Extensive lesions cause facial asymmetry.5Although most dentigerous cysts are considered to be of developmental origin, some examples seem to have an inflammatory pathogenesis, but it is impossible to determine histopathologically whether the inflammatory component is primary or secondary in nature.An 8-year-old girl reported to the Department of Pediatric Dentistry, Amrita School of Dentistry, with a painful, progressive, hard swelling of left side of the face and left molar area causing facial asymmetry . Clinica7It was decided to manage the lesion by marsupialization and an acrylic appliance to prevent the closure of the surgical opening, to attempt and salvage the involved permanent molar as a preliminary step in management with enucleation deferred until if need arises. Nowadays marsupialization of the cyst lining is the treatment of choice for dentigerous cyst in children in order to give a chance to the unerupted tooth to erupt.After routine blood investigations and preoperative serological evaluations, marsupialization of the lesion was done under general anesthesia  and 5.8 The cyst did not show any signs of infection, except that the patient had mild pain on presentation. The cystic contents were not purulent. The epithelial lining showed signs of inflammation. A part of the cystic lining was removed for histopathological evaluation. Impression of the surgical opening and mouth of the lesion was made. Antibiotics were prescribed to prevent secondary infection postsurgically. The regimen followed was amoxycillin 250 mg thrice daily for five days. Histopathological evaluation of the specimen showed a thick fibrocollageneous cyst wall showing sparse inflammation with an ulcerated squamous lining consistent with dentigerous cyst confirming the diagnosis. Acrylic appliance was fabricated and inserted on the tenth postoperative day to maintain the surgical window , which is not very common.Dentigerous cysts are odontogenic cysts arising from the reduced enamel epithelium after the formation of the tooth crown. The lower-third molars and the upper canines are the most affected, but any permanent tooth can be involved.25 Radioluscency often have a sclerotic border indicating bony reaction.14 An infected cyst may show ill-defined borders.5 The cyst to crown relationship in a dentigerous cyst can show different variations. The cyst surrounds the crown of the tooth with the crown projecting into the cyst in central variety. In the present case, the cyst appeared to be that of central variety with the crown of the tooth projecting into the cyst lumen.Radiographically the dentigerous cyst typically appears as a well-circumscribed, unilocular, usually symmetric radioluscency larger than that of a normal dental follicle around the crown of the tooth.5When the cyst grows laterally along the root surface surrounding the crown partially it is called the lateral variety as seen with mesioangular impacted mandibular third molars. In the circumferential variety, a considerable amount of root appears to lie within the cyst along with the crown that is surrounded by the cyst.1 During marsupialization of the cyst, it was observed that the cyst contents were watery yellow and not turbid or purulent, indicating that the cyst was not infected.Histologically the wall of the dentigerous cyst is composed of thin connective tissue containing varying numbers of islands of odontogenic epithelium with a layer of stratified squamous epithelium lining the lumen of the cyst. Inflammatory cell infiltration of the connective tissue is common. Presence of Rushton bodies which are peculiar, often curved, hyaline bodies probably of hematogeneous origin are seen within the epithelium lining. The content of the cyst lumen is a watery yellow fluid, which can occasionally be blood tinged.1 Except root resorption all other clinical features of an expanding dentigerous cyst were seen in this case.The dentigerous cyst is potentially capable of becoming an aggressive lesion. Expansion of the bone, facial asymmetry, extreme displacement of teeth, severe root resorp-tion of adjacent teeth and pain are all possible sequelae of continuous enlargement of the cyst.1Potential complications are development of ameloblasto-ma, epidermoid carcinoma or a mucoepidermoid carcinoma.Though the usual treatment for a dentigerous cyst is careful enucleation of the cyst together with the removal of unerupted tooth, if eruption of the unerupted tooth is considered feasible, the tooth may be left in place after partial removal of the cyst wall.58 Considering the age of the patient in this case, it was important to prevent the loss of a permanent tooth, which in turn will create the need for prosthesis. Hence, enucleation was decided against and the outcome turned out to be rewarding. The involved tooth erupted and was well aligned in the arch without any orthodontic assistance.This permits the decompression of the cyst with a resulting reduction in the size of the bone defect. Some patients may need orthodontic treatment to assist eruption. Large dentigerous cysts may also be treated by marsupialization.Marsupialization can be thought of as the first treatment option for dentigerous cyst, in children, when loss of viable permanent tooth buds can be prevented.Marsupialization allows for guided eruption of the developing teeth as the overlying cystic structure is decompressed.A radiographic review in every 6 months for the first 2 years is mandatory since the remnants of cystic lining can undergo ameloblastic changes.


Heart failure (HF) is defined by compromised contractile function and is associated with changes in excitation-contraction (EC) coupling and cardiomyocyte organisation. Tissue level changes often include fibrosis, while changes within cardiomyocytes often affect structures critical to EC coupling, including the ryanodine receptor (RyR), the associated protein junctophilin-2 (JPH2) and the transverse tubular system architecture. Using a novel approach, we aimed to directly correlate the influence of structural alterations with force development in ventricular trabeculae from failing human hearts. Trabeculae were excised from explanted human hearts in end-stage failure and immediately subjected to force measurements. Following functional experiments, each trabecula was fixed, sectioned and immuno-stained for structural investigations. Peak stress was highly variable between trabeculae from both within and between failing hearts and was strongly correlated with the cross-sectional area occupied by myocytes (MCSA), rather than total trabecula cross-sectional area. At the cellular level, myocytes exhibited extensive microtubule densification which was linked via JPH2 to time-to-peak stress. Trabeculae fractional MCSA variability was much higher than that in adjacent free wall samples. Together, these findings identify several structural parameters implicated in functional impairment in human HF and highlight the structural variability of ventricular trabeculae which should be considered when interpreting functional data. Causes of human HF are multifactorial and include structural changes that are directly associated with functional deficits. At the tissue level, interstitial fibrosis is widely reported throughout the diseased and failing myocardium3, leading to an increase in tissue stiffness that opposes active force generation2. This fibrosis may occur due to loss of cardiomyocytes (replacement fibrosis), or in the absence of necrotic cell death3. In addition to the reduction in proportional cardiomyocyte area as a result of fibrosis, the cardiomyocytes themselves often exhibit sub-cellular structural alterations in HF.Normal cardiac function is the ability of the ventricles to contract and efficiently pump blood. In heart failure (HF), there is a general loss of cardiac function such that there is an inability to meet the metabolic demands of the body4 which is vital for the synchronous activation of Ca2+ release from the sarcoplasmic reticulum (SR). Loss or disruption of t-tubules is commonly observed in human and animal models of HF8 and has been associated with compromised contractile function (development of force) as a result of sub-cellular delays in action potential propagation9 and desynchronized Ca2+ release11. Our group has previously identified that the extent of this t-tubule disorganisation is strongly correlated with the severity of cardiac dysfunction in the failing human heart12.These cellular level alterations often affect components essential for contractile activation and excitation-contraction (EC) coupling, which involves several key structures and proteins within the cell, including the transverse tubules (t-tubules). T-tubules are invaginations of the sarcolemma, creating an intracellular network capable of rapidly conducting action potentials into the cell interior14 which is important for the sarcolemma to closely associate with the terminal SR (jSR) to form junctions15. Junctions  are vital functional regions containing many of the proteins essential to EC coupling, including the SR calcium release channels – the ryanodine receptors (RyR)17. RyR form clusters at the junction, with their organisation tightly linked to Ca2+ handling properties and cardiomyocyte function18. Also present is the protein junctophilin-2 (JPH2), which spans the SR membrane and associates with the plasma membrane19. JPH2 has subsequently been implicated in the formation and maintenance of both the junctions and t-tubule structure in cardiomyocytes21. In addition, it has previously been suggested that the loss of full-length wild-type JPH2 plays a role in the development and progression of HF23, as well as t-tubule remodeling26. More recently, it has been proposed that microtubule densification (which is observed in HF)29 is responsible for altered trafficking of JPH2, such that it is displaced from the intracellular junctions, leading to t-tubule disruption and impaired cardiac function30.The t-tubules normally align to the z-disksin situ using cine magnetic resonance imaging (cMRI)12, cMRI generally lacks cellular resolution and does not allow for the direct measurement of force development. Likewise, isolated cardiomyocytes can be used to examine both cellular function and structure, but they lack information on the role of the extracellular matrix (ECM) and potential fibrosis in HF. An alternative approach is to use cardiac trabeculae which allow examination of cardiac function at the cellular level in the presence of non-myocyte cells (such as fibroblasts) that are present in the ECM and contribute to contractile dysfunction in HF.In order to elucidate the relationship between the various structural changes in HF and the development of contractile impairment, both contractile force and indicators of cell and tissue structure should be examined, ideally within a single muscle sample. Despite the insights provided by correlating ultrastructural remodeling within the cardiomyocytes and the degree of circumferential shortening 32. Other studies report peak stress is reduced33, or in some instances, increased, in human HF34. However, not all studies were performed using physiological temperature or stimulation frequency. Furthermore, many of these studies have been performed in failing patient groups from mixed etiologies, often including ischemic samples, which when sub-divided into cardiomyopathy type reveal different responses35. This further complicates the interpretation of the underlying mechanisms involved. It is also possible that several studies excluded preparations which demonstrated low or absent stress responses to stimulation. This would lead to experimental selection bias towards ‘healthy’ muscle preparations, despite originating from failing hearts, with some explicitly stating these preparations were excluded from their study36. While several previous studies have used cardiac trabeculae to examine functional changes in both human and animal models of HF39, investigation of alterations in trabeculae structure have generally received little attention in those studies. Here, we address this gap and directly examine the relationship between contractile force and structural alterations using ventricular trabeculae from human hearts in failure.Previous studies examining functional alterations in ventricular wall strips or trabeculae from failing human heart found little apparent difference in baseline peak stress development compared to non-failing samplesUsing a novel approach, we examined cardiac trabeculae from human hearts with idiopathic dilated cardiomyopathy (IDCM) in end-stage HF to directly correlate contractile function (stress measurements) with alterations in cardiomyocyte organisation within the failing heart. Hypothesizing that both tissue and cellular level alterations contribute to the contractile deficit, a range of structural parameters were examined using confocal microscopy. A striking finding was a high degree of variability in the myocyte content of trabeculae which was closely correlated with contractile performance. At the cellular level, trabeculae from failing hearts exhibited microtubule densification which was linked via JPH2 to an increased time-to-peak stress. The highly variable myocyte content of trabeculae excised from the same hearts complicates the interpretation of trabeculae stress measurements, unless these are closely associated with investigation of tissue structure, as shown here, which may explain some of the contradictory results between previous studies.2; see Supplementary Table 2) under similar experimental conditions33. It was therefore of interest to determine the key structural parameters contributing to the considerable functional variability observed in these trabeculae. At the tissue level, the composition of the trabeculae was found to be highly variable between samples , this was not statistically significant . Confocal imaging revealed a high degree of variability in the organisation of both RyR clusters and t-tubules in cardiomyocytes from human trabeculae, with t-tubules virtually absent in some cardiomyocytes Fig. . The remant Fig. . Interesant Fig. , suggest24. These disorganised t-tubules appeared to contribute to the decreased proportion of RyR clusters identified as being associated with t-tubules in the failing wall samples compared to non-failing  revealed a wide variability in the FFR from the samples we obtained  observed in the healthy human heart, a blunted or even negative FFR has been reported in human HF45 and is the most prevalent underlying cause of heart transplant procedures46. In this study, we have observed and quantified both tissue level sub-cellular structural changes to IDCM failing human myocardium, as well as explored the relationship between these changes and cardiac function. The defining feature of HF is the impairment of cardiac function, such that there is inadequate supply to meet metabolic demand. While, on average, we observed low active stress development in the trabeculae from failing human hearts, some trabeculae exhibited contractile strength at levels similar to those reported in trabeculae from the healthy human heart33. The contractile performance was strongly linked to a number of marked structural alterations at tissue and cellular levels. Importantly, the observed variability in contractile performance and myocyte content did not merely reflect differences between hearts as contractile performance also varied greatly between trabeculae from the same hearts.DCM is among the most common forms of heart disease2, with increased collagen deposition leading to higher ventricle wall stiffness and opposition to active force development3. Our data appear to support these previous findings, showing a reduction in the proportion of myocardium attributed to cardiomyocytes in the failing human heart . An additional observation was the inconsistency of cardiomyocyte orientation in several trabeculae  and the t-tubules in the failing human heart  is necessary for myocyte shortening to occur. In an elastic material, the strain developed is proportional to the applied stress and it has been shown that exposure to increased strain leads to remodeling of t-tubules in cardiomyocytes52. This provides a potential mechanism through which increased fibrosis and a corresponding decrease in cellular content, may contribute to the reduced association between RyR clusters and t-tubules in the remaining cardiomyocytes.Interestingly, we did identify a significant correlation between the myocyte content of trabeculae and the proportion of RyR clusters associated with t-tubules Fig. . This su2+ channel and impact Ca2+ spark properties20, contributing to spontaneous RyR opening and raised diastolic Ca2+, as observed in HF. However, we found no significant difference in either the RyR-JPH2 association in the failing heart compared to non-failing, nor in the labelling density of these proteins, despite previous studies reporting reduced protein expression levels reported43. However, JPH2 tissue density was positively correlated with increased association between RyR clusters and t-tubules in the trabeculae . It has been proposed that microtubules potentially modulate Ca2+ currents through both DHPR and RyR55, as well as their densification increasing resistive stresses within the myocyte, inhibiting sarcomere shortening56. Despite these proposed mechanisms, α-tubulin density was not correlated with active force development in the human HF trabeculae  or perhaps a more general property of human cardiac trabeculae also found in normal hearts. While the lack of trabeculae from non-failing human hearts is a limitation, it is notable that our comparison between trabeculae from failing hearts allowed circumvention of potentially confounding pharmacological differences between patient treatment regimes by using multiple trabeculae from each heart to provide within-heart comparisons.2+ transient, including the amplitude and temporal kinetics, which are thought to be involved in alterations of the FFR. As a result of limited trabeculae numbers and the time required for the force experimental protocol, the number of trabeculae able to be loaded with Ca2+ indicator in this study (n = 3) was insufficient for a systematic investigation of changes to Ca2+ handling in human HF; accordingly, we cannot currently dissect the reason for the variability of FFR in our samples. The blunted, or even negative, FFR in the failing heart is also thought to involve a shift towards Ca2+ extrusion via NCX, such that there is an increasing removal of Ca2+ from the myocyte with higher stimulation frequencies58. It would therefore be of interest to examine both the function and distribution of NCX and SERCA in relation to the FFR in further studies. While direct correlation between force, structure and Ca2+ handling in individual trabeculae from human hearts would be of great interest to pursue in future studies – it is clearly technically very challenging.A commonly reported alteration in HF are changes to the Ca59, such that force normalised to total CSA still remains acceptable for comparison between muscles. However, measurements from hypertrophied or failing hearts may over-estimate the degree of dysfunction unless force is normalised to MCSA. On this basis, we suggest that force studies should be augmented with structural assessments in order to draw mechanistic conclusions, particularly in HF studies. In addition to these tissue-level changes, several sub-cellular protein changes were identified and confirmed as occurring in HF compared to non-failing hearts. Trabeculae structural properties broadly mimicked features of the ventricular wall from the same hearts, confirming their usefulness in functional studies, particularly if effects of structural variability are eliminated by correlation with functional measurements.In conclusion, using a novel approach, we obtained both functional and structural measurements in individual muscle samples from the failing human heart. This enabled us to determine structural parameters directly correlated with functional variability, both within and between hearts. We observed highly variable cardiomyocyte content in trabeculae from failing human hearts, which was strongly correlated with contractile function, while total tissue CSA was not, contrary to assumptions generally made in trabeculae studies. Trabeculae from healthy hearts are almost entirely made up of myocytes2+20 mM 2,3-butanedione monoxime  to the laboratory. Suitable cardiac trabeculae were then micro-dissected from the endocardial surface of tissue blocks for functional experiments  for the collection of human samples, along with written, informed consent of the heart transplant recipients and the families of organ donors in the case of non-failing tissue samples. No samples were procured from prisoners, with all experiments performed in accordance with relevant guidelines and regulations. Samples from the septal and ventricular walls were collected from the explanted hearts of patients with non-ischemic IDCM in end-stage HF from Auckland City Hospital. These were immediately transported on ice in oxygenated Tyrode’s solution containing 0.25 mM CaCl61 and were comparable to sizes used in previous studies36. The experimental setup and procedures were adapted from those previously described62. In brief, dissected trabeculae with small ventricular tissue blocks on each end were transferred to a tissue bath positioned on the stage of an inverted Nikon Eclipse TE2000 microscope. Trabeculae were held at fixed length (LO) by mounting the tissue block at one end in a wire cradle which extended from a force transducer , while the tissue block at the opposite end was secured in a nylon loop. Both ends were attached to manipulators to enable control of muscle length. Trabeculae were continuously superfused with oxygenated Tyrode’s solution containing 2 mM [Ca2+]o, and 0 mM BDM at 37 °C, unless otherwise stated. Samples were field stimulated with supramaximal 5 ms electrical pulses at 1 Hz . Data was acquired using LabVIEW  and analysed offline.Suitable trabeculae were identified according to length and diameter (mean diameter ± SEM: 330 ± 150 µm) such that adequate oxygen diffusion could be maintained, based on tissue eccentricity40. Force measurements were therefore normalised to trabecula CSA and expressed as stress (mN/mm2) to enable comparison between trabeculae. Total trabecula CSA was determined by direct measurement from confocal images of fixed tissue transverse sections, rather than calculated from live preparations in which orientation may influence diameter measurements – particularly for elliptical tissue samples. While shrinkage may have occurred through tissue processing, this would be consistent across all samples. Confocal imaging revealed a large variability in the proportion of myocytes between trabeculae, which was found to positively correlate with developed force. Therefore, force was also normalised to myocyte CSA (MCSA) of the trabecula, with both stress per CSA and stress per MCSA determined for all trabeculae. Analyses performed on time-to-peak stress and and time to 50% relaxation (T50%), are presented in the supplementary material . A total of 14 trabeculae were analysed from the five failing hearts. It has been long recognized that developed force is proportional to muscle size, in particular cross-sectional area (CSA)63. Tissue sections were hydrated in PBS and permeabilised with 1% Triton-X100  for 10 min at room temperature, followed by blocking in Image-iT Signal enhancer (Life Technologies) for 1 h at room temperature. Immunolabelling was performed using rabbit anti-JPH2 64 combined with either mouse anti-RyR  or mouse anti-α-tubulin  as a microtubule marker53, or rabbit anti-collagen I  or anti-collagen VI  with mouse anti-collagen III  with the primary antibodies incubated overnight at 4 °C. Sections were washed and incubated with goat anti-mouse and goat anti-rabbit secondary antibodies conjugated to Alexa Fluor 488 or 568 . T-tubules and ECM were labelled using wheat germ agglutinin66 Alexa Fluor 647 , applied concomitantly to the secondary antibodies for 2 hours at room temperature. Collagen-labelled sections were also incubated with either DAPI  to stain nuclei, or phalloidin Alexa Fluor 647  to stain f-actin, concomitantly with secondary antibodies. All antibodies were dissolved in 1% BSA (w/v) + 0.05% NaN3 (w/v) + 0.05% Triton-X100 (v/v) in PBS. Samples were mounted onto slides using ProLong Gold (Life Technologies) and allowed to cure for ~72 h prior to imaging.Following dissection of trabeculae, blocks from the remaining tissue were fixed in 2% (w/v) paraformaldehyde for 1 h at 4 °C. Upon completion of functional experiments, the trabeculae were held at fixed length and fixed with the same protocol. After fixation, the trabeculae were cut in half transversely. All samples were cryoprotected in sucrose, frozen in methyl butane cooled in liquid nitrogen and stored at −80 °C, with the trabeculae halves embedded in TissueTek® O.C.T. compound. Frozen 16 µm thick sections of myocardium (failing and non-failing) and trabeculae were cut on a CM 3050 cryostat  and collected onto 0.05% poly-L-lysine (v/v) coated coverslips for immunohistochemistry. Immunolabelling procedures were adapted from those previously described14 to produce 32-bit image stacks. Tissue content was assessed in images of transversely orientated cardiomyocytes (acquired at 40× magnification) in all three sample groups, using ImageJ. Manual thresholding was applied to generate binary masks. JPH2 labelling was used to determine MCSA, while WGA labelling was used to measure total CSA. The outline of each WGA mask was manually traced to determine the total CSA of the trabecula tissue sections. Within each individual trabecula transverse section, all regions positive for JPH2 staining were combined to determine the CSA attributed to myocytes (MCSA). MCSA was also converted to a percentage of total CSA.A Zeiss LSM710 inverted confocal microscope was used to acquire images of the fluorescently labelled tissue sections using either a 40 × NA 1.3 oil-immersion or a 63 × NA 1.4 oil immersion objective. 8-bit images were generated with 173 µm/pixel or 67 µm/pixel, respectively. Fluorochromes were excited with either 405 nm (DAPI), 488 nm (Alexa 488), 561 nm (Alexa 568) or 633 nm (Alexa 647) laser excitation. Images were acquired of both longitudinally and transversely orientated cardiomyocytes, with confocal image stacks processed for deconvolution according to previously described protocols using a Richardson-Lucy algorithmet al.50. Briefly, the threshold was calculated from image statistics, i.e. the intensity histogram and and set to its mode+ one standard deviation. ImageJ particle analysis was used to identify RyR clusters as regions of interest (ROI), which were then overlaid on the corresponding WGA mask. The number of RyR clusters associated with t-tubule labelling could then be determined as ROI with mean pixel value > 0 (i.e. ROIs with overlap with the t-tubule mask) and we report the fraction of t-tubule positive ROIs as the percentage of total RyR clusters present.The extent of RyR-t-tubule association was assessed in RyR and WGA dual-stained images (acquired at with the 63× objective). Binary masks of labelling-positive regions were generated following thresholding using the value calculated from mode plus standard deviation of the grayscale image pixel values in ImageJ. This thresholding method used the algorithm described by Sachse 67. In brief, RyR-JPH2 dual-labelled images were thresholded using the mode plus standard deviation value in ImageJ, while JPH2-α-tubulin images had isodata thresholding applied and masks were generated of each protein label with Python software. From each protein label, the distance to the nearest labelling of the second protein mask was determined using distance maps to generate a distance distribution plot. Distances up to and including zero were summed as a measure of the fraction of protein co-localisation. Since WGA staining was used to identify both the t-tubules and ECM, this co-localisation method would not have been suitable for determining the association between RyR and t-tubules, as it would indicate the fraction of total WGA labelling associated with RyR, rather than t-tubules specifically.Co-localisation analysis was performed on the images of longitudinal cardiomyocytes that were dual labelled for JPH2-RyR and JPH2-α-tubulin, using previously established methods with custom written scripts in Python software53, the corresponding analysis was performed following selection of intra-cardiomyocyte regions (in 63x magnification-acquired images) to obtain cellular labelling density. Tissue labelling density was also assessed for RyR and JPH2.Labelling density of α-tubulin was assessed to determine the degree of microtubule densification. This was achieved by applying isodata thresholding to longitudinally orientated cardiomyocyte images (acquired with 40x magnification) to generate a binary mask. The fractional area of the total tissue section covered by the mask was determined and used as normalised global tissue labelling density measurement. As vascular smooth muscle cells have also been shown to express α-tubulin2. Since the design was unbalanced, statistical significance was examined (P < 0.05) using the Type III Sum of Squares. Differences in myocyte content were also evaluated with the same design except ‘Group’ had three categories . In this design p-values are reported for post-hoc procedure using a mutually-orthogonal set of contrast coefficients. Means are reported with standard error of the mean (SEM) as uncertainty, unless otherwise stated. All n-numbers are reported in corresponding figure legends.Statistical tests were performed using IBM SPSS Statistics v22 or SAS statistical package, with p < 0.05 considered significant. Pearson’s correlation analysis was performed on variables for cardiac trabeculae functional experiments. Differences in structural parameters between failing trabeculae and ventricle wall samples were analysed as a triply-nested design using the Generalised Linear Model (GLM) of the SAS statistical package. Variability within the data arising from each dependent variable of interest  was partitioned between ‘Group’ (‘trabeculae’ versus ‘ventricles’) and ‘Region’  and accounted for multiple samples from the same heart and same region using a third independent variable labelled ‘repetition’. Explicitly, ‘Hearts’ (n = 5) were nested with ‘Group’, ‘Region’ was nested within both ‘Group’ and ‘Heart’ and ‘repetition’ was nested within ‘Group’, ‘Heart’ and ‘Region’. This design accounted for all of the variability in the data, returning a value of zero for Error and a value of unity for rAll datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Supplementary Information


Implementation of the WHO Safe Childbirth Checklist with peer coaching resulted in >90% adherence to 35 of 39 essential birth practices among birth attendants after 8 months, but adherence to some practices was lower when the coach was absent. Implementation of the WHO Safe Childbirth Checklist with peer coaching resulted in >90% adherence to 35 of 39 essential birth practices among birth attendants after 8 months, but adherence to some practices was lower when the coach was absent. Adherence to evidence-based essential birth practices is critical for improving health outcomes for mothers and newborns. The WHO Safe Childbirth Checklist (SCC) incorporates these practices, which occur during 4 critical pause points: on admission, before pushing (or cesarean delivery), soon after birth, and before discharge. A peer-coaching strategy to support consistent use of the SCC may be an effective approach to increase birth attendants' adherence to these practices.We assessed data from 60 public health facilities in Uttar Pradesh, India, that received an 8-month staggered coaching intervention from December 2014 to September 2016 as part of the BetterBirth Trial, which is studying effectiveness of an SCC-centered intervention on maternal and neonatal harm. Nurse coaches recorded birth attendants' adherence to 39 essential birth practices. Practice adherence was calculated for each intervention month. After 2 months of coaching, a subsample of 15 facilities was selected for independent observation when the coach was not present. We compared adherence to the 18 practices recorded by both coaches and independent observers.Coaches observed birth attendants' behavior during 5,971 deliveries. By the final month of the intervention, 35 of 39 essential birth practices had achieved >90% adherence in the presence of a coach, compared with only 7 of 39 practices during the first month. Key behaviors with the greatest improvement included explanation of danger signs, temperature measurement, assessment of fetal heart sounds, initiation of skin-to-skin contact, and breastfeeding. Without a coach present, birth attendants' average adherence to practices and checklist use was 24 percentage points lower than when a coach was present (range: −1% to 62%).10.1056/NEJMoa1701075]. The results showed that the intervention had no significant effect on maternal or perinatal mortality or maternal morbidity, despite having positive effects on essential birth practices.)Implementation of the WHO Safe Childbirth Checklist with coaching improved uptake of and adherence to essential birth practices. Coordination and communication among facility staff, as well as behaviors with an immediate, tangible benefit, showed the greatest improvement. Difficult-to-perform behaviors and those with delayed or theoretical benefits were less likely to be sustained without a coach present. Coaching may be an important component in implementing the Safe Childbirth Checklist at scale. (After publication of this article, the impact results of the BetterBirth intervention were published in the New England Journal of Medicine [volume 377, pages 2313-2324, doi:  Kara.See related article by ––Quality of Care Framework for Maternal and Neonatal Health describes quality care as being safe, effective, timely, efficient, equitable, and people-centered.Childbirth and the first 24 hours postpartum remains a precarious time for both mother and newborn despite improvements in maternal and neonatal mortality over the past 2 decades.,,Essential birth practices that reduce harm and save lives during childbirth are well documented, but all too often, they are not performed. Other areas of health care have used a checklist-based approach to address this “know-do” gap.Essential birth practices that reduce harm and save lives during childbirth are well documented but often not performed.,Checklists are job aids designed to support routine adherence to evidence-based practices, and as such are intended to change health care workers' behaviors.We aimed to assess whether the behavior change intervention of this SCC-based peer-coaching program was associated with improved performance of essential birth practices during facility-based childbirth care. First, we examined birth attendants' adherence to a set of essential birth practices in 60 intervention facilities, as observed by a coach. Then, in a subset of these facilities, we employed independent observers to verify adherence to these practices in the absence of the coach.We aimed to assess whether implementation of the WHO Safe Childbirth Checklist with peer coaching was associated with improved performance of essential birth practices.Global Health: Science and Practice.The BetterBirth Trial was a matched-pair, cluster-randomized controlled trial conducted in Uttar Pradesh, India, to test whether the introduction of the SCC paired with peer coaching—the BetterBirth Program—could reduce maternal morbidity and mortality and perinatal mortality in 120 health care facilities (60 intervention facilities and 60 control facilities receiving standard of care) . The methodology of the trial is available elsewhere,Engagement with facility leaders and government officials at the district and state levelA 2-day motivational launch event of the SCC for facility staffSupport to birth attendants, facility leaders, and government officials through visitation of peer coaches to give regular feedbackThe BetterBirth Program supported adoption and use of the SCC using a 3-pronged implementation pathway 21:EngagThe BetterBirth Program used a 3-pronged approach to support adoption and use of the WHO Safe Childbirth Checklist: engagement with key stakeholders, a motivational launch event, and peer-coaching support to birth attendants.BOXTOOLSThe World Health Organization Safe Childbirth Checklist (SCC) comprises 28 essential birth practices to improve the quality of labor and delivery care.Pulse is an electronic data management system for quality improvement. Coaches entered their observations of birth attendants' behavior into Pulse via mobile-phone apps; Pulse then generated real-time “heat maps” to guide facility staff to identify gaps in care and find effective solutions.STRATEGY: COACHING FOR EMPOWERMENTMotivating birth attendants to change their practices.Observing, recording, and feeding back information about birth attendants' behavior.Supporting birth attendants to problem solve and resolve barriers to essential practices.Goals of coaching:Multilevel: Coaches worked with birth attendants, and coach team leaders worked with facility and district leadership to problem solve and facilitate change across the health system.Collaborative: Coaches and birth attendants had supportive, constructive, respectful, peer-to-peer relationships.Provider-centered: Coaches responded to the needs of the birth attendants and facility and district leaders with whom they worked rather than following a predetermined agenda.Principles of coaching:IMPLEMENTATION: ENGAGE-LAUNCH-SUPPORTEngage: Program goals and strategies were introduced to leadership at the national, state, and district level.Launch: A motivational event at each facility introduced the significance of the tools, explained the coaching strategy, and enlisted the participation of staff in a needs assessment.Support: Nurse coaches supported SCC adoption and quality improvement by making 43 visits to each facility over 8 months . Coach team leaders, who were physicians or public health professionals, accompanied coaches on half of the visits in order to address system-level and supply issues at the facility.SUSTAINABILITY PLANA motivated and respected staff member was selected by facility leadership to be a childbirth quality coordinator (CQC) at each facility. The CQC worked closely with team leaders to improve quality of care and champion the SCC beyond the BetterBirth Program.For a detailed description of the intervention, see Kara, Firestone, Kalita, et al. (2017).The intervention used a coaching strategy as the primary mechanism to encourage behavior change among health care workers. Coaches were trained nurses who worked directly with birth attendants. Coach team leaders were trained physicians or public health leaders who worked directly with facility leaders and government officials and who supervised the nurse coaches. Coaches visited the facility twice weekly during the early stages of the intervention, with the frequency of visits decreasing to once monthly by the end of the intervention, for a total of 43 visits over an 8-month period. Coaches did not provide patient care. Although coaches did not clinically intervene in emergencies, if needed they could escalate emergency situations to be addressed by a medical officer at the facility. Additionally, coaches had the discretion to encourage appropriate referral of a mother or a baby before, during, or after patient observation, as this is directly related to the SCC.On admissionBefore pushing or before cesarean deliverySoon after birth (within 1 hour)Before dischargeBirth attendants were coached to use the SCC at 4 critical “pause points” during childbirth:The paper-based checklist itself was commonly attached to a mother's chart or bedhead ticket for ease of reference and so completed tasks could be checked off for each patient at each pause point. Posters of the SCC were also posted on the wall in the delivery area.,,,The BetterBirth intervention used a behavior change framework to facilitate the adherence to checklist-based essential practices.Uttar Pradesh, India's most populous state, has some of the highest maternal mortality ratios and neonatal mortality rates.The BetterBirth Program was implemented in 60 public-sector facilities, including PHCs, CHCs, and CHC-FRUs, across 24 districts of Uttar Pradesh from December 2014 to September 2016. Site selection for the overall trial and batch-wise implementation is described elsewhere.For the primary analysis, we operationalized the 28 items on the SCC into 43 discrete measures  , as someFor the subanalysis of 15 facilities conducted after the first 2 months of coaching were completed, the outcome of interest was the difference between birth attendants' adherence to essential birth practices in the presence of a coach and birth attendants' behavior documented by independent observers in the absence of the coach. Since not all observed behaviors were recorded in the same way by the coaches and independent observers due to differences in data collection procedures, we included only the 18 overlapping checklist-related behaviors in the analysis .Coaches used standardized tools to document SCC use and adherence to essential birth practices at the 4 pause points listed on the SCC. Deliveries were selected for observation based on if a patient was present and a birth attendant was available to be observed for at least 1 complete pause point. Birth attendants could be observed for 1 or more pause points at each delivery. During the 8-month intervention, coaches attempted to observe each birth attendant at a facility multiple times. Data were first collected on paper forms while observing childbirth and subsequently entered into a mobile phone-based CommCare app  on the same day but after the coach left the patient care area. Practices were coded as either “completed” (green), “completed after prompt” (yellow), or “not completed” (red) and transformed into a standardized heat map report, displayable on a mobile device for coaches and team leaders to report back to birth attendants and facility leadership on adherence to the SCC and to essential birth practices.For the subanalysis, independent observers assessed SCC use and birth attendants' adherence to essential birth practices. Starting during the eighth week of coaching, independent observers visited facilities on non-coaching days to record adherence to essential practices. Independent observers collected data for a period of 6 to 12 weeks, depending on the delivery load of the facility, with a goal to reach 240 pause point observations per facility. Independent observers selected any case for which a pause point could be observed from start to finish. A mother was observed for as many pause points as possible. Data collected by independent observers were considered a proxy for birth attendant behavior under everyday conditions at the facility.Independent observers were nurses trained in childbirth who used a standardized tool to record behavioral data. Intensive training to ensure data quality was provided.We analyzed adherence to each of the 43 coach-observed practices by month over the 8-month intervention. First, we calculated an adherence proportion for each behavior based on the number of times a behavior was completed divided by the number of times that a behavior was expected to be completed at a given pause point. We then plotted this proportion across the 8 months of the intervention to understand how adherence to essential practices changed over time.Minimal improvement (<15 percentage-point absolute difference)Moderate improvement (15 to 24 percentage-point absolute difference)Major improvement (≥25 percentage-point absolute difference)Since coaches visited facilities only fortnightly or monthly by the seventh and eighth months of the intervention, data from these 2 months were combined (represented as Month 7). We used percentage-point differences to compare adherence during Month 7 versus Month 1 and allocated behaviors into 3 categories:P<.05.We developed a logistic regression model for each behavior to assess the probability of birth attendants' adherence to a given behavior across the 8 months of the intervention, controlling for facility clustering using dummy variables for facility. We calculated odds ratios and 95% confidence intervals to test how adherence changed over time. Results were considered statistically significant at Minimal difference (<15 percentage-point absolute difference)Moderate difference (15 to 24 percentage-point absolute difference)Major difference (≥25 percentage-point absolute difference)For the subanalysis, we calculated adherence to each of the 18 measured practices recorded by both nurse coaches and independent observers. We used percentage-point differences to compare adherence when a coach was present versus absent. Each behavior was categorized as having a:A Rao-Scott chi-square test was used to adjust for clustering within facility when comparing the overall proportion of practices completed between coaches and independent observers. We also compared adherence rates reported by coaches at the 15 sites of the subanalysis with the 45 sites not included in the subanalysis to assess differences between the 2 groups. All data analyses were conducted using Stata SE 13.1 for Mac and Microsoft Excel 2011.As a strategy to improve the quality and effectiveness of the BetterBirth Program, we used an explanatory qualitative study. We presented adherence rates documented by coaches and independent observers to members of the field implementation staff during a week-long workshop in September 2016. Successes and challenges related to birth attendants' adherence to individual behaviors were discussed and documented.The BetterBirth Trial, including data for this analysis, was approved by the Harvard T.H. Chan School of Public Health Institutional Review Board, the World Health Organization Ethics Review Board, the Population Services International Research Ethics Board, the Community Empowerment Labs Ethics Review Committee, and the Jawaharlal Nehru Medical College-Belgaum Ethics Review Board. All activities were conducted in partnership with the Government of Uttar Pradesh.For coaching, each facility and birth attendant formally agreed to participate in the BetterBirth Program as a quality improvement initiative at the beginning of the intervention. Coaches accompanied birth attendants during their work shift and documented practices during patient-care activities at the facility. Coaches did not collect any patient identifiers.For independent observation, each facility and birth attendant formally agreed to participate in the study. Since these data were collected solely for the purpose of research as part of the larger BetterBirth Trial, and identifying information of the mother was collected, a more rigorous consent process was used. Prior to each observation, independent observers verbally confirmed that the birth attendant agreed to be observed. Patients signed written consent to have independent observers present during their care.Supplement 1. By the final month of the intervention, 35 of 39 essential practices had achieved >90% adherence in the presence of a coach (Supplement 2), compared with only 7 of 39 practices that had achieved this level of adherence during the first month  and after delivery , measurement of baby's temperature , measurement of mother's temperature before  and after delivery , and measurement of fetal heart sounds on admission  (Supplement 2). More moderate increases were seen across other practices, including oxytocin administration within 1 minute of delivery , skin-to-skin infant care , and hand hygiene before delivery . Across all 39 behaviors, improvements ranged from an absolute increase of 2 percentage points to 51 percentage points from the first to the final month.Essential birth practices with the greatest absolute increase in adherence over time included explaining danger signs to the mother or to her birth companion on admission  and blood pressure  before delivery, as well as administration of bacille Calmette-Guérin (BCG) vaccine  and the oral polio vaccine (OPV)  to an infant before discharge.Behaviors that did not attain an adherence rate of 90% or above for any month of the intervention included measurement of a mother's temperature , before delivery , after delivery , and before discharge .Checklist use at each pause point increased between the first and final month of coaching, including checklist use on admission . Of the essential birth practices recorded by both coaches and independent observers, a minimal absolute difference (<15 percentage points) in levels of adherence was observed for preparation of supplies including cord ligature, neonatal bag and mask, mucus extractor, pads and clean towel, weighing of the baby, glove use during delivery, and immediate skin-to-skin care. A moderate absolute difference (15 to 24 percentage points) was observed across 3 behaviors, including practices such as oxytocin administration within 1 minute of birth and breastfeeding within 1 hour of birth. A major absolute difference (>25 percentage points) was seen with hand hygiene, measuring the baby's temperature, and measuring the mother's blood pressure and temperature. We found no major differences between the coach-recorded adherence rate in the 15 facilities of the subanalysis versus the 45 facilities not in the subanalysis.In the subset of 15 facilities, independent observers documented essential practices after 2 months of coaching on 1,277 deliveries at 1 or more pause points, while coaches observed 736 deliveries over the same 12-week period in the same facility. There was an absolute difference of 24 percentage points (range: −1 to 62 percentage points) in the proportion of the 18 practices completed when the coach was present versus absent  is an extremely busy time for birth attendants; birth attendants frequently regarded referring to a checklist as more of a burden or barrier to providing timely care at this point. Because the SCC was not always well integrated into the existing patient record (“bedhead ticket”), it was easily overlooked. Practices that improved at pause point 2, namely ensuring the necessary supplies were ready at the bedside, likely improved through advance preparation of birth trays with all necessary supplies at the start of the shift.This analysis had a number of limitations. Given the high level of adherence reported by coaches, it is possible that coaches may have overstated birth attendants' adherence due to social desirability or fear of bad reviews if improvement was not seen. Additionally, although we assume that adherence to essential practices recorded by an independent observer reflects normal day-to-day care, it is possible that we experienced a Hawthorne-like effect in our measurements. However, Leonard and Masatu found that while having an observer present will cause clinicians to positively change their behavior when first observed, clinicians return to their typical practices after 10 to 15 observations.Observations were structured to record only whether clinical practices occurred, such as taking blood pressure and listening for fetal heart sounds. We did not determine the accuracy of measures taken by birth attendants or whether clinical practices, such as neonatal resuscitation, were conducted correctly. A forthcoming analysis will look at whether the intervention was associated with reductions in maternal and neonatal mortality and maternal morbidity.Coaches and independent observers were not able to document adherence to essential practices at all pause points for all deliveries. For example, neither coaches nor independent observers made observations at night due to safety concerns for study staff; practices at night may be different than those during the day. Coaches worked with birth attendants and facility staff in all intervention facilities to standardize discharge procedures, to improve the quality of recovery room offerings, and to encourage women and their families to stay for the minimally recommended 24 hours post-birth. Nevertheless, many women left facilities within 6 hours after birth due to family pressures and lack of food availability, and these departures were generally against medical advice. Because women often leave without informing facility staff, it was not possible for independent observers to observe discharge procedures in a standard manner across all study facilities.Due to the small number of facilities included in the subanalysis, we were unable to confidently assess if particular facility characteristics or programmatic factors were associated with these differences. In comparing the coach-recorded adherence rate in the 15 facilities of the subanalysis versus the 45 facilities not in the subanalysis, we found no major differences between the 2 groups.While we advanced the understanding of how and when checklists are used in the presence or absence of a peer-coach, there are many areas to explore further. As part of the randomized controlled trial, the BetterBirth Program used a standard number of coaching visits with a prescribed frequency across all facilities. Further inquiry is needed on how the frequency and length of coaching can be structured to maximize sustained behavior change. Additional research is also needed to assess if there is a threshold level of adherence to essential practices that would be associated with improvement in health outcomes since consistent and complete adherence to the SCC and essential birth practices may not be likely in standard clinical practice. More research is needed to understand if there are specific programmatic, facility, or maternal characteristics that account for differences in adherence and how the SCC with coaching could operate as a team-based vs. individual-level intervention for health care workers. Sustainability will be assessed in a forthcoming analysis that explores adherence to essential practices 12 months after the start of the coaching program. Finally, a cost analysis of delivering this intervention in the context of Uttar Pradesh is currently underway.Further research is needed to discover how the frequency and length of coaching can be structured to maximize sustained behavior change.We conclude that coaching was effective in increasing the uptake of birth attendants' essential birth practices when a coach was present, but adherence to some behaviors was reduced when the coach was absent. These findings will help to optimize the use of peer coaches and improve overall implementation of the SCC at future facilities, to improve the quality of care available during childbirth, and to understand how to improve behavior change interventions with health care workers.


Temporal artery is superficially exhibited and easily traumatized. Rarely, a minor and blunt trauma, especially in elderly who are under anticoagulants, can cause a pseudoaneurysm. Diagnosis should be based, primarily, on history and physical examination and secondarily on duplex ultrasound scanning which will lead to confirmation and preoperative planning. The therapeutical plan consists of surgical ligation and excision of the aneurysm. Surgery can be performed under local anesthesia with no postoperative major or minor complications. Endovascular approach consists of catheter embolization and remains a second option due to the risk of complications and the inconclusive results. On this review, authors present a case of an 80-year-old male with a pseudoaneurysm of superficial temporal artery. Temporal artery is superficially exhibited and easily traumatized. Rarely, a minor and blunt trauma, especially in elderly who are under anticoagulants, can cause a pseudoaneurysm. The diagnosis is based on history and physical examination. However, duplex ultrasound scanning , contrasAn 80-year-old male presented to the emergency department of our hospital with a mass on the left frontal bone, after a minor fall on the ground ten days ago. The patient was under anticoagulants for his chronic atrial fibrillation at therapeutic levels. Examination revealed pale skin with necrotic components overlying a 1.56 × 1.12 cm pulsatile mass on the temporal side of the left frontal bone  layers of the arterial wall. Pseudoaneurysm of the superficial temporal artery, caused by minor blunt trauma, was first described by Bartholin in 1740 and ever since represents the 1% of all traumatic aneurysms .The diagnosis is based on history taking and physical examination. The typical case starts with a minor blunt trauma to the temple one to six weeks ago , 7. The Differential diagnosis includes abscess, cyst, lipoma, simple hematoma, vascular tumor, and true aneurysm , 3. In oThe standard therapeutic plan is surgery. However, “watch and wait” treatment was suggested, but the long duration of symptoms, headaches, and risk of rupture discouraged its use . SurgicaThe case presented illustrates and binds the preoperative ultrasound image of the pseudoaneurysm with the real, operative one.Temporal artery pseudoaneurysm is a rare clinical presentation. Diagnosis should be based, primarily, on history and physical examination and secondarily on duplex ultrasound scanning which will lead to confirmation and preoperative planning.The therapeutical plan consists of surgical ligation and excision of the aneurysm.We conclude the following:


The outcome of preterm infants has been varied in different hospitals and regions in developing countries. Regular clinical monitor are needed to know the effects of health care. This study aimed to describe the survival and morbidity rates of extreme to very preterm infants in 15 neonatal-intensive care hospitals in China.Data were collected from January 1, 2013 to December 31, 2014 for preterm neonates with gestational age (GA) between 24 and 31 complete weeks born in hospitals from our collaborative study group. The primary outcomes were survival and major morbidities prior to hospital discharge. Major morbidities included bronchopulmonary dysplasia (BPD), intraventricular hemorrhage (IVH), necrotizing enterocolitis (NEC), retinopathy of prematurity (ROP), patent ductus arteriosus (PDA) and sepsis. Mutivariate logistic regression was used to analyze the risk factor influencing on the outcomes.P < 0.05). Infants at the lowest GAs had a highest incidence of morbidities. Overall, 58.5 % had respiratory distress syndrome, 12.5 % bronchopulmonary dysplasia, 3.9 % necrotizing enterocolitis, 15.4 % intraventricular hemorrhage, 5.4 % retinopathy of prematurity, 28.4 % patent ductus arteriosus, and 9.7 % sepsis. Mortality and morbidity were influenced by gestational age , birth weight , SGA , Apgar score <7 at 5 min . The survival rate was increased with more prenatal steroid use .The preterm birth rate was 9.9 % (13 701/138 240). The proportion of extreme to very preterm infants was 1.1 % and 11.8 % respectively. The survival rate prior to discharge was increased with increasing GA . Rate of survival and without severe morbidity according to GA were 0 at 24 weeks, 8 % at 25 weeks, 60.6 % at 26 weeks; 53.2 % at 27 weeks; 62.3 % at 28 weeks; 67.9 % at 29 weeks; 79.1 % at 30 weeks, 85.8 % at 31 weeks respectively. Rate of antenatal steroid use was 56 %. The antenatal steroid use was lower in GA < 28 weeks infants than that in GA between 28 and 32 weeks (28–44.3 % vs 49.7–60.1 %, Although most of the preterm infants with GAs ≥26 weeks survived, a high complication in survivors still can be observed. Rate of survival of GAs less than 26 weeks was still low, and quality improvement methods should be used to look into increasing the use of antenatal steroids in the very preterm births. In 2010, the national preterm birth rate was estimated at 14.9 million infants or more than one in ten of all infants in 184 countries –5. In orAlthough China is one of the most populous countries in the world, the outcomes of newborn infants and the associated risk factors are only estimated by vital statistics. It may address public health concerns with regards to overall mortality and related causes, but does not answer questions pertaining to specific perinatal and neonatal care, gestation specific mortalities and morbidities, risk factors, as well as resuscitation and postnatal care of these high risk newborns . In ChinWe established a collaborative study group including nine general hospitals and six maternal and children’s hospitals from major cities of 11 provinces in China. All the participating hospitals are tertiary neonatal care centers and have their own maternity wards in local region, which are representatives of medical units providing newborn intensive care in their respective areas. Nine neonatal intensive care units (NICU) of all the participating hospitals provide regional perinatal and neonatal retrieval services. In this prospective study design, our study was approved by the Institutional Ethics Committee of the Army General Hospital, the People’s Liberation Army (PLA). The approval of data collection was also obtained at each of the collaborating hospitals. This study was also conducted as part of the Neonatal Research Network of the Chinese Neonatologist sub-association of the Chinese Medical Doctor Association. Coordination for this study was based at the Army General Hospital of PLA.+0 to 31+6 weeks were studied, excluding those with congenital anomalies. Outborn infants were also excluded in this investigation. Researchers collected maternal pregnancy/delivery data soon after birth and infant data from birth to death or discharge. To minimize bias among centers and investigators, we provided comprehensive and systematic training for the staffs committed to the survey. Data collection by research staff at each of the collaborating hospitals was supervised by the NICU director, who was responsible for the quality assurance. The quality control was mainly focused on completeness and accuracy of the contents in records by each unit investigators, along with visiting, telephone or email communication for verification and correction of the data. Records were also subjected to collaborative center to check for accuracy and completeness and returned for correction if needed.Infants born alive at hospitals of our collaborative study group in 2013–2014 with GAs of 24GA was determined by the most experienced obstetric physicians estimated by the date of last menstrual period. Preterm infants was defined as a GA <37 completed weeks. Extremely preterm infant (EPI) was defined as GA < 28 weeks and very preterm infants (VPI) was defined as GA < 32 weeks. Small for gestational age (SGA) infant was defined as birth weight of <10th percentile for gender and GA, by using growth charts published by Fenton and Kim [Morbidities were defined according to the earlier publications , 9–11, iMortality was defined as death in the delivery room or in NICU before discharge. Infants were considered as having a major neonatal morbidity if they had at least one of the following complications before discharge: moderate to severe BPD, severe IVH, high grade NEC, severe ROP and sepsis. Combined outcome refers to infants who died or survived with a major neonatal morbidity. Infants who died in delivery room were excluded from analyses focus on morbidity diagnosed later after birth.χ2 or Fisher’s exact test wherever appropriate.All statistical analyses were conducted by using SPSS 16.0 software . Continuous variables are described as means and SD. Categorical variables are presented as rates and odds ratio with 95 % confidence intervals (CI). Comparison between continuous variables was conducted by using a One-way ANOVA. Univariate analyses on categorical variables were made by using a 2-tailed Pearson P < 0.05 was considered statistically significant.Multivariate analyses were performed separately by using logistic regression to analyze the risk factors of mortality and morbidity in preterm infants. GA, gender, maternal perinatal complications such as: Gestational diabetes, Placenta previa, placental abruption, hypertensive disorder complicating pregnancy (HDCP) and Premature rupture of membrane (PROM), multiple birth, delivery mode, Small for GA, Apgar score and antenatal steroid use were included in the analysis. During a two-year period, there were 138 240 alive born infants including 13 701 preterm infants from the 15 participating hospitals. The incidence of preterm birth rate was 9.9 %. Of these, there were totally 1 760 infants born before 32 weeks . The number of preterm infants increased with increasing GA. See Table p = 0.000). The majority (89.7 %) of women received antenatal care. Rate of antenatal steroid use increased with advancing GA, from 0 at 24 weeks to 44.3 % at 27 weeks and 49.7 % to 59.6 % at 28 to 31 weeks. The rate of complete antenatal steroid use was 32.3 % and also increased with GA.  male infants, 514(29.2 %) multiple births, and 116(6.6 %) small for gestational age infants. Infants with Apgar scores of <7 at 5 min represented 9.0 % of all EPI and VPI, and the incidence was high in lower GA infants. Caesarean sections were done in 710 (40.3 %) infants and the rate was increased with increased GA, with 9.1 % at 24 weeks and 50.3 % at 31 weeks  1.149–1.187; 54/1 760 (3.1 %) live born infants died during the first week after birth, ranging from 54.5 % at 24 weeks, 3.5 % at 28 weeks to 0.3 % at 31 weeks of gestation. In 22.2 % of the early deaths, a discontinued treatment policy was made according to parents’ opinion before birth: 45.5 % at 24 weeks of gestation, 15.2 % at 25 weeks, 3.0 % at 26 weeks of gestation. In total, there was no infant survived at 24 weeks gestation. Rates of survival before discharge increased with the increase of GA, from 28 % at 25 weeks, 84.8 % at 26 weeks, 83.5 % at 27 weeks, 87.4 % at 28 weeks, 90.7 % at 29 weeks, 93.9 % at 30 weeks and to 96.0 % at 28 weeks . There was a large increase in survival of greater than 50 % from 25 week to 26 gestational weeks. The survival rate was over 90 % in 29 weeks, 30 weeks and 31 weeks of gestation. (Table p = 0.0047), birth weight , and more antenatal steroids use . Infants with SGA and Apgar < 7 at 5 min died more often than infants with infants who have no these risk factors .  and birth weight . SGA, HDCP, placental abruption, placenta previa, multiple birth, Apgar < 7 at 5 min and maternal fever before delivery were associated with increased risk of morbidity.Mutivariate logistic regression analysis indicated that the morbidity of GA < 32 weeks preterm infants was decreased with increasing gestational age  as the reference for morbidity and mortality, the current study assessed outcomes in accordance with GA. BW specific data may not be accurate because of the limited growth of more mature infants. Appreciation of GA-based outcomes is especially valuable for prenatal counseling and physician/family decision-making . GA was 6. In Guangdong province of China, the survival rate of EPI was 20 % at 24 weeks、15.9 % at 25 week、27.2 % at 26 week and 48.4 % at 27 weeks. According to birth weight the survival rate of ELBW infants were also vary from 38.5 to 87 % in China [This study on outcome of 24 to 31 weeks premature infants in China showed a survival rate of 91.2 %  among live born infants. 74.6 % of infants had discharged home without any severe morbidity during their hospital stay. The survival rate was slightly increased than that of reported in previous data in China, although the survival rate of EPI and VPI vary greatly. A study from Jiangsu province in China in 2010 had reported the survival rate of 25 weeks to 31 weeks was 0, 0, 58.3 %、66.7 %、60.3 %、82.9 % and 79.2 % respectivelyin China . Isayamain China . New Souin China . Our datThe incidence of hospital morbidity rate was still high among EPIs, and morbidities contribute to poor neurodevelopmental outcomes. Our data showed the combined outcome of death or ≥1 major neonatal morbidity in survivors was 100 % at 24 weeks and 92 % at 25 weeks, decreased to 39.4 % among 26 weeks and 14.2 % at 31 weeks. This is a big gap compared to data from other countries , 19. In Many studies showed the major neonatal morbidities are predictive of long-term neurodevelopmental disorders such as cerebral palsy, cognitive impairment, hearing loss, and visual problem –22. We cMultiple factors were associated with neonatal mortality and morbidity. In current study, it was identified that perinatal factors including antenatal steroids, gender, SGA, low Apgar score at 5 min, HDCP, placental abruption, placenta previa and multiple birth have an impact on the survival rate or morbidity rate. Previous study showed antenatal steroids use can improve the perinatal health in preterm infants , 25. OurThe strengths of this study are the prospective, GA-based and a multicenter survey. The design of the study makes it was comparable to other GA-based cohort studies of extremely and very premature infants and provides information on the international position. Adopting methods from other countries in China might improve outcomes by analyzing multicenter data periodically for benchmarking, and assessing quality improvement initiatives. Our results may be valuable in counseling families and making novel interventions in China. We acknowledge inevitably there are several limitations. We only presented short-term outcomes in this study. The long-term neurodevelopmental outcome analysis is needed for this cohort study. The other disadvantage of this dataset is that the analysis is restricted to the information collected. We did not present any other data that might influence the outcomes of preterm infants such as maternal age, socio-economic status and details in clinical management strategy. Finally, this study was limited to liveborn preterm neonates of 24 to 31 complete weeks admitted to NICU, and we did not take into account any stillbirths and more immature babies less than 24 weeks. It hard to distinct if the cause of stillbirth at 24–25 weeks gestation was not influencing by the decisions of parents.In summary, we presented the hospital outcomes of extreme to very preterm infants from well-selective regional hospitals in China. Our study highlighted that many perinatal factors were associated with the mortality and morbidity besides gestational age and birth weight. Infants born at 24 and 25 weeks’ gestation were at higher risk of death than infants born at more than 26 weeks’ gestation. There is a need for further discussions on management for infants born at 24 and 25 weeks’ gestation.


Chiranjeevi Yojana (CY) was implemented in 2005. Institutional birth is provided free of cost at accredited private health facilities to women from socially disadvantaged groups (eligible women). CY has contributed in increasing facility birth and providing substantially subsidized  birth care; however, the retention of mothers in this scheme in subsequent child birth is unknown. Therefore, we conducted a study aimed to determine the effect of previous utilization of the scheme and previous out of pocket expenditure on subsequent child birth among multiparous eligible women in Gujarat.In Gujarat, India, a state led public private partnership scheme to promote facility birth named This was a retrospective cohort study of multiparous eligible women (after excluding abortions and births at public facility). A structured questionnaire was administered by trained research assistant to those with recent delivery between Jan and Jul 2013. Outcome of interest was CY utilization in subsequent child birth (Jan–Jul 2013). Explanatory variables included socio-demographic characteristics (including category of eligibility), pregnancy related characteristics in previous child birth, before Jan 2013,  and type of child birth in subsequent birth. A poisson regression model was used to assess the association of factors with CY utilization in subsequent child birth.n = 289), 182 (63%) subsequently utilized CY and 33 (11%) gave birth at home; whereas those who did not utilize CY (n = 708) had four times higher risk (40% vs. 11%) of subsequent child birth at home. In multivariable models, previous utilization of the scheme was significantly associated with subsequent utilization (adjusted Relative Risk (aRR): 2.7; 95% CI: 2.2–3.3), however previous out of pocket expenditure was not found to be associated with retention in the CY scheme.Of 997 multiparous eligible women, 289 (29%) utilized and 708 (71%) did not utilize CY in their previous child birth. Of those who utilized CY (Women with previous CY utilization were largely retained; therefore, steps to increase uptake of CY are expected to increase retention of mothers within CY in their subsequent child birth. To understand the reasons for subsequent child birth at home despite previous CY utilization and previous zero/minimal out of pocket expenditure, future research in the form of systematic qualitative enquiry is recommended. Of the total health expenditure in India, out-of-pocket payments contribute 51%, the Government contributes 33% and minor proportion is paid by third party such as insurance companies , 3. ReseJanani Suraksha Yojana (JSY or safe motherhood program)”implemented nationwide. It is a conditional cash transfer scheme where the mother is paid a fixed amount after giving birth in a public health facility   .To date there are several studies on various aspects of CY including its utilization –19. In oRetention of mothers in this scheme in subsequent child birth is unknown. It would be pertinent for the state to know how many continue with subsequent CY utilization after previous CY utilization and the factors associated with it. Therefore, the present study aimed to determine the effect of previous utilization and previous OOPE on subsequent utilization among eligible CY beneficiaries in Gujarat, India. Specific objectives were to determine the i) number (proportion) with previous CY utilization stratified by subsequent CY utilization; ii) median OOPE in previous child birth stratified by subsequent CY utilization; iii) factors associated (including previous utilization and OOPE) with subsequent CY utilization.This retrospective cohort study was part of a large scale community-based Maternal Health India (MATIND) project aimed to study the CY scheme in the state of Gujarat . A subseGujarat state (population 60.4 million) is comprised of 33 districts, the average population of a district is two million. Districts are further divided into 10–20 blocks (sub-districts) of approximately 100,000 to 200,000 people. These districts have varying human development indices and different population compositions , 22. TheThe government pays Indian Rupee ₹4000 per child birth (approximately USD$ 67) directly to the provider, irrespective of its type i.e. normal or operative (assisted or Caesarian section). Eligible beneficiaries have to visit the accredited CY facility within district with eligibility documentation to utilize the CY services free of cost. As of now, about 1 million births have been covered under CY , 15, 18.Community-based MATIND survey was conducted in three heterogeneous districts from the western, central and eastern belts of the Gujarat state   Fig. 1)1). The dThe study population included eligible women in the study districts who delivered between July 2013 and November 2014. As CY providers were not evenly distributed geographically throughout the district, blocks in each district were selected to represent areas where there were no, low (one or two providers) or high number (more than two per block) of CY providers. In brief, in each of the 3 districts, 3–5 blocks were selected purposively. A list of all the villages in these blocks was compiled using the criteria: village population more than 1,000 and less than 2,500, greater than 40% BPL population and scattered all over the block. From the list, 142 villages were selected randomly to cover approximately 300,000 populations. The sampling methodology of MATIND survey has been described elsewhere .n = 990), abortions (n = 39) and births in public health facility (n = 447), the study population included in this study were 997 multiparous eligible beneficiaries  and ‘previous child birth’ (Gave child birth prior to January 2013) in the paper to denote the same.CY utilization was defined as giving child birth in an accredited facility and receipt of either fully or partially subsidized intra-partum care. OOPE was defined as the total expense made by the woman/or her family including pregnancy care cost and other costs related to the birth . OOPE was expressed in USD ($) after discounting for inflation between year of child birth and 2016.CY non-utilization comprised three different groups of women: (i) those who did not receive any benefit but delivered in a CY accredited facility, (ii) those who delivered in a non-accredited private facility or (iii) those who gave birth at home.Details of data collection in MATIND survey has been described elsewhere . House tData were double-entered and validated in REDCap . The datp-value of <0.2 in the unadjusted analysis were included in the regression model. Variables with high collinearity with birth type in subsequent child birth were excluded from the model. Adjusted RRs were reported with 95% confidence intervals (CI).Frequencies and proportions  were used to summarize CY utilization. The median and interquartile ranges (IQR) were used to summarize the OOPE in the previous child birth. Unadjustedanalyses were performed to assess the association  of factors with CY utilization in subsequent child birth. Variables with n = 289), 182 (63%) utilized CY and 33 (11%) delivered at home in their subsequent child birth. Of those who did not utilize (n = 708), 136 (19%) utilized CY in their subsequent child birth. Women with previous CY utilization had three time higher incidence (63% vs. 19%) of subsequent CY utilization. Women with previous CY non-utilization had four times higher incidence (40% vs. 11%) of subsequent child birth at home  utilized and 708 71%) did not utilize CY in their previous child birth. Of those who utilized CY (% did notMedian (IQR) OOPE in previous child birth among those with subsequent CY utilization was $0  USD and among those with subsequent CY non-utilization was $17  USD Table .Table 2PPrevious CY utilization was significantly associated with subsequent CY utilization after adjusting for potential confounders . Zero OOPE in previous child birth was associated with subsequent CY utilization; however, after adjusting for confounders, there was no significant association. Higher Education (secondary or more), belonging to both eligibility categories (BPL and ST) and subsequent vaginal birth were also significantly associated with subsequent CY utilization  and this was intriguing. This is a very important finding that our quantitative analysis could not explore further. Our study was not designed to explore the reason of shifting towards home birth despite utilizing CY in previous child birth.Third, even though previous OOPE was not found to have an effect on subsequent CY utilization, there was some expenditure which has scope for further minimization. This is supported by the finding in our previous study that OOPE among CY beneficiaries was higher than OOPE among women with child birth in public facility [facility . Zero OOfacility , 7, 26. facility , 18, 19.Fourth, we found that women who had dual eligibility criteria had higher incidence of CY utilization which is in line with finding of our previous study [us study . Presentus study .This study had several strengths. This is the first time that a cohort design has been used to study a state led public private partnership scheme’s utilization. Previous experience is known to have a significant effect on the heath belief system of an individual ; the samOur study has few limitations. The cohort was in built into data from the community-based survey. Hence, data regarding the previous child birth was collected retrospectively at the time of the survey and recall bias on OOPE cannot be ruled out .First, the CY scheme is performing well in retaining women and reducing OOPE. However, the overall demand side utilization has scope to improve. Steps to increase uptake of CY are expected to increase retention of mothers within CY in their subsequent child birth. In our previous study “failure to provide the required documentation” at the time of child birth was one of the major factors associated with non-utilization [e-Chiranjeevi” to overcome the issue of “failure to provide the required documentation”. This was also recommended in our previous paper [e-Chiranjeevi” may be an extension of the existing Mother & Child Tracking System (eMAMTA) of Gujarat where mother’s information from antenatal registration to child birth to 5 years post child birth is tracked against a unique MAMTA ID. During antenatal registration, the eligibility documentation pertaining to either CY or JSY may be collected and uploaded. These documents may be accessed by the accredited private providers on entering the unique MAMTA ID. This way, the mother may visit a facility of her choice (either public or accredited private) with her unique MAMTA ID. “E-Chiranjeevi” by overcoming “failure to provide the required documentation” at child birth may have two fold benefits: i) increasing the utilization of the scheme and ii) retaining mothers within CY in subsequent delivery.There are some implications for policy and practice. lization . We recous paper . “e-ChirSecond, systematic qualitative inquiry may be essential to understand both attrition from CY and more importantly the persisting phenomenon of home child birth despite previous CY birth, minimal previous OOPE and substantial financial support in these communities covered by CY. Factors associated with home births have been studied before. However, unknown factors [ factors , 32 relaPrevious CY program utilization resulted in women using the program for their subsequent child birth. However it is essential to address the significant proportion of women who were previously not covered under CY and underwent child birth at home. Systematic qualitative enquiry is recommended to understand why women subsequently delivered at home despite previous CY utilization.


Birth by cesarean section is associated with increased risks of immune disorders. We tested whether establishment of immune function at birth relates to mode of delivery, taking other maternal and infant characteristics into account.Using a prospectively collected database, we retrieved information on maternal and infant characteristics of 6,014 singleton infants delivered from February to April 2014 in Stockholm, Sweden, with gestational age ≥35 weeks, Apgar scores ≥7, and without congenital malformations or any neonatal morbidity. We linked our data to blood levels of T-cell receptor excision circles (TREC) and κ-deleting recombination excision circles (KREC), determined as part of a neonatal screening program for immune-deficiencies, and representing quantities of newly formed T- and B-lymphocytes. Multivariate logistic regression was used to calculate odds ratios (OR) with 95% confidence intervals (CI) for participants having TREC and KREC levels in the lowest quintile. Multivariate models were adjusted for postnatal age at blood sampling, and included perinatal , and maternal characteristics .Low TREC was associated with cesarean section before labor (adjusted OR:1.32 [95% CI 1.08–1.62]), male infant sex (aOR:1.60 [1.41–1.83]), preterm birth at 35–36 weeks of gestation (aOR:1.89 [1.21–2.96]) and small for gestational age (aOR:1.67 [1.00–2.79]). Low KREC was associated with male sex (aOR:1.32 [1.15–1.50]), postterm birth at ≥42 weeks (aOR:1.43 [1.13–1.82]) and small for gestational age (aOR:2.89 [1.78–4.69]). Maternal characteristics showed no consistent associations with neonatal levels of either TREC or KREC.Cesarean section before labor was associated with lower T-lymphocyte formation, irrespective of maternal characteristics, pregnancy, and neonatal risk factors. The significance of a reduced birth-related surge in lymphocyte formation for future immune function and health remains to be investigated. The worldwide rate of Cesarean section (CS) has quadrupled in less than two decades, making CS the most common surgical procedure performed in women of child-bearing age , 2. WherCompared with vaginal delivery, CS delivery is associated with increased risks of immune disorders later in life, such as asthma and allergies –10, typeWe have previously discovered that hematopoietic stem cells (CD34+) could have a cellular memory of birth, expressed as an epigenetic imprint . AccordiQuantitative data on T-cell receptor excision circles (TRECs) and κ-deleting recombination excision circles (KRECs) are used as part of a neonatal screening program for detection of primary immune deficiencies  and reflThis population-based cohort was based on 7,174 singleton, live-born infants, delivered in February through April 2014 in Stockholm county, Sweden. We restricted the study population to infants born at 35 completed weeks of gestation and later, with Apgar scores ≥7 at 1, 5 and 10 minutes, and without any record of ICD-10 diagnoses for congenital malformations or neonatal morbidity (the Q- and P-chapters in ICD-10). After these exclusions of 1,160 infants, the study population comprised 6,014 infants and their mothers, Using the Stockholm-Gotland Obstetric Data Base, we retrieved maternal, pregnancy, delivery and infant characteristics. Information in this data base was prospectively collected; from the first antenatal visit that occurred during the first 13 gestational weeks in 90% of pregnancies, until postpartum hospital discharge.Mode of delivery was categorized as non-instrumental vaginal delivery, instrumental vaginal delivery, emergency or elective CS. Elective CS was defined as CS before onset of labor.2) in early pregnancy was calculated from self-reported height and measured weight at the first antenatal visit. Maternal diabetes or hypertensive disease was defined as recorded ICD-10 diagnoses: diabetes included pre-gestational (ICD-10 codes E10-E14 and O240-O243) and gestational diabetes (ICD-10 code O244). Hypertensive diseases included pre-gestational  and gestational hypertension (ICD-10 code O13), and preeclampsia (ICD-10 codes O14-O15). Gestational age was determined by the following hierarchy: a) date of embryo transfer (2.8%), b) early second trimester ultrasound (96.1%), c) date of last menstrual period (1.1%), and d) from a postnatal assessment (<0.1%). Birth weight for gestational age was estimated according to the sex-specific Swedish reference curve for normal fetal growth. A normal birth weight for gestational age was defined as a birth weight between the 3rd and 97th percentiles. Postnatal age at blood sample was calculated by subtracting sampling date and time from birth date and time.Maternal age was computed from the national identification number (mother’s birth date) at the date of delivery. Parity and self-reported smoking habits were based on information obtained at the first antenatal visit. Maternal body mass index  in relation to TREC- and KREC-quintiles. In addition, linear regression models were used to investigate whether the log-transformed TREC- or KREC-values were associated with our perinatal covariates. We also used logistic regression to calculate odds ratios with 95% confidence intervals for the risk of having a TREC- or KREC-value within the lowest quintile (Q1), with the other quintiles (Q2-Q5) as the reference category. As postnatal age for blood sampling was strongly associated with TREC- and KREC-values, we investigated whether postnatal age for sampling modified associations. A statistical interaction was pre-defined as an interaction term with p-value of less than 0.05. Finally, sensitivity analyses were performed in the subset of infants with mothers without recorded diabetes and hypertensive disease.The SAS software package version 9.4  was used for statistical analyses. The study was approved by the Regional Ethical Review Board in Stockholm, Sweden (No. 2014/1292-31/4). Informed consent to the study was waived as all participants previously had agreed on participation in the neonatal screening program and to data collection in the Stockholm-Gotland database, both for clinical and research purposes.Neonatal blood levels of TREC ranged from 16–622 copies/3,2 mm punch with a median value of 166 copies/3,2 mm punch; KREC blood levels ranged from 2–638 copies/3,2 mm punch, with a median of 97 copies/3,2 mm punch.In the study population of 6,014 singleton infants born between 35–42 gestational weeks, low TREC- and KREC-levels (within the first quintile) were more commonly found after elective CS, in male infants, after preterm birth (35–36 weeks), in infants with low birth weight for gestational age, and when the blood sample was taken within the first 3–4 postnatal days . In addiThe proportions of infants with TREC- and KREC-levels in the lowest quintile decreased with infant postnatal age and therefore, all logistic regression models were adjusted for postnatal age at blood sampling.In the adjusted models, elective CS, male infant sex, preterm birth at 35 to 36 weeks gestation and smallness for gestational age (SGA) were associated with increased odds for a TREC-level in the lowest quintile . Male inGiven the strong and inverse association between postnatal age and low TREC- and KREC-levels, we investigated whether postnatal day for blood sampling modified the associations between other perinatal covariates with regard to risks of low TREC- or KREC-levels. However, we found no statistical interactions between postnatal day for blood sampling and mode of delivery, gestational age, infant sex or birth weight for gestational age, respectively.Since rates of low TREC- and KREC-values were higher in infants to mothers with diabetes and hypertensive disease , we repeIn this study, we found that de novo production of neonatal lymphocytes was associated with mode of delivery and other perinatal characteristics. Compared to vaginal delivery, CS was associated with 32% higher risk of having an infant with a lower number of newly formed T-lymphocytes . In addition, male sex, delivery at 35 to 36 gestational weeks, and being born SGA were also associated with a shift towards lower T-lymphocyte formation. Infants born SGA were also at increased risk of low levels of B-lymphocytes  compared to infants with normal birth weight for gestational age. Associations between KREC values and mode of delivery or other infant characteristics were less pronounced or absent. Importantly, maternal characteristics or complications during pregnancy were not associated with levels of TRECs or KRECs in the offspring. To our knowledge, these associations have not been previously reported and the selective contributions from each of these risk factors to the neonatal immune phenotype have not been possible to disentangle before.Birth has previously been associated with acute leukocyte recruitment –29. ThisThe present findings extend our knowledge on how mode of delivery affects the immune system. We found that elective CS was associated with a reduced differentiation into T-lymphocytes after birth. Whether or not this is just a transient neonatal phenomenon remains to be established. Differentiation of immature T-, and B-cells gives rise to a pool of long-lived lymphocytes . If thisAccumulating evidence links early fetal-neonatal living conditions to later health in adult life . Recent In addition to mode of delivery, we identified male sex as a risk factor for low TRECs after birth. Consistent with our findings, cord blood from female infants contains a higher number of CD4+ T-lymphocytes, higher CD4/CD8 T-lymphocyte ratios and lower numbers of CD8+ T-lymphocytes and NK cells than cord blood from male infants, whereas the number of B- lymphocytes is comparable in males and females , 38. SexPreterm born infants have functional deficiencies in their immune system and are more vulnerable to infections . The funStrengths of this study include its population-based design, minimizing selection bias and ensuring generalizability to pregnant women and their infants in high-resource countries like Sweden. We studied a relatively large cohort with numbers that exceeded most previous studies of the neonatal immune system, and with detailed exposure and outcome data. The sample size enabled us to adjust for potential confounders, and to disentangle the selective contributions from important covariates. The method for TREC and KREC determinations has been validated .This study also has limitations. We measured TREC and KREC levels in blood representing the number of newly formed lymphocytes, but not in tissues. Stress induces a mobilization of lymphocytes into the blood, followed by a trafficking of these cells out of the blood into different body compartments . TherefoIn conclusion, this study provides another piece of knowledge on establishment of immune function at birth and its relation to various perinatal risk factors, such as gestational age, mode of delivery and birth weight. The rapidly increasing rates of CS worldwide and the greater risk for immune disorders later in life add fuel to efforts trying to resolve these issues. Our study has identified several important risk factors for reduced number of T- and B-lymphocytes in newborn infants, and CS delivery without a clear medical indication may be the one that can be modified most rapidly. Future research on elective CS and health effects in offspring should focus on longitudinal aspects targeting lymphocyte numbers and function, as well as on how low neonatal lymphocyte numbers relate to infections as well as disorders related to immune function later in life.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.