Neuronal phenotyping showed that the activated neurons were predominantly vasopressinergic, and vasopressin mRNA increased fivefold in the hypothalamus during the 2–3 days before birth. Copeptin, a surrogate marker of vasopressin, was elevated 30-to 50-fold in plasma of perinatal mice, with higher levels after a vaginal than a C-section birth. We also found an acute decrease in plasma osmolality after a vaginal, but not C-section birth, suggesting that the difference in vasopressin release between birth modes is functionally meaningful. When vasopressin was administered centrally to newborns, we found an ~ 50% reduction in neuronal cell death in specific brain areas. Collectively, our results identify a conserved neuroendocrine response to birth that is sensitive to birth mode, and influences peripheral physiology and neurodevelopment.Long-standing clinical findings report a dramatic surge of vasopressin in umbilical cord blood of the human neonate, but the neural underpinnings and function(s) of this phenomenon remain obscure. We studied neural activation in perinatal mice and rats, and found that birth triggers activation of the suprachiasmatic, supraoptic, and paraventricular nuclei of the hypothalamus. This was seen whether mice were born vaginally or via Cesarean section (C-section), and when birth  The brain houses the control centers of peripheral functions that undergo changes at birth, and the sudden exposure to the many novel stimuli that occur upon delivery requires neural integration and processing. Surprisingly, however, little is known about whether and how birth affects brain activity.Birth is an extraordinary event for the placental mammalian fetus, involving major physiological changes in key peripheral organs that are required to survive ex utero4, whereas the newborn piriform cortex and amygdala show spontaneous oscillatory bursting that results from coupled respiratory activity in the brainstem5. However, the ability to capture changes in neural activity triggered by birth using this approach is limited, due to the time and ex vivo procedures required to prepare and record from brain slices .Several studies based on electrophysiological recordings of rodent brain slices in vitro demonstrate perinatal changes in neural activity that may prepare the fetal/newborn brain for birth. In the hippocampus and neocortex, for example, neural network activity is reduced perinatallyc-fos or its protein product8. This method has the important advantages of allowing for pre- and postnatal measures, as well as simultaneous analyses of many brain regions. Most existing reports, however, either used whole brain homogenates6, which cannot address the site(s) of activation, or examined a single time-point9, which cannot establish whether the activation is actually induced by birth. Thus, the extent to which specific brain nuclei and neural subtypes are selectively activated at delivery or what role they play in the fetus/newborn is unknown.In vivo analyses of brain activity at birth have been performed by examining changes in the expression pattern of the immediate early gene 12. Clinical reports spanning four decades have noted that a vaginal birth is accompanied by a massive surge of VP in umbilical cord blood of the human newborn, and that this surge is muted in neonates delivered by Cesarean section (C-section)15. Nonetheless, what triggers the VP surge, its neuroanatomical basis, and the functional significance of the surge (if any) remain unknown. Here, we examined these questions in mice and rats and report that the transition to ex utero life elicits a neuroendocrine response that is conserved across species, sensitive to birth mode, and may have important consequences for peripheral physiology and neurodevelopment.We therefore examined expression of c-Fos at several time points before and after birth in the rodent brain. Given the dramatic nature of a vaginal birth, we anticipated widespread cell activation. Instead, we found striking activation limited to discrete hypothalamic nuclei, including the suprachiasmatic nucleus (SCN), supraoptic nucleus (SON), and paraventricular nucleus (PVN), which constitute the main sites of production of the neuropeptide vasopressin (VP)16, we examined brains on embryonic day (E)18.5 (the day before expected birth), and at 1 h, 3 h, and one day postnatal  after a vaginal delivery. We found a striking pattern of activation in several hypothalamic nuclei, including the SCN, SON, and PVN. In all three regions, the density of c-Fos+ cells was low at E18.5, high at 3 h postnatal, and returned to baseline levels at P1   Fig. . By contse) Fig. . Moreove3/F3,41–43 > 12.17, P < 0.005 in each case): c-Fos+ cell density was again low at E18.5, high at 3 h postnatal, and returned to baseline levels at P1  and very little activation in most other regions , and at 3 h and P1. Because expression of c-Fos protein generally peaks 1-3 h after stimulus onsetons Fig. .19, and the SON is comprised primarily of magnocellular VP and oxytocin (OT) neurons that project to the posterior pituitary and release their contents directly into the bloodstream20. The PVN contains (i) magnocellular VP and OT neurons that project to the posterior pituitary or to other brain regions via axon collaterals; (ii) parvocellular VP, OT, and corticotropin releasing hormone (CRH) neurons that project to the median eminence as well as to other brain regions; and (iii) neurons containing many other peptides24.The brain regions with the greatest activation at 3 h postnatal  all house prominent groups of neuropeptide-containing neurons. VP is the main output peptide of the SCN26. In addition, VP and OT have been implicated in protecting the neonatal rodent brain from the stress of birth by decreasing perinatal neural activity4, and CRH neurons in the PVN are activated around the time of birth in sheep27. Therefore, in a new cohort of mice we phenotyped the activated neurons in the perinatal SCN, SON, and PVN, focusing on VP, OT, and CRH. Double-label immunofluorescence was used to examine co-labeling of c-Fos with VP and OT, and c-Fos immunofluorescence was performed in a CRH-tdTomato reporter mouse.The pattern of neural activation at birth was intriguing because long-standing clinical reports show that human newborns experience extremely high circulating levels of VP2/F2,32 > 6.709, P < 0.004), although the overall density of c-Fos+ cells was lower in this experiment than in Fig. We again saw peak c-Fos+ cell number in the SCN, SON, and PVN at 3 h    .We also performed the reverse analysis, calculating the percent of VP+, OT+ or CRH+ neurons that were c-Fos+ in each nucleus  were placed on a warming pad immediately after birth to control for absence of the dam following a C-section. The activation did not differ significantly whether pups were delivered vaginally or by C-section , and the percent of c-Fos/VP neurons was nearly identical after a C-section delivery  , neural phenotype , and an age-by-neural phenotype interaction  in a two-way ANOVA].It remained possible that the increase in c-Fos labeling at 3 h postpartum was not related to birth at all, but occurred instead due to a developmentally-programmed induction at 19 days post-conception. To test this, we examined the neural activation in animals that were delivered one day early (E18). We reasoned that if birth triggers the neural activation, then c-Fos+ cell density should increase 3 h after an early birth, even though labeling in utero on E18 is normally low  Fig. C. In faces) Fig.  and 1. M31. As mentioned above, human neonates experience extremely high circulating levels of copeptin/VP, and levels are higher after a vaginal birth than after a C-section delivery15. To determine whether birth is associated with a peripheral release of VP in the mouse, we measured plasma levels of copeptin at three perinatal timepoints , and compared them to levels in pregnant or non-pregnant adults. We also evaluated the effect of birth mode on peripheral VP release in the mouse by collecting plasma 0 h and 3 h after a C-section delivery.Copeptin is widely used as a surrogate marker of VP because it is derived from the same precursor molecule, is released into systemic circulation in equimolar amounts, and is a larger and more stable peptidePlasma copeptin was below the detection limit of the assay (4.36 pg/mL) in all non-pregnant adults. By contrast, levels ranged from 21.75 to 287.7 pg/mL in all fetuses and vaginally-born neonates , with higher levels at 0 h, and an age-by-birth mode interaction . Copeptin levels were similar between groups at 0 h, but were lower in the C-section group than the vaginal group at 3 h (P < 0.04).Using two-way ANOVA to compare copeptin levels between the vaginal and C-section groups at 0 h and 3 h, we found a significant main effect of age (FP < 0.04)  at the same timepoint  Fig. A. Thus, To better understand the underpinnings of the VP surge at birth, we examined VP mRNA perinatally  and at P23 in hypothalamic brain punches that included the PVN. We found a striking, 500% increase in VP gene expression between E16.5 and E18.5/E19 that dropped back down to prenatal levels by weaning , and at 1 h, 3 h, and P1 after a vaginal delivery, as well as 3 h after a C-section delivery. Osmolality of fetuses (E18.5 and E19) was in the expected normal range35  which normalized by P1  . This suggests that the high circulating copeptin/VP after a vaginal birth may transiently boost water retention, thereby decreasing osmolality. Moreover, the difference in copeptin/VP release between vaginal and C-section delivery  in the blood by increasing water reabsorption from the kidneyse35 Fig. . Interes37, indicating that birth is associated with concomitant peripheral and central VP release. In rodents, the SCN and PVN are the main source of VP in cerebrospinal fluid38, and these regions were highly activated at birth . We found that intracerebroventricular injections of VP reduced cell death by over 50% in the PVN (P < 0.04) and the AHA (P < 0.03), but had no effect in the SON or the LHb  neurons is triggered by the transition to life 43, and is not induced by all types of stimuli46. Nonetheless, its cellular resolution and low basal expression, as well as the possibility to perform brain-wide mapping of activity in utero and ex utero, make c-Fos a powerful tool for the study of neural activation patterns in response to birth. Our observation that prominent activation of VP neurons was associated with elevated peripheral vasopressin further supports the notion that c-Fos reflected neural activity.One limitation of the current findings is that c-Fos expression does not invariably match other techniques measuring neural activity47. Our finding that VP neurons in the SON and PVN are activated at birth provides a neuroanatomical correlate for these long-standing observations. In addition, we found a remarkable ramping up of hypothalamic VP mRNA expression in the 2–3 days before birth, and much higher expression at birth than three weeks later. This pattern is in accord with a previous report on the ontogeny of the vasopressin system in mice48. Because synthesis and release of VP are not necessarily coupled processes51, it is possible that high levels of VP mRNA perinatally allows for the production and storage of VP peptide, which support the extraordinary VP release at birth.Human and sheep fetuses show extremely high copeptin/VP levels during labor and at parturition15. VP mRNA levels were similar at 3 h postpartum in C-section and vaginally-delivered pups, suggesting that release, not production, of VP is sensitive to birth mode. However, neural activation  was also similar between birth modes. While c-Fos labeling  correlates with stimulus intensity52, differences between groups may not be detectable past a neural activity and/or protein expression threshold. Therefore, it is possible that c-Fos labeling is at ceiling in vaginally-born mice at 3 h, or that production and release of copeptin/VP are similar by birth mode, but that metabolism of the peptides are accelerated following a Cesarean delivery.We found very high levels of circulating copeptin in perinatal mice that dropped by 3 h postnatal in vaginally-delivered pups, and more precipitously in pups delivered by C-section. This is consistent with clinical reports that the copeptin/VP surge drops rapidly after birth and is dampened in babies born by C-sections54. Birth is an inflammatory event, with the fetus exposed to maternal cytokines during late gestation and to antigens from the plethora of microbes encountered at parturition60. The PVN coordinates the central response to peripheral infection or inflammation, and shows c-Fos immunoreactivity within 3 h of a peripheral immune challenge63. Therefore, it would be interesting to determine if neural activation in the newborn is altered by the suppression of immune/inflammatory signals or in sterile (germ-free) mice.Activation of VP neurons could occur in response to, or in preparation for, the stressors that newborn mammals experience, including immune challenge and hypoxia65. Hypoxia increases peripheral and central copeptin/VP release in rats, and umbilical cord copeptin is higher at birth in human newborns suffering from birth asphyxia66. Thus, the hypoxia associated with birth may trigger activation of VP neurons and consequent VP release. Because hypoxia is greater in premature than in full-term infants68, this may explain why we observed even greater neural activation 3 h after an early birth than after an on-time delivery in mice.Newborn mammals, including humans, almost invariably experience hypoxia in association with both a vaginal and C-section birth33. In addition, VP neurons of the SCN play a crucial role in osmolality and thirst in mice via central projections70. Despite high copeptin concentrations in E18.5 fetuses, osmolality was within the normal range. This was expected, due to maternal control of osmolality via the shared maternal–fetal blood supply73. However, 1-3 h postnatal, we found an acute decrease in plasma osmolality in pups born vaginally which, to our knowledge, has not previously been reported. We hypothesize that this results from the abrupt severing of the maternal blood supply in the face of elevated fetal VP. We cannot rule out the possibility that the drop in osmolality is the cause of higher neonatal plasma copeptin/VP in vaginally-delivered pups74; if so, however, it would be necessary to explain what causes an abrupt osmolality change at birth. The transient decrease in plasma osmolality was attenuated in pups delivered by C-section, which is consistent with the lower copeptin levels measured in C-section delivered mouse pups (current study) and human babies15.VP released by magnocellular SON and PVN neurons controls blood osmolality and electrolyte balance77. Whether this is related to birth-mode differences in VP release and resultant effects on plasma osmolality is not known, and is an important area for future study.An acute decrease in plasma osmolality after a vaginal birth could be an important adaptation to conserve water until lactation is established. Human babies delivered via C-section are more likely to suffer from dehydration and/or hypernatremia in the postnatal period than babies delivered vaginally37, indicating concomitant peripheral and central release. The SCN and PVN are the main contributors of VP in cerebrospinal fluid38, and VP is released from the dendrites of magnocellular VP neurons in the rat hypothalamus in an activity-dependent manner79. Thus, it is likely that birth causes VP release within the mouse brain. We found that cell death in the PVN and neighboring AHA was markedly suppressed in C-section-delivered pups treated intracerebroventricularly with VP at birth, with no effect in the SON and LHb. This suggests that central release of VP may alter the magnitude of neuronal cell death in newborns. The regional differences we observed may reflect differences in the distribution of vasopressin receptors at birth80, but could also reflect the fact that following an icv injection, regions adjacent to the ventricles  may have experienced greater concentrations of VP than those at a distance.Copeptin/VP levels in cord blood correlate closely with those in cerebrospinal fluid of human newborns81 and a recent study demonstrates an inverse correlation between copeptin levels in umbilical cord blood of newborns and sensitivity to a mildly painful stimulus82. Central VP may also affect thermoregulation, social behavior, and other functions84.In addition to neurodevelopment, several other functions could be altered by central VP release at birth. For example, VP has been reported to promote neonatal analgesiaThe ubiquitous nature of birth may have led us to overlook its importance as a developmental signal. Birth is a dramatic event for placental mammals, and it would be surprising if the fetal/newborn brain was not equipped with mechanisms to face the upcoming challenges. Collectively, our data suggest that birth triggers a neuroendocrine response that may underlie elevated peripheral and central VP, and cause fluid retention and neuroprotection in the newborn. The finding that some of these processes are altered by birth mode has implications for current obstetric practices in which non-vaginal delivery has become increasingly common across the world.(CRH-ires-Cre x Ai14D) were generated by breeding C57BL/6J mice expressing Cre recombinase under the control of endogenous promoter/enhancer elements of the CRH locus  with mice containing a conditionally activatable fluorescent locus [Gt(ROSA)26Sor] that included transcriptional stop cassettes flanked by loxP sequences . Offspring express tdTomato specifically in CRH-producing cells, as has been previously validated for this cross85.Adult C57BL/6J mice were purchased from The Jackson Laboratory  or obtained from our colony. Adult Wistar rats were purchased from The Charles River Laboratory . A total of 204 mice and 35 rats were used for all reported experiments. Mice and rats were kept in a 12 h:12 h light dark cycle with ad libitum access to water and food. All procedures were approved by the Georgia State University Institutional Animal Care and Use Committee (protocol #A18062) and performed in accordance with ARRIVE and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. CRH reporter mice 86, C-sections on E19 were yoked to vaginal births as reported previously29. Immediately after one female delivered her first pup vaginally, another dam not showing signs of labor was selected for C-section delivery to match time of day and total gestation length. In addition, all brains collected on P1 were harvested at 27 h after birth . Dams were euthanized using 2% CO2 followed by rapid decapitation. An aseptic abdominal incision was made to expose the uterine horns, and the fetuses were removed one by one from their gestational sacs. The brains of C-section-delivered pups were collected immediately after C-sections (0 h), or pups were kept on a heating pad at 32 °C until brain collection at 3 h postnatal on E18 or E19. Offspring from vaginal births that were yoked to C-section births were also kept on a heating pad at 32 °C until brain collection at 3 h postnatal on E19.We established timed-pregnancies by housing breeding pairs together within 2 h of lights off. Males were removed the next morning, 1–2 h after lights on, and that day was designated embryonic day (E) 0. Starting at E18.5, expectant females were checked hourly around the clock for any signs of labor until vaginal delivery. Brains of male and female pups were collected at 1 h, 3 h, or one day postpartum (P1). We also collected brains of pups delivered via C-section at E18 and E19. Because c-Fos may be expressed in a circadian fashionBrains were immersion-fixed in 4% paraformaldehyde for 24 h, transferred into 30% sucrose solution, and coronally frozen-sectioned into four, 40 µm series. Sections were then processed for immunohistochemical detection of c-Fos, or immunofluorescent double labeling of c-Fos and VP, or c-Fos and OT. Unless otherwise noted, all rinses and incubations were performed in 0.01 M phosphate-buffered saline .Free-floating sections were rinsed and submerged in concentrated blocking solution  for 1 h. Sections were then incubated overnight in primary antibody solution  at room temperature. The next day, sections were washed and incubated in secondary antibody solution  for 1 h. Next, sections were washed and then incubated in an avidin–biotin complex solution [1:500 ] for 1 h. Following rinses in sodium acetate buffer, sections were incubated for 30 min in diaminobenzidine-nickel solution, rinsed, mounted onto slides, counterstained with neutral red, and coverslipped.2O2) for 1 h, and then incubated overnight at 4 °C in primary antibody solution against c-Fos . The next day, sections were washed in a dilute blocking solution , and incubated for 2 h at 4 °C in secondary antibody solution . Sections were washed in dilute blocking solution, and then incubated overnight at 4 °C in primary antibody solution against VP or OT . To reduce background auto-fluorescence, sections were washed and incubated in 100 mM cupric sulfate in 50 mM ammonium acetate for 1.5 h at room temperature. Finally, sections were rinsed in 1X Tris-Buffered Saline and mounted onto microscope slides using Fluoromount medium .Alternate free-floating sections were rinsed and incubated in 0.05 M sodium citrate at 70 °C for 1 h. Sections were then rinsed, placed in 0.1 M glycine for 30 min, rinsed, incubated in a concentrated blocking solution . The overall cross-sectional area and the number of c-Fos+ or AC3 + cells within each trace were recorded. The volume of each brain region was determined by summing the areas of all sections and multiplying by section thickness. c-Fos+ and AC3 + cell density was calculated by dividing the total number of positive cells per animal by the volume of the brain region in mm3.Brain regions were identified using well established anatomical landmarks88. Z-stacks of images in areas of interest were captured to quantify c-Fos/VP, c-Fos/OT, or c-Fos/CRH double-labeled cells; each z-step was 2 μm with an average of about 12 optical sections per image. We using the Cell Counter plugin of Image J  to mark each subtype within z-stacks of the areas of interest. All analyses were performed by researchers blind to experimental conditions.Fluorescently labeled sections were magnified to 100X and imaged using structured illumination microscopy  and Stereo Investigator software (MBF Bioscience Inc.). The Apotome system eliminates the out-of-focus blur from other focal planes computationally, and achieves spatial resolution comparable to that of standard confocal microscopy2 followed by rapid decapitation.Copeptin concentrations in perinatal mouse plasma were measured using a commercially-available sandwich ELISA kit , following the manufacturer’s instructions. Trunk blood was collected from male and female offspring at E18.5, immediately after birth (0 h) and at 3 h following a vaginal delivery, or at 0 h and 3 h following a C-section delivery. C-sections were yoked to vaginal deliveries to match the time of day and gestation length, as above. We also collected blood from six pregnant dams , as well as three non-pregnant adults  that were euthanized using 2% COg at 4 °C for 15 min, and plasma was collected and stored at − 80 °C until analysis (< 1 month). Because very low volumes could be obtained from perinatal mouse pups, plasma from 2 to 3 pups of the same sex from different litters was pooled to form single samples for analysis. All plasma samples were diluted fourfold in PBS and assayed in duplicate following the manufacturer’s instructions. The plate was read immediately using a multimode plate reader at 450 nm . The intra- and inter-assay coefficients of variability (CV) were ≤ 10%, and the CV cutoff was 20% for all data replicates. The sensitivity of the assay was 4.36 pg/mL. The manufacturer reports no significant cross-reactivity or interference between mouse copeptin and analogues. Curve fit and concentration values were calculated using the Microplate Manager 6 Software.Blood was collected in EDTA-coated capillary tubes  containing protease inhibitors . Samples were then transferred into clean microcentrifuge tubes, spun at 1000×2 followed by rapid decapitation. Brains were immediately frozen and stored at − 80 °C. PVN punches of 0.8 mm inner diameter were taken in a cryostat and homogenized in TRIzol . RNA was precipitated and reverse transcription was performed with a Superscript IV kit  in a thermal cycler . Real-time PCR was performed in the LightCycler 96 System  using FastStart Essential DNA Green Master Kit (Roche), according to the manufacturer’s instructions. Validated primers were used to amplify VP and GAPDH mRNA , and average quantitative cycle (Cq) values were used to quantify expression of VP relative to the control gene (GAPDH) for each animal using the 2-ΔΔCq method. Values for E16.5 were used as the calibrator group.Brains of male and female offspring were collected via rapid decapitation on E16.5, E18.5, E19, and at 3 h postnatal following a vaginal or C-section delivery. C-sections and vaginal deliveries were yoked, as above. We also collected the brains of vaginally born offspring at P23 after euthanasia with 2% COg at 4 °C for 5 min. Plasma was collected and stored at -80 °C until analysis. Plasma samples were diluted twofold in nanopure water and osmolality was measured using the Advanced Micro Osmometer .Trunk blood of male and female offspring was collected at E18.5 or E19, and postnatally at 1 h, 3 h, or P1 following a vaginal delivery or at 3 h following a C-section delivery. Blood was collected in heparin-coated capillary tubes (Fisher Scientific), transferred into clean microcentrifuge tubes, and centrifuged at 1680×90, and ICV injections were performed as described previously91. Briefly, a 30-gauge needle attached to a 5 µl Hamilton syringe was lowered 2 mm below the skull, at approximately 1 mm rostral to lambda and 1 mm lateral to the sagittal suture. Solutions were injected at a rate of 33 nL/s into the brain of each pup using a Micro4 micro syringe pump . Pups were kept on a heating pad at 32 °C until brain collection 3 h later.We injected 250 ng of VP , or the vehicle alone, into each lateral ventricle of cryoanesthetized pups within 30 min of C-section delivery at E19. The dose was based on previous reports in adult mice and infant ratsSex was included as a factor for all analyses but had an effect on only one measure. Therefore, results are reported for both sexes combined, unless noted otherwise. Parametric statistics (one- and two-way ANOVAs or t-tests) were used for datasets that passed tests of normality (Shapiro–Wilk) and homogeneity of variance (Bartlett’s test). If normality was not met, data were transformed (square root or logarithmic), and if normality was still not met, non-parametric statistics were used . A mixed-effect ANOVA was used to examine effects of age (between-subjects factor) and neural phenotype (within-subjects factor) on c-Fos labeling. When applicable, ANOVAs were followed by Fisher’s least significant difference post hoc tests, whereas Kruskal–Wallis tests were followed by Dunn’s post hoc tests. All analyses were performed using GraphPad Prism.Supplementary Information.


Over the past 40 years access and effectiveness of assisted reproductive technologies (ART) have increased, and to date more than 8 million children have been conceived after ART globally. Most pregnancies resulting from ART are uncomplicated and result in the birth of healthy children. Yet, it is well known that pregnancies following ART are more likely to be affected by obstetric complications such as hypertensive disorders in pregnancy, preterm birth, and low birth weight compared with spontaneously conceived pregnancies. ART children are also at increased risk of birth defects. The majority of the problems arise as a result of multiple pregnancies and can be reduced by transferring a single embryo, thereby avoiding multiple pregnancies. New ART technologies are constantly introduced, and monitoring of the health of ART children is crucial. To date, more than 8 million children have been conceived after ART globally , and up ICSI is the more advanced method, where a single sperm is injected into the cytoplasm of the oocyte. ICSI was originally used for severe male-factor infertility, but nowadays it is also used to treat mild male-factor infertility, mixed infertility, unexplained infertility, and fertilisation failures. There has been an increasing global use of ICSI, with 71.3% of fresh IVF/ICSI cycles performed with ICSI in Europe in 2014, as shown in the latest reports from European Society of Human Reproduction and Embryology (ESHRE)  has gained popularity. In Europe, cryopreservation constituted 27.4% of all cycles in 2014, with the highest rate in Switzerland, 41.1% , and more recent methods such as blastocyst culture and vitrification. The review is based on recent systematic reviews (SRs) and large cohort studies. In case of rare outcomes, cohort studies with limited numbers of children have been included. Studies on perinatal outcomes after medically assisted reproduction including intrauterine insemination, preimplantation diagnostics, preimplantation genetic testing for aneuploidy, and oocyte vitrification have been excluded.Several SRs and large cohort studies have analysed perinatal outcomes in ART singletons versus singletons from the general population ,11–17. MResults from the SRs consistently show that singletons born after ART have more adverse perinatal outcomes compared with singletons born after spontaneous conception, even after adjustment for relevant confounders . ART sinPreterm birth is a syndrome with multifactorial aetiology. ART pregnancies have more placental complications  which entail an increased risk for indicated preterm birth . An incrAnalogously, ART singletons have higher rates of low birth weight (adjusted risks 1.6–1.7) and very low birth weight (adjusted risks 1.8–3.0) compared with spontaneous conception ,17 Tabl. Most stper se under the assumption that maternal factors  remain constant across pregnancies. In a meta-analysis by Pinborg et al., the risk of preterm birth in a singleton sibling born after ART was higher than in a singleton sibling born after spontaneous conception  (Sibling studies where the same mother has given birth to both an ART and a spontaneously conceived singleton is a way to examine ART methods 08–1.49) .In summary, it is well documented that ART singletons have an increased risk for preterm birth and low birth weight compared with singletons born after spontaneous conception. Both parental characteristics, such as age and underlying subfertility, and the ART technique per se may contribute to the more adverse outcome in ART children.When comparing ICSI with standard IVF, most large studies have found similar or lower risks of preterm birth, low birth weight, and peri/neonatal mortality in singletons born after ICSI. Pinborg et al. analysed singletons born after ICSI (fresh or frozen/thawed cycles) versus singletons born after IVF (fresh or frozen/thawed cycles) . Five stIn summary, children born after ICSI have a better perinatal outcome compared with standard IVF.Blastocyst culture (day 5–6) compared with cleavage stage culture (day 2–3) is considered to improve the selection of the most viable embryo and to increase pregnancy and live birth rates per transfer and potentially result in more healthy infants . Yet, SRTwo recent systematic reviews and meta-analyses on perinatal outcomes after blastocyst transfer versus cleavage transfer included more than 100,000 singletons born after fresh cycles ,26. BothAbnormal placentation and implantation may cause the increased risk of preterm birth after blastocyst transfer. A population-based registry study found increased rates of placenta previa and placental abruption after blastocyst transfer . BlastocIn summary, blastocyst transfer compared with cleavage transfer is associated with a small increased risk of adverse perinatal outcomes, particularly preterm birth. Further, a higher rate of MZT and an altered sex-ratio have been observed after blastocyst transfer.Several SRs and meta-analyses have observed that perinatal outcomes are better in children conceived following frozen/thawed embryo transfer (FET) compared with fresh embryo transfers, with reduced risks of preterm birth and low birth weight ,34–36.Maheshwari et al. performed an updated SR and meta-analysis  and confirmed that singletons conceived from FET were at lower risk of preterm birth , low birth weight , and SGA  compared with those conceived from fresh embryo transfers . Yet, thTheories of the underlying mechanisms behind this over-growth include a selection of better embryos surviving the freezing and thawing procedure. Further, epigenetic modifications may occur at the early embryonic stages and affect the growth potential of the foetus. A third suggested theory is that the uterine environment in a FET cycle is more natural than in fresh IVF, as most FET cycles do not use the ovarian stimulation which is used in fresh IVF cycles.n = 1446) were associated with higher rates of hypertensive disorders in pregnancy, postpartum haemorrhage, postterm birth, and macrosomia compared with natural cycles (n = 6297). There were no differences regarding preterm birth and low birth weight. The results support the hypothesis of a link between absence of corpus luteum in programmed cycles and adverse perinatal outcomes. With the increasing number of ART cycles worldwide performed as FET, this finding is important and may support the use of natural cycles in FET.Recent studies have shown a link between the absence of corpus luteum and a higher risk of pre-eclampsia ,39. The A recent SR (11 randomised controlled studies) evaluated perinatal outcome after eFET versus fresh embryo transfers (5379 women) . There wVitrification is an ultrarapid cryopreservation method, which instead of slow freezing has become the dominant method for cryopreservation in recent years. It has been associated with higher post-thaw survival rates and higher clinical pregnancy rates when compared with slow freezing ,42. HoweIn summary, the change of strategy from slow freezing to vitrification seems to be reassuring.The number of OD treatments has increased during recent years, and a total of 17,259 deliveries after OD was reported in 2014 from ESHRE . The higIn summary, OD may constitute an independent risk factor for a more adverse perinatal and maternal outcome than pregnancies after ART with autologous oocytes.Large registry-based cohort studies and SRs with meta-analyses have assessed birth defects in ART singletons compared with spontaneously conceived singletons. Most studies have found an increased rate of birth defects in ART children, ranging between 30% and 70%  6,7,14,,50–52. Tn = 62,379) with singletons born after spontaneous conception  observed an increased risk for major birth defects  (A Nordic cohort study from the CoNARTaS comparing ART singletons (08–1.20) . Increas08–1.20) .Neither Lie et al. (4 studies) nor Wen et al. (24 studies) in their meta-analyses including both singletons and multiples found any increase in the risk of birth defects in ICSI compared with standard IVF  ,56. In cn = 359 children) was assessed in a nationwide Norwegian study (There seems to be no difference in the rate of birth defects in children conceived by ICSI using non-ejaculated sperm compared with ICSI using ejaculated sperm . The ratan study . Birth dn = 827) was similar to that after fresh cycles (n = 1374)  .Earlier studies have claimed that blastocyst transfer is associated with an increased risk of birth defects compared with cleavage stage ,66. Two In summary, ART is associated with a modestly increased risk of birth defects, when compared with spontaneous conception. There seems to be no increased risk after cryopreservation, while the risk after ICSI is still unresolved. On an individual level the increase in birth defects is small.de novo, non-inherited chromosomal abnormalities in ICSI children (n = 1586) compared with the rate in the general population (1.6% versus 0.5%)   versus ejaculated sperm (n = 2516) (Early studies form the Belgian group have shown a higher rate of us 0.5%) . This was 0.24%) . The inc = 2516) .In summary, based on few studies, ICSI may be related to a modestly increased risk of chromosomal abnormalities associated with sperm parameters.n = 62,379) versus a control group of spontaneously conceived singletons   . The rat362,215) .Another study from the CoNARTaS group assessed the risk of major birth defects and the risk over time between 1988 and 2007 in ART singletons compared with spontaneously conceived singletons . The ratIn summary, singletons born after ART have a higher risk for adverse perinatal outcomes compared with singletons born after spontaneous conception. There is a positive trend with improved outcomes, mainly for rates of preterm birth during recent years.The conclusion from numerous studies is that ART is a safe and successful treatment for infertility. Further, perinatal outcomes have improved over time. The increased use of single embryo transfer (SET), thus avoiding multiple pregnancies, is the main contributor to the better outcome seen during recent years. Nordic countries, Sweden in particular, have been the leading countries in reduction of multiple pregnancies by implementing SET as the main strategy . SeveralThere are several pitfalls and methodological limitations when birth defects are studied. The definition of birth defects, mode and frequency of pre- and postnatal surveillance, length of follow-up, inclusion or exclusion of pregnancy terminations for birth defects, source of data, etc. may differ between ART pregnancies and spontaneous conception. Most early studies included small sample sizes with inadequate power to assess differences in rates of birth defects.The increasing and often unnecessary use of ICSI worldwide is a matter of concern, as there are still conflicting results concerning the risk of birth defects. Therefore, until further research can demonstrate safety, ICSI should mainly be reserved for its original intended use, male-factor infertility.Caution should be applied about embarking on a policy of electively freezing all embryos in ART as there seems to be increased risks for hypertensive disorders of pregnancy, macrosomia, and LGA babies after FET. The implications of the findings of macrosomia and LGA after FET, and the consequences for future health and risk of obesity in offspring, are unclear. Therefore, eFET should be used in specific cases such as high risk of ovarian hyperstimulation syndrome, fertility preservation, or in the context of randomised trials.Concerning management during pregnancy, closer surveillance during pregnancy and prophylactic treatment for pre-eclampsia with low-dose aspirin may be indicated in high-risk pregnancies such as pregnancies after OD . The higContinuous supervision after ART is needed to ensure safety and quality, especially when new techniques are introduced. National ART registries such as those existing in the Nordic countries enable follow-up studies of ART children and should be encouraged. Furthermore, research collaborations between countries should be supported as well .


Preterm infants are at risk for structural disruption of brain connectivity due to perinatal complications encountered during the fetal and neonatal periods. This study aimed to investigate the development of connectivity using diffusion tensor imaging at near-term age and the effect of grade 1 intraventricular hemorrhage on it.A total of 86 infants  without apparent brain injury underwent diffusion magnetic resonance imaging (MRI) between 36 and 41 weeks post-menstrual age. The diffusion-MRI based connectomics were constructed from 64-segmented regions by using the Johns Hopkins University neonate atlas and were weighted with fractional anisotropy. The connectomes were quantified in the structural networks and investigated using network metrics, such as the clustering coefficient, local efficiency, characteristic path length, global efficiency, and small-worldness. We compared the differences in the brain networks of preterm infants with or without grade 1 intraventricular hemorrhage in binary and fractional anisotropy-weighted (wFA) connectomes.p = 0.027) and local efficiency , implying the potential for segregation. However, the preterm infants with intraventricular hemorrhage revealed a longer path length  and lower global efficiency , indicating a decreased integration in the wFA connectivity matrix than those without germinal matrix hemorrhage, after correcting for gestational age, sex, bronchopulmonary dysplasia, and age at scan.The 55 preterm infants had a mean gestational age at birth of 29.3 ± 4.1 weeks and the 24 term-born infants, 38.1 ± 1.1 weeks. A total of 13 of the 55 preterm infants (23.6%) were diagnosed with grade 1 intraventricular hemorrhage. The development of connectivity of the brain network in preterm infants without intraventricular hemorrhage was comparable at near-term age to that in term infants. The preterm infants with germinal matrix hemorrhage exhibited higher clustering (0.093 ± 0.015 vs. 0.088 ± 0.007, Grade 1 intraventricular hemorrhage in preterm infants may enhance the capacity for local information transfer and the relative reinforcement of the segregation of networks at the expense of global integration capacity. Advances in neonatal intensive care have markedly enhanced the survival rate and decreased the rates of intraventricular hemorrhage (IVH) in preterm infants , 2. AlthPreterm infants are at risk for structural disruption of brain connectivity due to the perinatal complications encountered during the fetal and neonatal period. Research has been conducted on the value of structural brain networks ascertained with diffusion MRI to predict the functional impairments and neurodevelopmental outcomes in very preterm children , 8. RelaIVH is an important cause of neurological and cognitive complications and can significantly impair brain development . SeveralThis study is part of a prospective observational cohort involving the postnatal follow-up of preterm infants at Hanyang Inclusive Clinic for Developmental Disorders in Hanyang University College of Medicine between December 2016 and June 2018. The institutional review board at Hanyang University Hospital approved the study protocol and scanning procedures, and informed consent was obtained from the parents of all children included in this study. The inclusion criteria for preterm infants consisted of the following: birth at < 35 weeks of gestation without major congenital malformations, no evidence of intraventricular or intracranial hemorrhage greater than grade I, no evidence of intrauterine growth retardation, and availability of MRI at near term age between 36 and 41 weeks postmenstrual age (PMA). Neonates with grade 1 IVH were included in our study. All cranial ultrasound scans and subsequent MRIs were evaluated for brain injury by a single pediatric radiologist. Early cranial ultrasound scans were performed within 3 days of birth and at 1 and 3 weeks later. Subsequent MRI scans conducted at near-term age were assessed for brain injury in preterm infants, including IVH, overt white matter injury, and cystic periventricular leukomalacia (PVL). We excluded 14 infants with evidence of intraventricular or intracranial hemorrhage greater than grade I, 1 infant with cerebellar hemorrhage, 2 infants with punctate white matter lesions, and 11 infants with poor image quality due to motion artifacts.We performed MRI imaging in healthy, term infants (between 37 and 42 weeks gestation) with uneventful deliveries from the same hospital, after informing the parents in writing and obtaining their consent. The 24 healthy, full-term infants with normal MRI and neurologic examination results were included as controls. The following exclusion criteria for full-term infants were used: prolonged intensive care (> 7 days), neonatal asphyxia, congenital infection, and congenital heart disease. Controls and preterm infants were imaged during natural sleep without sedation. Prenatal and neonatal data were prospectively recorded, including maternal details, gestational age (GA), birth weight, delivery type, sex, head circumference at scan, germinal matrix hemorrhage, culture-proven sepsis, necrotizing enterocolitis, and bronchopulmonary dysplasia (BPD). IVH was determined using the grading system established by Papile et al. . and was2; number of diffusion gradient directions, 15; TR/TE, 5243/76 ms; 40–50 axial slices; 2.0-mm thickness; field-of-view, 150 mm; matrix size, 1.97 × 1.97; total acquisition time, 6.5 min.T1- and T2-weighted imaging and DTI scans were acquired with a 3.0 T MRI scanner  and a 16-channel SENSE head coil. The MRI data were obtained at the age of 36–41 weeks PMA in preterm infants and at 37–40 weeks PMA in full-term infants. The T1- and T2-weighted images were obtained with sagittal T1 Turbo field-echo sequences (repetition time [TR]/echo time [TE] 8.2/3.8 ms) and Turbo spin-echo (TR/TE 4800/90 ms) sequences, respectively. The DTI sequence parameters consisted of the following: SENSE factor, 2; b value, 800 s/mmhttp://www.fmrib.ox.ac.uk/fsl). The eddy current distortion and head motion for individuals were corrected using FMRIB’s Linear Registration Tool (FLRIT) [dsi-studio.labsolver.org) [Diffusion-weighted imaging (DWI) data were preprocessed using the Diffusion Toolbox (FDT) from the FMRIB’s Software Library ( (FLRIT) , 21. The (FLRIT) . The dif (FLRIT) . The scaver.org) . The FA ver.org) . We usedver.org) , 27. Finver.org)  while thver.org) .Fig. 1Tp) and global efficiency (Eglob). The shorter path length and increased global efficiency in structural networks suggest the potential integration for effective connections. In contrast to network integration, network segregation refers to the degree to which the network is organized as clusters of sub-networks. Segregation indicates the relative strength of within-network connections compared to integration, and the most popular measures of segregation are clustering coefficient and local efficiency. Finally, small-worldness reflects both integrated and segregated information processing. The descriptions and definitions of these network metrics for a given graph G with N nodes are provided in Table http://www.brain-connectivity-toolbox.net).At a global level, connectomes were quantified in the structural networks and investigated using various network metrics, including integration measures, segregation measures, and small-worldness. Topological organizations of neonatal structural networks were characterized using the Brain Connectivity Toolbox in MATLAB  . The netSPSS 21.0  software was used for statistical calculations. We used the Mann-Whitney U-test and Fisher’s exact test to compare the clinical variables between groups. The preterm infants were sub-categorized into three groups as extremely preterm (< 28 weeks), very preterm (28 to 32 weeks), and moderate to late preterm (33 to 36 weeks). Mean network parameters in the preterm sub-groups were compared using the analysis of variance and general linear models with sex, BPD, and age at scan as covariates. Analysis of covariance was performed to confirm the differences in networks between preterm and full-term infants. Additional statistical analysis between preterm infants with and without grade 1 IVH was performed using general linear models with GA, sex, BPD, and age at scan as covariates to compare the structural network metrics, including clustering, local efficiency, path length, global efficiency, and small-worldness.p < 0.001) and featured a lower proportion of males .The total population consisted of 55 preterm infants 26 males) with a mean GA at birth of 29.3 ± 4.1 (range 23–35) weeks and a mean age at scan of 37.2 ± 1.4 (range 36–41) weeks, and 24 term-born infants  with a mean GA at birth of 38.1 ± 1.0 (range 37–40) weeks and a mean age at scan of 39.8 ± 0.9 (range 37–41) weeks. Table 6 males wp = 0.002) and birth weight . However, the age at the MRI scan and head circumference at age of MRI scan were not significantly different between preterm infants with and without grade 1 IVH. During the neonatal period, preterm infants with grade 1 IVH differed in that they exhibited a high rate of BPD  and a longer duration of total parenteral nutrition days . This is associated with chronic intubation and poor nutritional support for proper brain growth; however, these differences were insignificant.Relative to the preterm infants without grade 1 IVH, preterm infants with grade 1 IVH exhibited a low mean GA , very preterm , and moderate to late preterm . These exhibited no significant differences among preterm sub-groups in the statistical analyses, controlling for sex, BPD, and age at MRI scan as covariates in preterm infants. Table p < 0.001) and featured a lower proportion of male infants . The preterm neonates without grade 1 IVH exhibited slightly lower wFA clustering coefficients , and wFA local efficiency  than full-term infants. However, the clustering, local efficiency, path length, global efficiency, and small-worldness in preterm infants without grade 1 IVH were comparable to those in full-term infants after adjusting for GA, sex, BPD, and age at scan. Remarkably, the preterm infants with grade 1 IVH exhibited higher clustering  and local efficiency , implying the potential for segregation. However, they exhibited longer path length  and lower global efficiency , indicating a decreased integration in the wFA connectivity matrix among preterm infants with grade 1 IVH than those without grade 1 IVH, after correcting for GA, sex, BPD, and age at scan. In contrast, we observed no significant differences in the binary network parameters with and without grade 1 IVH.Table Developmental connectomics obtained from advanced neuroimaging techniques have emerged as an important tool for the characterization of the structural and functional brain connectivity during the early period of life in neonates. We explored potential alterations in structural network properties related to grade 1 IVH in preterm infants. The measures of structural segregation, such as modularity and local efficiency, were observed to increase, while global efficiency decreased in preterm infants with grade 1 IVH compared with those without grade 1 IVH, indicating a specialized reinforcement of network segregation at near-term age.Previous studies have reported the effects of grade 1 IVH on the developmental white matter microstructure in preterm infants. Morita et al.  observedThe beginning of the development of the microstructure of white matter connections and the emergence and establishment of small worldness reportedly occurs throughout 30 PMA , 10, 26.We explored the significance of the weighted networks and the differences between binary and FA-weighted networks. Discrepancies between unweighted or weighted connectome pipelines might be attributed to the differences in the speed at which networks change during dynamic developmental phases along with the abundant formation of new connections. To a lesser extent, differences in edge weights may account for the discrepancies. Compared to the binary approach, the FA-weighted approach could be more sensitive to the network organization of atypical early neural development in preterm infants than to the early development of the full-term brain . Our obsBall et al.  demonstrDespite these limitations, knowledge of the neonatal human connectome may provide insight into preterm development and the influence of grade 1 IVH on brain development at near-term age. Further follow-up studies based on structural brain networks should examine whether long-term neurodevelopment in preterm infants with grade 1 IVH is mitigated by delayed or altered integration and segregation compared to that in full-term infants.


The early initiation of breast feeding is a high-impact intervention that gives newborns a better chance of survival. We assess the barriers and facilitators influencing the practice of early breast feeding of newborns in public primary healthcare facilities (PHCs) in Northeast Nigeria, to influence the planning of programmes targeted at improving newborn care in the region.We used an explanatory mixed-method approach. We conducted case observation of childbirths and newborn care for the quantitative arm, and interviewed mothers and birth attendants 1 hour after childbirth for the qualitative arm. The analysis for the quantitative arm was done with SPSS V.23. For the qualitative arm, we transcribed the audio files, coded the texts and categorised them using thematic analysis.We observed 393 and 27 mothers for the quantitative and qualitative arms of the study, respectively. The quantitative arm shows that 39% of mothers did not breastfeed their newborns within 1 hour of birth. The qualitative arm shows that 37% of mothers did not breastfeed within 1 hour of birth. Themes that describe the barriers to early breast feeding in public PHCs are: birth attendants’ unwillingness or inability to accommodate mothers’ safe traditional practices, ineffective rooming-in practices, staff shortages, lack of privacy in the lying-in ward and poor implementation of visiting-hour policy in public PHCs. The pregnant women denied safe traditional birth practices like chanting, praying or reading religious books during delivery are five times more likely not to breastfeed newborns within the first hour of birth  compared with pregnant women allowed these practices.Stakeholders must increase their focus on improving breastfeeding practices in public PHCs. Instituting policies that protect mothers’ privacy and finding innovative ways to accommodate and promote safe traditional practices in the intrapartum and postpartum period in PHCs will improve the early breast feeding of newborns in these PHCs. We used a mixed-method study design.Also, we used purposive sampling technique to select the public PHC, focusing on information-rich cases.Data collection included direct observation of mothers’ newborn breastfeeding behaviour in the first hour after childbirth.We observed a high number of childbirths.The sampling approach may limit the generalisability of the findings to places not similar to our study setting.Every year, about 77 million 50%) newborns do not get breast fed in the first hour of birth globally.0% newbor14 15The practice of early breast feeding of newborns differs between and within countries.21 23–25To improve early breastfeeding and avert newborn deaths, one approach is for more births to occur in health facilities.18 29The Northeast region of Nigeria is witnessing an increase in health investments by governments and non-governmental organisationsIn this study, we assess the barriers and facilitators influencing early breast feeding of newborns in public PHCs in Northeast Nigeria. Most studies assessing breastfeeding practice in health facilities have used secondary data from demographic health surveys.We used an explanatory mixed-method approach for this study. Mixed-method study designs deepen how we understand a research phenomenon.We conducted the study in Gombe State, in the centre of Northeast Nigeria on latitude 9″ 30′ and 12″ 30′N, longitude 8″ 5′and 11″ 45′E. It borders Borno, Yobe, Adamawa, Taraba and Bauchi State. It has 11 local government areas (LGAs) and 114 political wards.49Fifty percent of these priority PHCs have staff trained in providing basic emergency obstetric and newborn care services. About 36% of them have labour rooms and lying-in wards. None has a medical doctor, 4% have at least one nurse, and 19% have at least one midwife. Each PHC has an average of six health workers not categorised as skilled attending labour and delivery. These include CHEWs, environmental health technicians, hospital assistants (cleaners) and students. The majority (34%) of the staff in these PHCs have no medical training.We selected 10 of the 114 priority PHCs using a purposive sampling technique. The 10 had the most deliveries per day on the average in the 6-month period prior to starting the study, thus suited to provide rich information on our study objective. They also had labour wards and lying-in wards. The budget for the study dictated the number of health facilities chosen.51We selected three3 of the 10 priority PHC in the quantitative arm of the study. The three had the most deliveries per day on the average. They also had labor wards and lying-in wards. The intensity of the childbirths in the three PHCs over the study period, will illuminate the barriers and facilitators of early initiation of breast feeding in public PHCs in the region.We included all mothers who delivered in the selected PHCs and gave their consent to take part in the study in both arms of the study. For the qualitative arm: we also recruited all consenting healthcare providers who attended deliveries; and the mothers with a live birth for interviews.We recruited and trained nurses and nurse–midwives who are not a part of the health facility staff to complete the assessment tool. They observed and documented the time of events from when a pregnant woman in labour entered the health facility to when she leaves after childbirth or referral. During data collection, they only observed and did not take part or comment in the care offered to the pregnant woman. We only told them to intervene or offer help during a life-threatening situation to the mother and/or baby. The data collectors used separate assessment tools in cases of twin deliveries.The health facility staff and pregnant women knew of the nurse and nurse–midwives but were unaware of what aspect of care was being observed. Data on the cadre of the health worker, events during the first-to-third stage of labour, and newborn care activities in the first hour after birth were collected. The nurses and nurse–midwives were available for 24 hours in each of the health facilities throughout the study period on a shift schedule. They observed all deliveries.We also recruited and trained female nurses and midwives who are not part of the health facility staff to observe and complete the assessment tool under this arm of the study. Observation for each pregnant woman started during the second stage of labour and ended 1 hour after childbirth under this arm of the study. The data collector then interviewed the mother and attendant health worker, after the observation period. The assessment tool used for the observation is an extract from the tool used for the quantitative arm. For the mothers and birth attendants, we asked questions around how they felt about the childbirth process and knowledge of when newborn babies should breastfeed during the interview. We also asked the mothers why they chose to or not to breastfeed their newborns within the hour depending on if they did or not. Also, we asked the attending healthcare workers why they think breastfeeding occurred or did not occur within the first hour for each newborn. We also asked the attending healthcare workers about the strategies they used to encourage mothers to breast feed their newborns within the hour. The data collectors attended every birth in the health facilities and interviewed respondents who could not speak or understand English in Hausa. They interviewed the mother and attending healthcare worker separately. The mothers’ interviews occurred at their bedside in the lying-in ward. The data collectors excused relatives or visitors in the lying-in room during interviews.2 test as appropriate.We analysed the data using SPSS V.23. We present nominal variables as percentages. We also determined associations and relative risks (RRs) between initiation of breast feeding and predictor categorical variables using two-by-two contingency tables. To assess significant associations, we used Fisher’s or Pearson’s χWe transcribed the interviews from their audio files and analysed the data with Saturate, an online qualitative software. Two people on the team analysed a subset of the data and generated codes. The two people then came together to review and agree on the codes generated and their meaning, eliminating less useful codes. The two reviewers held regular face-to-face meetings to discuss their codes. We then analysed the rest of the dataset using the agreed code-framework developed by the two. We generated themes from the codes using an inductive approach.Resources for patient and public involvement statement were unavailable, so we could not involve patients. The development and dissemination of a policy brief of the study findings will involve patients.All pregnant women recruited under the two arms of the study consented to be observed. Under the quantitative arm, we observed 393 pregnant women. Most (54%) were between 15 and 24 years old with a median age of 23 years. A quarter were below the age of 20, half below the age of 23 and three quarters below the age of 30. The childbirths were through spontaneous vaginal delivery (SVD). Twin delivery occurred in only six (1.5%) cases. Also, 39% (95% CI 34% to 44%) of the new mothers did not breast feed their newborns in the first hour after delivery. Thirty-three health workers attended the deliveries under this arm of the study. The number of deliveries attended by each health worker ranged from 1 to 37, with an average of 12 (SD 10) deliveries each and a median of eight during the observation period. At least two health workers attended about 61% of the 393 deliveries. Also, JCHEWs attended the majority (36%) of the 393 deliveries .Under the qualitative arm, 27 pregnant women were delivered of their babies by 16 health workers. The pregnant women consented to be observed and interviewed. The 16 health workers also consented to be interviewed after the observation. Most (63%) of the pregnant women were between 15 and 24 years old, with a median age of 22 years. A quarter were below 20 years of age and a quarter above 30 years of age. None had a twin delivery. Also, 37% (95% CI 19% to 56%) of the mothers did not breast feed the newborn within 1 hour of birth under this arm of the study. We also interviewed all 27 mothers and 16 health workers 1 hour after childbirth. Of the 16 healthcare workers interviewed under the qualitative arm, most (63%) were hospital assistants. About a third (33%) were CHEWS, 17% (2) were students, 6% (1) were nutritionist and 6% (1) environmental health technician. Of the 27 deliveries under this arm of the study, in 44% of cases, the attending healthcare worker was assisted by another health worker .The qualitative arm shows that health workers know when mothers should breastfeed newborns. Ninety-two percent of them responded that breastfeeding should start between 0 to 60 min .They attributed their knowledge to on-the-job training sessions on newborn care with visiting clinical mentors. They believe their knowledge is sustained via peer-to-peer discussions while on the job and during staff meetings. One JCHEW said:Breastfeeding should commence immediately after birth …we have clinical mentors that visit and remind us of these things. We also have staff meetings where we remind ourselves of these practices (birth attendant 1)The word ‘immediately’ is more used by lower cadre health workers to describe when breastfeeding should start. The CHEWs are more specific about when breast feeding should start. They also link the time breast feeding should start with its benefits. The quote below is a typical response from a senior CHEW when she was asked when breastfeeding should startIt is very important because it helps the child to suck the yellowish nutrient in the breast milk. It boosts the child’s immunity. It also helps the mother’s uterus to shrink and close …helping to stop bleeding. It should start by 30 min to 1 hour after delivery (birth attendant 2).The quantitative study shows that pregnant women denied safe traditional birth practices such as praying or reading religious texts during the second and third stages of labour are five times more likely not to breastfeed within the first hour  compared with pregnant women allowed these practices .When mother’s state of health after childbirth is not an issue, a typical response given by some mothers for not breast feeding in the first hour was the need to first wash the breast or have a bath to feel clean.He (the baby) has to exercise patience until we get home (before he is breastfed). I can’t breastfeed him before I take my bath (mother 1)The quantitative study suggests that the mother’s need to be clean may not be influenced by the cleanliness of the environment. Initiating breast feeding is not related to the cleanliness and comfort provided by the delivery room. Mothers who had their babies when the labour room was clean and comfortable were just as likely not to initiate breast feeding early as those who had their babies when the labour room was not .Postdelivery pains and fatigue are barriers to breast feeding within the first hour after birth. Even when mothers show a good knowledge of when breast feeding should start, some still express the need to regain strength and wellness first before they breast feed the newbornbreastfeeding should commence immediately after birth… I did not commence it because I was feeling after pains. The health worker said she will bring the baby to suck. I told her to allow me to have some relief ZA (mother 2)The quantitative study shows that pregnant women not encouraged to consume fluids or food at least once during labour are twice as likely not to breastfeed within the first hour compared with those encouraged to do so . There is no evidence from the study that blood loss greater than 500 mL during labour and delivery influence early breastfeeding .The qualitative study shows that shortage of health workers in PHCs introduces a delay in carrying out newborn care activities. Sometimes, health workers have to attend to other ill patients when there is no one else to assist. When we asked some health workers why a mother under their care did not breastfeed early, a typical response given was:You see, if you have someone that will assist you, you will assign the person to carry the baby to the mother and initiate the breast feeding, or weigh the child, or apply chlorhexidine to the baby’s cord or any other thing needed while you continue with the remaining work and management of others …but most of the time you are on duty alone. It is because we have shortage of manpower here in this facility (birth attendant 3)The human resource shortage also affects rooming in in the PHCs. Placing the mother and newborn in the same room after delivery is rooming-in.I wanted to transfer the mother and baby to the postnatal ward… before she commences breastfeeding (of the newborn). The (lying-in) room is not set (for use yet). I have to clean and make the room so she is comfortable to commence breastfeeding (birth attendant 4)When rooming-in happens, the quantitative study reveals that mothers who do not have skin-to skin contact with their newborns in the first hour after birth are twice as likely not to breastfeed early, compared with mothers who did . Just keeping the mother and newborn in the same room (rooming-in) without skin-to-skin contact has no influence on early breastfeeding .The PHCs have open rooming-in rooms that does not guarantee privacy. There are also no defined visiting hours in the PHCs. The qualitative arm of the study shows that male and female relatives visit the new mother in the hospital after childbirth. During the visit, the relatives pray for the newborn baby and congratulate the mother. Some relatives sit around after prayers for long. When relatives come visiting, the mothers have to dress up to receive them and do not breast feed during this time. The birth attendants also delay supporting the mother to breast feed. One birth attendant suggested that addressing the issue puts them in bad light in the community.you know, the people in the community have a unique character or attitude. The moment you try to talk to them about this kind of issue they feel you are molesting them or depriving them of coming close to their relatives. They do not know you are trying to ensure their relative (mother and baby) gets what is beneficial to them (birth attendant 5).From the qualitative study, we find that antenatal clinics (ANC) helps pregnant women to learn about breastfeeding newborns within 1 hour of birth. The knowledge they gain during these clinics influences their behaviour after childbirth. Most mothers who practised early breastfeeding said what they learnt from ANC influenced their decision to do so. The typical response they gave is:I used to give my children water (after childbirth). I did not know the importance of breastfeeding early. I used to think breast feeding could start at any time of the day (of birth). I started breastfeeding early because the health workers tell us (of the importance of starting breastfeeding immediately after birth) during ANC (mother 3)From the qualitative study, we find that when the birth attendants encourage some reluctant mothers to breast feed their newborns within 1 hour of childbirth, they do.I was told  to give (the baby breast to suck early enough). Normally, I won’t (mother 4)The encouragement process takes the form of a negotiation between the birth attendant and the mother. We find that the information passed by the birth attendants to the mothers around the benefits of early breast feeding facilitate early breast feeding in the PHCs.I commenced breastfeeding early because of what the health worker said …she explained how it is important to the health of my baby. That’s why I commenced it (mother 5)Sometimes, showing the mothers how to place and breast feed the newborns also facilitates early breast feeding after childbirth in these health facilitiesI gently encourage them and tell them to give (breast milk) early… I put the baby on her laps and remove the breast, and demonstrate to her how to breastfeed (birth attendant 6)In our study setting, we find that close to 4 out of every 10 newborns do not get breastfed within the first hour of birth. This doubles their risk of dying in the first 28 days of their lives.The northeast region of Nigeria has a shortage of skilled health workers. The Boko-Haram insurgency has made this worse.61 62In our study, we find that the mothers denied safe traditional birth practices like praying, reciting religious texts or reading religious books during deliveries were five times more likely not to breastfeed the newborns than the mothers not denied. This supports the findings from other settings that shows that the unwillingness of birth attendants to accommodate safe traditional birth practices affects mothers’ adoption of supportive care.67Washing of the breast with water after childbirth is a hygiene-related practised in parts of the world.60 69The other barriers that influence early breast feeding of newborn in PHCs that our study finds are post-delivery pains and fatigue within the first hour after birth, delay in skin-to-skin contact during rooming in and poor visiting hour policy in the PHC. These findings reinforce what other studies have reported.Helping mothers to breast feed within an hour of birth is an international recommendation practised by healthcare providers in most of the world.Only about 60% of babies born in public PHCs in Northeast Nigeria get breastfed in the first hour of birth. This means the rest miss important nutrition that saves lives. The stakeholders in the region must increase their focus on improving the breastfeeding practices in public PHCs. This will improve the survival of newborns and impact of their investments. Instituting policies that protect mothers’ privacy, and finding innovative ways to accommodate and promote safe traditional practices in the intrapartum and postpartum period in PHCs will improve the early breast feeding of newborns in these PHCs. Birth attendants in PHCs must also be trained on effective rooming-in to further improve early breast feeding of newborns in these public PHCs, however.


Total knee arthroplasty (TKA) is commonly performed around the world. Implant designs include fixed-bearing and mobile-bearing. Mobile-bearing design was developed as a rotating platform that allows axial rotation of the insert around the longitudinal axis. This phenomenon may limit full exploitation of the characteristics of the mobile-bearing insert, which may cause wearing and reduce longevity. However, there is limited knowledge on rotational behavior of the polyethylene mobile-bearing insert under weight-bearing conditions. We aimed at determining the rotational motion of each component at full extension and flexed positions during a squatting activity after TKA.This study was a cross-sectional study (level 4) involving patients with severe knee osteoarthritis scheduled to receive TKA. We examined 13 knees of 11 patients after mobile-bearing TKA  at 10 weeks and 1 year postoperatively. Four identical metallic beads were embedded into the insert. Wide-base squatting was chosen for analyses. Three-dimensional in vivo poses of the prostheses were created using a 3D-to-2D registration technique. During flexion, rotation of the femoral component relative to the insert (FEM/INS) and tibial component (FEM/TIB) as well as insert rotation relative to the tibial component (INS/TIB) were computed. Repeated measure 2-way ANOVA and post hoc test was used.p = .013) and 1 year , respectively. During the squatting activity, rotation motions of FEM/TIB, FEM/INS, INS/TIB were 5.7°, 5.9°, and 1.8° at 10 weeks and 6.3°, 5.5°, and 1.6° at 1 year, respectively. Rotation motion of FEM/INS was significantly greater than that of INS/TIB at both 10 weeks (p < .001) and 1 year (p < .001).In the fully extended position, FEM/INS was significantly smaller than INS/TIB both at 10 weeks (− 0.3° vs. 6.3°, The mobile-bearing insert enhances the compatibility of FEM/INS in extension; the amount of INS/TIB rotation is significantly smaller than that of FEM/INS during a squatting activity. This information will inform surgeons to take caution to perform TKA with a fixed insert in which 6.3° of rotational offset would be added to the rotational alignment at FEM/INS at full extension.UMIN000024196. Retrospectively registered on 9 September 2016.UMIN-CTR,  The design of the insert of the artificial joint can be roughly divided into two types, fixed-bearing and mobile-bearing. Mobile-bearing design was developed as a rotating platform that allows axial rotation of the insert around the longitudinal axis. It was theorized that mobile-bearing total knee arthroplasty (TKA) should allow self-alignment of the polyethylene insert with the femoral component in order to lessen polyethylene surface stresses, minimize post-cam impingement, and possibly increase polyethylene longevity . HoweverSeveral studies have used embedded tantalum beads to analyze the behavior of the mobile-bearing insert by X-ray fluoroscopy during in vivo knee motion –17. ThesThe objective of this study was to clarify the tibiofemoral kinematics and rotation of the polyethylene mobile-bearing insert relative to the femoral and tibial components under weight-bearing conditions in patients implanted with mobile-bearing TKA. The hypotheses of this study were as follows: (1) the mobile-bearing insert can rotate on the tibial tray to reduce the offset at FEM/INS at knee extension position and (2) intra-prosthetic axial rotation between the tibial tray and mobile-bearing insert is greater than that between the femoral component and mobile-bearing insert.This study was a cross-sectional study (Level 4) involving patients with severe knee osteoarthritis scheduled to receive TKA at a single regional hospital. The study protocol was approved by the institutional review board of Hiroshima International University (12-025), and informed consent was obtained from all subjects included in the study prior to the initiation of this study. All subjects who met specific selection criteria were enrolled. Inclusion criteria were (a) Japanese males and females and (b) between 50 and 85 years old, primary medial KOA with radiographic severity of grade III or IV in the Kellgren-Lawrence system, with or without lateral or patellofemoral degradation. Exclusion criteria were (a) lateral compartment knee osteoarthritis; (b) history of knee surgery; (c) any other disease involving the knee joint; and (d) history of rheumatoid arthritis, cerebral disorders, neuropathy, or gout.All osteoarthritic knees underwent TKA with posterior stabilizing (PS) components and mobile-bearing insert . This prosthesis was designed to safely allow 155° flexion and has a mobile bearing that allows up to 25° internal or external rotation on an anteriorly placed rotation center. A senior surgeon (YK) performed arthroplasty using his own technique, which is summarized by a paramedian approach for minimal exposure, achieving 6° valgus for the femoral cut and 90° to the tibial shaft, modified ligament dependent cut for femoral rotational alignment.Because the polyethylene insert is transparent during X-ray fluoroscopy, three-dimensional pose estimation of the mobile-bearing insert was achieved using tantalum beads embedded in the insert. Using a custom-made bead injector, four identical metallic tantalum beads (diameter 0.8 mm) were inserted into predefined non-weight-bearing areas of the polyethylene insert during surgery Fig. . The fouWe measured active and passive knee flexion angles, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) , and artWeight bearing and passive knee range of motion (ROM) were measured using sagittal images from a movie taken with a commercial video camera and analyzed using the ImageJ software . The video camera was positioned 3 m away from the subject to bilateral sides and adjusted the camera so that the center of the knee joint was at the center of the viewing screen. For the weight bearing ROM, subjects stood sideway to the camera and were instructed to squat down from full extension to maximal flexion with maximal effort. They were allowed to hold handrail for safety. For passive knee flexion measurement, subjects were instructed to lie supine on a bed, and a senior physical therapist bent their knee to maximum flexion.Symptoms and physical function were measured using the WOMAC index at 10 weeks and again 1 year postoperatively. Subjects responded to all sections including subscales of pain, stiffness, and physical function during daily living. All measurements were performed by an 11th year physical therapist with research experience of 4 years as a graduate student.o  for continuous values. The assumption of normality and equality of variance was assessed using the Shapiro-Wilk test. If the data met parametric assumptions, we used repeated measure ANOVA and Tukey post hoc test for pairwise comparisons. Paired 2 at 10 weeks postoperatively)  were evaluated in this study  28.4  kg/mp = 0.173) or passive ROM (p = 0.196). Median WOMAC scores for pain, stiffness, physical function, and total score using 100 mm VAS were 41, 44, 337, and 468 at 10 weeks and those at 1 year were 5, 0, 167, and 228, respectively. All scores at 1 year were significantly less than those at 10 weeks  . FEM/TIB, FEM/INS, and INS/TIB angles at 1 year were 4.0° , − 0.8° , and 4.9° , respectively. FEM/INS was significantly smaller than INS/TIB (p = 0.011). FEM/TIB at 1 year was significantly smaller than that at 10 weeks (p = 0.041)  were − 2.0°  at 10 weeks and − 3.2°  at 1 year, respectively. FEM/TIB, FEM/INS, and INS/TIB angles at 10 weeks were 6.0° , − 0.3° , and 6.3° , respectively. FEM/INS was significantly smaller than INS/TIB (p < 0.001). At 1 year, rotation motion during squatting from − 3.2  to 53.9  degrees were 6.3° , 5.5° , and 1.6° , respectively. Total rotation motion of FEM/INS was significantly greater than that of INS/TIB (p < 0.001). There were no statistically significant differences between data obtained at 10 weeks and those at 1 year   the use of a 3D-to-2D registration technique with fluoroscopy, which is an accurate and well-established technique to measure in vivo knee kinematics , 24. OurThe mobile-bearing insert improves the rotational offset of the femoral and tibial components at the starting position, but the amount of rotation motion during squatting at the INS/TIB interface is very small. This study is thought to provide useful data for improving surgical procedures, developing future implant designs and understanding the implant wear pattern in TKA with a mobile-bearing. In a future study, it will be necessary to analyze whether the amount of mobile-bearing insert movement is affected by the presence or absence of weight-loading.