanced NASH with liver fibrosis and without diabetes. This study aimed to assess the effects of empagliflozin on hepatic and metabolic outcomes in a diet-induced obese (DIO) and insulin-resistant but non-diabetic biopsy-confirmed mouse model of advanced NASH. Male C57BL/6JRj mice with a biopsy-confirmed steatosis and fibrosis on AMLN diet  for 36-weeks were randomized to receive for 12 weeks: (a) Empagliflozin (10 mg/kg/d p.o.), or (b) vehicle. Metabolic outcomes, liver pathology, markers of Kupffer and stellate cell activation and lipidomics were assessed at the treatment completion. Empagliflozin did not affect the body weight, body composition or insulin sensitivity , but significantly improved glucose homeostasis as assessed by oral glucose tolerance test in DIO-NASH mice. Empagliflozin improved modestly the NAFLD activity score compared with the vehicle, mainly by improving inflammation and without affecting steatosis, the fibrosis stage and markers of Kupffer and stellate cell activation. Empagliflozin reduced the hepatic concentrations of pro-inflammatory lactosylceramides and increased the concentrations of anti-inflammatory polyunsaturated triglycerides. Empagliflozin exerts beneficial metabolic and hepatic (mainly anti-inflammatory) effects in non-diabetic DIO-NASH mice and thus may be effective against NASH even in non-diabetic conditions. Non-alcoholic fatty liver disease (NAFLD) is a metabolic disease with a continuously increasing prevalence that is currently affecting approximately 30% of the general population and 40–50% of patients with type 2 diabetes (T2DM) and obesity ,2. EarlyDespite the high prevalence of NASH, no specific treatment for the disease exists to date ,2,3. DueConsequently, the results from human studies raise the following questions: (a) whether empagliflozin, apart from liver fat and markers of liver fibrosis, can improve liver inflammation and fibrosis stage as assessed by the gold standard, which is liver histology; (b) whether—and if yes, through which mechanisms—empagliflozin can be an effective treatment for NAFLD in non-diabetic conditions, i.e., in pre-diabetes obesity. Animal studies with empagliflozin have been performed so far in animal models of overt diabetes ,12,13,14Both DIO-NASH mice treated with a vehicle as well as DIO-NASH mice treated with empagliflozin gained approximately 5% body weight during the 12 weeks of treatment whereas mice fed a chow diet gained significantly less weight . The higDIO-NASH mice treated with a vehicle had slightly higher fasting glucose levels at the treatment start (week 0) and a similar response in the oral glucose tolerance test (OGTT) at week 7 compared with the chow mice thus suggesting the presence of a mild hyperglycemia but no overt diabetes . DespiteAll DIO-NASH mice included in both groups in our study had a NAS ≥ 4 (in most cases 5 or 6) and liver fibrosis. Treatment with empagliflozin resulted in a significant reduction in NAS compared with treatment with the vehicle in the post- vs. pre-treatment biopsy . InteresAlthough empagliflozin did not significantly affect the lipid content, we performed a hepatic lipidomic analysis in order to assess whether empagliflozin affected the lipid composition. The lipidomic analysis of the liver identified 987 lipid species. The partial least squares-discriminant analysis (PLS-DA) showed that, based on the lipid measurements, the DIO-NASH + empagliflozin group and the DIO-NASH + vehicle group formed two distinct clusters with some overlap in their 95% confidence interval . This suWe demonstrate herein that treatment with empagliflozin improved glucose metabolism and liver pathology in biopsy-confirmed DIO-NASH mice with insulin resistance without the presence of diabetes.(−/−) mice [Previous studies in different animal models of diabetes with obesity  mice  and OLET/−) mice ) have sh/−) mice ,13,17 as/−) mice , lipogen/−) mice ,18 and g/−) mice . In thesIn our study, we used a model that developed diet-induced obesity, insulin resistance and NASH with steatosis, inflammation and liver fibrosis but no overt diabetes. Despite the fact that treatment with empagliflozin did not reduce body weight and improved only slightly blood glucose levels, it still led to a modest but significant reduction in NAS in the liver pathology compared with the vehicle. Interestingly, the reduction in NAS by empagliflozin was driven by lower scores in liver lobular inflammation and not in liver steatosis. Treatment with empagliflozin had, in these conditions, no effect on liver fibrosis. To our knowledge, only one study has previously assessed the effects of empagliflozin alone or combined with dulaglutide in a non-diabetic mouse model of NAFLD with inflammation (NASH) and did not find any major impact of empagliflozin on liver histology . HoweverWhen assessing the possible mechanisms mediating the effects of empagliflozin, we did not observe any changes in the markers of the hepatic Kupffer (hepatic macrophages) and stellate cell activation as well as in the amount of intrahepatic lipid content. We did, however, observe changes in the intrahepatic lipid composition with empagliflozin, which were characterized by a reduction in the hepatic concentrations of lactosylceramides and an increase in the hepatic concentrations of several unsaturated triglyceride species. Hepatic lactosylceramides are elevated in NASH in humans  and are Although the hepatic effects of empagliflozin are more modest compared with other medications under the same conditions, they are mediated through distinct mechanisms, which supports the notion for the future evaluation of empagliflozin as part of a combination therapy. Furthermore, our findings supported the evaluation of empagliflozin in humans with NAFLD, obesity-insulin resistance and pre-diabetes who may profit from the combined beneficial effects of the medication in the cardiovascular, renal and metabolic system as well as in the hepatic function.Our study has several strengths and limitations. A strength of the study is the use of an established mouse model that develops all of the components of human NAFLD. Another strength is the use of a liver biopsy both before and after treatment completion, which allowed us to include our study mice with advanced NAFLD and to perform comparative evaluations. A limitation of our study is the lack of direct assessment of liver autophagy or SPM levels.In conclusion, we have shown that empagliflozin exerts beneficial metabolic and hepatic (mainly anti-inflammatory) effects in non-diabetic mice with diet-induced obesity, insulin resistance and NASH, possibly by downregulating the intrahepatic concentrations of lactosylceramides and by increasing hepatic unsaturated triglycerides . Future n = 13, DIO-NASH + empagliflozin) at a dose of 10 mg/kg/day and we compared the findings with DIO-NASH mice treated with the vehicle  for 12 weeks. Finally, in order to compare the metabolic status of the DIO-NASH mice, we included in our analysis a group of mice fed a normal chow diet . All mouse experiments were performed according to Gubra bioethical guidelines that fully comply with international accepted principles for the care and use of laboratory animals.The study design and methodology have been previously described ,20. BrieThe measurements and procedures are described with detail in the supplemental information in . BrieflyDetails about the liver pathology and IHC staining have been described in ,20. BrieThe body composition was assessed by an EchoMRI 3-1 body composition analyzer  in non-anaesthetized mice.A mass-spectrometric analysis of hepatic lipids (lipidomics) was performed by Metabolon Inc.  as described in detail in ,20.n = 13 for DIO-NASH + vehicle and DIO-NASH + empagliflozin, the study had 80% power to detect differences ≥ 20% of the mean with an SD of 20% of the mean and at an α = 0.05 two-tailed. A one- or two-way (for OGTT and ipITT) ANOVA and mixed-models  for changes in body weight were performed with parameters Time (corresponding with days for body weight and with minutes for OGTT and ipITT) and Treatment . Post-hoc Dunnett tests for the comparison of DIO-NASH + empagliflozin vs. DIO-NASH + vehicle or for Chow + vehicle vs. DIO-NASH + vehicle were performed when three groups were involved and when p by the interaction of Time with Treatment (Time*Treatment) was <0.05. An unpaired t-test was performed for the parameters where only two groups  were assessed and after the confirmation of the normal distribution for the involved parameters with a D’Agostino–Pearson test. One-tailed p-values in the unpaired t-test were reported for the histologic outcomes of DIO-NASH + empagliflozin  and MetaboAnalystR . With n liflozin  based onescribed ,20,27,28The present study demonstrates the beneficial metabolic and hepatic (mainly anti-inflammatory) effects of empagliflozin in non-diabetic obese mice with insulin resistance and NASH. Further research is required to elucidate the underlying mechanisms and to evaluate whether empagliflozin could be effective in the treatment of NAFLD/NASH in humans even without diabetes.


Strong correlation between PTX3 and PCSK9 levels was observed . Multiple stepwise backward regression analysis with PCSK9 as dependent variable revealed that PTX3, apo B100, apo A1, apo B48, and BMI were significantly positive and the presence of NAFLD and HbA1c negatively associated with PCSK9 concentrations. These variables together explain 57% of PCSK9 variability; the strongest relationship was observed between PCSK9 and PTX3 and apo B100. Our results indicate that circulating PCSK9 is significantly associated with inflammation marker PTX3 as well as atherogenic lipids and apolipoproteins C3, B100, and B48, which might be of value in understanding interactions between development of atherosclerosis and inflammatory state in DM2 patients.Proprotein convertase subtilisin/kexin type 9 (PCSK9) is involved in the regulation of LDL metabolism. There is evidence that circulating PCSK9 is a cardiovascular risk factor. In this study, we determined factors associated with circulating PCSK9 in a group of patients with type 2 diabetes mellitus (DM2). Material included 116 consecutive patients with DM2 from outpatient diabetes clinic. Circulating PCSK9, PTX3, apolipoprotein (apo) B100, apo B48, and apo C3 levels were determined by ELISA, apo A1 by immunoturbidimetry. The mean (sd) age of patients was 59.1 (11.1) years, the mean (sd) values of serum PCSK9 were 255.4 (106.97) ng/ml. Circulating PCSK9 correlated negatively with age ( Q152H variant carriers, apart from marked hypocholesterolemia, exhibited normal liver function despite their lifelong endoplasmic reticulum PCSK9 retention. Hepatoprotective effects of this variant could be explained by its cochaperone function [Proprotein convertase subtilisin/kexin type 9 (PCSK9) has been gaining major attention after the emergence of data on the role of this protein in lipid homeostasis through regulation of low-density lipoprotein receptors and in atherosclerosis process. Increased PCSK9 is associated with decreased clearance of LDL-C and consequently with increased LDL-C serum concentration , 2. Therfunction .Increasing number of data indicate that NAFLD is associated with cardiovascular risk , 21. In The results of clinical, experimental, and genetic data indicate that PCSK9 might be influenced by several metabolic and lipid risk factors, including atherogenic lipoproteins, obesity, and insulin resistance , 14.In this study, we aimed to determine factors associated with circulating PCSK9 in a group of patients with DM2.In this observational, retrospective cross-sectional study, we enrolled consecutive patients with DM2, treated in outpatient diabetes clinic. Material included 116 consecutive patients, referred to diabetes outpatient clinic because of poor glycemic control. Diagnosis of diabetes was made based on EASD criteria. Patients with acute or chronic infections or devastating diseases were excluded. In each patient, a standardized questionnaire, including data on past history, treatment, presence of diabetes complication, diabetes duration, family history, and habits, was evaluated. In all patients, physical examination and anthropometric measurements were performed, using standardized methods.LOUD-CLONE CORP., Houston, USA; Human LDLr ELISA kit SK00418-01, AVISCERA BIOSCIENCE Inc., Santa Clara, CA, USA) and PTX3 by sandwich ELISA using a commercial reagent kit following the manufacturer's instruction .Serum fasting lipid concentrations were determined by enzymatic methods  and HbA1c by HPLC . Human proprotein convertase subtilisin/kexin type 9 (PCSK9) and soluble low-density lipoprotein receptor (sLDLr) were determined by quantitative sandwich ELISA using a commercial reagent kit following the manufacturer's instruction  according to the manufacturer's instructions. Fatty liver index (FLI) was calculated according to a formula using BMI, triglyceride concentration, GGTP, and waist circumference [apo A1 was determined by immunoturbidimetry  and apo C3, apo B100, and apo B48 by sandwich ELISA kits  of studied variables between patients with and without NAFLD and treated or not treated with statins. For variables, normally distributed analysis of variance ANOVA and Tukey's test were used to determine the differences between means in PCSK9 tertiles. For other variables, the Kruskal-Wallis test and test for multiple comparisons were used for comparison of distribution between PCSK9 tertiles. Regression analysis with PCSK9 as dependent variable, adjusted for age and sex, has been calculated in the whole examined group. The independent variables in regression analysis were body mass index (BMI), waist/hip ratio (WHR), systolic (SBP) and diastolic (DBP) blood pressure, serum concentration of total cholesterol (TC), triglycerides (TG), HDL-C, LDL-C, apolipoprotein B100, A1, C3, B48, sLDLr, glucose, creatinine, HbA1c, activity of alanine and asparagine aminotransferases , and gamma-glutamyltranspeptidase (GGTP) for each variable separately. Multiple stepwise backward regression analysis was performed to determine the associations between examined variables and PCSK9, calculated in the whole examined group and in the subgroups divided according to the presence of NAFLD and statin treatment.Statistical analysis included calculations of means (sd) or medians (interquartile range IR), when a variable was not normally distributed, and calculations of Spearman's  or Pearson's correlation coefficients. Student's Written informed consent was obtained from each patient included in the study. The study protocol conforms to the local ethical guidelines and has been approved by the Jagiellonian University Collegium Medicum Ethics Committee on research on humans.2. Mean (sd) values of serum LDL-cholesterol were 2.60 (1.16) mmol/l and of HDL-cholesterol 1.14 (0.30) mmol/l. Median (IR) of triglyceride concentration was 1.8(1.0) mmol/l. The more detailed biochemical characteristics of the patients were presented earlier [We examined 116 patients with DM2, among them 57.8% were men. Mean (sd) age of patients was 59.1 (11.07) years, median (IR) duration of diabetes was 9  years, m earlier .100 patients (86.2%) had arterial hypertension, 79 (68.1%) hyperlipidemia, 79 (68.1%) NAFLD, and 43 (37.3%) coronary artery disease (CAD). 67 patients (59.8%) were treated with statins. Low physical activity was reported by 66 (56.9%) of patients, and 18 patients (15.5%) reported active smoking habit.p < 0.05). The mean PCSK9 concentrations did not differ between patients with and without NAFLD, smokers vs. nonsmokers, on statin therapy vs. nonstatin takers, nor with or without CAD.p < 0.001), while concentrations of LDL-C, apo B100, apo B48, apo C3, and PTX3 increased between tertiles 1-3 and 2-3, and apo A1 between the lowest and 2nd and 3rd tertile. Triglyceride and apo C3 concentrations, as well as FLI values, were highest in the third PCSK9 tertile. sLDLr concentration decreased from the lowest to the highest PCSK9 concentration tertiles (p < 0.001).r = −0.21, p < 0.05) and HbA1c  and positively with BMI , total cholesterol (TC) , LDL-cholesterol , and triglyceride (TG) . Negative correlation of PCSK9 with sLDLr concentration  was noted. We also found significant correlation between PCSK9 and apolipoproteins: apo B100 , apo B48 , apo A1 , and apo C3  concentrations. Interestingly, strong positive correlation between PTX3 and PCSK9 was observed . We did not find any associations between PCSK9 and CK-18 fragments, nor liver enzymes, glucose, creatinine, and WHR; however, positive correlation between FLI, marker of liver steatosis, and PCSK9 was observed   .r = 0.53, p < 0.001) and apo B48  and negatively with serum creatinine . In patients not taking statins, negative correlation between HbA1c and PCSK9 was found   .r = 0.44, p < 0.001) and apo B48  was observed only in NAFLD patients and between PCSK9 and BMI  was present only in a group without NAFLD (In the NAFLD as well as non-NAFLD, patient associations between PCSK9 and total cholesterol, triglycerides, cholesterol LDL-C, sLDLr, apo B100, apo A1, PTX3, and FLI were found. Significant association between PCSK9 and apo C3 (ut NAFLD .Regression and partial correlation analysis between PCSK9 and examined variables, after adjustment for age and sex, revealed that total and LDL-cholesterol, triglycerides, PTX3, apo B100, apo B48, apo C3, apo A1, and DBP were significantly positively associated with PCSK9 serum concentration . sLDLr cIn the whole group of patients, multiple stepwise backward regression analysis with PCSK9 as dependent variable and age, apo B100, apo B48, apo A1, apo C3, PTX3, BMI, HbA1c, NAFLD, and statin treatment as independent variables revealed that PTX3, apo B100, apo A1, apo B48, and BMI were positively and presence of NAFLD and HbA1c negatively associated with PCSK9 concentrations . These vThe multiple stepwise backward regression analysis was performed also in groups of patients treated and not treated with statins. In the group of patients treated with statins, the crude model consisted of PCSK9 as dependent variable and age, apo C3, apo B48, apo B100, apo A1, and PTX3 as independent variables. It was shown that PTX3, apo A1, and apo B100 were significantly associated with PCSK9 . These vIn the group of patients with NAFLD, multiple stepwise backward regression analysis with PCSK9 as dependent variable and age, apo C3, PTX3, apo B48, apo B100, and apo A1 as independent variables revealed that apo A1, apo C3, apo B100, and PTX3 were significantly associated with PCSK9 . These vIncreasing number of data indicate that high levels of PCSK9 are associated with proatherosclerotic processes; therefore, the factors associated with PCSK9 concentrations are the subjects of intensive research –4. In ouWe confirmed observed by others positive associations between PCSK9 and total cholesterol, LDL-cholesterol, and triglyceride , 24–26, Correlations between apolipoprotein B100, the main apolipoprotein of LDL, and other liver-derived proatherogenic lipoproteins were also observed in patients with stable angina, in patients with high cardiovascular risk and in population of young patients , 28, 29.Apolipoproteins B48 and C3, markers of triglyceride-rich lipoproteins, are involved in atherosclerotic/inflammatory processes and increase vascular complications –34. apo In type 2 DM patients, we observed significant positive association between PCSK9 and apo A1, the main apolipoprotein of HDL lipoproteins. Positive correlations between PCSK9 and HDL-C and apo A1 were observed in adult populations; however, we did not observe in opposite to others any associations between HDL-C and PCSK9 , 28, 29.In this study, PCSK9 correlated significantly with PTX3 in the total group as well as in all subgroups of patients. Recent observations suggest that PCSK9 is involved in inflammation processes with implications in atherosclerosis and its major consequence—myocardial ischemia , 35, 36.Statins have been found to upregulate PCSK9 expression and thereby reducing their own lipid-lowering effects . In our In our patients, sLDLr concentration correlated strongly negatively with PCSK9 levels, in opposite to the observation of Mayne et al. who observed in a white Canadian cohort significant positive association between sLDLr and PCSK9 . On the There are some data that PCSK9 is involved in NAFLD development , 17. We The recent literature underlines that PCSK9 plays an important role in glucose homeostasis, influencing the survival and normal function of pancreatic islet beta cells . Our obsPCSK9 levels were positively associated with BMI in the whole group of our patients, similarly like in other studies , but notIn the literature data, serum PCSK9 concentration correlated positively with age in patients suspected for NAFLD and in adolescents , 29. In The limitation of this study is relatively small number of patients. We did not perform other inflammation markers; we plan to perform them in future studies. The strength of the study is a representative group of DM2 patients, who are at high risk of vascular disease and NAFLD and use of apolipoprotein, which are according to some data better than lipoprotein risk markers associated with atherosclerosis.The results of our study indicate that there are strong positive associations between circulating PCSK9 and atherogenic apolipoproteins B100, C3, and B48, as well as unexpectedly antiatherogenic apo A1 serum concentrations. These data might be of value in understanding the pathophysiology of lipid metabolism according to statin treatment and presence of NAFLD in patients with diabetes and could be of value in modulation of CVD risk in DM2 patients. Association of PCSK9 with PTX3, an inflammatory marker, might suggest the important role of PCSK9 in inflammatory atherosclerotic processes.