Although preterm birth predisposes for cardiovascular disease, recent studies in children indicate normal blood pressure and arterial stiffness. This prospective cohort study therefore assessed blood pressure and arterial stiffness in adolescents born very preterm due to verified fetal growth restriction (FGR).n = 24) and two control groups born with appropriate birth weight (AGA), one in similar gestation  and one at term  were included. 24-hour ambulatory blood pressure and aortic pulse wave velocity (PWV) and distensibility by magnetic resonance imaging were acquired.Adolescents (14 (13–17) years; 52% girls) born very preterm with FGR . In boys, diastolic and mean arterial blood pressures increased from term AGA to preterm AGA to preterm FGR with higher daytime and 24-hour mean arterial blood pressures in the preterm FGR as compared to the term AGA group. In girls, no group differences were observed .There were no group differences in prevalence of hypertension or in arterial stiffness (all Very preterm birth due to FGR is associated with higher, yet normal blood pressure in adolescent boys, suggesting an existing but limited impact of very preterm birth on cardiovascular risk in adolescence, enhanced by male sex and FGR.Very preterm birth due to fetal growth restriction was associated with higher, yet normal blood pressure in adolescent boys.In adolescence, very preterm birth due to fetal growth restriction was not associated with increased thoracic aortic stiffness.In adolescence, very preterm birth in itself showed an existing but limited effect on blood pressure and thoracic aortic stiffness.Male sex and fetal growth restriction enhanced the effect of preterm birth on blood pressure in adolescence.Male sex and fetal growth restriction should be considered as additional risk factors to that of preterm birth in cardiovascular risk stratification. True FGR, on the other hand, can with high sensitivity be verified by abnormal fetal blood flow as measured by fetal Doppler velocimetry using structured clinical protocols.14 Further, whether one sex is more susceptible to the effect of preterm birth on blood pressure and arterial stiffness is not clear.15Preterm birth and low birth weight both predispose for cardiovascular disease later in life.16 and magnetic resonance imaging (MRI) is the reference standard to assess aortic stiffness using pulse wave velocity (PWV) and distensibility as surrogate markers.19We hypothesized that very preterm birth due to verified FGR is associated with elevated blood pressure and increased arterial stiffness in adolescence and that FGR exacerbates the effect of preterm birth. We secondly hypothesized that sex has a modifying effect on relationships between FGR, very preterm birth and cardiovascular outcomes. The current study therefore examined adolescents born very preterm due to FGR, verified with abnormal fetal blood flow velocimetry, using sensitive measures of blood pressure variations and aortic stiffness. 24-hour ambulatory blood pressure measurements (24-hour ABPM) is the preferred method for assessing blood pressure variations,The specific aims were to assess the impact of very preterm birth and FGR on (1) 24-hour ABPM; (2) PWV and distensibility in the ascending and descending thoracic aorta using MRI; and (3) possible modifying effects by sex.20Examinations were performed at Skåne University Hospital, Lund, Sweden, between 2014 and 2019. The Regional Ethical Review board in Lund, Sweden, approved the study (Dnr 2013/244). All participants, and their guardians when appropriate, provided written informed consent before participation. Participants underwent 24-hour ABPM and MRI, and blood were sampled for measures of kidney function. Weight and height were measured in conjunction with 24-hour ABPM or MRI. Body surface area (BSA) was calculated using the Mosteller formula.21 absent or reversed end-diastolic blood flow in the umbilical artery as determined by a standardized protocol using Doppler velocimetry, and were delivered with cesarean section between 24–29 gestational weeks (preterm FGR). Two control groups with birth weight appropriate for gestational age (AGA) born in the same time-period (1998–2004) were identified and a total of 102 participants were included prospectively and divided into three groups forming 34 matching triplets  of all children (n = 371) who were born very preterm and who were admitted to the neonatal intensive care unit at Skåne University Hospital, Lund, Sweden. The causes of very preterm birth in this group included preterm premature rupture of the membranes, ablatio placentae, chorioamnionitis and being delivered as the healthy matched twin to a same sex individual in the preterm FGR group.9 This group was selected to match the preterm FGR group for sex, gestational age at delivery, and year of birth and thus consisted of those born very preterm without FGR (preterm AGA). Twins formed a matched pair if the following criteria were met; one twin was born with FGR, the other twin was born AGA, was of same sex, had normal blood flow in the umbilical artery, and twin-to-twin transfusion had been excluded.9 The second control group consisted of children born at term after normal pregnancy and were matched to both groups for sex and year of birth, forming the third triplet (term AGA). Detailed peri- and neonatal data and follow-up studies of cardiovascular, neuro-cognitive and pulmonary outcomes during childhood have been reported previously.24As part of a prospective cohort study, the current study included adolescents born very preterm with early onset FGR actively delivered due to fetal blood flow velocity abnormality between the years 1998 and 2004. Subjects had a birth weight <2 standard deviations,All individuals from the original cohort were contacted via mail and by phone and asked to participate in this prospective cohort study in adolescence. Potential differences in the presence of peri- and neonatal confounders, e.g., maternal smoking, preeclampsia and bronchopulmonary dysplasia in those who participated versus those who opted out from follow-up were evaluated.2 >0.30) at an age corresponding to 36 gestational weeks.Preeclampsia was defined as diastolic blood pressure >90 mmHg on two or more consecutive occasions >4 h apart, arising after 20 weeks of gestation, and proteinuria >300 mg/L in two random clean-catch midstream urine specimens collected ≥4 h apart. Bronchopulmonary dysplasia was defined as need for supplemental oxygen .25 Mean systolic and diastolic blood pressures were graded as normal (<90th percentile), prehypertension (≥90th to <95th percentile) or hypertension (≥95th percentile) for daytime and nighttime separately. A nocturnal decrease in mean arterial blood pressure <10% was graded as pathological and in addition to reference values for children,25 systolic or diastolic blood pressures above adult reference values were graded as hypertension. Normal values for adults were defined for 24 hours as <130/80 mmHg, for daytime <135/85 mmHg, and for nighttime <120/70 mmHg.16Quality assessment and evaluation of the activity diary together with blood pressure readings was performed by a physician (PS) with 10 years of experience. Generally, >80% of successful readings was deemed sufficient for inclusion. Evaluation was in accordance with European Society of Cardiology (ESC) guidelines, using reference values for children and adolescents based on sex, age, and height.Participants were imaged in supine position using a 1.5T MR scanner . Flow data were acquired in the ascending aorta and descending aorta at diaphragm level using a 2D phase-contrast gradient recalled echo sequence with retrospective ECG gating. Typical parameters were 35 timeframes per cardiac cycle, temporal resolution 23 ms, TR/TE = 9/6 ms, flip angle = 15°, 1.2 × 1.2 × 6 mm (Philips), and TR/TE = 10/3 ms, flip angle = 20°, 1.5 × 1.5 × 5 mm (Siemens). Velocity encoding was 150–250 cm/s to optimize for individual velocity resolution and to avoid aliasing. Respiratory-gated 3D angiography of the thoracic aorta was acquired using a T2-prepared balanced steady-state free precession sequence with isotropic resolution 0.88 mm (Philips) or 0.55 mm (Siemens).http://segment.heiberg.se, Medviso AB, Lund, Sweden).26 Two observers with 5 (JL) and 6 years (DR) of MRI experience performed analyses.Images were analyzed in Segment 3  at 1500 G for ten minutes, pipetted  into cryotubes, and stored at −80 °C . Cystatin C was measured at Skåne University Hospital Clinical Chemistry laboratory using validated routine clinical assays. Estimated glomerular filtration rate (eGFR) was calculated using the Caucasian, Asian, Pediatric and Adult (CAPA) equation.https://inkscape.org/). For 24-hour ABPM data, boys and girls were analyzed separately as per clinical routine whereas MRI data were analyzed both with both sexes combined and separately. Data are expressed as median (range). Kruskal-Wallis with Bonferroni’s multiple comparison test assessed group differences. The Jonckheere-Terpsta and Kendall’s Tau-b tests assessed trends between groups. Pearson’s chi-squared test or Fisher’s Exact Test assessed categorical variables. P values <0.05 were considered to show statistically significant differences.Statistical analyses were performed using SPSS 26.0  and GraphPad Prism 9 . Artwork was performed using the free and open-source vector graphics editor Inkscape (p = 0.003). Of those who underwent pharmacological treatment for persistent ductus arteriosus (PDA), 2/10 individuals in the preterm FGR group and 3/9 individuals in the preterm AGA group also underwent surgical treatment for closure of the PDA while one individual in the preterm FGR group underwent surgical treatment alone (Table p = 0.99) and 23, 25 and 27 of the fathers (p = 0.80) having gone through upper secondary school education. The respective numbers for university education were 11, 13 and 13 for mothers (p = 0.99) and 7, 8 and 10 for fathers (p = 0.80). The number of families who were cohabited at time of follow-up in childhood (~7 years) were 18, 18 and 25 for the respective group (p = 0.10).22A total of 79 adolescents agreed to participate in the current study (14 (13–17) years, 52% girls). Figure Table p = 0.37) or diastolic (p = 0.08) office blood pressure measurements (Table p ≥ 0.38). There were no differences in the prevalence of prehypertension, hypertension, or pathological day-to-night ratio in mean arterial blood pressure between groups  . In boys, the preterm FGR group had higher daytime mean arterial blood pressure  and 24-hour mean arterial blood pressure  as compared to the term AGA group. Further, in boys, trend analyses showed increasing diastolic and mean arterial blood pressures from term AGA to preterm AGA to preterm FGR for 24-hour and for daytime blood pressure   .Table p ≥ 0.5). When stratifying for sex, median PWV was higher in girls in the preterm AGA group as compared to girls in the term AGA group .Figure Interobserver variability for aortic centerline distance and PWV, reported as median , were −6 (−2 to −12) mm and −0.2 (−0.6 to 0.02) m/s, respectively. Median aortic centerline distance between observes was 195 mm whereas median PWV was 3.9 m/s.2, for the preterm AGA group 100 [IQR 85–118] ml/min/1.73 m2 and for the term AGA group 100 [IQR 89–110] ml/min/1.73 m2 (p = 0.35).Median cystatin C-based eGFR were for the preterm FGR group 94 [IQR 78–109] ml/min/1.73 mThe current study shows that very preterm birth due to early onset fetal growth restriction is associated with subtle blood pressure increases in adolescent boys. Further, arterial stiffness was increased in girls born very preterm with appropriate birth weight. Renal function did not differ between groups.11 however the effect of FGR or birth weight deviation on blood pressure was not studied. As in the current study, kidney function was normal in the previous study,11 indicating a limited negative effect of very and extremely preterm birth on kidney function and blood pressure in childhood and adolescence.There were no group differences in prevalence of prehypertension or hypertension as determined by 24-hour ABPM in adolescence. Similar observations, also using 24-hour ABPM, have been noted in a recent cohort of extremely preterm children (~8 years old) born between 2008 and 2011,12 which showed that boys but not girls have higher but still normal systolic and diastolic office blood pressure compared to controls. The current study, using 24-hour ABPM, indicates an additive effect of FGR to that of very preterm birth on blood pressure in boys. The ‘EXPRESS’ study lacked the definition of true FGR as the study did not have access to fetal blood flow velocity measurements. Further, the ‘EXPRESS’ study used office blood pressure, which in the current study showed no difference between groups, as opposed to the observed differences based on 24-hour ABPM. Trends in higher blood pressures for boys born preterm FGR as compared to preterm AGA may be due to differences in peri- and neonatal morbidity as both maternal hypertension and the presence of bronchopulmonary dysplasia, with higher prevalence in the preterm FGR group, are associated with increased blood pressure and arterial stiffness.33In absolute numbers, with no adjustment for age or height, there were no differences in systolic blood pressure for either daytime or nighttime for either sex. However, boys born preterm FGR had higher diastolic and mean arterial blood pressures in comparison with boys in the term AGA group. This is partly in line with the nation-wide follow-up study of the ’Extremely Preterm Infants in Sweden Study’ (EXPRESS) cohort,12 indicate that preterm birth associates with higher but still normal blood pressure in childhood and adolescence, with male sex and FGR as possible exacerbating factors. Earlier studies show strong associations between preterm birth and hypertension,34 with a possible lessened effect of preterm birth on blood pressure in those born after 1990.34 These earlier studies, in contrast to the current study and more recent studies, suggest female sex as an exacerbating factor.15 Whether the current and other recent cohorts, born in late 1990s and later, continues to show a limited and sex-specific effect of preterm birth and FGR on blood pressure when reaching adulthood remains to be studied. Nevertheless, even a small increase in blood pressure as observed for boys in the preterm FGR group in the current study, may have negative effects on future incidence of cardiovascular disease.36The current and other recent studies.37 Other factors indicative of socioeconomic differences,38 such as parental educational status and cohabitation in childhood that are known to impact blood pressure,39 were similar between groups. Socioeconomic factors could thus not explain blood pressure differences between groups in the current study.Although not significantly different, maternal smoking was almost three times as common in the preterm AGA group as compared to the preterm FGR group and could be hypothesized to be associated with increased blood pressure in this group.Further, the differences found using 24-hour ABPM were not observed using standard office blood pressure, underscoring the increased sensitivity of 24-hour ABPM in the assessment of future cardiovascular risk after very preterm birth and FGR.41 Whether low birth weight due to FGR exacerbates the effect of preterm birth is not clear from these earlier studies. Contrary to Cheung et al.,41 the current study showed no differences in arterial stiffness between groups and thus no additive effect of FGR to that of very preterm birth. More in line with the current study, however, the recent ‘EXPRESS’ studies showed normal office blood pressure,12 lower carotid stiffness, and no increase in aortic stiffness assessed by ultrasonography.13 in children born extremely preterm compared to those born at term.The current study showed that PWV and aortic distensibility were similar between groups, irrespective of preterm birth and FGR. This is in contrast to earlier studies indicating increased arterial stiffness after either preterm birth or low birth weight.5 In contrast to that previous study, however, the current study population had normal blood pressure, possibly indicating improved cardiovascular outcome in this more recent study population.When stratifying for sex, PWV was higher for girls in the preterm AGA group as compared to girls in the term AGA group. Increased arterial stiffness has previously been shown in adolescent girls born very or extremely preterm with no additional effect of low birth weight for gestational age to that of preterm birth.42 Differences in arterial stiffness and aortic dimensions between the preterm groups in the current study suggest differences in vascular development and subsequent response to extrauterine life. Whether differences in aortic dimensions or arterial stiffness are indicative of future cardiovascular disease in the current population remain to be shown.Finally, both the ascending and descending aorta were smaller in the preterm FGR group as compared to the term AGA group. Although not significant after BSA adjustment, most likely due to sample size, a visual trend of smaller vessels was observed for both groups born very preterm. A smaller and stiffer arterial tree increases afterload and is hypothesized to predispose for hypertension and cardiovascular disease both in those born preterm and after FGR.43Not all individuals from the original cohort participated in the current study, limiting statistical power. However, the strict inclusion criteria and close matching between groups and minimal differences in peri- and neonatal data observed between those participating and those who opted out, limit the potential negative effects of the small sample size. Finally, puberty may affect results and onset of puberty was not determined. It is however likely that most if not all participants were in puberty.Very preterm birth due to early onset fetal growth restriction is associated with higher, yet normal blood pressure in adolescent boys. This difference suggests an existing but limited impact of very preterm birth on cardiovascular risk in adolescence, possibly enhanced by male sex and fetal growth restriction.


The association between birth weight and ambient temperature during pregnancy remains inconclusive, and data from Chinese populations are scarce. We conducted a cross-sectional study to investigate the association between birth weight and ambient temperature during pregnancy among the residents of Suzhou Industrial Park, Suzhou, China.Information regarding 10,903 infants born between January 2018 and December 2018 who were born at the hospitals in Suzhou Industrial Park, Jiangsu province was obtained via public birth records.This study found that the ambient temperature during the first trimester of pregnancy was negatively correlated with birth weight, suggesting that elevated temperature may be related to lower birth weight. However, the ambient temperatures during the second and third trimesters of pregnancy were positively correlated with birth weight. Moreover, when the ambient temperature was below 15°C during the second trimester of pregnancy, the birth weight increased with temperature. However, when the temperature was higher than 15°C, the birth weight decreased with temperature. The relationship between ambient temperature in the third trimester and birth weight presented an inverted “U” curve. When the ambient temperature was lower than 20°C, the birth weight increased with ambient temperature, but when the ambient temperature was higher than 20°C, the increase of ambient temperature showed no significant relationship with the increase of birth weight.The ambient temperature was correlated with birth weight. The ambient temperature during the first trimester of pregnancy was negatively correlated with birth weight. The relationship between ambient temperature in the third trimester and birth weight presented an inverted “U” curve. Birth weight is an important indicator for evaluating fetal growth and development, which is related to early cognition, metabolism, and risk of cardiovascular disease during life . It is aThe known influencing factors of birth weight include race, socioeconomic situation, maternal energy intake during pregnancy, and maternal weight gain during pregnancy, maternal body size, and maternal disease status, maternal nutrition before pregnancy, gestational weeks, and maternal smoking history. Numerous studies have focused on the effects of seasonal or ambient temperature on birth weight, although the results have been inconsistent. Some investigators have found seasonal variations in birth weight , while others have not –13. TherInformation regarding 10,903 infants born between January 2018 and December 2018 who were born at the hospitals in Suzhou Industrial Park, Jiangsu province was obtained via public birth records. Pregnant women in our study are all permanent residents. The establishment of cards during pregnancy, prenatal examination, post-natal visit, and child health care of the subjects were completed in the local hospital. The information included date of birth, sex, birth weight (g), and gestational age (weeks). Information for infants who had died since the time of birth was not included. Each participant signed an informed consent form at the interview. According to the definition of WHO, infants whose birth weight is < 2,500 g are called low birth weight infants.Meteorological data were collected from China Meteorological Data Network including daily average temperature (°C) and daily average relative humidity (%) in Suzhou from March 20, 2017 to March 27, 2018. The data of Suzhou Air Quality Index (Daily Average) are from the National Environmental Monitoring Station. The baby weight measuring instrument is used for birth weight measurement. The unit is Kg, accurate to 2 decimal places, and the measuring accuracy is ±10 g. The measuring instrument is sent to the measurement department for calibration every year. The average temperature, relative humidity, and air quality index of early pregnancy (the last menstruation to the 12th week of pregnancy), middle pregnancy (the 13th week to the 27th week of pregnancy) and late pregnancy (the 28th week to the delivery) were calculated according to the daily average of the air quality index in Suzhou.t-test or ANOVA. A value of P < 0.05 for two-sided tests was considered statistically significant. A generalized linear model and a logistic regression model were used respectively to evaluate the effects of ambient temperature during different trimesters on the birth weight of newborns. Model 1: Adjust maternal age, pregnancy order and baby sex. Based on Model 1, the gestational age was adjusted in Model 2. Based on Model 2, environmental relative humidity and air quality index are adjusted in Model 3. A natural cubic spline with three degrees of freedom were defined for the environmental mean temperature and relative humidity in the generalized linear model. The exposure-effect curves of the environmental mean temperature and birth weight in different pregnancies were fitted. Data analyses were conducted using the SPSS Statistics 21.The clinical characteristics of the continuous variables were expressed as the mean ± SD and were tested using a two-sample p = 0.013), and mothers aged > 30 years had newborns with the highest birth weight. Moreover, significant differences in birth weight were found between women with 1, 2 or more pregnancies (p < 0.001), and women with more pregnancies had a higher newborn weight. Significant differences in birth weight were also found between newborn genders (p < 0.001), and males had higher birth weight than females. Furthermore, significant differences in birth weight were found between different modes of delivery  (p < 0.001), and the birth weight was higher in cesarean section. Significant differences in birth weight were also found between the newborns with gestational weeks < 37, 37–41, and > 41 weeks (p < 0.001), and the newborns with longer gestational weeks had higher birth weight. Among the low-birth-weight newborns, no significant difference was found in maternal ages, gestational weeks, infant gender, or delivery mode (p > 0.05). There was a significant difference between the newborns with gestational weeks < 37 and 37–41 weeks (p < 0.001), and newborns with longer gestational weeks had a higher birth weight.The basic information of pregnant women and newborns in the Suzhou Industrial Park 10,903 is shown in The daily mean temperature, daily mean relative humidity, and air quality index of pregnant women are shown in p < 0.001), wherein the birth weight decreased by 3.134 g (1.271–4.997 g) for every 1°C increases in temperature. There was no significant correlation between ambient temperatures in the second and third trimesters of pregnancy and birth weight.The results of logistic regression analysis between ambient temperature during pregnancy and birth weight in 10,471 newborns with normal birth weight are shown in The results of logistic regression analysis between ambient temperature during pregnancy and birth weight in 476 newborns with low-birth weight are shown in p-values of correlation coefficients of 0.0066 and 0.0318, respectively (The relationship between birth weight and ambient temperatures during first, second and third trimesters of pregnancy was analyzed by the generalized non-linear curve in 10,471 pregnant women and newborns. The results showed no non-linear relationship between birth weight and ambient temperature in the first trimester of pregnancy. A significant non-linear correlation was found between birth weight and ambient temperatures in the second and third trimesters of pregnancy, with ectively .This study found that the ambient temperature during the first trimester of pregnancy was negatively correlated with birth weight, suggesting that elevated temperature may be related to lower birth weight. However, the ambient temperatures during the second and third trimesters of pregnancy were positively correlated with birth weight. Moreover, when the ambient temperature was below 15°C during the second trimester of pregnancy, the birth weight increased with temperature. However, when the temperature was higher than 15°C, the birth weight decreased with temperature. The relationship between ambient temperature in the third trimester and birth weight presented an inverted “U” curve. When the ambient temperature was lower than 20°C, the birth weight increased with ambient temperature, but when the ambient temperature was higher than 20°C, the increase of ambient temperature showed no significant relationship with the increase of birth weight. The results of this study were consistent with several large cohort studies worldwide. Lawlor et al.  studied An animal study indicated that chronic heat stress during pregnancy may reduce blood flow in the uterus and umbilical cord and placental weight, resulting in lower birth weight of many species . HoweverThis study had some limitations. First, the indoor and outdoor activity times and real-time ambient temperatures of pregnant women were not monitored, which may have led to inaccurate estimation of real exposure temperature. Second, confounding factors, such as maternal living habits and nutritional status, were unavailable. Third, our study didn't analyze the relationship between humidity and newborn birth weight. Humidity is known to reduce the body's ability to release heat, its potential independent effects on health are unclear , 25. TheIn conclusion, our study has examined the association between the ambient temperature and birth weight based on normal and low birth weight population analyses. We found that the ambient temperature during the first trimester of pregnancy was negatively correlated with birth weight, suggesting that elevated temperature may be related to lower birth weight. Moreover, the ambient temperatures during the second and third trimesters of pregnancy were positively correlated with birth weight. Additionally, the relationship between ambient temperature in the third trimester and birth weight presented an inverted “U” curve. Independent replications in large sample sizes are needed to confirm the role of the ambient temperature with newborn birth weight found in this study for normal and low birth weight population.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.YD and LW conceived and designed the research. YD wrote the manuscript. LW, YD, MT, YM, and XC revised it critically for important intellectual content. YD and HZ performed the data analysis. All authors contributed to the interpretations of the findings, reviewed the manuscript, contributed to the article, and approved the submitted version.


We describe the use of serial block-face scanning electron microscopy (SBF-SEM) to resolve the three-dimensional architecture of cells and organelles in the cyst at ultrastructural resolution. We present a deep learning-based pipeline for high-throughput quantitative analysis of three-dimensional confocal datasets of cysts in vivo. We provide a method for laser ablation of cellular components for manipulating cyst cells in ovaries. These methods will facilitate the investigation of the cyst cellular organization, expand the toolkit for the study of the zebrafish ovary, and advance our understanding of female developmental reproduction. They could also be further applied to the investigation of other developmental systems.Oocytes develop in the germline cyst, a cellular organization in which germ cells are tightly interconnected and surrounded by somatic cells. The cyst produces oocytes for follicle formation and is a hub for essential processes in meiosis and oocyte differentiation. However, the formation and organization of the cyst, and their contribution to oocyte production in vertebrates remain unclear. Here, we provide tools for three-dimensional and functional  Summary: A description of a set of imaging-based tools for automated 3D analyses and functional live manipulations in vivo of the germline cyst, a cellular hub for oocyte production, in the zebrafish ovary. Oogenesis is a dynamic process that is essential for sexual reproduction. From insects to mammals, early oocytes develop in a cellular organization called the germline cyst, in which germ cells are clustered, interconnected, and collectively enveloped by somatic cells . The gerThe germline cyst serves as a hub for key events in oogenesis. Crucial events in meiotic prophase, including the induction of double-strand breaks and chromosomal pairing, occur in the cyst  Fig. 1B, 2023 F see als Fig. 1C.In addition to meiosis, the formation of a conserved oocyte organelle, called the Balbiani body (Bb)  begins iDrosophila model. In Drosophila ovaries, oogonia undergo exactly four rounds of mitotic divisions, forming a cyst of 16 cells  in ovaries and provide methods for segmenting and rendering SBF-SEM data to characterize the spatial organization of the germline cyst in 3D and at ultrastructural resolution. We present the implementation of the deep-learning algorithms StarDist  with three-dimensional image acquisition. In this setup, the sample is embedded in a block and after each scan, a microtome scrapes off a 70 nm-thick surface layer of the sample, exposing a new surface for imaging. From multiple iterations of image acquisition and sectioning cycles in a region of interest, 3D images at electron microscopic resolution are constructed.n=30 cells from three cysts and two ovaries). Segmented labels were then used to generate volume rendering of each component of the germline cyst  using TrakEM2 software  Fig. 2)Fig. 2). ine cyst  Movie 1). We previously identified a symmetry-breaking event in the oocyte at the zygotene stage , in two cysts we detected one oocyte with three CBs per cyst (Movie 2), indicating that at least one cell in the cyst can undergo three rounds of divisions. Although, typically for SBF-SEM, this analysis is limited to a small sample size (see Discussion), a scenario wherein most oocytes in a cyst contain one or two CBs, and few contain three, best fits the branched network class of cysts, but this remains to be determined. Whether the zebrafish cyst forms by synchronous or asynchronous divisions needs to be addressed by live time-lapse imaging. Nonetheless, these data provide the first indication of the organization of the zebrafish germline cyst as a branched network.We were previously able to reliably measure the size of the CB using SBF-SEM, resulting in an average diameter of 567±172 nm . We therDrosophila transfers material, including mRNA and proteins, from nurse cells to the oocyte through CB ring canals . Using this approach, we executed instance segmentation on high volumes of raw datasets in a robust and high-throughput manner (Movie 3) and a 16-cell cyst of zygotene-stage oocytes (Movie 4).We show here that cellular complexities in the juvenile ovary can be addressed and overcome robustly by using deep learning-assisted instance segmentation algorithms with custom models for cell types . We ran our pipeline on ovaries labeled with each of the above markers, and segmented cell borders and nuclei in 25 cysts of various stages from 13 ovaries.We show the 3D segmentation of nuclei and cell cortexes from a raw dataset of ovaries A-E. CellOur segmentation detected individual cells in cysts with high accuracy. In some cases, because of the inevitable minimal variability of transgenic expression, dye detection or antibody staining, the labeling signal was weaker along a cell border. These specific cases of insufficient labeling quality resulted in the false merging of cells in a few optical sections. We tested the accuracy in our hands, and manually supervised the calling of cells in cysts and compared the automated calling to manual calling.n=146 cells from eight cysts; n=207 cells from nine cysts; n=120 cells from eight cysts; n=473 cells from 25 cysts and 13 ovaries) of total cell calling from all cell border labels (n=335 cells in 25 cysts from 13 ovaries), which is consistent with the accuracy originally reported for StarDist , cultured ovaries are mounted in 1% low-melt agarose in a glass-bottom culture dish and reinforced with another layer of low-melt agarose to minimize movements A. On theTg(bact:Arl13b-GFP); Tg(bact:Cetn2-GFP)], which simultaneously visualizes cilia and centrosomes , we used the double transgenic line Tg(bact:Arl13b-GFP); Tg(bact:Cetn2-GFP), as described above. We marked an ablation ROI (Movie 5). Using the laser power parameters described above, we successfully excised the cilium (compare pre- and post-ablation panels in Movie 5), without cellular damage (Movie 4)  .Movie 6), we used ovaries of the single transgenic line Tg(bact:Cetn2-GFP), which visualizes centrioles. We imaged cysts pre-ablation, ablated a single centrosome (Movie 6) using same parameters as above, and monitored the cyst by subsequent time-lapse imaging for∼10 min, recording every ∼30 s (Movie 6). To confirm centrosome ablation and rule out its translocation to a different position, we imaged a stack of optical sections through the cyst z-axis as shown in the sum-projection images in Movie 6. We were able to ablate the selected centrosome specifically, without affecting non-ablated control centrosomes . Cysts appeared to remain vital post-ablation and during the course of recording, consistent with our previous experiments (To ablate the oocyte centrosome (eriments .Movies 7-9), we used ovaries of the Tg(h2a:H2A-GFP) transgenic line, which labels Histone 2A, visualizing nuclei in ovaries. Using the same settings, we ablated a single nucleus (Movies 7-9) in cysts, and monitored them by time-lapse imaging, acquiring z-stacks of cyst images every ∼60 s for 20-40 min (Movies 7-9). We were able to ablate a single nucleus specifically, whereas the non-ablated control nuclei were unaffected and cysts appeared to remain vital post-ablation and during the course of imaging . Movies 7-9 show examples of successful ablations of single nuclei in a 2-cell oogonia cyst (Movie 7), as well as in meiotic leptotene-zygotene cysts , demonstrating consistent results in cysts of different cell number and stage.To ablate the oocyte nucleus (These experiments demonstrate that our ablation protocol is suitable for ablating different organelles of various cellular and extracellular positions in the cyst (cytoplasmic centrosome versus the cell-protruding cilium), as well as sizes (centrosome versus nucleus). This versatility suggests that this protocol can be extended to be efficiently performed in ovaries expressing other transgenic reporters or labeled by vital dyes of interest, for manipulation of other organelles. Our method could thus be utilized to manipulate various cellular components of interest, paving the way for numerous possibilities for analyzing cellular developmental dynamics in real time.From insects to mammals, key events in oogenesis are executed in the context of the germline cyst. Deciphering the cellular architecture and functions of the cyst is required for understanding the developmental mechanisms that underlie female reproduction, but these remain unclear in vertebrates. We report powerful protocols for understanding the spatial-temporal development and 3D morphology of the germline cyst in zebrafish, and provide them in reproducible step-by-step protocols.Movies 1, 2). Our protocol utilizes ultrastructural resolution images for generating 3D views of the morphology of the germline cyst, including the intercellular organization of cyst cells and their subcellular structures. This tool provides valuable information for deciphering the cyst functional architecture and dynamics, as we show for the zygotene cilia, for mitochondria subcellular distribution during oocyte polarization dynamics (Movie 1), and for CB morphology, number (Movie 2) and content . It can be demanding to identify the ROI deep in tissues while keeping it intact for imaging, and there is a trade-off between the Movies 3, 4). Automatically analyzed datasets provide unbiased characteristic features of cyst cells, including their physical parameters, such as volume and surface area, and how they change during development. Applying this tool to datasets in which additional cellular structures are visualized by transgenic reporter lines, vital dyes, or immunostaining will provide ample information and an unprecedented quantitative understanding of the germline cyst. For example, visualizing the midbody in CBs in a Cep55l transgenic line . This protocol enables the experimental manipulation of cellular components of interest to study their real-time effects on cyst cell development over time. Genetic approaches and data from fixed samples can be limited in providing direct and precise functional information in real time and at high resolution. Even sophisticated conditional genetics resulting in tightly controlled mutant or loss-of-function conditions and combined with live imaging can be limited in providing information on rapid and highly dynamic phenotypes. Our protocol for laser ablation of cellular structures of interest in the zebrafish ovary comprises a complimentary approach to provide exactly this type of information and offers a new tool for functional analyses in the germline cyst.Our protocol for laser-induced ablation of organelles in germline cyst cells outlines a method for laser-assisted ablation of subcellular features in the germline cyst (Movies 5-9). The only requirements for this methodology are the use of a multiphoton microscope and the availability of a transgenic line that visualizes cellular features of interest for live imaging. To transfer our live-culturing protocol of ovaries from confocal settings to imaging by multiphoton microscopy, we only needed to minimally adjust our mounting set-up. When applying our protocol in other systems, we recommend that users carefully adjust the settings of the ablating laser stimulation according to the tissue, laser wavelength and ROI. Our reported settings provide an excellent reference point.We demonstrate that this protocol is useful for manipulating cellular organelles of various positions and sizes in cyst cells  (Tg(β-act:Cetn2-GFP) (Tg(h2a:H2A-GFP) (Tg(β-act:LifeAct-GFP) (.Juvenile ovaries were collected from 5–7 week post-fertilization (wpf) juvenile fish. Fish had a standard length (SL) measured according to 13b-GFP) , Tg(β-actn2-GFP) , Tg(h2a:H2A-GFP)  and Tg(βAct-GFP) .x and y, 75 nm in z. Images were binned by two giving a final pixel size in x and y of 12 nm 3. Open a new TrakEM2 (blank) workspace.4. Select a directory for saving TrakEM2 temporary files.5. In the TrakEM2 workspace panel, right-click on empty space, import the image stack.6. While importing, in the Slice Separation dialog box, manually enter the voxel depth and check the box ‘One Slice Per Layer’.7. In the TrakEM2 organizer panel, select the Template tab. Create a new ‘Area_list’ under ‘Add new child’.8. Drag and drop the area_list from the Template tab to the Project Object tab.9. In the Project Object tab, select the area_list and rename it to the intended structure to be segmented.10. Select the area_list from step 8, under the ‘Z Space’ tab.11. From the TrakEM2 workspace, select the ‘Brush Tool’ and the desired size.12. Draw a contour on the structure of interest on alternate slices A, middleMenu>Areas>Interpolate All Gaps13. Once all the slices are marked, fill the empty slices by interpolating the contours.14. Repeat steps 7-13 for all the structures of interest.Menu>Export>Arealists as labels (tif)15. Export the area_lists from the TrakEM2 workspace. Set the scale to 100.File>Save as>Tiff16. Save the exported labels image to the local directory.1. From the Imaris homepage, open the working directory and import the labels in the Imaris Arena Tab.File>Image Preferences2. Set the voxel size according to the raw image data.3. In the Surpass Tree Item Menu, select the ‘Create New Surface’ tool from the Surpass Tree Item Menu.4. In the Creation dialog box, uncheck the ‘Classify Surfaces’ and ‘Track Surface over time’ options.5. Select ‘Absolute Intensity’ and select he area around the peak in the histogram to segment.6. Set the desired color and transparency for the surface created in step 5 A, right.7. Repeat steps 3-6 for all surfaces of interest in the labeled image. Save the surface by exporting as ‘Scenes’.File>Import Scenes8. Import the raw dataset in the Surpass workspace and the surfaces saved in step 7.9. Add ‘Orthoslicer’ from the Surpass Tree Item Menu. Set the slice of choice.10. Note: Uncheck ‘Volume’ in Surpass Tree Items to hide the raw dataset.11. Go to the ‘3D Animation’ tab from the Surpass workspace menu.12. Adjust the scene in the desired orientation.13. Add the Animation option and the total number of frames.14. Hit ‘Record’, and select the directory for saving the animation B-G.xy resolution=1104×1104 pixels, 12-bit, 2× sampling averaging, pixel dwell time=0.59 s, zoom=0.8×, pinhole adjusted to 1.1 μm of z thickness, increments between images in stacks 0.53 μm, laser power and gain set in an antibody-dependent manner to 7-11% and 400-650, respectively, and below saturation conditions.Sample preparation for live time-lapse imaging and immunostaining was previously described . Images https://www.anaconda.com/).1. Install Anaconda environment manager  and Cellpose (https://github.com/MouseLand/cellpose.git) from the respective GitHub repositories.4. Download the Jupyter notebooks for StarDist ’. The closer to 1, the better the performance. 6. Evaluate the quality of the model by looking at:7. Enter the path to unseen datasets in Jupyter Notebook B. Choose8. Run the program to make predictions on the unseen dataset using the above created model C.Once the model is trained it can be further used to make predictions on unseen datasets.1. Launch a new Jupyter browser in the Cellpose environment using the Anaconda environment manager.2. Open a Cellpose notebook in the Jupyter notebook.3. Provide the directory path for images to be predicted B and sav4. Choose the provided model.5. Set do_3d=True for segmentation done using 3D image or set do_3d=False for 2D segmentation and stitching of labels.6. Set the minimum diameter of cell in pixels, if do_3d=True in step 5.7. Proceed to segmentation.1. Import the predicted label images into Fiji.Fiji>Image>Properties2. Set the voxel size to raw data voxel size from properties.3. Correct the labels for misidentification using the label editor from the MorphoLibJ plugin .4. Extract the features from the 3D label images using plugin ‘Analyze Regions 3D’ from MorphoLibJ  Fig. 3EEFig. 3E.Fiji>Image>Lookup Tables>Glasbey on Dark5. Set the Glasbey colormap LUT on the predicted label image from LUT menu.Fiji>Plugins>3D viewer6. Open 3D viewer from the Fiji Plugins menu.7. Select the filename from the drop-down menu in the import dialog box. Import as Volume.8. Visualize the spatial arrangement of segmented structures in volume reconstruction.9. Volume reconstruction can also be generated by importing the label images in to the Imaris Workspace followed by creating a surface using the ‘Surface Creation Tool’ D.2HPO4, 0.44 mM KH2PO4, 1.3 mM CaCl2, 1.0 mM MgSO4, 4.2 mM NaHCO3) for single-use dishes], micro-scissors and Forceps #5 (Sigma-Aldrich) were used for ovary dissection , ∼150 µl of mounting solution was added (agarose layer 1) and left until it started to solidify. Ovaries were transferred carefully to the mounting solution (agarose layer 1) in the glass-bottom dish using forceps A3. The oLaser excisions were performed using a Leica TCS SP8 MP two-photon microscope with a 25× objective and equipped with an incubation chamber set to 28°C. The glass-bottom dish with the cultured ovaries was mounted on the microscope stage inside the incubator chamber. The region of interest (ROI) was located using a 25× objective. The desired zoomed view of the ROI was obtained using ‘Digital Zoom’ and ‘Capture a Live View’. and draw The ROI for ablation was drawn on the above acquired image using ‘ROI tools’. Once the ROI was marked, the imaging time parameters were set as follows: pre-ablation timelapse acquisition 60 s; laser stimulation of the ROI 60 s at laser power 2.0-8.0% out of a power source of ∼3 W; post-ablation timelapse acquisition 600 s. These steps were repeated for each ROI. Note that only one ablation per cyst should be performed to avoid cell and tissue damage. Ovaries (4-6 wpf) should be mounted in the cell culture dish towards the center, leaving enough space for the lens to move around.Fiji was used for the preprocessing of image datasets and post processing of labeled images. Anaconda, an open-source distribution of Python was used specifically to maintain a dedicated virtual environment with the desired versions of Python packages installed. Jupyter Notebook, a web-based interactive computational environment for creating and sharing documents, was used to run deep-learning algorithms. Cellpose  is an an10.1242/develop.201349_sup1Supplementary informationClick here for additional data file.


Is skin-to-skin contact (SSC) between parents and their very preterm infants immediately after birth more effective than standard incubator care in enhancing mother-infant interaction at 4 months?In this secondary analysis of a randomized clinical trial including video recordings of 71 mother–very preterm infant dyads in interaction during free play, SSC provided by a parent during the first 6 hours after birth significantly enhanced child interactive behaviors and positive affect at 4 months.These findings suggest that alongside necessary nursing and medical care, clinical practices should support the mother-infant relationship by promoting immediate SSC with a parent after birth. This secondary analysis of the IPISTOSS randomized clinical trial examines the effect of immediate skin-to-skin contact at birth for very preterm infants on mother-infant interaction quality at 4 months of corrected infant age. Good-quality parent-infant interactions have protective effects on infant socio-emotional and behavioral development. These interactions are especially critical for very preterm infants at risk of vulnerabilities related to immaturity. Skin-to-skin contact (SSC) has been found to improve mother–preterm infant interaction behaviors, but few studies exist regarding its benefits when initiated immediately after birth.To determine the effect of immediate SSC at birth for very preterm infants on mother-infant interaction quality at 4 months of corrected age.This secondary analysis used data from the Immediate Parent-Infant Skin-to-Skin Study (IPISTOSS), a randomized clinical trial conducted between April 1, 2018, and June 30, 2021, at 3 neonatal units in Sweden and Norway. Participants included very preterm infants  and their parents. Four-month follow-up was concluded in December 2021. Data analyses were performed on March 16 and September 18, 2023.Infants were allocated to standard incubator care or SSC with either parent initiated at birth and continued throughout the first 6 hours after birth.The primary outcome was mother-infant interaction quality as measured with the Parent–Child Early Relational Assessment (PCERA), based on video recordings of a 5-minute free-play situation with mother-infant dyads at 4 months of corrected age. A multilevel regression analysis was performed.d = 0.67 ; P = .01). This effect remained significant when adjusting for primiparity, child sex, and observation setting.This analysis included 71 infants (31 twins [44%]) and 56 mothers. Infants had a mean (SD) gestational age of 31 weeks 3 (1.3) days, and more than half were male (42 [59%]); mothers had a mean (SD) age of 32 (4.9) years. There were 37 infants allocated to standard care and 34 to SSC with either parent after birth. During the first 6 hours after birth, fathers provided more SSC than mothers, with a median (IQR) of 3.25 (2.25-4.5) and 0.75 (0-2.5) hours, respectively. A statistically significant difference in 1 of 5 PCERA subscales  was observed, with higher-quality mother-infant interaction in the SSC group at 4 months (Cohen In this study of the effect of immediate parent-infant SSC after very preterm birth, SSC was beneficial for the mother-infant relationship. These findings suggest that immediate SSC should be supported in the clinical setting.NCT03521310ClinicalTrials.gov Identifier:  The SSC method is routinely practiced intermittently for preterm infants in the neonatal unit. Because of its multiple benefits, SSC between newborn infants and mothers provided in the immediate period after birth is considered the standard of care.11 and are critical for preterm infants with inherent vulnerabilities related to immaturity.13 Mother-infant interaction has been described as an act of reciprocity,14 in which both participants modify their behaviors depending on feedback provided by the other. It is often more difficult for parents to interpret and respond to the behavioral cues of preterm infants because these cues are weaker.15 Moreover, becoming a parent to a preterm infant can be stressful and has been linked to compromised psychological well-being in parents16 and reduced maternal sensitivity,17 which may affect interaction quality. Differences in dyadic interaction behaviors between mothers and preterm infants, compared with mother and full-term infant dyads, are most evident during the first 6 months of life.18 Early interventions that foster close parent-infant contact are therefore warranted.20Good-quality early parent-infant interactions have protective effects on infant social-emotional, cognitive, and behavioral development22 When in SSC, parents can respond more promptly to infant cues, providing opportunities for co-regulation.23 In a systematic review,24 SSC improved parent–preterm infant interaction patterns, but the timing of SSC was not investigated. Indeed, the first hour or hours after birth have been described as an early sensitive period,26 characterized by neuroendocrine changes that provide a biological and behavioral basis for social interaction, bonding, and attachment.29 In term infants, 2 hours of SSC after birth positively influenced mother-infant interaction  1 year later.30 In preterm infants, SSC onset within the first 3 days favored the development of infant interactive behavior at 6 months,31 and SSC within the first day of life predicted mothers’ sensitivity to their infant.32 Very preterm infants placed in SSC with their mother for 60 minutes at 1 hour after birth demonstrated better responses during mother-infant interaction at 6 months compared with dyads with only visual contact.33 To our knowledge, no study has evaluated the effect of SSC with either parent immediately after birth on mother-infant interaction.Skin-to-skin contact provides an environment that is sensitive to the infant’s needs and gives opportunities for emotionally supportive interactions that stimulate brain growth and development.There seem to be critical time points after birth when SSC is especially important for the developing parent-infant relationship. Yet evidence remains scarce as to whether parent-infant SSC initiated immediately after birth for preterm infants is more beneficial than standard incubator care (with later initiation of SSC), in terms of supporting the mother-infant relationship. Thus, the main objective of this study was to determine the effect of immediate SSC at birth for very preterm infants on mother-infant interaction at 4 months of corrected infant age. An exploratory and secondary objective was to investigate whether the potential relationship between immediate SSC after birth and mother-infant interaction at 4 months would be mediated through accumulated SSC in the early postpartum period (within 72 hours and 8 days after birth). We hypothesized that SSC provided to the very preterm infant within the first hours after birth improves mother-infant interaction quality in infancy.34 Electronic randomization was performed in uneven block sizes, with a 1:1 ratio, and stratified by site and gestation (28 weeks 0 days to 30 weeks 6 days and 31 weeks 0 days to 32 weeks 6 days). Ethical approval was obtained from the Swedish Ethical Review Authority and the Norwegian Regional Ethical Committee. Research staff informed parents meeting the IPISTOSS inclusion criteria, and written informed consent was obtained from both parents before birth. The study followed the Consolidated Standards of Reporting Trials (CONSORT) reporting guideline.This secondary analysis reports on a secondary outcome from the Immediate Parent-Infant Skin-to-Skin Study (IPISTOSS), a randomized clinical trial with 2 parallel, nonblinded groups conducted between April 1, 2018, and June 30, 2021.The IPISTOSS study was conducted at 2 neonatal units at Karolinska University Hospital in Stockholm, Sweden, and at the neonatal unit at Stavanger University Hospital in Stavanger, Norway. Screening was performed for women admitted to obstetric units with threatening preterm labor. This study included inborn infants (singletons or twins) with a gestational age of 28 weeks 0 days to 32 weeks 6 days, regardless of birth mode. Infants with congenital infection, major malformations, or other conditions deemed contraindicating to participation were excluded.34The intervention consisted of SSC between either parent and their very preterm infant (or infants) initiated immediately after birth (SSC group) and continued throughout the first 6 hours after birth and was compared with conventional care in an incubator or cot (control group). Electronic randomization was performed by research staff when birth was imminent. Twins were allocated to the same study group. During the intervention period, only the place of care differed; all other monitoring, nursing, and medical care were identical in both groups, as previously described in the IPISTOSS research protocol in After vaginal birth, SSC was initiated immediately, or as soon as possible, on the mother’s chest, with positioning assisted by the neonatal team. The infant was cared for initially in the birth unit and later transferred to the neonatal unit while maintaining SSC with either parent. After cesarean delivery, SSC was initiated with the father until the mother could be transferred to the neonatal intensive care unit. Twins were either cared for with one parent each or placed together with one of the parents.Infants allocated to the control group were stabilized in a warmer  or in an incubator and then transported to the neonatal unit in an incubator. Intermittent SSC was initiated after the first 6 hours. Parents in the control group were allowed to stay at their infant’s bedside and were able to touch the infant in the incubator or cot.35 and Spielberger State-Trait Anxiety Inventory36) and parental stress (Swedish Parenthood Stress Questionnaire37) were collected. Mother-infant interaction was video recorded according to a standard procedure.38A follow-up visit was conducted at 4 months (±2 weeks) of corrected infant age in the clinic or at participant homes . The visit was made in the morning at a time when the infant preferably had slept and was newly fed. Baseline data regarding maternal mental health  was recorded with the Parent-Infant Closeness Diary38 during video-recorded free play. Mothers were instructed to play with their infant as usual, with preselected toys available. The PCERA includes 29 parent items, 28 child items, and 8 dyadic items. Based on a 5-minute interaction,40 each item is rated on a 5-point Likert scale. Higher scores indicate more positive quality in interaction or lack of negative affect or behavior. Scores 1 and 2 describe an area of concern, score 3 an area of some concern, and scores 4 and 5 an area of strength. Two blinded certified coders (including J.L.) rated the data, and 21% of the data were double scored. The coders agreed on 82% of the items on a categorical level. The 5-point scale was used in the analyses, and items were combined into subscales following the general guidelines in the PCERA manual.38 Two dyadic scales were combined into 1 global dyadic scale, producing 5 conclusive subscales: (1) maternal positive affect, sensitivity and responsiveness; (2) maternal negative affect and behavior; (3) infant positive affect, communicative and social skills; (4) infant dysregulation and irritability; and (5) dyadic emotional tone, reciprocity and regulation 41 Data analysis was performed according to intention to treat. Descriptive statistics were used to present baseline variables. Crude and adjusted effects of the dichotomous SSC vs control treatment variable on the PCERA subscales and on accumulated time of skin contact were estimated with multilevel regression analyses, in which children were nested within mothers, thereby controlling for the dependence of data from twins with the same mother. Effect sizes were estimated with Cohen d. Adjustments were made for statistically significant imbalances found between groups in terms of primiparity, child sex, and observation setting (home vs clinic). Through bootstrapping 1000 subsamples from the study sample, we estimated the size and statistical significance of mediated effects of SSC vs controls on PCERA subscales via accumulated time of SSC. The mean of the mediated effect across the 1000 bootstrapped subsamples was divided by the SD of the mediated effect across the subsamples. This gave an estimated z score of the mediated effect, used to estimate a P value of the effect. P < .05 was considered statistically significant with a 2-sided hypothesis test. Analyses and illustrations were conducted with IBM SPSS Statistics, version 28 (IBM Corp), and R statistical software, version 4.1.3 (R Core Team) employing the effsize42 and beanplot43 packages. Data analyses were performed on March 16 and September 18, 2023.The IPISTOSS sample size was calculated for the main outcome variable of infant cardiorespiratory stability.This study included 71 infants (31 twins [44%]) and 56 mothers in the PCERA analysis at 4 months. Infants had a mean (SD) gestational age of 31 weeks 3 (1.3) days and a mean (SD) birthweight of 1535 (408) g; 42 (59%) were boys and 29 (41%) were girls. Mothers had a mean (SD) age of 32 (4.9) years; 32 (57%) were primiparous. A total of 37 infants were allocated to standard care and 34 to SSC with either parent after birth. Baseline characteristics of infants and mothers, including depression symptoms, anxiety symptoms, and parenting stress, were distributed equally between groups, except there were more boys and first-time mothers in the SSC group . Figure For infants in the SSC group, SSC was initiated at a median (IQR) time of 15 (0-62) minutes after birth. During the intervention period of 0 to 6 hours, the median (IQR) SSC duration was 5.25 (4.5-5.5) hours, and fathers provided more SSC than mothers during this period . d = 0.67 ; P = .01), with higher-quality interaction in the SSC group . For PCERA subscale 5 , there was no significant difference in the unadjusted analysis ; however, the difference became significant  after adjustment for observation setting. No other significant differences between the study groups were observed.SC group . After ad = 1.04 ; P < .001). This group’s accumulated time of SSC was also higher during the first 8 days, with a median (IQR) of 51.75 (36.5-70) vs 36.5 (24.75-44) hours . Two mediation analyses were performed to test whether the effect of SSC vs the control on PCERA subscale 3 was mediated through the accumulated SSC time within the first 72 hours and 8 days after birth. No significant effects were observed.After the intervention, infants’ accumulated time of SSC was higher in the SSC group during the first 72 hours, with a median (IQR) of 17 (10.5-25) vs 10 (5.25-13.5) hours in the control group  to further support their physiological transition after birth,49 and SSC was provided by both mothers and fathers.Sensitive periods have been defined as opportunities that exist in early environments. These opportunities are biobehavioral experiences between parents and infants that trigger specific neuroendocrine systems influencing gene expression, brain development, and parent-infant attachment.50 In a meta-analysis,51 SSC generally had a positive effect on preterm infant self-regulation skills, such as emotion regulation, which is important for social interaction. The positive development in infant interactive behaviors may contribute to more positive dyadic interaction, because these infants may appear more engaging to their mother.In this study, enhanced interaction quality in the SSC group was observed for infant PCERA subscale 3, which describes positive affect and communicative and social skills. Furthermore, after adjustment for observation setting, a between-group difference was observed for dyadic PCERA subscale 5, which describes emotional tone, reciprocity and regulation between mother and infant. Thus, immediate SSC seems to be especially beneficial for interactive behaviors of very preterm infants, and it may also benefit the dyadic aspects of interaction. One possible interpretation is that infants exposed to SSC at birth became more mature social partners, making it easier for their mother to respond to and interact with them. As reported previously, preterm infants have an increased risk of less synchronous interactions due to their immature nervous system and diffuse behavioral cues.52 and it also points to the triadic nature of family relationships.53Interestingly, in our study, fathers provided the most SSC at birth. This finding reflected the clinical situation, with mothers often unavailable for SSC during the first hours after a cesarean birth, for example. The beneficial effect of SSC on interaction was assessed in the mother-infant dyad 4 months later. Our findings showed that the higher-quality mother-infant interaction in the SSC group was driven by the infants’ enhanced social skills. Thus, an important conclusion of the present data might be that from a developmental perspective, time spent in SSC after birth is valuable and may be provided by either parent. This finding highlights the role of fathers in supporting the development of their very preterm infant immediately from birth when the mother is not available,55 and it allowed for an objective assessment of mother and infant behaviors. Interrater reliability between the 2 blinded coders was high, as was the internal consistency of the PCERA subscales.This study had several strengths, including its randomized design, which ensured substantially decreased selection bias. The study was well controlled, since only the place of care differed during the intervention. At the 4-month follow-up, 20 (22%) of the originally randomized infants were lost to the PCERA analysis; however, no differences between analyzed infants and dropouts were found, minimizing the risk for attrition bias in this study. Another strength was the robust observational measurement tool used. The PCERA is well used in studies worldwide and suitable for the preterm population,41 The current exploratory analysis of accumulated SSC was not prespecified, with a chance of a type 2 error given the sample size. We applied the same exclusion criteria for both groups, but 4 infants in the intervention group were excluded due to health conditions; this may have influenced the results either way. Interactions were video recorded both in the clinic and in participant homes; we could not always use the preselected toys because they were not allowed into homes due to COVID-19 restrictions. These limitations may have caused some variability in PCERA scores, so we adjusted for setting in the analysis. Also, dyadic interaction was observed only in a free-play situation, which might limit the generalizability of these findings to other  caregiving situations.56 The study population is from a high-income setting offering high-quality neonatal care, and the results should be interpreted within this context. Finally, although most time in SSC after birth was spent between fathers and infants, the effect of SSC was evaluated only with the mother-infant dyad. Still, the early exposure to SSC was clearly associated with higher scores of communication skills and positive affect in infants. Future research should investigate the effects of immediate SSC on the father-infant relationship as well as on parent-infant interaction in diverse caregiving settings and situations.This study had limitations. The sample size was small, due to the main trial being terminated earlier because of benefit of the intervention.In this secondary analysis of the IPISTOSS randomized clinical trial, SSC practiced between a parent and a very preterm infant in the immediate postpartum period after birth enhanced child interactive behaviors and positive affect at 4 months of corrected infant age. Skin-to-skin contact may also benefit the dyadic aspects of interaction. These findings support the existence of a sensitive period after very preterm birth, during which close contact between parent and infant may induce a long-term positive effect on the parent-infant relationship. To support infant long-term development, clinical practices should consider the place of care to be in immediate direct SSC with a parent after birth, alongside other necessary nursing and medical care.


Ixodes trianguliceps Birula lives in the nests and burrows of its small mammalian hosts and is—along with larvae and nymphs of Ixodes ricinus or Ixodes persulcatus—one of the most commonly collected tick species from these hosts in its Eurasian range. Ixodes trianguliceps is a proven vector of Babesia microti. In this study, up-to-date maps depicting the geographical distribution and the climate preference of I. trianguliceps are presented. A dataset was compiled, resulting in 1161 georeferenced locations in Eurasia. This data set covers the entire range of I. trianguliceps for the first time. The distribution area between 8I. trianguliceps, the georeferenced locations were superimposed on a high-resolution map of the Köppen–Geiger climate classification. The Köppen profile for I. trianguliceps, i.e., a frequency distribution of the tick occurrence under different climates, shows two peaks related to the following climates: warm temperate with precipitation all year round (Cfb), and boreal with warm or cold summers and precipitation all year round . Almost 97% of all known I. trianguliceps locations are related to these climates. Thus, I. trianguliceps prefers climates with warm or cold summers without dry periods. Cold winters do not limit the distribution of this nidicolous tick species, which has been recorded in the European Alps and the Caucasus Mountains up to altitudes of 2400 m. Conversely, I. trianguliceps does not occur in the Mediterranean area with its hot and dry summers.The Eurasian shrew and vole tick  Ixodes trianguliceps Birula, the Eurasian shrew and vole tick , is a proven vector of Babesia microti  habitats in deciduous, mixed and coniferous forests. In Russia, most I. trianguliceps findings are located within the dark-coniferous forest of the Central and Southern Taiga. It is less often found in pine, broad-leaved and aspen-birch forests. Occasionally, the tick penetrates into the Northern Taiga and the forest-steppe , whose distribution area can be retrieved from the International Union for Conservation of Nature  is provided on I. trianguliceps ticks and also research on the vector competence of I. trianguliceps for any pathogens. Because this tick species is endophilous and it is usually not possible to collect its unfed stages by flagging, infections of I. trianguliceps with microorganisms have been detected only in individuals removed from hosts. However, this approach leaves it open whether the found microorganisms were freshly taken by that tick with the current bloodmeal or whether the unfed tick had already carried that infection. As a consequence, even a positive result leaves the critical eco-epidemiological question open whether or not I. trianguliceps is a vector of the found microorganism.Finally, the literature search included the occurrence of microorganisms or their DNA/RNA in I. trianguliceps and a higher resolution section of the Greater Alpine Region (GAR) is shown in Fig. I. trianguliceps in cooler climate regions such as the European Alps. There the tick has been found both in Switzerland at Göscheneralp  and boreal with precipitation all year round Df (41%). Thus, a total of 99% of all I. trianguliceps locations was reported in these climates, and it is evident that I. trianguliceps prefers precipitation all year round. This agrees surprisingly well with the Köppen profile for Dermacentor reticulatus .A key result is the determination of the climate preference of I. trianguliceps findings near Göscheneralp and Obergurgl described above are located in the so-called Alpine belt above the tree and forest line. This altitudinal belt—for details see Rubel et al.  and hemorrhagic fever renal syndrome (HFRS), which motivated the early work of Korenberg and Lebedeva (I. trianguliceps, and it is now known that HFRS is caused by hanta viruses, which are transmitted through aerosolized excrement of rodents. However, numerous pathogenic bacteria and protozoa have been found in I. trianguliceps. It must be pointed out that the finding of any tick-borne pathogens in ticks removed from hosts is no proof of vector competence. Without proven capability of transmission the vector function of a given tick species for a given pathogen is not substantiated (Kahl et al. I. trianguliceps are only available for Babesia microti, which were carried out for the first time by Young (B. microti between the tick vector I. trianguliceps and the host M. glareolus. In experimental infection studies, transovarial transmission of B. microti could be ruled out and the natural transmission cycle explained by host-to-vector, vector-to-vector, and vector-to-host transmission. Without experimental transmission carried out, but indicated by extensive field studies, I. trianguliceps is a putative vector for Anaplasma phagocytophilum (Bown et al. Borrelia burgdorferi s.l. spirochaetes in I. trianguliceps removed from hosts. They would also justify experimental transmission studies, but for the time being this tick species can only be called a possible vector of B. burgdorferi s.l. (Eisen Table Lebedeva , was notby Young  in the Uby Young  quantifil. Eisen .I. trianguliceps, a dataset of 1161 locations was collated to compile a geographical map covering its whole distribution range from Ireland and the Spanish Atlantic coast in the west to Lake Baikal in the east. Although there are numerous recent studies on I. trianguliceps (Keskin and Selcuk I. trianguliceps were assigned climate classes using digital data from the global Köppen–Geiger climate classification (Kottek et al. I. trianguliceps, according to which the tick species occurs primarily in the warm temperate and boreal climate zones with precipitation in all seasons. However, I. trianguliceps has also occasionally been found in Alpine tundra climates. Looking at the list of pathogens found in feeding I. trianguliceps ticks (Table B. microti, it becomes clear that there is a great need for having more such studies to prove or disprove its vector competence for some tick-borne pathogens (Bonnet and Nadal To summarize the current knowledge of the distribution of ks Table  and the Dermacentor reticulatus (Brugger and Rubel I. trianguliceps have already been compiled and can be downloaded as a preview from the following link https://epidemic-modeling.vetmeduni.ac.at/.The here presented data might be a suitable basis for compiling maps based on species distribution models as already applied for


Population‐based administrative data have rarely been used to compare the birth prevalence, risk factors for occurrence, and mortality of congenital diaphragmatic hernia (CDH) subtypes.We used a national birth cohort to identify CDH subtypes and compared their birth prevalence, relationship with maternal age after accounting for sociodemographic factors, and 1‐year mortality rates.Linked hospital admission and death records were used to identify isolated and complex CDH cases  among singleton livebirths in England between 2002 and 2018. The prevalence of each CDH subtype per 10,000 livebirths was estimated overall and by infant, birth and maternal characteristics. The relationship between maternal age and each subtype relative to no CDH was examined using multivariable log‐binomial regression to estimate risk ratios (RRs). One‐year mortality rates were examined using Kaplan–Meier curves and the hazard ratio (HR) of complex versus isolated CDH was calculated using Cox regression.Among 9.5 million livebirths, we identified 1285 with isolated CDH and 1150 with complex CDH. The overall prevalence of isolated and complex CDH was 1.4  and 1.2  per 10,000 livebirths, respectively. Only complex CDH was associated with maternal age. Compared with maternal age 25–34 years, complex CDH risk was elevated for maternal age < 20 years . Risk was highest for maternal age ≥ 40 years  although accounting for chromosomal anomalies attenuated the risk . The 1‐year mortality rate for complex CDH  was slightly higher than for isolated CDH  .Mechanisms of occurrence differed between and within CDH subtypes and 1‐year mortality of complex CDH was slightly higher than for isolated CDH. How do congenital diaphragmatic hernia (CDH) subtypes compare in their birth prevalence, occurrence across different maternal age groups and mortality?Few epidemiological studies have described the birth prevalence of subtypes of CDH or examined potential risk factors for their occurrence. Current mortality estimates for subtypes are limited by the use of registry data, which may be subject to ascertainment bias.We identified differences in the association of CHD subtypes with maternal age after accounting for sociodemographic factors which suggest different mechanisms of occurrence. We estimated 1‐year mortality rates for subtypes and examined mortality before and after surgery.1Congenital diaphragmatic hernia (CDH) is a rare condition with high mortality that affects 2.2–4.9 of every 10,000 births in England.Birth prevalence estimates for CDH subtypes have rarely been reported.Subtype aetiology is uncertain although genetic and maternal factors are likely involved.Despite frequent assertions that CDH subtypes differ in mortality rates, the evidence base is sparse. A specialist registry study using data from UK paediatric surgical centres did not find evidence of an association between additional anomalies and 1‐year mortality of CDH but would miss cases born elsewhere that died before transfer.To address these limitations, we analysed all livebirths in England between 2002 and 2018 using linked hospital admission and death records which, unlike registry data, can explore the epidemiology of rare CDH subtypes at a national level while avoiding ascertainment bias. We described the birth prevalence of subtypes overall and by infant, birth and maternal characteristics. We assessed maternal age as a risk factor for each CDH subtype relative to no CDH after accounting for other sociodemographic factors. We also examined subtype mortality at 1 and 10 years of age, by birth period and by surgery status.22.1International Classification of Diseases 10th Revision (ICD‐10) and Classification of Interventions and Procedures 4th Revision (OPCS‐4), respectively. Linked death records include dates and all causes of death encoded using ICD‐10.We used linked Hospital Episode Statistics (HES) and Office for National Statistics (ONS) death records. HES contains data from all state‐funded hospital admissions in England; 97% of births (livebirths and stillbirths) are captured and subsequent admissions of live‐born infants are linked.2.2We carried out a retrospective study using a national cohort of singleton livebirths delivered between April 2002 and March 2018 to linked mothers resident in England.2.3Infants were defined as having CDH if a diagnosis and repair were recorded in HES, or if CDH was recorded on the death certificate Table . InfantsRecords of infants with CDH were searched for additional congenital anomaly diagnoses. The ICD‐10 codes used to define these were part of a wider code‐list developed by Hardelid et al (2013) to identify conditions requiring clinical follow‐up Table .23 The2.4Data on birth year as well as infant, birth and maternal characteristics were available. Birth year was grouped into 4‐year periods. Infant characteristics included sex and ethnic group. Birth characteristics included gestational age at delivery and birthweight. Maternal characteristics included maternal age, maternal deprivation quintile ,2.5Infant, birth and maternal characteristics of the study population were described by CDH status . Overall birth prevalence per 10,000 livebirths was calculated for any, isolated and complex CDH. Birth prevalence of isolated and complex subtypes were reported by birth period, infant, birth and maternal characteristics.n = 7,807,310) of the original population  and, thus, could be affected by selection bias. We, therefore, also performed multiple imputation (further details below); however, because missingness affected all variables similarly and is thought to relate to poor infant healthRisk ratios (RRs) for isolated or complex CDH compared with none were estimated for maternal age groups using univariable and multivariable log‐binomial regression, with the latter accounting for birth period, infant ethnicity , maternal deprivation and region Box . The anaMulticollinearity between ethnicity, deprivation and region was evaluated by monitoring the standard errors of their coefficients. To assess how much of the association between maternal age and complex CDH was accounted for by chromosomal anomalies we repeated the analysis excluding cases with chromosomal conditions.The cumulative probability of death up to 1 year  for infants with isolated and complex CDH was estimated using Kaplan–Meier curves to account for varying follow‐up. The mortality hazard ratio (HR) and 95% confidence interval (95% CI) for complex versus isolated CDH was calculated using univariable Cox regression. One‐year mortality rates by birth period were examined by plotting separate Kaplan–Meier curves. Ten‐year mortality rates were also estimated.The proportion of infants with each CDH subtype who died with and without surgical repair was calculated with 95% CIs. To assess the impact of surgery on mortality rates we fitted a proportional hazard model where surgery was treated as a time‐varying covariate.2.6Missing data in the study population were observed for the following characteristics: infant sex (0.1%), infant ethnicity (15.9%), maternal age (2.1%), maternal deprivation (0.3%), gestational age (19.2%) and birth weight (14.6%). Analyses of maternal age and CDH occurrence were repeated using multiple imputations (under an assumption of missingness at random) with 10 imputed datasets generated using chained equations. The imputation model included all variables involved in the analysis of maternal age and CDH occurrence as well as gestational age and birth weight as missingness in birth characteristics was correlated with missingness of maternal age.2.7Missingness of infant, maternal and birth characteristics in HES may correlate with poorer infant health as clinical staff may have fewer opportunities to record these characteristics when infants have high‐care needs.2.8We have a data sharing agreement with National Health Service (NHS) Digital to use a de‐identified extract of HES linked to ONS death records; therefore, we did not require ethics approval to use English data sets.33.1n = 1150) were considered complex due to the presence of additional congenital anomalies outside of the respiratory and digestive systems (Table n = 973) of infants with CDH, followed by nervous system  and chromosomal anomalies   and 1.2  per 10,000 livebirths, respectively  occurred among infants with isolated CDH while a further 1.1% (n = 381) occurred among complex CDH cases. The overall 1‐year mortality rate for all livebirths with isolated CDH was 29.7%  and for complex CDH was 33.1% , with an indication of greater hazards in complex versus isolated CDH  . The association between advanced maternal age and complex CDH was largely explained by chromosomal anomalies. The 1‐year mortality rate was only slightly higher for complex compared with isolated CDH. Mortality for both subtypes was similar in the absence of surgery and in the neonatal period, when most deaths occurred, but was higher for complex than isolated CDH after surgery.3.6.2A key strength of our study was the use of national, routinely‐collected administrative data. This enabled robust estimates of CDH prevalence and mortality among live‐born infants at a national scale. Our study avoided the bias associated with specialist registries that only include live‐born infants who attend tertiary centres and miss severely affected livebirths that die earlier. While population‐based regional registry studies can address this bias, these were only available for some areas in England until recently and studies were limited in size.3.6.3Severe CDH cases are more likely to result in fetal death or termination but could not be examined using HES as antenatal diagnoses are unreliably recorded and stillbirth certificates are not linked. Population‐based registry data on CDH cases in England during the study period suggested 3.9% were stillborn and 23.8% resulted in termination.Incomplete data were a further limitation. Our findings from multiple imputation analyses and sensitivity analyses restricted to infants with completely recorded infant, maternal and birth characteristics suggest we cannot preclude the possibility that in the wider population of complex CDH (which includes infants with incomplete records), the risk of complex CDH is not increased for young maternal age. However, although infants with more complete records may represent a healthier subset of CDH infants, comparison of mortality or repairs between CDH subtypes produced similar results when restricted to infants with completely recorded characteristics.3.6.4The prevalence estimate of any CDH was consistent with previous estimates, as were findings of a preponderance among males, preterm and low‐birthweight infants and findings that cardiac, nervous system and chromosomal anomalies were among the most frequent additional anomalies.Birth prevalence of complex CDH increased over time, consistent with a European registry study.Advanced maternal age was not associated with isolated CDH but was associated with complex CDH, which suggested different mechanisms of occurrence. The genetics of isolated CDH remain elusive but studies report it is more likely than complex CDH to be associated with inherited, non‐age‐dependent genetic variants.UK studies that compared 1‐year mortality for isolated and complex CDH reported conflicting results.4We quantified the national prevalence of isolated and complex CDH among livebirths and described differences in their occurrence and prognosis. Young and advanced maternal age were associated with an increased risk of complex but not isolated CDH, with chromosomal anomalies accounting for some of the increased risk associated with advanced maternal age. The prognosis of subtypes differed after surgical repair, with complex cases experiencing higher mortality rates. Administrative data could be usefully enhanced through linkage to specialist registry data  in order to help further our understanding of the prevalence, aetiology and prognosis of subtypes.MP, BLD, SL, RG and PDC conceptualised and designed the study. MP and SL carried out initial analyses. MP, BLD, SL, AZ, RG and PDC contributed to the interpretation of the data. MP drafted the manuscript. All authors critically reviewed the manuscript for important intellectual content. All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work.This project is funded by the National Institute for Health Research Great Ormond Street Hospital Biomedical Research Centre (NIHR GOSH‐BRC). NIHR GOSH‐BRC had no role in the design, conduct, analysis or interpretation of the study and no role in drafting the manuscript.The authors have no conflicts of interest to disclose.Appendix S1.Click here for additional data file.