Background: Changes in both circulating cytokines and neurochemical concentrations have been observed in aging. Patterns of change across these factors are associated with age-related pathologies, including neurodegenerative disease. More evidence to define patterns of change that are characteristic of healthy aging is needed, as is an investigation into how age-related changes in blood cytokines and brain neurochemicals may relate to one another in a healthy older adult population.Methods: Single voxel 1H-proton magnetic resonance spectroscopy was collected in medial frontal and medial parietal regions. Phosphocholine and glycerophosphocholine (Cho), myo-inositol (MI), N-acetylaspertate and N-acetylasperglutamate (NAA), creatine and phosphocreatine (Cr), and glutamate and glutamine (Glx) were measured in a sample of 83 healthy, cognitively normal adults aged 52–89. Blood data were collected to quantify 12 cytokines: interleukins (IL-) 2, 5, 6, 7, 8, 10, 12, 13, IL-1 β, tumor necrosis factor α (TNF-α), interferon γ (IFN-γ), and IL-17 α. Correlation analyses were performed to assess age relationships between each of these factors. Backward linear regressions were performed. Cytokine data and age were used as predictors of each cerebrospinal fluid (CSF)-corrected metabolite concentration in both voxels.Results: Associations were identified between a variety of cytokines and concentrations of frontal NAA, Cr, and Glx, and of parietal MI, Cho, NAA, and Cr. In the frontal voxel, NAA was predicted by more IL-1B and less TNF-α, Cr by less TNF-α and more IL-5, and Glx by less TNF-α. In the parietal voxel, MI was predicted by more IL-10 and IL-8 and less IL-2, Cho by more TNF-α and less IL-2, NAA by more IL-1B and TNF-α and less IL-13, IL-2, and IL-7, and Cr by more IL-10 and less IL-2.Conclusions: Associations were identified between circulating cytokines and neurometabolite concentrations in this sample of older adults. The present results serve as the initial evidence of relationships between circulating cytokines and neurophysiology. Findings invite further investigation to understand the physiological consequences of aging, and how peripheral inflammatory markers may relate to neurochemical concentrations in healthy aging. There is well-documented evidence of age-related changes in a variety of physiological processes that impact health and functional outcomes. Several biological theories of aging may contextualize these observations. The free radical theory of aging Harman, , for exaCytokines are immune signaling molecules that can be measured through blood. Cytokines modulate inflammatory responses, and circulating cytokines are useful inflammatory markers for study. Specific cytokines have more often been investigated in the context of aging, in particular interleukin 6 , and decreased markers of brain health or function . The opposite is hypothesized for the greater presence of anti-inflammatory cytokines.The present study explores age relationships with circulating cytokines and cerebral metabolites to identify whether previously observed age relationships exist in this sample. Greater pro-inflammatory cytokines  and cerebral metabolites measured by Eighty-three older adults  were recruited from the local community as volunteers for a study on healthy aging . Participants were cognitively assessed as part of the study procedure; summary statistics for these tests and participant demographics are described in 1H spectra PRESS MRS were collected in two voxels, in the anterior and posterior regions (for placement see 3 isotropic voxels) was acquired for MRS voxel placement and segmentation. GannetCoRegister software . The PRESS sequence had a repetition time of 2 s and an echo time of 68 ms. Acquisition variables included: 320 transients with editing ON-OFF scans alternating every two transients, a 16-step phase cycle with steps repeated for on and off, 2048 data points acquired at a spectral width of 2 kHz, and variable pulse power and optimized relaxation delays (VAPOR) water suppression at a width of 80 Hz. No fat suppression was used. Editing-OFF spectra were acquired for use in the present dataset, and research has been done to validate this method , Glx (glutamate plus glutamine), and Cho (glycerophosphocholine plus phosphocholine). Water scaling and eddy-current correction settings were set to true; other LCModel settings were kept as default . The MRS metabolites measured, along with their proposed roles, are described in Scanning was performed on a 3T Philips Achieva scanner , using a 32-channel head coil. Single-voxel Once blood samples were acquired, plasma from each sample was separated, aliquoted, and immediately stored at −80°C. Aliquots were used to measure cytokine levels using an xMAP multiplexed bead array. Using the Luminex-100 system , multiple cytokines were simultaneously quantified by capturing them onto antibody-coated, spectrally distinct fluorescent microspheres and measuring fluorescence intensity. In some cases, cytokine measurements fell below a detectable value, and in those cases, the threshold for detectability was used. The cytokines measured in picograms permilliliter (pg/ml), along with their proposed functions, are described in IBM SPSS Statistics version 25  was used to perform all analyses. Absolute values of metabolites were corrected for cerebrospinal fluid (CSF) by computing [Metabolite/(1-CSF)] for each. This was performed to address the potential contribution of individual differences in tissue fraction within voxels to metabolite quantities. Tissue correction is particularly relevant in an older adult population where age-related cortical atrophy may drive differences in uncorrected metabolite values. Normality was assessed, and cytokine data were log-transformed to assess skewness. All variables included in the models were standardized by z-scoring to facilitate interpretation of the results. Simple bivariate Pearson correlations were performed to explore the relationships between age and each cytokine and cerebral metabolite in the current sample. Cytokine concentrations and age were entered as predictors of MRS concentrations in the anterior and posterior voxels in backward stepwise regression models. Independent analyses were conducted for each MRS concentration in both voxels. MRS metabolites were entered into each model as the dependent variables; independent variables across all models included circulating cytokines, sex, and age.p-values for correlations presented here were not corrected for multiple comparisons, and when corrected no significant age associations survived.These values represent the original data prior to the statistical transformations described above. Findings in Present findings contribute to the body of literature describing age-associated changes across both cytokines and cerebral metabolites. The broad hypothesis that associations exist between cytokines and cerebral metabolites was supported by the evidence presented. However, data from this sample failed to provide consistent evidence in support of hypotheses about directionality. Also of interest are the unexpected parallel effects of cytokines across different and in some cases functionally oppositional, cerebral metabolites concentrations. The differences observed in effects across frontal and parietal metabolites are noteworthy as well. Changes in both peripheral inflammation and neurophysiology have been observed both in pathological , and healthy aging  that inflammatory changes occur in healthy aging and drive the development of age-associated morbidities, including pathological brain changes; (b) that peripheral and cerebral inflammatory processes may be parallel such that greater pro-inflammatory cytokines circulating peripherally would correspond with greater metabolic indications of cerebral neuroinflammatory processes; and (c) that greater anti-inflammatory cytokines circulating may relate to some compensatory processes that would correspond with greater metabolic indications of brain health. Evidence has supported that increases in circulating pro-inflammatory cytokines are related to worse outcomes for cardiovascular health and neurodegenerative disease  may be important for characterizing brain changes occurring in non-pathological aging.The present study had several limitations worth considering which may help guide future research endeavors. First, the study design was cross-sectional, preventing any causal interpretation of the results. It is possible to speculate about the differences between previous studies’ methodologies or samples in the context of inconsistent evidence across these markers; however, longitudinal data would be useful in determining both how cytokines and MRS-measured cerebral metabolites may change and relate to one another in healthy aging. One methodological consideration is the use of absolute values rather than ratios. The present study assessed only absolute values; however, the use of ratios in future research may add valuable information about the nature of neurochemical alterations in aging.Another limitation may have been the use of absolute values of individual cytokines. An important direction for future work, considering the sometimes counterintuitive effects of cytokines found here, is to investigate the presence of non-linear relationships. Modeling relative rather than absolute concentrations of cytokines also may be helpful in fully understanding their collective impact, as many are pleiotropic and regulate the production or proliferation of other cytokines. Further, future incorporation of other related factors would be helpful in understanding the complex relationships between cytokines and cerebral metabolites in older adults. Some factors of interest may be objective cardiovascular and general medical health measures to provide context for inflammatory markers, as well as diffusion tensor measures of white matter integrity and quantification of central nervous system cytokines in CSF samples. It would be valuable to compare levels of peripheral and central circulating cytokines, and to assess whether central circulating cytokines have more direct relationships to cerebral metabolites. Prior research has identified relationships between CSF chemokines and MRS-measured metabolites in an HIV population . The findings in this sample also provide a basis for allocation of research exploring a broader collection of cytokines in healthy aging, as there are implications of a greater number of inflammatory markers for brain health in this population. Further exploration of this topic is relevant to building more well-informed risk profiles for cognitive decline and dementia, if patterns of circulating cytokines may be useful in predicting neurochemical changes characteristic of preclinical neurodegenerative disease and pathological aging.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by University of Florida Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.Each author made substantial contributions to the formation of the project, and the execution of the manuscript. 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.


This research aimed to determine the effects of body weight, the body condition score(BCS), body measurements, birth type (single offspring or twin birth), birth weight and sex on the gestational length in sheep( Karayakasheep are a breed with a low milk and offspring yield, a fine tail and a coarse mixed fleece. They are known tohave good-quality meat. In Karayaka sheep, the number of lambs at birth is 1.05 . For this reason, it is beneficial for breeders to increase the fertility rate in this breed via various breedingmethods . The period spend in the uterushas an important effect on the development of the offspring. In Karayaka sheep, the pregnancy rate,return rate, twinning rate and triplet rate of females from twin births mated with rams from twin births were determinedto be 100, 16.22, 52.25 and 3.60 respectively. Thus, the selective breeding of twin animals in Karayaka sheep may increase therate of twinning .The body condition score (BCS) can vary depending on the environment and husbandry conditions. The gestational length in sheep is influenced by the interaction of maternalage, offspring sex, genotype, nutrition, environmental temperature, year, lambing season,location of the enterprise and geographical location. It is stated that approximately two-thirds ofthe variation seen among sheep breeds in terms of gestational length is caused by the genotype ofthe fetus . The average gestational length of Kari sheep inPakistan has been determined to be The BCS is a method that can be easily applied by the breeder and gives information on the nutritional status of theanimals. At the same time, it is a system based on the grading of the differences that can beobserved in the fattening of the organism with the help of identifiable physical properties. Sheepin good condition during the mating period show a higher value in terms of reproductiveefficiency. Determining the BCS of the herd and bringing it to an optimum level during themating period in sheep increases the number of lambs. The body weight and BCS at the time of mating havepositive effects on some reproductive traits, such as ovulation rate and the number of lambs born . At thesame time, the mother's bodily reserves have an effect on the lamb's feeding and on the mother's mammary glands afterbirth . In Karya-type sheep, the average respective BCS and body weight values during themating period were found to be In this study, the effects of age, BCS, body weight and body measurements, birth weight, sex,and birth type on the gestational length were examined. It is thought that the results obtained will contribute to expanding upon existing knowledge in the literature.2This study was carried out in accordance with the ethical principles and rules of decision number 1 of theOrdu University Animal Experiments Local Ethics Committee dated 27 January 2016.2.1The research was carried out on Karayaka sheep raised on a private farm in Bolaman Gölbaşı village in the Fatsa district of Ordu. Animals were selected for the study, their ear tagnumbers and ages were recorded, and their reproductive yield records were evaluated.The results obtained during the mating period are given in Table 2. The effect of age on the BCS insheep was found to be significant . Gestational length wasdetermined as 151.11 d for male offspring, 150.81 d for female offspring . However, the findings of this study did not concur with Cam et al. (2018), who reported that the length of gestation was positively affectedby the live weight and BCS of sheep. This divergence may be due to individual differences stemming from the farm, region and animals used in the studies.In the province of Tokat, the rate of infertility, the birth rate, the twinning rate, the abortion rate and the numberof lambs of per ram and per ewe in Karayaka sheep were 22.7 %, 73.0 %, 3.3 %,2.1 %, 0.75 and 1.03 respectively . These values divergefrom the reports by Belgüzar (2011) and Cam et al. (2017), which may be due toregional differences and breeding practices.The findings in this study that the gestational length in sheep was not affected by age, BCS,body measurements, sex, birth weight or birth type .Differences between the literature and the results of this study may be due to genotype, management,animal nutrition, breeders' practices and regional differences.5It was determined that the length of pregnancy did not change according to physicalcharacteristics. However, it was observed that the gestational lengths of male and single offspring births werelonger. The lowest gestational length was seen in animals with a BCS


Solanum tuberosum). The in silico model was also performed with the tumor electroporation model, which is more conductive than the vegetal model. We do not recommend using deflected electrodes. We have found that a deflection of ± 2 mm is unsafe for treatment. Inward deflection can cause dangerous electrical current levels when treating a tumor and cannot be described with the in silico vegetal model. Outward deflection can cause blind spots in the electric field.Electrochemotherapy is a selective electrical-based cancer treatment. A thriving treatment depends on the local electric field generated by pairs of electrodes. Electrode damage as deflection can directly affect this treatment pillar, the distribution of the electric field. Mechanical deformations such as tip misshaping and needle deflection are reported with needle electrode reusing in veterinary electrochemotherapy. We performed in vitro and in silico experiments to evaluate potential problems with ESOPE type II electrode deflection and potential treatment pitfalls. We also investigated the extent to which the electric currents of the electroporation model can describe deflection failure by comparing in vitro with the in silico model of potato tuber ( EP is a physical phenomenon of cell permeability-increasing by exposition to high-intensity pulsed electric fields2. Studies from science, technology, engineering, and mathematics play a key role in EP-based treatment safety improvement. Investigations include analysis of electric field diffraction due to adjacent tumor structures, such as vascular4, eyelid-periocular skin5, and auxiliary methods to maximize the tumor treatment7. The pulsed electric fields are generated by dedicated hardware and delivered to the tissue using arrangements of plate or needle electrodes. The European Standard Operating Procedures on Electrochemotherapy (ESOPE)—first released in 20068 and updated in 20189—standardizes four main types of electrodes for ECT procedures.Electrochemotherapy (ECT) is an anti-cancer procedure that uses electroporation (EP) to improve traditional chemotherapeutic treatments11. Solid tumors increase the collagen fiber density in the extracellular matrix and adjust tissue stiffness12. Reuse of electrode produces mechanical deformations due to mechanical stress, especially during the needle electrode penetration on tumor tissue12, as illustrated in Fig. 16. Needle tip misshaping introduces immediate perforation risk to the safety of procedures. Needle deflection is a greater concern when analyzed in an electrical scope as field distribution will differ from the engineering design.Although ESOPE does not report instructions about electrode refurbishment, it is commonly practiced in veterinary procedures reusing the electrode after autoclave sterilization or during multiple perforations in the same treatment17. To guarantee that the tissue is electroporated, it must receive a minimum electric field intensity called reversible EP threshold (ERE)18. The electric field is a physical quantity related to the applied potential and the distance between the charges, in the case of ECT electrodes, to the oppositely charged needles. Under needle deflection, the entire electric field distribution on the tissue may change and compromise treatment safety and success. Inward deflection  can introduce higher electric fields, higher electric current, and more undesired ablation in greater tissue extension by irreversible EP (IRE). IRE occurs when the electric field intensity surpasses the irreversible EP threshold (EIRE), directly killing the cell by osmotic imbalance or homeostasis loss19. Outward deflection (when opposite charge needles become farther than predicted) could reduce the treatment effectiveness by lowering the electroporated volume, inducing non-electroporated regions, and increasing tumor recurrence risks9. Because the effects of EP in animal tumor tissue are not directly observable, their outcome could take days or weeks 21, it is common to study this effects using both in vitro experiments with vegetal tissue and in silico computational models.The success of ECT procedure depends on the electric field distribution on the tissue23. The ethics committee on the use of animals does not recommend in vivo animal testing for some scenarios involving suffering and pain, particularly for failure testing and effects analysis24. Tissue-mimicking phantoms such as potato tubers (Solanum tuberosum) are alternatives under the 3Rs  practice24. Also, computational models can calculate field distribution, optimal electrode positioning and geometries, and other relevant treatment parameters such as maximum demanded electric current, an equipment design requirement. A study may require that a comprehensive simulation of electrode positioning and geometries be performed to cover a possible ECT scenario. Nevertheless, computational modeling requires attention because each tissue may have a specific nonlinear electrical conductivity σ(E) model, where E is the local electric field25. The EP provokes an increase in the permeability of cells, which changes the electrical conductivity of the tissues27.Vegetal models are used to assess EP volume and equipment testing as they show visual feedback through enzymatic browning when electroporatedIn this work, we performed in vitro and in silico experiments to investigate the interference of needle deflection in the electric field distribution and the risky limits of mechanical deformation for treatment success. By comparing the in vitro and in silico results, we evaluated whether the computational model can describe needle deflection cases. Additionally, we used an in silico tumor case model to demonstrate the effects that electrode deflections could impose in clinical scenarios. The simulation-based design may support recommendations on mechanical deformation tolerances. We evaluate the electric field distribution and maximum electric current, which are technical parameters or requirements used in ECT equipment design.in silico model may not describe the in vivo experiment.We observed ECT unsafe parameters during needle deflections. When performing +2 mm outward deflections, we show a potentially dangerous electric field indentation. When performing −3 mm inward deflections, we show a device-damaging electric current. Moreover, the findings indicate that the Solanum tuberosum tissue. Figure 30. The darkly stained region on in vitro results represents electroporated tissue. We observed stains starting from approximately 40 kV/m. The electric field indentation was observed in vitro and in silico . We observed that when using the 4-pair electrode, the total electrical currents are 30.6 A, 25.0 A, 21.5 A and 18.7 A for −3 mm, 2 mm, 1 mm, and 0 mm deflection, respectively . Figure 32. Exceeding the equipment limits may cause the hardware shutdown or damage, provoking a treatment failure.The electric field distribution see Fig.  shows th.22 is adequate for an electrode deflection of ± 1 mm (4-pair electrode) and ± 2 mm (3-pair electrode). Solanum tuberosum has already been validated as in vitro electroporation electric fields model-dependent for plates and needle electrodes29. However, when the deflection overpasses a certain level, the in silico experiments do not describe the in vitro experiments. The mismatch could be explained by the fact that the model was built to best fit the ESOPE recommendations (40 to 120 kV/m). However, severe deflections can induce a wide range of electric field amplitude into the tissue. In the inward deflection, especially for the 4-pair electrode variant : inward (Δx < 0) and outward (Δx > 0) deflection. In those cases, the anode needles were deflected, whereas the cathode ones were kept fixed, as shown in Fig. We performed both in silico and in vitro experiments to evaluate the electric field distribution and electric current from different arrangements of ESOPE Type II needle electrodeElectrode dimensions follow the ESOPE standards with 3 and 4 parallel pairs of needles. Each pair consists of two needles with 1 mm diameter, 20 mm height, and separated by 4 mm on inner edges in its standard configuration . In both 3 and 4 pairs variations, the inner edges of identically charged needles are 3 mm apart. All needles were fully inserted into the biological tissue (20 mm insertion). EP protocol also follows ESOPE recommendations with eight square-wave electric pulses of 400 V (100 kV/m electric field is expected as needles inner edges are at a 4 mm separation gap), 100 µs of pulse duration and 1 Hz of repetition rate. Because the current EP equipment do not adjust the voltage, we do not realize any voltage compensation due to electrode deflection.22 to describe potato tissue conductivity changes through EP.The computational model was performed by the finite element method (FEM) software COMSOL Multiphysics v.5.1 . The mathematical model that describes the electric field distribution in biological systems can be modelled by the Laplace equation in the steady-state regime, as shown in Eq.  were performed for each standard electrode and deflections cases. Calculations were run on a cluster server  with Ubuntu Linux  operating system.39. Therefore, we modeled the geometry of a subcutaneous tumor neighboring the epidermis and dermis tissues to mimic such an environment. Equation  were purchased from local stores. The producer  is certified by the Brazilian Ministry of Agriculture, Livestock and Food Supply (MAPA) audit for organically grown products. The Organic Conformity Assessment Organization accredited by MAPA provides the traceability of the product at https://conecta.paripassu.com.br/ (traceability code PPGHO390M0FIHIHB). This study complies with relevant institutional, national, and international guidelines and legislation on experimental research and field studies on plants or plant material.Potato tubers  per number of pairs (3 and 4) were performed with ten samples per group. We developed an external apparatus to guarantee electrode insertion with respective deflections. The apparatus can be seen in Supplementary Fig. 41, thus, minimizing misalignment effects when inserted in potato tissue. Along with the electrodes, we designed external spacers. Spacers are 20 mm in height and consist of two fiberglass boards with 1.1 mm diameter orifices perforated on each board.The vegetables were not peeled and received a minimum number of cuts, followed by drying the electroporated surfaces before and after the pulse application to minimize artefacts unrelated to the EP processThe electrode parts were placed in the tissue through orifices of spacers. For all bottom boards, the orifices distances mimicked ESOPE type II electrodes, as in real scenarios, the configuration is maintained at the base of the electrode. To ensure the desired Δx, we shifted the perforation places of anode orifices at each upper board spacer (1 mm step), as shown in Supplementary Fig. To deliver a consistent analysis of the in vitro distribution of electric fields, we also performed Δx measurements on another set of samples (nonelectroporated ones), eight for each Δx configuration. After insertion into tubers, we cut slices precisely at each needle pair insertion and measured each Δx using an analogic caliper Mitutoyo 530–312 . Finally, we calculated each average and 95% confidence interval  and electric current probe Tektronix A622 . We registered the average electric current of the last pulse protocol. The average current was measured in the top of the square wave and disregarding the firsts and lasts 10 µs from the pulse edges to ensure that there are no transient effects.., the percentage difference between the means. We note that comparing multiple groups is not appropriate  because they are electrically different situations that should not be compared.In our data set, we need to know how the in silico electrical current samples compare to our population (the corresponding in vitro results). We have 16 situations to test. For each situation, there is an in vitro group and an in silico sample. They are based on the Δx  and the number of electrode pairs (3 and 4). We first tested whether the in vitro groups were approximately normally distributed using Shapiro–Wilk normality test. The data must be normally distributed to test means. We found that all in vitro groups were approximately normally distributed. No data transformation was performed to adjust for normality. We used a one-sample t-test to test the probability that the in vitro mean was equal to the in silico value. The t-test was chosen because of the small sample size (10 samples). Variance in the in vitro electric current data is expected due to the heterogeneity of the tissue. We found no tolerable error consent in the ECT area. We included a 95% confidence interval (CI = 0.95) and a 5% significance level (α = 0.05) in all statistical calculations. In silico and in vitro were also compared from another perspective, namely the relative error, i.eWe also applied the Shapiro–Wilk normality test to the in vitro measurements of Δx uncertainty. Again, all groups presented normal distribution, and the results were expressed by averages. All data analyses were performed using the RStudio statistical software package .Supplementary Information.


To examine which elements of an obstetric anal sphincter injury (OASI) care bundle were protective for OASI. Several interventional trials showed that application of a care bundle involving a hands-on approach to perineal protection may reduce the risk of OASI. Previously, we found that only the element “hand on the fetal head” in itself was protective, although the risk of a type 2 error was calculated to be 50%.A prospective follow-up study in an obstetric department in Denmark with 3200 deliveries per year. We included a cohort of 10,383 women giving birth vaginally from gestational week 22 + 0 from 2016 through 2019. We documented on a person-level the five elements of the care bundle together with maternal and obstetrical characteristics. The elements were 1) communication, 2) visible perineum, 3) hand on fetal head, 4) perineal support and 5) certification. Regression analysis was used for analysis of associations. The primary outcome measure was OASI.The total rate of OASI in vaginally delivering women was 1.9%. The incidence was 3.2% in nulliparous women giving birth vaginally. The rate of cesarean section was 16.5% and for episiotomy 2.4%. The reduction in the incidence of OASI was sustained since 2013. Hand on the fetal head and perineal support both were protective factors for OASI. In case of a nulliparous woman with a neonate weighing 3500 g giving birth spontaneously, the relative risk (RR) for OASI was 0.50 (95% CI 0.49- 0.51) with use of hand on the fetal head together with perineal support against no use. Similarly, with a nulliparous woman giving birth to a neonate of 3500 g by vacuum extraction, the RR for OASI was 0.65 (95% CI 0.62-0.68) against no use.Both hand on the fetal head and perineal support were associated with a reduced risk of OASI. Obstetric anal sphincter injury (OASI) is a serious complication of vaginal delivery. The severity of OASI is documented , 2 and cSeveral interventional trials show that application of a care bundle involving a hands-on approach to perineal protection may reduce the risk of OASI –11. FurtPreviously, we showed that only the element of having a hand on the fetal head resulted in a statistically and clinically significant reduction in OASI , albeit We aimed at analyzing the individual elements of an OASI care bundle including episiotomy and several known risk factors in a large cohort. Even though episiotomy may be viewed as a grade 2 laceration in itself, there is the potential that its use may reduce the risk of OASI . MoreoveThe present follow-up study arose from a quality improvement project that began in June 2013 at the delivery ward in Herning Hospital, Denmark in which we successfully reduced the incidence of OASI from 7.0 to 3.4% in vaginally delivering nulliparous women . In thisAll women were included who gave birth vaginally in our department or at home with support by a midwife from our department from gestational age 22 + 0 in 2016 through 2019.In our ward we have approximately 3200 deliveries per year from gestational week 28 + 0. Very preterm births before this gestational age are transferred to a nearby university hospital at Skejby whenever feasible. Ninety full-time midwives and 20 doctors are working in our department. Information is given to the pregnant woman about what may be done to reduce the risk of OASI in consultations with the midwife in gestational week 29. A shared decision-making takes place about giving birth including preferred treatment of pain from contractions, birth position etc. In our department, instructions for midwives and doctors are that the naturally occurring extension of the head should be supported when the head is delivered through the introitus during spontaneous vaginal delivery (SVD) and operative vaginal delivery (OVD). In case of OVD, we use a vacuum extractor and we advocate that OVD is done as a team work with a midwife supporting the perineum while the doctor performs the extraction. Communication is maintained with the woman at all times. When the fetal head is crowning the doctor controls the speed of delivery using one hand on the extractor while the other regulates the necessary force and direction of the pull. Episiotomy is either by medio-lateral or by the lateral method. The latter is recommended in our department.Information from each birth was entered into the electronic maternity records immediately after birth by the attending midwife. This included the use of each of the five elements of the care bundle. There was no registration of the time that each of the elements were applied. Satisfaction with the birth experience was recorded from September 2016 on a visual analogue scale from 1 to 10 where 10 is the highest level of satisfaction. The registration was carried out within the first couple of days after delivery. From December 2017 we started documenting birth position.The primary outcome was OASI, which comprises grade 3 and 4 anal sphincter injury according to the international classification used in our department for several years . The attCompared with the initial project we wanted to reduce the risk of a type-2 error and aimed at halving the variance. This meant that we needed at least four times the size of the population compared with the initial cohort of 1622 nulliparous women. Thus, all women giving birth vaginally from gestational week 22 + 0 from January 2016 through December 2019 were included in the cohort.p < .05 was the level of significance. Values are given as mean ± SD if Gaussian distributed or otherwise stated. We have adhered to the STROBE guideline when reporting this study.Statistics were performed with IBM SPSS Statistics 24. The difference between two means was tested with Student’s t-test if data followed Gaussian distribution; otherwise, Mann-Whitney’s test was used. χ2- test was used for categorical variables. Categorical variables were analyzed separately and by 5 elements present and absent combined. When the continuous variables age, gestational age, birth weight were categorized they were analyzed combined and for trend for linear association. Relative risk (RR) of OASI was calculated from a chosen baseline and stated with 95% confidence intervals (CI). Regression analysis was performed with OASI as dependent variable. Linear regression was used on the continuous variables age and birth weight and logistic regression on parity, the five elements individually , all five elements present , epidural analgesia, oxytocin drip, episiotomy, and vacuum delivery as they are dichotomous. All independent variables were tested individually against OASI and those with significant results and all five elements separately were added to a full model. Separate analyses were performed on all five elements present versus not all five elements present at birth. For multiparous women previous cesarean was added to the full model with all five elements individually. A two-sided p < 0.04). From the total group we received feedback on birth experience from 6528 women; thus, the response rate was 61%. Satisfaction with birth experience was rated between 8 and 10 by 98%. In the group of vaginally delivering women, the rate of Apgar score < 7/5 min was less than 1% and the rate of umbilical cord pH < 7.0 was less than 0.5%. In the group with vaginal birth, 30% did not receive all five elements of the OASI care bundle. For instance, in 11% of deliveries the attending midwife was not previously certified to the care bundle. Likewise, 17% of deliveries took place without perineal support, while communication, visible perineum and hand on the fetal head were used in 90% of vaginal deliveries or more  was 2047 resulting in a total rate of 16.5% with 17.3% in nulliparous women and 15.9% in multiparous (P < 0.0001). Similarly, the subgroup of multiparous women with a previous CS had a rate of OASI of 4.9%, which was 9.8 times higher compared with multiparous women without previous CS . The OASI rate was increased by nulliparity, epidural analgesia, dystocia treated with oxytocin, episiotomy, OVD, and higher birth weight. Birth weight increased the risk of OASI most prominently in nulliparous women. Standing birth position in nulliparous women was complicated by more cases with OASI  and OR = 3.76 (2.7-5.2)). Women with OVD had a 7.2 times higher risk of OASI compared with SVD .Presence of the whole care bundle  was associated with a significant reduction in the OASI rate in the whole group and in nulliparous women. To investigate further the association of the whole care bundle, we compared the event “all five elements” being present and “not all five elements” present with the incidence of OASI. In the group of nulliparous, the incidence of OASI was 5.3% vs. 2.5% in favor of the presence of the whole care bundle. Similarly, in the case of OVD, the OASI was 15.6% compared to 7.7% in favor of all five elements present Table .Table 4IIn the univariate regression analysis nulliparity, higher birth weight, epidural, dystocia treated with oxytocin, episiotomy, and OVD were associated with a higher risk of OASI. In contrast, hand on the fetal head, perineal support and the OASI care bundle with all five elements present were associated with a lower risk Table .Table 5SFor evaluation in the full regression model, all significant variables were entered from the univariate regression analysis and the five individual elements of the care bundle. Thus, dystocia treated with oxytocin and epidural were no longer significant factors. Nulliparity and OVD remained as factors increasing the risk of OASI. Still, hand on the fetal head and perineal support were associated with a reduction of the OASI rate, when adjusted for all other factors except age and birth weight.From the full regression model, we could calculate the potential benefit of using the care bundle. For instance, in a case of a nulliparous woman with a neonate weighing 3500 g giving birth with SVD the RR for OASI is 0.50  with use of hand on the fetal head together with perineal support against no use. Similarly, with a nulliparous woman giving birth to a neonate of 3500 g with OVD the RR is 0.65  against no use.The emphasis on documenting process indicators on a person-level enabled us to gain novel information on the quality of obstetrical care concerning the relation between the individual elements and the outcome. Both hand on the fetal head and perineal support were associated with less OASI as did the 5 elements of the care bundle together. This finding was in alignment with previous interventional programs using similar care bundles . The comThe OASI care bundle has been used in our department since 2013. In the original project, we reduced the OASI rate to 3.4% in nulliparous women delivering vaginally. The incidence was 3.2% in the present period from 2016 through 2019. Thus, corresponding with previous studies on robustness –14 the rRisk factors for suffering OASI in our study as well as in other’s were nulliparity, OVD and higher birth weight , 21. TheThe tendency towards less OASI when a non-certified midwife took care of the delivery  should preferably be described in addition to presenting and analyzing the exposure and the outcomes in an interventional trial. In the present project, the time was not registered where the hands-on elements were in use because of the anticipated short period. This may constitute a weakness of the study. We advocate that the elements should only be applied during the last couple of contractions in order to avoid undue delay of the birth. In a case with suspected asphyxia, an expedite delivery will, of course, have first priority. In the beginning of the project, we noticed that hand on the fetal head and perineal support were applied for a longer period compared with later on in the project. This may still be an issue for untrained staff members. A too long application time of the hands may cause tiring of the hands besides possibly leading to an unwanted delay of the birth. For these reasons, we included this as a learning point for untrained midwives and doctors. A possible strength of these observations is an increased attention on being especially alert when the head is descending through the birth canal in order to avoid a sudden, uncontrolled fast delivery.Another weakness in this study was the potential risk of residual confounding that was not taken into account by the statistical analysis. Causal relationships should be interpreted with caution because of the observational design, although, previously, we argued that the Bradford-Hill criteria support a causal relation between the OASI care bundle and a reduced risk of OASI . The stuWe acknowledge that all the elements of OASI care bundles should be seen as integrated parts of the combined bundles , 18. In In the present study only around 70% of the women received all five elements of the OASI care bundle while 83% received perineal support Table . Some woIn fact, the lack of analyzing per protocol may partly explain that no difference was found in the risk of OASI in the RCTs between the hands-on versus hands-off techniques. Three recent meta-analyses on randomized trials –33 incluAnother issue in the discussion between hands-off versus hands-on is about the definition of the hands-on intervention. In the three larger RCT’s the supposed aim in the hands-on technique was to “increase flexion of the head” , 35 or “The present study confirms that a lesser risk of OASI is associated with use of both hand on the fetal head and perineum. We suggest documenting on a person-level the specific elements of the intervention aimed at reducing the incidence of OASI. Future studies should focus on the question of flexion or extension of the head of the child during the passage of the introitus in order to achieve a further reduction in OASI.


Anganwadi centres (AWC) (meaning ‘courtyard shelter’), the Indian government runs a nationwide intervention providing nutrition supplement to pregnant mothers to improve the health of their children. Using two successive rounds of the nationally representative cross-sectional National Family Health Survey data (collected during 2005–2006 and 2015–2016) of India, we assessed whether nutrition supplements given to pregnant mothers through AWC were associated with select child health indicators – extremely low birth weight (ELBW), very low birth weight (VLBW), low birth weight (LBW) and neonatal mortality (death during day 0–27) stratified by death during day 0–1, day 2–6 and day 7–27. A total of 148 019 children and 205 593 children were eligible for analysing birth weight and neonatal mortality, respectively. OR with 95% CI, estimated from multivariate logistic regression models, suggest that receipt of nutrition supplements was associated with decreased risk of VLBW , LBW , but not ELBW . Women who always received nutrition supplements during their pregnancy saw lower risk of death of their neonates , including death on day 0–1 , day 2–6  and day 7–27 . Therefore, nutritional supplementation to pregnant mothers appears to be helpful in deterring various stages of neonatal mortality, VLBW and LBW, though it might not be effective in mitigating ELBW. Findings were discussed considering possible limitations of the study.With over 1·3 million  In low-and-middle-income countries, inadequate intake of energy, protein, vitamins and minerals to meet maternal and fetal needs is rampant and dietary intake of vegetables, meat, dairy products and fruit is often insufficient to meet these essential requirements–5. India is no exception–10, where an unacceptably high undernutrition among pregnant women is modelled to be a risk factor for poor birth outcomes–13. The Global Burden of Disease estimates highlight an unacceptably high burden of various indicators of undernutrition among women in India. For example: in 2016, Fe-deficiency anaemia accounted for 11 % of all disability among women in India. A high burden of Fe-deficiency anaemia among pregnant mothers has severe consequences on the health of their children,16, and nutrition supplements could be helpful in treating Fe-deficiency anaemia.Benefits of maternal nutrition during pregnancy are well documented. A healthy and balanced nutrition among pregnant mothers leads to healthy birth outcomes and, consequently, better offspring health in later life,6,17,18. In line with the evidence, the Indian government established Anganwadi centres (AWC) (meaning ‘courtyard shelter’) in 1975 as part of the Integrated Child Development Services programme to combat hunger and malnutrition among women and children,20. India currently has over 1·3 million operational AWC managed by Anganwadi workers, and among their various responsibilities, Anganwadi workers encourage pregnant mothers to eat locally available nutritious foods; they provide education and counselling on exclusive breast-feeding practices to mothers and advise them on supplementary food required for the healthy development of their children,21. The need of the Integrated Child Development Services programme was confirmed in the 2013 National Food Security Act (NFSA) where distribution of supplementary nutrition to pregnant and lactating mothers was mandated as part of the right to food, and it was instructed that Anganwadi workers will continue providing daily ‘meal, free of charge, during pregnancy and six months after the child birth, through the local Anganwadi, so as to meet the nutritional standards’ – 2510 kJ of energy content/energy and 18–20 g of protein. Empirical study on the effect of NFSA on reduction of undernutrition in India is non-existent, but some preliminary studies indicate that NFSA is unlikely to greatly affect food consumption, and even if it does, it could only make a small dent to reduce undernutrition. Pregnant women registered with AWC are supposed to receive hot cooked meals and micronutrient-fortified and energy-dense food as take-home ration across the country. NFSA instructs all states and union territories of India to develop a menu of commonly accepted nutritious food which will provide the required nutrition. However, there is significant variation in the take-home ration provided across states, reflecting how take-home ration is frequently not aligned with Integrated Child Development Services guidelines for micronutrient composition, and a systematic lack of accountability at AWC remains a key barrier to programmatic effectiveness.To mitigate the burden of undernutrition among pregnant women, providing nutrition supplements to pregnant mothers is recommended to be a scalable intervention and has positive effects on a range of health outcomesTo our knowledge, no up-to-date studies exist on whether nutritional supplementation provided to pregnant mothers has helped to improve the health of children born to them. Against this knowledge gap, using two successive rounds of the nationally representative cross-sectional National Family Health Survey data (collected during 2005–2006 and 2015–2016) from India, we investigated if nutrition supplement given to pregnant mothers through AWC was associated with select child health indicators – extremely low birth weight (ELBW), very low birth weight (VLBW), low birth weight (LBW) and neonatal mortality (death during day 0–27) stratified by death during day 0–1, day 2–6 and day 7–27. This study’s findings aim to be helpful for guiding national policies on the role of nutrition intervention during pregnancy in improving child health.,26. NFHS2005–2006 (NFHS-3) and NFHS2015–2016 (NFHS-4) are cross-sectional surveys and widely used for programme and policymaking in India. NFHS-3 covered twenty-nine states/union territories, whereas NFHS-4 included thirty-six states/union territories. For NFHS-3, 2001 Census of India and, for NFHS-4, 2011 Census of India sampling frame were used to draw the sample for both rural and urban areas using two-stage stratified random sampling. Villages in rural areas and census enumeration blocks in urban areas served as the primary sampling units or clusters. By virtue of their sampling design, estimates from both rounds of surveys are comparable. Details of sampling procedures are furnished elsewhere,26. Six administrative regions  were not part of NFHS-3, leaving twenty-nine common states/union territories covered in both waves of the NFHS, representing over 99·6 % of India’s population. A household response rate of over 95 % was registered in both NFHS rounds.Two successive rounds of India’s Demographic and Health Survey (collected during 2005–2006 and 2015–2016), commonly known as the National Family Health Survey (NFHS), were usedTo analyse birth weight, 148 019 children were eligible for this study – 14 856 children from NFHS-3 and 133 163 children from NFHS-4. The denominator for analysing neonatal mortality was 205 593, consisting of 33 263 samples from NFHS-3 and 172 330 samples from NFHS-4. NFHS records information on birth weight for children born in the 5 years preceding the survey date, and age at death was recorded for children ever born. However, this study includes the record of birth weight or child mortality only for index children, for two reasons – it would minimise recall errors, and information on primary variable and covariates used in this study was collected only for the index birth.. In both rounds of NFHS, women were asked if the children born to them were weighed at birth, and if the response was affirmative, the weight at birth was recorded in kg. Enumerators were advised to record birth weight from the health card when available; otherwise reporting of birth weight was based on mother’s recall which could have been affected by recall errors, and digit preference which would cause heaping.Two sets of outcome events were investigated. For the first set of events, three child health indicators related to LBW were considered. Following the guideline developed by the WHO, children’s LBW (in kg) was categorised into three groups: LBW with weight of <2·5 kg, VLBW with weight of <1·5 kg and ELBW with weight of <1·0 kg.As a second set of outcome events, neonatal mortality (death during day 0–27) stratified by death during day 0–1, day 2–6 and day 7–27 was analysed. The NFHS asked women about the birth and death history of children ever born to them. In case of death of a child, the age at which the child died was reported. Age at death was recorded in days if the child died within the first month of life, in months if the child died between one month and the second birthday or otherwise in years. In this study, death of children during the first 28 d of life (0–27 d) was defined as neonatal mortality. Neonatal mortality was further investigated by age at death: day 0–1, day 2–6 and day 7–27. This categorisation of age of death is critical as neonatal death varies greatly with daysAnganwadi Centre during this pregnancy?’ If the response was affirmative, a follow-up question was posed: ‘During this pregnancy, were you always able to get the supplementary nutrition from the Anganwadi Centre?’ Responses were coded into ‘yes, always’ and ‘no’. Using this information, the primary variable of interest was computed into three groups about receipt of supplementary nutrition – never received, received but not always and always received. As nutritional supplementation during pregnancy is crucial, one may expect that women who always received supplements will have higher odds of experiencing favourable health outcomes of children born to them, compared with women who only sometimes received  supplements.As a primary variable of interest, this study assessed if women received nutritional supplementation during their last pregnancy. In NFHS, for their last birth in the 5 years preceding the survey date, eligible mothers were asked: ‘Did you receive any supplementary nutrition from the Select covariates representing socio-economic characteristics are current age group (in completed years) of mother , mother’s age at marriage , education of mother , sex of child , birth order , place of residence , social group , religion , economic group  and state of residence (non-high-focus states and high-focus states). In addition, covariates on maternal healthcare service utilisation included number of antenatal care visits (≥4 and <4), institutional delivery (yes and no) and maternal nutrition status represented by BMI of the mother . Variable on sources of birth weight data  was used to analyse LBW, and waves of NFHS (2005–2006 and 2015–2016) were used to capture variation of outcome events by time of survey., Scheduled Castes, Scheduled Tribes and Other Backward Classes are historically socially and economically disadvantaged populations; the ‘Others’ category represents the population that has historically been relatively more privileged. NFHS provides the variable of wealth index in its data set. The wealth index is calculated using household assets and durables. The method of constructing the wealth index representing economic groups is presented elsewhere. Due to both high fertility and high mortality indicators, nine states are regarded as high-focus states in need of special attention; these are Bihar, Chhattisgarh, Jharkhand, Madhya Pradesh, Odisha, Rajasthan, Uttarakhand, Uttar Pradesh and Assam. According to the WHO, a BMI of <18·5 kg/m2 is considered a measure of underweight, 18·5–22·99 kg/m2 as optimum weight and ≥23 kg/m2 is labelled as overweight, including obesity, for the Asian population.Primary education refers to studying ≤8th grade, and secondary education refers to study of 9th–10th grade. For social group, as per the Constitution of India. Adjusting NFHS year for all the regression models helped in accounting for secular time trend. Before executing the multivariate analysis, the variables included in the model were tested for multicollinearity by estimating the variance inflation factor and all variance inflation factors were <5·0, indicating a low probability of multicollinearity.This study used a combination of descriptive and multivariate analyses. To run the analysis, data from both NFHS were pooled to increase the power of the study, totalling 148 019 children to study LBW, and 205 593 children for studying neonatal mortality. Bivariate analysis was run to understand the proportional difference of outcome events – ELBW, VLBW, LBW and neonatal mortality (death at age 0–27 d) – stratified by age at death . Multivariable logistic regression analyses were conducted for all outcome variables coded in binary terms (0 and 1). For each outcome, four regression models were run. Model I included the primary variable of interest – receipt of nutritional supplementation during pregnancy and waves of the NFHS survey; model II included all variables from model I and variables representing socio-economic characteristics; model III included all variables of model II and variables representing maternal healthcare service utilisation (ANC and delivery care); model IV included all variables from model III and maternal BMI. This four-step modelling helped separate the possible role of other variables while understanding the association between receiving nutritional supplementation during pregnancy and select outcome events. For all the outcomes on birth weight, one additional variable representing sources of birth weight data was adjusted as birth weight reporting could differ between health card and maternal recall which leads to heaping. To check the sensitivity of this, an alternate analysis with alternate definition of ELBW of ≤1·0 kg, VLBW ≤ 1·5 kg and LBW of ≤2·5 kg was run (data not shown separately), hoping the magnitude of negative association of nutritional supplementation received during pregnancy would be smaller than the standard definition of respective categories. Appropriate sample weighting available with the NFHS data set was used, and ‘svy’ suite available to adjust sample weighting with the statistical software Stata, version 14, was used. P value (two-tailed) of <0·05 obtained from logistic regression models was included in the Results and Discussion.Recording of birth weight through mother’s recall is likely to have digit preference, often in multiples of 500 gPrior to conducting the NFHS, ethical approval was obtained by the implementing institute, the International Institute for Population Sciences, from an independent ethics review committee constituted by the Ministry of Health and Family Welfare, Government of India. NFHS data sets are available in the public domain with all participant identifiers removed. Thus, no separate ethical approval was required for this study.S1). ELBW was not associated (>0·05) with mothers receiving nutritional supplementation, irrespective of covariates added in the model (model 1 through model IV). Receipt of supplementary nutrition  appears to have helped reduce VLBW and LBW (model II through IV). For neonatal mortality (stratified by day of death), role of nutrition supplement is presented in S2). As shown in model IV, compared with women who never received nutritional supplementation during their pregnancy, women who always received nutrition supplements were less likely to experience neonatal mortality . Women always receiving supplementation were also less likely to experience death of the infant in various day increments: day 0–1 , day 2–6  and day 7–27 , and the unadjusted (model I) association also showed similar results. However, women who only sometimes received nutrition supplements had no association (P ≥ 0·05) with prevention of neonatal mortality on day 2–6 and day 7–27, whereas a protective association was observed for death on day 0–1 and neonatal mortality for women who sometimes received nutrition supplements during pregnancy.Prior to running multivariable logistic regression analysis, variance inflation factor was estimated to check the presence of multicollinearity and all estimates were <5·0 indicating low possibility of multicollinearity (data not shown separately). Whether nutritional supplementation to mothers was associated with ELBW, VLBW and LBW was tested in a logistic regression model, presented with OR in Anganwadi Workers of the AWC under the Integrated Child Development Services programme are given responsibility for distributing nutritional supplementation, primarily in the form of take-home ration, to pregnant mothers. In absence of a nationwide evaluative study, using nationally representative NFHS data, this study assesses whether nutritional supplementation given to women during their last pregnancy was associated with select child health indicators – ELBW, VLBW and LBW, and neonatal mortality as well as day of neonatal mortality .Nutritional supplementation to women during their pregnancy is crucial for health of their children. In India, P ≥ 0·05) with ELBW. The primary causes of ELBW are pre-term birth and intra-uterine growth restriction, and prevention of pre-term birth and intra-uterine growth restriction is multi-factorial where biological pathways and preventive measures for these two conditions are different. But whether the role of nutrition supplements during pregnancy outweighs the biological pathways and preventive measures requires further investigation. On the other hand, multivariable analysis revealed that providing nutritional supplementation  during pregnancy might be helpful in reducing VLBW and LBW. This finding is consistent with interventions confirming the role of supplementary nutrition in mitigating LBW,40, but evidence on the role of supplementary nutrition in VLBW in India is absent, and this study offers some insight into the issue.Multivariate analysis revealed that receipt of nutrition supplements during pregnancy is not associated (P ≥ 0·05) with prevention of mortality on day 2–6 and day 7–27. These estimates indicate the benefits of systematic and intense nutrition supplement intervention in reducing neonatal mortality and unorganised and untimely intervention of nutritional supplementation may not be useful in preventing mortality on day 2–6 and day 7–27. In India, about 57 % of all neonatal deaths occur in the first 3 d after birth and two-thirds of these deaths occur on the first day, that is, within 24 h of birth. Prevention of neonatal mortality would require quality prenatal care including nutrition-sensitive interventions, coupled with prevention strategy of death due to prematurity, malformations and sepsis. While nutrition supplements such as Fe-and-folic-acid, Ca and other supplements to pregnant mothers are crucial for mitigating neonatal death, the prevention of overall death also had to do more with quality of emergency obstetric care of pregnant women in health facilities as well as special care to the new-born unit and postnatal care,43. Well-organised home-based postnatal care could be useful for preventing neonatal death on day 2–6 and day 7–27.In the case of neonatal mortality, women who always received nutrition supplements during their last pregnancy experienced higher odds of survival of their neonates of all ages. However, women who sometimes received nutrition supplement had no association (S1). Fifth, while the association between nutritional supplementation to pregnant mothers and outcome events should be interpreted cautiously as detailed information on nutrition supplements  were not available with NFHS data. Sixth and finally, the response against receipt of nutritional supplementation from AWC is heavily contingent upon the pregnant mother and her family member’s knowledge, awareness and nutrition supplement-seeking behaviours. Thus, reporting about receipt of supplementation could be variable, and a careful interpretation of study findings is needed. Despite these limitations, with a coverage of 99·6 % of India’s children aged 6–59 months and high external validity (no sample selection bias was recorded), findings of this study successfully demonstrated the importance of nutritional supplementation for pregnant mothers in reducing the burden of various stages of LBW and neonatal mortality in India.This study acknowledges certain limitations in light of the findings. First, data on all possible determinants of birth weight and neonatal mortality were not available in the NFHS, thus not included in regression modelling. Second, the most information was self-reported or reported by mothers on behalf of their children, thus reporting might be affected by recall errors and social desirability bias. Third, information on birth weight was based on data from the health card and mother’s recall, which reduced the sample size and may underestimate the association. However, the prevalence of LBW in this study being comparable to the general population offers confidence about generalisability of the study findings. Fourth, supported by the sensitivity analysis (data not shown separately), the multivariable models adjusted for recording of birth weight data indicated that mothers’ recall had higher likelihood to record ELBW, VLBW and LBW (online supplementary Table Abhiyaan. Additionally, the 2017 National Health Policy emphasised the prevention of neonatal mortality by ensuring required nutrition to mothers and children. Health has been a state subject in India, dictated by the constitution of India. Thus, central government must encourage every state and union territory to follow NFSA guidelines and strengthen the performance of AWC while exercising appropriate monitoring and evaluation. Pregnant mothers enrolled with AWC should be encouraged to receive and consume nutritional supplementation as directed. Failure to improve child health through the nutritional supplementation programme designed for pregnant mothers would not only compromise the targets set under POSHAN Abhiyaan and the National Health Policy, but also erode the foundation of health for coming generations.Reduction of LBW at the rate of 2 % per annum has been a prime target of the Prime Minister’s Overarching Scheme for Holistic Nutrition or POSHAN


Birth asphyxia is the major public health problem in the world. It is estimated that around 23% of all newborn deaths are caused by birth asphyxia worldwide. Birth asphyxia is the top three causes of newborn deaths in sub-Saharan Africa and more than one-third of deaths in Ethiopia. Therefore, the aim of this study was to identify determinants of birth asphyxia which can play a crucial role to decrease the death of newborns.p-value less than and equal to 0.05 were used to assess the level of significance.Unmatched case-control study design was implemented among 276 (92 cases and 184 controls) newborns from January 1st to March 30th, 2020. A systematic sampling technique was used to select the study participants. Data were collected by using a semi-structured interviewer-administered questionnaire and document review by trained nurses and midwives who work at the delivery ward of the hospitals. Bivariate logistic regression analysis was done to identify determinants of birth asphyxia. Adjusted odds ratios with 95% confidence intervals and In this study, maternal education of being can’t read & write , ante-partum hemorrhage , prolonged labor , meconium stained amniotic fluid , breech fetal presentation  and preterm birth  were factors which showed significantly associated with birth asphyxia among newborns.In this study, maternal education can’t read & write, antepartum hemorrhage, prolonged labor, stained amniotic fluid, breech fetal presentation, preterm birth were significantly associated with birth asphyxia. So, educating mothers to enhance health-seeking behaviors and close monitoring of the labor and fetus presentation were recommended to reduce birth asphyxia.The online version contains supplementary material available at 10.1186/s12887-022-03223-3. It is also an existence of umbilical cord arterial pH < 7; APGAR score of 0–3 for longer than 5 min; neurological manifestations such as seizures, hypercapnia, metabolic acidosis and hypoxic-ischemic encephalopathy  were 4.7 times higher to develop birth asphyxia compared from mother who have college diploma and above. The odds of developing birth asphyxia among mothers who faced antepartum hemorrhage was 7.7 times higher compared from the counterparts . This study revealed that prolonged labor was the main predictor of birth asphyxia. A mother who had prolonged labor was more than 13 time higher risk compared from normal labor on the outcome of birth asphyxia . Preterm babies were 4.1 times higher risk than term babies to development birth asphyxia   was 4.1 times more likely asphyxiated than the term newborns. This finding was consistent with those of previous studies conducted in Pakistan , Nepal , and Eth, both of the study was conducted in metropolitan city of Ethiopia at which the maternal healthcare services were more advanced than the study area.Newborns with the breech presentation were 4.5 times the odds of developing birth asphyxia than the cephalic presentation. This finding was consistent with previous studies conducted in Uganda , CameronIndia [, Malawi [Prolonged labour was significantly associated with birth asphyxia. This finding was consistent with studies conducted in India , Malawi , Malawi , Cameron, Malawi , Pakista, Malawi , and Eth, Malawi , 26–28. In this study, induced labor doesn’t show an association with birth asphyxia when compared to Spontaneous labor. This result was congruent with the study conducted in West Shewa Zone, central Ethiopia [. But it was inconsistent with the Meta-analysis study conducted in Ethiopia [. This difference may be due to the difference in the study setting at which both this study and the central Ethiopia study were conducted in a single institution. This induction of labor may cause hyper-stimulation of the uterine constriction that could cause fetal heart rate changes. When constrictions are too fast and strong, the placenta, which helps carry oxygen-rich blood to the baby, often cannot recharge with an adequate supply of this blood for the baby [.Ethiopia . But it Ethiopia . This dithe baby .The study also have other limitation that variables in intrapartum events like umbilical cord status, uterine rupture, placental abruption, shoulder dystocia and major maternal haemorrhage, trauma, cardiorespiratory arrest or maternal seizures immediately preceding delivery were not addressed.In this study, we have limitations that should be noted. The study was done in single health facility therefore; it is difficult to generalize for the whole country with this small sample. This study also subjected to recalling bias of mothers when they remembered their previous history. Less than or equals to two ANC follow up by the mothers were 4.6 times higher odds of developing asphyxiated newborns than mothers who have more than four ANC follow up. This finding was inconsistent with the study conducted in Ethiopia , 23, 27.to enhance health seeking behaviors and close monitoring of the labor and fetus presentation were recommended to reduce birth asphyxia.In this study, maternal education can’t read & write, antepartum hemorrhage, prolonged labour, stained amniotic fluid, breech fetal presentation, preterm birth were significantly associated with birth asphyxia. So, educating mothers Additional file 1.


Lipidome-wide metabolites may be useful biomarkers of pregnancy outcomes. We sought to characterize maternal lipidomic signatures associated with preterm birth and neonatal anthropometric parameters. Plasma samples were collected 24–28 weeks gestation, and lipidomic profiling was quantified using high-performance liquid chromatography tandem mass spectrometry. Lipid metabolites were analyzed individually and as whole lipid classes and subgroups based on degree of hydrocarbon chain saturation. Associations were estimated using linear and logistic regression. After false discovery adjustment (q < 0.15), four plasmenyl-phosphatidylethanolamines and three free fatty acids associated with increased risk for spontaneous preterm birth. Five phosphatidylinositols, two phosphatidylglycerols, and one phosphatidic acid were associated with large for gestational age neonates. The saturated plasmenyl-phosphatidylethanolamines held the association with increased risk for spontaneous preterm birth. Both the mono- and poly-unsaturated free fatty acids held the association for increased risk for spontaneous preterm birth. Mono- and poly-unsaturated phosphatidylinositols were associated with large for gestational age neonates. Whole lipid classes (plasmenyl-phophatidylcholines and plasmenyl-phosphatidylethanolamines) were associated with increased risk for large for gestational age at delivery. This study provides evidence that finer omics-scale analysis of the maternal lipidome may be more informative biomarkers of pregnancy outcomes compared to whole class level lipid analysis. Phospholipids on the other hand have polar heads covalently bound to non-polar hydrocarbon tails, making them a core structural component for lipid membranes and associated integral peptides2. Within the sterol class of lipids, cholesterol contributes towards membrane structure maintenance and signal transduction3. Finally, sphingolipids are unique in that they contain an amino group bound to hydrocarbon chains, and some are involved in regulating inflammation and immune responses4. In addition to these differences across lipid classes, there are also functional differences within the same lipid classes. For example, unique polar head groups differentiate phospholipids. Phospholipids with inositol head groups contribute towards key cellular processes such as migration, proliferation, senescence, and apoptosis3. Whereas phosphatidylserines in brain tissue are critical for facilitating neuronal signaling pathways and neurotransmitter release5. The diversity of lipids and their functions underscore the importance of investigating them in detail to better understand their signatures in pregnancy.Adequate regulation of maternal lipid metabolism is essential for the progression of a healthy pregnancy and fetal growth. Lipids are generally defined as insoluble organic compounds, and their unique chemical structure is used to classify individual lipid compounds into organizational groups. At the broadest level, some lipid classes of biological interest include phospholipids, glycerides, sterols, and sphingolipids. The structural differences across lipid classes partly define unique biological functions. For example, polymerized glycerides such as triglyceride are stored in adipocytes and released through hormone signaling for energy production7. Conversely, during the third trimester, there is an upregulation of lipolysis, resulting in catabolism and decomposition of lipid stores7. In both processes—accumulation and decomposition of lipid stores—there is a complex matrix of circulating free and vesicular lipids that facilitate crosstalk between the maternal immune-endocrine systems and placenta8. Lipidomic analysis affords greater insight into the vast repertoire of lipid metabolites and has potential utility towards defining biomarker signatures of disease states such as inflammation or metabolic dysfunction. Furthermore, individual lipid metabolites can be leveraged to construct broader levels of organizational hierarchies, including whole lipid classes and subgroups of lipid classes based on the degree of saturation in hydrocarbon chains.Maternal lipid profiles change dynamically across the duration of gestation. For example, maternal adipose tissue is increasingly active in the anabolism of circulating lipids during the first and second trimesters, resulting in accumulation and storage of fatty acids, triglycerides, phospholipids, and cholesterols9. Furthermore, social factors such as environmental injustice, income inequality, and systemic racism have led to disparities in rates of adverse pregnancy outcomes10. In Puerto Rico, the rate of preterm birth in 2019 was 11.9%11. It is critical for emerging omics-scale studies to include underrepresented communities that are historically neglected in order to better understand and prevent health disparities. Consequently, characterization of lipidomic signatures among preterm birth cases in Puerto Rico can provide insight on early markers of disease and inform behavioral and therapeutic interventions. Additionally, maternal lipid profiles are responsive to extracellular vesicles and signaling molecules secreted from the placenta8. Therefore, lipidomic biomarker signatures can also yield utility in estimation of fetal growth and neonatal anthropometric parameters.Estimation of lipidomic biomarker signatures can have predictive utility for a wide range of pregnancy outcomes. For example, preterm birth—delivery prior to 37 weeks gestation—remains a persistent public health concern and affects approximately 10% of live birthsThe primary objective of this study was to estimate lipidome-wide associations for multiple pregnancy outcomes, including preterm birth, spontaneous preterm birth, gestational age at delivery, birthweight, and small and large for gestational age neonates. The secondary objective of this study was to construct hierarchies of lipid organization: whole lipid classes and subgroups based on hydrocarbon chain saturation. Our overarching hypothesis is that physiological states underlying adverse pregnancy outcomes would be accompanied by unique lipidomic signatures. For example, systemic inflammation and hormone synthesis may be linked to lipid metabolism, both of which are critical for ensuring adequate progression of a healthy pregnancy. Thus, the motivation to investigate lipid signatures of adverse pregnancy outcomes will have broad applicability towards future studies seeking to characterize mechanisms and early biomarkers of reproductive health outcomes.2. Most participants did not report smoking (98%) or drinking (92%) during pregnancy.Table Supplementary Table Spearman’s ρ ranged from − 0.74 to 0.97 among all lipid molecules  for each lipid in association with pregnancy phenotypes. All annotated estimates in Fig. Figure For the continuous outcome birthweight z-score, one lipid metabolite from the phosphatidic acid class was associated with higher birthweight z-score while one metabolite from the phosphatidylethanolamine class was associated with lower birthweight z-score Fig. D. ContexFigures Figure Sparse-group lasso showed that the most important and predictive lipid class for overall preterm birth was free fatty acids  individual lipidome-wide metabolites, (2) subgroup clustering based on hydrocarbon chain saturation, and (3) whole lipid classes. Using these organizational hierarchies of lipids, we sought to explore their signatures in association with multiple pregnancy phenotypes: preterm birth and its subtype spontaneous preterm birth, gestational age at delivery, birthweight, and neonatal size relative to gestational age at delivery. We found that at the broadest level of whole lipid classes, there is not substantive evidence of associations in this sample. Upon evaluation of lipid saturation subgroups, we observed more pronounced relationships with multiple phenotypes. At the lipidome-wide level of individual metabolites, we observed multiple single metabolites associated with each pregnancy phenotype. Phospholipid classes appeared to have the greatest number of metabolite specific signatures with multiple pregnancy outcomes. The notable theme in our findings was the variation in signatures among distinct phospholipid classes . These findings support the need to measure lipid metabolites at a finer omics-scale through lipidome-wide measurement to identify important lipid-derived biomarkers of adverse pregnancy outcomes not captured with the cruder yet more common class-level measurements.12. The increased risk for preterm birth among a lipidome-wide quantification of free fatty acids is circumstantially consistent with previous findings where we observed increased risk for preterm birth in association with eicosanoids13. Other studies have also observed that free fatty acids measured at a general non-specific level were associated with preterm delivery15. When spontaneous preterm birth was disaggregated from the overall preterm birth phenotype, associations involving free fatty acids remained, but the signature of plasmenyl phospholipids was substantially stronger and more significant. Additionally, sparse-group lasso also selected this lipid class as an important predictor of spontaneous preterm birth. This suggests that the metabolism and circulation of plasmenyl phospholipids may be more closely linked to spontaneous labor or premature rupture of the membranes. Overall, these findings provide further evidence that biomarker signatures are differentiated when evaluating general preterm birth compared to more precise etiologically linked phenotypic subclasses.The dominant lipid signature for overall preterm birth was from lipid metabolites belonging to the free fatty acid class. This class of lipids was also selected by sparse-group lasso, further underscoring its importance relative to other lipid classes. Free fatty acids include several essential compounds such as arachidonic acid, a major precursor for immuno-potent metabolites such as eicosanoidscases = 16; ncontrols = 32) based in Ireland, Morillon et al. (2020) observed that several metabolites (measured at 20 weeks’ gestation) within the phosphatidylcholine and phosphatidylethanolamine classes were significantly lower among cases. This study also found that multiple sphingolipids were downregulated in cases of spontaneous preterm birth16. Another lipidomic study preterm birth focusing exclusively on Black women  in the U.S. Midwest observed that lipid metabolites (measured 9–25 weeks’ gestation) in the phosphatidylcholine class were downregulated in preterm birth cases while triglycerides were upregulated17. Potential factors influencing differences in exact lipid signatures could be due to demographic differences between our study and the comparative studies. Furthermore, the timing of lipidome measurement was different as well. We focused on samples collected in the second and third trimesters, while the comparison studies measured the maternal lipidome earlier in pregnancy, during the first and second trimesters.In a lipidomic profiling study of spontaneous preterm birth  and non-anthropometric outcomes . This finding is interesting because it gives initial evidence that neonatal anthropometric parameters may be more sensitive to different maternal lipid profiles compared to preterm birth. The significance of this finding is that potential therapeutic and dietary interventions may need to consider different lipid metabolite biomarkers when evaluating risk factors for neonatal versus maternal pregnancy phenotypes.cases = 52, ncontrols = 28)19. Unlike other comparison studies cited earlier, this study quantified lipids using nuclear magnetic resonance, which differed from our quantitative approach and could also explain differences in observed associations19. Another study based in the United Kingdom measured maternal lipid metabolites at 15 weeks gestation and observed higher concentrations from the lysophosphatidylcholine and sphingolipid classes among small for gestational age neonates 20. The inconsistencies in findings for small for gestational age neonates across existing studies and our current study may also be influenced by timing of lipidomic measurements and differences in geographic and social factors.In small for gestational age neonates, we only observed lower levels of one maternal ceramide metabolite. This finding parallels the lack of lipids associated with decreased birthweight z-score. Perhaps the timing of lipidomic measurement in this study is not sensitive at displaying lipid signatures for these particular neonatal outcomes. Additionally, the sample size of cases that were small for gestational age was low (n = 16), limiting power to detect notable lipid signatures. One previous study based in Spain measured maternal lipids at the time of delivery, and detected lower concentrations of circulating cholesterols, triglycerides, and phosphatidylcholines in women with small for gestational age neonates , among the neonatal outcomes, we observed the greatest lipid signature when evaluating this outcome—multiple phosphatidylinositols and one phosphatidylglycerol were associated with increased risk. Giving birth to large for gestational age neonates is an important phenotype to focus on, given that these pregnancies are associated with co-morbidities such as maternal obesity, gestational diabetes, and gestational weight gainThe major limitation in our study design is the modest sample size. However, a majority of the previously conducted lipidome-wide association studies cited were focused on smaller sample sizes. This exploratory analysis builds on the methodological foundation of previous studies and provides evidence for scaling up future larger replication studies. Another limitation in our study is that there are unmeasured confounders that may affect the estimation of associations in our study. For example, our study did not evaluate maternal dietary patterns, which could be driving both lipidomic signatures, pregnancy outcomes, and fetal growth. We partially accounted for this unmeasured confound by adjusting for maternal pre-pregnancy BMI and weight gain during pregnancy. Additionally, adjustment for diet may not be appropriate in this setting, given that internal lipid profiles are directly dependent on dietary intake, in which case mediation analysis may be a more appropriate method to account for the relationship between diet, lipidomic profiles, and pregnancy outcomes. Furthermore, adjustment for weight gain during pregnancy provides a more precise measurement of physiological changes due to diet, rather than dietary food intake questionnaires. Another limitation in our study is that the lipidomic biomarkers we measured were restricted to maternal circulation, therefore we are unable to evaluate tissue specific lipid profiles. Future studies should evaluate other tissues such as placenta and amniotic fluid, to evaluate the degree of consistency or divergence in lipid signatures for pregnancy phenotypes. Our study was also limited by having only one time point for measurement of the maternal lipidome, therefore we were unable to evaluate windows of vulnerability during pregnancy. Given that lipid profiles change across the duration of pregnancy, certain periods may be more insightful for the etiology of preterm birth and neonatal anthropometric outcomes.Our study contained several strengths that should be underlined. First, a unique attribute about our study is the demographic representation of our study sample. We focused on an underrepresented U.S. community which not only diversifies modern omics-scale study populations, but also critically informs data driven policy development to reduce health disparities within the U.S. Another strength of our study was the exhaustive repertoire of metabolites that were measured within the maternal lipidome, which has the potential to yield important biomarkers of adverse pregnancy outcomes. In the lipidomic biomarker space, we also pursued creative approaches to develop hierarchical organization of metabolites based on whole lipid classes and hydrocarbon saturation subgroups. This approach informs future studies that may be limited in their ability to measure the entire lipidome, and therefore must prioritize specific lipid classes and subgroups.In conclusion, this study provides evidence that finer omics-scale analysis of the maternal lipidome may be more informative biomarkers of adverse pregnancy outcomes compared to cruder levels of lipid quantification. Lipidomic measurement may advance precision health profiling of maternal and fetal outcomes, which warrants subsequent replication studies. Furthermore, this study discusses major gaps in lipidomic research, chiefly, the need to evaluate repeated measurements of the maternal lipidome and also quantification across multiple tissue types. Future studies that pursue these research gaps will advance scientific discovery in characterizing the etiology of adverse pregnancy outcomes.This study sample is an exploratory subset of the Puerto Rico Testsite for Exploring Contamination Threats (PROTECT) cohort. From the larger cohort of 812 pregnant women, we randomly sampled 100 women for lipidome-wide analysis with the goal of achieving a 1:2 case–control ratio for preterm birth. The PROTECT cohort began recruitment in 2010 through funding from the National Institute of Environmental Health Sciences Superfund Research Program. Study participants were recruited in the first or early second trimester of pregnancy (median 14 weeks gestation). Inclusion criteria for recruitment included: participant age between 18 and 40 years; residence in the Northern Karst aquifer region; disuse of oral contraceptives three months before pregnancy; disuse of in vitro fertilization; and no indication of medical records for major obstetrical complications, including pre-existing diabetes. The research protocol was approved by the Ethics and Research Committees of the University of Puerto Rico and participating clinics, the University of Michigan, Northeastern University, and the University of Georgia. The study was described in detail to all participants, and informed consent was obtained prior to study enrollment. All methods within this study were performed in accordance with the relevant guidelines and regulations approved by the Ethics and Research Committees.24 were used in our study as previously described26. Per common practice, preterm birth was defined as delivery < 37 completed weeks of gestation. Based on the presentation of preterm delivery, preterm birth was further classified as spontaneous preterm birth  and non-spontaneous preterm birth . We included overall and spontaneous preterm birth as two of the birth outcomes in our analysis.All of the birth outcome data were extracted from medical records. The American Congress of Gynecologists recommendations for best obstetrical estimate to calculate the gestational age for complete pregnancies27. Gestational age- and sex- specific birthweight z-score were constructed according to the INTERGROWTH-21st standards28. Small for gestational age (SGA) births were defined as those below the 10th percentile of birthweight z-scores. Large for gestational age (LGA) births were defined as those above the 90th percentile of birthweight z-scores.Birthweight z-scores (defined as the number of standard deviations by which a birthweight is above or below the mean) are commonly used to compare individual birthweights within the cohort29 using liquid–liquid extraction at room temperature after spiking with internal standards. Analysis of lipids was performed on reversed phase high-performance liquid chromatography (HPLC), followed by mass spectrometry (MS) analysis that alternates between MS and data-dependent MS2 scans using dynamic exclusion in both positive and negative polarity and yields excellent separation of all classes of lipids. The lipids are quantified using Multiquant and normalized by internal standards.Plasma lipids are extracted from blood samples provided by women between 24 and 28 weeks gestation using a modified Bligh-Dyer method30.For chromatography, a Shimadzu CTO-20A Nexera X2 UHPLC systems equipped with a degasser, binary pump, thermostated autosampler, and a column oven for chromatographic separation were used. A linear gradient using solvents was created over the 20-min total run time. Solvent A contained acetonitrile/water  with 10 mM ammonium acetate. Solvent B contained acetonitrile/water/isopropanol  with 10 mM ammonium acetate. For the first ten minutes, a 60% Solvent A and 40% Solvent B was used. For the next seven minutes, the gradient increased in a linear fashion to 100% Solvent B. Thereafter the system was switched back to 60% Solvent B and 40% Solvent A for 3 min. The flow rate used for these experiments was 0.4 mL/min and the injection volume was 5μL. The column was equilibrated for three minutes before the next injection and run at a flow rate of 0.4 uL/min for a total run time of 20 min/z. Measurements are semi-quantitative and are based on relative abundance of peak intensities.Samples were ionized in positive and negative ionization model using a Triple TOF 5600 . The source voltage was set to 5500 V for positive ionization and 4500 V for negative ionization mode, the declustering potential was set to 60 V, and the source temperature to 450 °C for both modes. The instrument performed one TOF MS survey scan (150 ms) and 15 MS/MS scans with a total duty cycle time of 2.4 s. The mass range in both modes was 50–1200 m31.Quality controls are prepared by pooling equal volumes of each sample, in addition to a well characterized plasma pool, and are injected at the beginning and end of each analysis and after every 10 sample injections, to provide a measurement of the system’s stability and performance as well as reproducibility of the sample preparation method32 library , by matching the product ions MS/MS data. The method allowed us to identify and quantify 587 lipids belonging to 19 different lipid classes, including acylcarnitine (AcyICN), ceramides (CER), cholesterol esters (CE), diacylglycerols (DG), glucosylceramides (GlcCer), free fatty acids (FFA), fatty acid esters of hydroxy fatty acids (FAHFA), lysophosphatidylcholine (LysoPC), lysophosphatidylethanolamine (LysoPE), phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), plasmenyl-phosphatidylcholine (PLPC), plasmenyl-phosphatidylethanolamine (PLPE), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin (SM), and triacylglycerol (TG).Lipids are identified using the LipidBlastGroup sum of the individual lipids’ relative abundance that belong to each lipid class were calculated to construct a total measurement for whole lipid classes. As chain length and number of double bonds in the lipid are of biological interest, sub-group  sums of lipids’ relative abundance were also created. Descriptive statistics for lipids were computed to examine their distributions. Spearman correlation coefficients were calculated between individual lipids, lipid class, and lipid sub-groups.2 and Akaike information criterion. Final covariates included maternal age, maternal education, fetal sex, pre-pregnancy BMI, and pregnancy weight gain. Individual models were performed for each log-transformed and standardized relative abundance of lipid, creating 587 models for each birth outcome. False discovery rate (FDR) adjusted p-values , a commonly used method of adjusting for multiple comparisons in lipidomics studies, were used to account for multiple comparisons. Sums for each lipid class (pc = 19) and lipid sub-group (pg = 46)  were then regressed on each birth outcome.Multiple statistical strategies were used to assess relationships between lipid profiles and birth outcomes. The relative abundance of individual lipids were log-transformed due to their skewed distribution. Subsequently the log-transformed individual lipids, sub-group sums, and group sums were normalized among individuals (rows) by standard normal variate normalization. Multiple linear and logistic regression models were fitted to evaluate the association between continuous and binary birth outcomes and the lipidome. Models were adjusted for covariates based on a priori assessment and through model fit diagnostic parameters such as adjusted RRegression results were transformed and presented as estimated percentage changes for continuous outcomes  and odds ratios for dichotomous outcomes  with the corresponding 95% confidence intervals and p values per unit change in lipids z score.SGL package, version 1.3). In this setting, lipids were given a group-level identity according to their class. Sparse-group lasso operates as a variable selection method that utilizes tuning parameters to control for groupwise and within group sparsity among lipids to determine which lipid classes are most predictive of individual outcome variables.Models were also constructed with all lipids simultaneously using sparse-group lasso (Supplementary Information 1.Supplementary Information 2.