The "toxic window" of amoxicillin exposure during pregnancy on long bone development in fetal mice.

AIMS: Amoxicillin is a broad-spectrum beta-lactam antibiotic used to treat infectious diseases in pregnant women. Studies have shown that prenatal amoxicillin exposure (PAmE) has developmental toxicity on fetal development. However, the effect of PAmE on long bone development has not been reported. This study aimed to investigate the "toxic window" of PAmE on long bone development and explore its possible mechanism in fetal mice. MATERIALS AND METHODS: Pregnant mice were administered amoxicillin by gavage at different stages (gestational day (GD)10-12 and GD16-18), different doses (150 and 300 mg/kg·d) and different courses (single and multiple courses). Fetal femurs were collected at GD18 and bone development related indicators were detected. KEY FINDINGS: The results showed that PAmE significantly reduced the length of the femur and primary ossification center of fetal mice, and inhibited the development of fetal growth plate. Meanwhile, PAmE inhibited the development of bone marrow mesenchymal stem cells, osteoclasts and endothelial cells in fetal long bone. Further, we found the fetal long bone developmental toxicity induced by PAmE was most significant at late-pregnancy (GD16-18), high dose (300 mg/kg·d) and multiple-course group. Besides, PAmE inhibited the expression of Wnt/ß-catenin signaling pathway in fetal long bone. The ß-catenin mRNA expression was significantly positively correlated with the development indexes of fetal long bone. SIGNIFICANCE: PAmE has toxic effects on long bone development, and there was an obvious "toxic window" of PAmE on the long bone development in fetal mice. The Wnt/ß-catenin signaling pathway may mediate PAmE-induced fetal long bone development inhibition.

Antenatal Antidepressant Prescription Associated With Reduced Fetal Femur Length but Not Estimated Fetal Weight: A Retrospective Ultrasonographic Study.

PURPOSE/BACKGROUND: Antidepressants are among the most frequently prescribed medications during pregnancy and may affect fetal weight. Associations between antenatal antidepressant use and ultrasonographic measures of fetal development have rarely been examined. We hypothesized that the prescription of an antenatal antidepressant would be associated with lower estimated fetal weight (EFW). METHODS/PROCEDURES: A retrospective analysis of routine ultrasonographic data extracted from electronic medical records was performed on a cohort of pregnant women with psychiatric diagnoses and grouped according to the presence of an antenatal antidepressant prescription (n = 32 antidepressant-prescribed and n = 44 antidepressant prescription-free). After stratifying for gestational age, comparisons included 13 ultrasonographic parameters, frequency of oligohydramnios and polyhydramnios and growth deceleration, and maternal serum protein markers assessed per routine care, including α-fetoprotein, free ß-human chorionic gonadotropin, and unconjugated estriol levels, using t tests, nonparametric and Fisher tests, and effect sizes (ESs) were computed. FINDINGS/RESULTS: No statistically significant EFW differences between groups at any time point were detected (P > 0.05). Antenatal antidepressant prescription was associated with lower femur length at weeks 33 to 40 (P = 0.046, ES = 0.75) and greater left ventricular diameter at weeks 25 to 32 (P = 0.04, ES = 1.18). No differences for frequency of oligohydramnios or polyhydramnios or growth deceleration were observed (P > 0.05). We did not detect group differences for maternal proteins (P > 0.05). IMPLICATIONS/CONCLUSIONS: Our evidence suggested a lack of association between antenatal antidepressant prescription and lower EFW but indicated an association with lower femur length and greater left ventricular diameter in mid-late gestation. Future research should examine the clinical implications of these findings.

Differences in fetal fractional limb volume changes in normal and gestational diabetic pregnancies: an exploratory observational study.

OBJECTIVE: Fetal fractional limb volume has been proposed as a useful measure for quantifying fetal soft tissue development. The aim of this study was to investigate the growth of fractional arm volume (AVol) and fractional thigh volume (TVol) of fetuses with maternal gestational diabetes (GDM) compared with those of fetuses with normal glucose tolerance (NGT). We hypothesised fetal fractional limb volume would be larger in the GDM group than in the NGT group in late gestation. DESIGN: Exploratory observational study. SETTING: Saitama Municipal Hospital. SAMPLE: A total of 165 (125 NGT and 40 GDM) singleton Japanese pregnant women. METHODS: AVol and TVol were assessed between 20 and 37 weeks' gestation as cylindrical limb volumes based on 50% of the fetal humeral or femoral diaphysis length. Women were diagnosed as GDM based on the criteria of the Japan Society of Obstetrics and Gynecology. MAIN OUTCOME MEASURES: AVol and TVol were compared between women with NGT and those with GDM at each gestational age period (2-week intervals from 20 to 37 weeks' gestation). RESULTS: Overall, 287 ultrasound scans were performed (NGT group, 205 scans; GDM group, 82 scans). There was no significant difference of AVol between the groups before 32 weeks' gestation. AVol was significantly larger in the GDM group than in the NGT group after 32 weeks' gestation (P < 0.05). TVol was not statistically different between the groups across gestation. CONCLUSIONS: Detection of variations in fetal AVol may provide greater insight into understanding the origins of altered fetal body proportion in GDM. TWEETABLE ABSTRACT: AVol, but not TVol, is significantly larger in fetuses with GDM than in those with NGT after 32 weeks' gestation.

Fetal growth velocity standards from the Fetal Growth Longitudinal Study of the INTERGROWTH-21st Project.

BACKGROUND: Human growth is susceptible to damage from insults, particularly during periods of rapid growth. Identifying those periods and the normative limits that are compatible with adequate growth and development are the first key steps toward preventing impaired growth. OBJECTIVE: This study aimed to construct international fetal growth velocity increment and conditional velocity standards from 14 to 40 weeks' gestation based on the same cohort that contributed to the INTERGROWTH-21st Fetal Growth Standards. STUDY DESIGN: This study was a prospective, longitudinal study of 4321 low-risk pregnancies from 8 geographically diverse populations in the INTERGROWTH-21st Project with rigorous standardization of all study procedures, equipment, and measurements that were performed by trained ultrasonographers. Gestational age was accurately determined clinically and confirmed by ultrasound measurement of crown-rump length at <14 weeks' gestation. Thereafter, the ultrasonographers, who were masked to the values, measured the fetal head circumference, biparietal diameter, occipitofrontal diameter, abdominal circumference, and femur length in triplicate every 5 weeks (within 1 week either side) using identical ultrasound equipment at each site (4-7 scans per pregnancy). Velocity increments across a range of intervals between measures were modeled using fractional polynomial regression. RESULTS: Peak velocity was observed at a similar gestational age: 16 and 17 weeks' gestation for head circumference (12.2 mm/wk), and 16 weeks' gestation for abdominal circumference (11.8 mm/wk) and femur length (3.2 mm/wk). However, velocity growth slowed down rapidly for head circumference, biparietal diameter, occipitofrontal diameter, and femur length, with an almost linear reduction toward term that was more marked for femur length. Conversely, abdominal circumference velocity remained relatively steady throughout pregnancy. The change in velocity with gestational age was more evident for head circumference, biparietal diameter, occipitofrontal diameter, and femur length than for abdominal circumference when the change was expressed as a percentage of fetal size at 40 weeks' gestation. We have also shown how to obtain accurate conditional fetal velocity based on our previous methodological work. CONCLUSION: The fetal skeleton and abdomen have different velocity growth patterns during intrauterine life. Accordingly, we have produced international Fetal Growth Velocity Increment Standards to complement the INTERGROWTH-21st Fetal Growth Standards so as to monitor fetal well-being comprehensively worldwide. Fetal growth velocity curves may be valuable if one wants to study the pathophysiology of fetal growth. We provide an application that can be used easily in clinical practice to evaluate changes in fetal size as conditional velocity for a more refined assessment of fetal growth than is possible at present (https://lxiao5.shinyapps.io/fetal_growth/). The application is freely available with the other INTERGROWTH-21st tools at https://intergrowth21.tghn.org/standards-tools/.

GR/Sp3/HDAC1/UGDH signaling participated in the maternal dexamethasone-induced dysplasia of the rat fetal growth plate.

Maternal dexamethasone decreases the body length of the newborn. However, whether dexamethasone inhibits the development of the growth plate of the fetal long bone is still unknown. Here, we found that lengths of fetal femur and growth plate were both shorter in the fetuses with maternal dexamethasone (0.2 mg/kg.d from gestation day 9 to 20), with a decreased proteoglycan content of the growth plate in the fetal rat. Notable decreases in both the gene expression and H3K9 acetylation of UDP-glucose dehydrogenase (Ugdh) gene, which codes a key enzyme in the proteoglycan biosynthesis in the chondrocyte, were also observed. Meanwhile, up-regulation of glucocorticoid receptor (GR), specific protein 3 (Sp3), and histone deacetylase 1 (Hdac1) gene expression were detected in the fetal growth plate. Similar changes were also observed in the chondrogenic rat bone marrow stromal cells (BMSCs) with excessive exogenous dexamethasone. However, antagonizing GR with RU486 and silencing Hdac1 or Sp3 with specific siRNAs could all stimulate the H3K9 acetylation and gene expression of Ugdh previously inhibited by dexamethasone. Meanwhile, dexamethasone also induced the nuclear translocation of GR, which further directly bound to the Ugdh promoter and interacted with HDAC1 and Sp3, respectively. Collectively, our study revealed that maternal dexamethasone induced the direct binding of GR to the Ugdh promoter of the chondrocyte in the rat fetal growth plate, which recruited HDAC1 and Sp3, induced deacetylation of the H3K9, and subsequently inhibited Ugdh gene expression. Such changes further led to attenuated proteoglycan synthesis in the developing chondrocyte and therefore disrupted the development of growth plate and fetal long bone.

Ultrasonographic assessment of fetal thyroid in Japan: thyroid circumference and distal femoral and proximal tibial ossification.

PURPOSE: The present study established a nomogram of fetal thyroid circumference (FTC) and the appearance timing of fetal distal femoral and proximal tibial ossification to assess fetal thyroid function in Japan. METHODS: Between April 2015 and July 2019, normal pregnant women at our hospital were recruited for the study. FTC was measured by the automatic ellipse outline and plotted against gestational age (GA). Fetal distal femoral and proximal tibial ossification measurements were obtained with standard electronic calipers from outer-to-outer margins (> 1 mm as the presence of ossification). RESULTS: A total of 199 pregnant women were examined. FTC increased logarithmically to GA. A nomogram of FTC was expressed by a logarithmic formula: [Formula: see text]. The respective 5-95th percentiles of FTC at each GA were 20.2-36.2 mm at 22 weeks, 25.0-44.8 mm at 26 weeks, 29.2-52.3 mm at 30 weeks, and 32.9-59.0 mm at 34 weeks. The fetal distal femoral epiphysis was not visualized before 30 weeks, but was visualized in 100% of fetuses after 35 weeks of gestation. The fetal proximal tibial epiphysis was not visualized before 33 weeks, but was visualized in 73.7% of fetuses at 37 weeks of gestation. CONCLUSION: We generated a GA-dependent FTC nomogram for Japanese fetuses. We also confirmed the appearance timing of fetal distal femoral and proximal tibial ossification to assess bone maturation. These assessments may be very useful for evaluating fetal thyroid function in Japan.

Ultrasound prediction of Zika virus-associated congenital injury using the profile of fetal growth.

Zika virus (ZIKV) is a mosquito-transmitted flavivirus, recently linked to microcephaly and central nervous system anomalies following infection in pregnancy. Striking findings of disproportionate growth with a smaller than expected head relative to body length have been observed more commonly among fetuses with exposure to ZIKV in utero compared to pregnancies without ZIKV infection regardless of other signs of congenital infection including microcephaly. This study's objective was to determine the diagnostic accuracy of femur-sparing profile of intrauterine growth restriction for the identification of ZIKV-associated congenital injuries on postnatal testing. A retrospective cohort study of pregnant women with possible or confirmed ZIKV infection between January 1, 2016 and December 31, 2017 were included. Subjects were excluded if no prenatal ultrasound was available. A femur-sparing profile of growth restriction determined using INTERGROWTH-21st sonographic standard for head circumference to femur length (HC: FL). Congenital injuries were determined postnatally by imaging, comprehensive eye exam and standard newborn hearing screen. A total of 111 pregnant women diagnosed with ZIKV infection underwent fetal ultrasound and 95 neonates had complete postnatal evaluation. Prenatal microcephaly was detected in 5% of fetuses (6/111). Postnatal testing detected ZIKV-associated congenital injuries in 25% of neonates (24/95). A HC: FL Z-score ≤ -1.3 had a 52% specificity (95% CI 41-63%), 82% negative predictive value (NPV, 95% CI 73-88%) for the detection of ZIKV-associated congenital injuries in the neonatal period. A more stringent threshold with a Z-score ≤ -2 was associated with a 90% specificity (95% CI 81-95%), 81% NPV (95% CI 77-85%). Excluding cases of fetal microcephaly, HC: FL (Z-score ≤ -2) demonstrated a similar specificity (89%, 95% CI 81-95%) with superior NPV (87%, 95% CI 84-90%). The sonographic recognition of a normally proportioned fetus may be useful prenatally to exclude a wider spectrum of ZIKV-associated congenital injuries detected postnatally.

Development of the Proximal-Anterior Skeletal Elements in the Mouse Hindlimb Is Regulated by a Transcriptional and Signaling Network Controlled by Sall4.

The vertebrate limb serves as an experimental paradigm to study mechanisms that regulate development of the stereotypical skeletal elements. In this study, we simultaneously inactivated Sall4 using Hoxb6Cre and Plzf in mouse embryos, and found that their combined function regulates development of the proximal-anterior skeletal elements in hindlimbs. The Sall4; Plzf double knockout exhibits severe defects in the femur, tibia, and anterior digits, distinct defects compared to other allelic series of Sall4; Plzf We found that Sall4 regulates Plzf expression prior to hindlimb outgrowth. Further expression analysis indicated that Hox10 genes and GLI3 are severely downregulated in the Sall4; Plzf double knockout hindlimb bud. In contrast, PLZF expression is reduced but detectable in Sall4; Gli3 double knockout limb buds, and SALL4 is expressed in the Plzf; Gli3 double knockout limb buds. These results indicate that Plzf, Gli3, and Hox10 genes downstream of Sall4, regulate femur and tibia development. In the autopod, we show that Sall4 negatively regulates Hedgehog signaling, which allows for development of the most anterior digit. Collectively, our study illustrates genetic systems that regulate development of the proximal-anterior skeletal elements in hindlimbs.

The effect of maternal and paternal height and weight on antenatal, perinatal and postnatal morphology in sex-stratified analyses.

INTRODUCTION: Low birthweight is associated with diseases later in life. The mechanisms for these associations are not well known. If the hypothesis concerning "maternal constraint" is correct for humans, as shown in animal experiments, we expect the maternal, not paternal, body proportions to influence antenatal growth and those of both parents to influence postnatal growth. We aimed to study the effect of maternal and paternal height and weight on fetal femur length antenatally (gestational weeks 20 and 30) and body length and weight at birth and postnatally (12 and 24 months old) in both sexes. MATERIAL AND METHODS: In this prospective cohort study, 399 healthy pregnant women aged 20-42 years were recruited at The Mercy Hospital for Woman, Melbourne, Australia from 2008 to 2009. Fetal femur length was measured using antenatal ultrasound (gestational weeks 20 and 30). Body length and weight were measured for parents and offspring at birth and postnatally (12 and 24 months). RESULTS: Each standard deviation (SD) rise in maternal weight (15.5 kg) was associated with 0.24 SD (0.5 mm) and 0.18 SD (0.4 mm) longer femur length in female and male fetuses at week 20 and 0.17 SD (0.5 mm) and 0.38 SD (1.1 mm) longer femur length in female and male fetuses at week 30, respectively. In girls, each SD rise in paternal height (7.2 cm) was associated with 0.29 SD (0.6 cm) longer birth length. In boys, each SD rise in maternal height (6.7 cm) was associated with 0.23 SD (0.5 cm) longer birth length. In both sexes, parental height and weight were associated with offspring length and weight at 12 and 24 months (SD ranging from 0.20 to 0.38, length from 0.7 to 1.5 cm and weight from 0.3 to 0.6 kg). The multivariable linear regression analyses were adjusted for parental age, height and weight, maternal smoking, alcohol intake, parity, and ethnicity, all P < 0.05. CONCLUSIONS: Maternal, not paternal, body proportions determined fetal growth in both sexes. Paternal height predicted birth length in girls. In contrast, maternal height predicted birth length in boys. Both parents predicted postnatal body proportions at 12 and 24 months in both sexes.

Gestational vitamin D concentration and other risk factors versus fetal femur length.

Introduction: Vitamin D plays a crucial role in the development of healthy fetal bone tissue. Analysis of fetal bone parameters versus maternal vitamin D status is necessary to shed some light on the matter. The aim of the study was to investigate the relationship between maternal and cord vitamin D concentrations and other factors which might affect fetal bone development and femur length.Material and methods: The study included 94 term pregnancies, delivered at the Department of Obstetrics, Gynecology and Oncology, Medical University of Warsaw. Fetal femur length was measured during an ultrasound examination on admission to the delivery unit. Total 25(OH)D concentrations were measured in maternal and cord blood. Direct interview with a dietician was used to collect lifestyle and nutrition data. Multiple regression analysis was used for statistical analysis.Results: No statistically significant relationship was found between vitamin D concentrations and fetal femur length, either in maternal (p = .7709) or cord (p = .7751) blood samples, despite the fact that low vitamin D concentrations, which might indicate aberrations in fetal bone development, were detected in 50.0% of the mothers and 28.7% of the newborns. Also, no relationship was confirmed for the remaining parameters, including nutritional factors (calcium or caffeine intake, vitamin/mineral supplements).Conclusion: Low vitamin D concentrations during pregnancy and lifestyle factors had no negative associations with fetal femur length.

Expected-value bias in routine third-trimester growth scans.

OBJECTIVES: Operators performing fetal growth scans are usually aware of the gestational age of the pregnancy, which may lead to expected-value bias when performing biometric measurements. We aimed to evaluate the incidence of expected-value bias in routine fetal growth scans and assess its impact on standard biometric measurements. METHODS: We collected prospectively full-length video recordings of routine ultrasound growth scans coupled with operator eye tracking. Expected value was defined as the gestational age at the time of the scan, based on the estimated due date that was established at the dating scan. Expected-value bias was defined as occurring when the operator looked at the measurement box on the screen during the process of caliper adjustment before saving a measurement. We studied the three standard biometric planes on which measurements of head circumference (HC), abdominal circumference (AC) and femur length (FL) are obtained. We evaluated the incidence of expected-value bias and quantified the impact of biased measurements. RESULTS: We analyzed 272 third-trimester growth scans, performed by 16 operators, during which a total of 1409 measurements (354 HC, 703 AC and 352 FL; including repeat measurements) were obtained. Expected-value bias occurred in 91.4% of the saved standard biometric plane measurements (85.0% for HC, 92.9% for AC and 94.9% for FL). The operators were more likely to adjust the measurements towards the expected value than away from it (47.7% vs 19.7% of measurements; P < 0.001). On average, measurements were corrected by 2.3 ± 5.6, 2.4 ± 10.4 and 3.2 ± 10.4 days of gestation towards the expected gestational age for the HC, AC, and FL measurements, respectively. Additionally, we noted a statistically significant reduction in measurement variance once the operator was biased (P = 0.026). Comparing the lowest and highest possible estimated fetal weight (using the smallest and largest biased HC, AC and FL measurements), we noted that the discordance, in percentage terms, was 10.1% ± 6.5%, and that in 17% (95% CI, 12-21%) of the scans, the fetus could be considered as small-for-gestational age or appropriate-for-gestational age if using the smallest or largest possible measurements, respectively. Similarly, in 13% (95% CI, 9-16%) of scans, the fetus could be considered as large-for-gestational age or appropriate-for-gestational age if using the largest or smallest possible measurements, respectively. CONCLUSIONS: During routine third-trimester growth scans, expected-value bias frequently occurs and significantly changes standard biometric measurements obtained. © 2019 the Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of the International Society of Ultrasound in Obstetrics and Gynecology.

Impact of biometric measurement error on identification of small- and large-for-gestational-age fetuses.

OBJECTIVES: First, to obtain measurement-error models for biometric measurements of fetal abdominal circumference (AC), head circumference (HC) and femur length (FL), and, second, to examine the impact of biometric measurement error on sonographic estimated fetal weight (EFW) and its effect on the prediction of small- (SGA) and large- (LGA) for-gestational-age fetuses with EFW < 10th and > 90th percentile, respectively. METHODS: Measurement error standard deviations for fetal AC, HC and FL were obtained from a previous large study on fetal biometry utilizing a standardized measurement protocol and both qualitative and quantitative quality-control monitoring. Typical combinations of AC, HC and FL that gave EFW on the 10th and 90th percentiles were determined. A Monte-Carlo simulation study was carried out to examine the effect of measurement error on the classification of fetuses as having EFW above or below the 10th and 90th percentiles. RESULTS: Errors were assumed to follow a Gaussian distribution with a mean of 0 mm and SDs, obtained from a previous well-conducted study, of 6.93 mm for AC, 5.15 mm for HC and 1.38 mm for FL. Assuming errors according to such distributions, when the 10th and 90th percentiles are used to screen for SGA and LGA fetuses, respectively, the detection rates would be 78.0% at false-positive rates of 4.7%. If the cut-offs were relaxed to the 30th and 70th percentiles, the detection rates would increase to 98.2%, but at false-positive rates of 24.2%. Assuming half of the spread in the error distribution, using the 10th and 90th percentiles to screen for SGA and LGA fetuses, respectively, the detection rates would be 86.6% at false-positive rates of 2.3%. If the cut-offs were relaxed to the 15th and 85th percentiles, respectively, the detection rates would increase to 97.0% and the false-positive rates would increase to 6.3%. CONCLUSIONS: Measurement error in fetal biometry causes substantial error in EFW, resulting in misclassification of SGA and LGA fetuses. The extent to which improvement can be achieved through effective quality assurance remains to be seen but, as a first step, it is important for practitioners to understand how biometric measurement error impacts the prediction of SGA and LGA fetuses. © 2019 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of the International Society of Ultrasound in Obstetrics and Gynecology.

Morphogenesis of the femur at different stages of normal human development.

The present study aimed to better characterize the morphogenesis of the femur from the embryonic to the early fetal periods. Sixty-two human fetal specimens (crown-rump length [CRL] range: 11.4-185 mm) from the Kyoto Collection were used for this study. The morphogenesis and internal differentiation process of the femur were analyzed in 3D using phase-contrast X-ray computed tomography and magnetic resonance imaging. The cartilaginous femur was first observed at Carnegie stage 18. Major anatomical landmarks were formed prior to the initiation of ossification at the center of the diaphysis (CRL, 40 mm), as described by Bardeen. The region with very high signal intensity (phase 5 according to Streeter's classification; i.e., area described as cartilage disintegration) emerged at the center of the diaphysis, which split the region with slightly low signal intensity (phase 4; i.e., cartilage cells of maximum size) in fetuses with a CRL of 40.0 mm. The phase 4 and phase 5 regions became confined to the metaphysis, which might become the epiphyseal cartilage plate. Femur length and ossified shaft length (OSL) showed a strong positive correlation with CRL. The OSL-to-femur length ratio rapidly increased in fetuses with CRL between 40 and 75 mm, which became moderately increased in fetuses with a CRL of ≥75 mm. Cartilage canal invasion occurred earlier at the proximal epiphysis (CRL, 62 mm) than at the distal epiphysis (CRL, 75 mm). Morphometry and Procrustes analysis indicated that changes in the femur shape after ossification were limited, which were mainly detected at the time of initial ossification and shortly after that. In contrast, femoral neck anteversion and torsion of the femoral head continuously changed during the fetal period. Our data could aid in understanding the morphogenesis of the femur and in differentiating normal and abnormal development during the early fetal period.

Uncoupling of in-vitro identity of embryonic limb derived skeletal progenitors and their in-vivo bone forming potential.

The healing of large bone defects remains a major unmet medical need. Our developmental engineering approach consists of the in vitro manufacturing of a living cartilage tissue construct that upon implantation forms bone by recapitulating an endochondral ossification process. Key to this strategy is the identification of the cells to produce such cartilage intermediates efficiently. We applied a cell selection strategy based on published skeletal stem cell markers using mouse embryonic limb cartilage as cell source and analysed their potential to form bone in an in vivo ectopic assay. FGF2 supplementation to the culture media for expansion blocked dedifferentiation of the embryonic cartilage cells in culture and enriched for stem cells and progenitors as quantified using the recently published CD marker set. However, when the stem cells and progenitors were fractionated from expanded embryonic cartilage cells and assessed in the ectopic assay, a major loss of bone forming potential was observed. We conclude that cell expansion appears to affect the association between cell identity based on CD markers and in vivo bone forming capacity.

Live-imaging of Bioengineered Cartilage Tissue using Multimodal Non-linear Molecular Imaging.

Coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) are non-linear techniques that allow label-free, non-destructive and non-invasive imaging for cellular and tissue analysis. Although live-imaging studies have been performed previously, concerns that they do not cause any changes at the molecular level in sensitive biological samples have not been addressed. This is important especially for stem cell differentiation and tissue engineering, if CARS/SHG microscopy is to be used as a non-invasive, label-free tool for assessment of the developing neo-tissue. In this work, we monitored the differentiation of human fetal-femur derived skeletal cells into cartilage in three-dimensional cultures using CARS and SHG microscopy and demonstrate the live-imaging of the same developing neo-tissue over time. Our work conclusively establishes that non-linear label-free imaging does not alter the phenotype or the gene expression at the different stages of differentiation and has no adverse effect on human skeletal cell growth and behaviour. Additionally, we show that CARS microscopy allows imaging of different molecules of interest, including lipids, proteins and glycosaminoglycans, in the bioengineered neo-cartilage. These studies demonstrate the label-free and truly non-invasive nature of live CARS and SHG imaging and their value and translation potential in skeletal research, regeneration medicine and tissue engineering.

Estimating Gestational Age from Ultrasound: External Validation of the NICHD Formula with Comparison to the Hadlock Regression.

OBJECTIVE: To externally validate the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) formula developed from the National Fetal Growth Studies-Singletons and compare with 1984 Hadlock regression in a general obstetrical population. STUDY DESIGN: Cross-sectional study of nonanomalous singletons with a crown-rump length (CRL) and ≥1 additional ultrasound (US) with complete fetal biometrics. CRL established the referent estimated due date to calculate the error at every examination from both formulas. Error was the difference between the CRL-derived gestational age (GA) and each method's predicted GA. Comparisons were also made in three GA intervals: 1 (140/7-206/7), 2 (210/7-286/7), and 3 (≥290/7). Odds ratios evaluated the likelihood of errors outside the prespecified (±) day ranges. Repeated measures analysis of variance and generalized estimating equations controlled multiple US in the same patient. RESULTS: A total of 6,043 patients produced 16,904 USs for evaluation. The NICHD formula yielded significantly smaller mean errors in all GA ranges compared with the Hadlock formula (p < 0.01). In interval 3, the NICHD formula had significantly lower odds of discerning examinations outside the prespecified error range (odds ratio: 1.27). CONCLUSION: The NICHD formula is a valid estimate of estimating GA in a general obstetrical population and was superior to the Hadlock formula, most notably in the third trimester.

Effect of human embryonic morphological development on fetal growth parameters: the Rotterdam Periconceptional Cohort (Predict Study).

RESEARCH QUESTION: Is embryonic morphological development according to the Carnegie stages associated with pregnancy outcome? DESIGN: In a tertiary hospital-based cohort, 182 singleton non-malformed pregnancies were selected. Serial transvaginal three-dimensional ultrasound (3D-US) scans were carried out between 6+0 and 10+2 gestational weeks. Embryonic development was annotated according to the morphological criteria of the Carnegie classification using a virtual reality system. Second-trimester biparietal diameter, head circumference, abdominal circumference and femur length measurements were retrieved from medical records. Z-scores were calculated for mid-pregnancy estimated fetal weight (EFW) and newborn birth weight. Associations between longitudinal Carnegie stages and fetal growth parameters were investigated using linear mixed models, with subgroup analysis based on fetal gender. RESULTS: A total of 576 first-trimester 3D-US scans were analysed (median of three scans per pregnancy). Embryonic development was positively associated with EFW z-score (ß = 0.69; 95% CI 0.51 to 0.86; P < 0.001), biparietal diameter and femur length, but not with head circumference, abdominal circumference and birth weight z-score. After stratification for fetal gender, positive associations for both males and females were confirmed between embryonic development and EFW z-scores. Moreover, opposite gender-specific associations were detected between embryonic development and birth weight z-scores (males: ß = 0.37; 95% CI 0.04 to 0.70; P < 0.05; females: ß = -0.36; 95% CI -0.62 to -0.10; P < 0.01). CONCLUSIONS: Human embryonic development according to the Carnegie stages is associated with fetal growth parameters with gender-specificity of birth weight. These results emphasize the importance of the first-trimester of pregnancy, raising the morphological staging of the embryo as a new methodology for early risk assessment and improvement of subsequent fetal growth parameters.

Prenatal growth map of the mouse knee joint by means of deformable registration technique.

Joint morphogenesis is the process during which distinct and functional joint shapes emerge during pre- and post-natal joint development. In this study, a repeatable semi-automatic protocol capable of providing a 3D realistic developmental map of the prenatal mouse knee joint was designed by combining Optical Projection Tomography imaging (OPT) and a deformable registration algorithm (Sheffield Image Registration toolkit, ShIRT). Eleven left limbs of healthy murine embryos were scanned with OPT (voxel size: 14.63µm) at two different stages of development: Theiler stage (TS) 23 (approximately 14.5 embryonic days) and 24 (approximately 15.5 embryonic days). One TS23 limb was used to evaluate the precision of the displacement predictions for this specific case. The remaining limbs were then used to estimate Developmental Tibia and Femur Maps. Acceptable uncertainties of the displacement predictions computed from repeated images were found for both epiphyses (between 1.3µm and 1.4µm for the proximal tibia and between 0.7µm and 1.0µm for the femur, along all directions). The protocol was found to be reproducible with maximum Modified Housdorff Distance (MHD) differences equal to 1.9 µm and 1.5 µm for the tibial and femoral epiphyses respectively. The effect of the initial shape of the rudiment affected the developmental maps with MHD of 21.7 µm and 21.9 µm for the tibial and femoral epiphyses respectively, which correspond to 1.4 and 1.5 times the voxel size. To conclude, this study proposes a repeatable semi-automatic protocol capable of providing mean 3D realistic developmental map of a developing rudiment allowing researchers to study how growth and adaptation are directed by biological and mechanobiological factors.

Chinese fetal biometry: reference equations and comparison with charts from other populations.

OBJECTIVE: This study aimed to construct new reference charts and equations for fetal biometry from Chinese fetuses at 15-40 weeks and to compare them with previous references. METHOD: This was a prospective, cross-sectional study of singleton pregnancies in patients with a confirmed gestational age who underwent ultrasound examinations between the 15th and 40th weeks of gestation. Each woman was selected only once for this study. Biparietal diameter (BPD), head circumference (HC), abdominal circumference (AC), and femur length (FL) were recorded. For each measurement, separate regression models were fitted to estimate the mean and standard deviation at each menstrual age. Z-scores were calculated with our reference equations for comparison with other references. RESULTS: New charts and reference equations involving 7553 Chinese women were created for BPD, HC, AC, and FL. Reference equations were cubic models. Prediction intervals for the new reference charts were similar to those of previous references, but with some slight differences. The main difference in our fetal biometric measurements compared with those of Korea and Hong Kong was the FL. CONCLUSIONS: We report new Chinese reference charts and equations for fetal biometry. These reference charts and equations are available for clinical use in obstetric ultrasound studies for the Chinese population.

Are selection criteria for healthy pregnancies responsible for the gap between fetal growth in the French national Elfe birth cohort and the Intergrowth-21st fetal growth standards?

BACKGROUND: The Intergrowth-21st (IG) project proposed prescriptive fetal growth standards for global use based on ultrasound measurements from a multicounty study of low-risk pregnancies selected using strict criteria. We examined whether the IG standards are appropriate for fetal growth monitoring in France and whether potential differences could be due to IG criteria for "healthy" pregnancies. METHOD: We analysed data on femur length and abdominal circumference at the second and/or the third recommended ultrasound examination from 14 607 singleton pregnancies from the Elfe national birth cohort. We compared concordance of centile thresholds using the IG standards and current French references and used restricted cubic splines to plot z-scores by gestational age. A "healthy pregnancy" sub-sample was created based on maternal and pregnancy selection criteria, as specified by IG. RESULTS: Mean gestational age-specific z-scores for femur length and abdominal circumference using French references fluctuated around 0 (-0.2 to 0.1), while those based on IG standards were higher (0.3-0.8). Using IG standards, 2.5% and 5.2% of fetuses at the third ultrasound were <10th centile for femur length and abdominal circumference, respectively, and 31.5% and 16.7% were >90th. Only 34% of pregnancies fulfilled IG low-risk criteria, but sub-analyses yielded very similar results. CONCLUSION: Intergrowth standards differed from fetal biometric measures in France, including among low-risk pregnancies selected to replicate IG's healthy pregnancy sample. These results challenge the project's assumption that careful constitution of a low-risk population makes it possible to describe normative fetal growth across populations.