Embryonic morphological development is delayed in pregnancies ending in a spontaneous miscarriage.

STUDY QUESTION: Is there a difference in embryonic morphological development between ongoing pregnancies and live pregnancies ending in a miscarriage? SUMMARY ANSWER: Embryonic morphological development, assessed by the Carnegie stages, is delayed in live pregnancies ending in a miscarriage compared to ongoing pregnancies. WHAT IS KNOWN ALREADY: Pregnancies ending in a miscarriage tend to have smaller embryos and slower heart rates. STUDY DESIGN, SIZE, DURATION: Between 2010 and 2018, 644 women with singleton pregnancies, in the periconception period, were enrolled in a prospective cohort study with follow up until 1 year after delivery. A miscarriage was registered as a non-viable pregnancy before 22 weeks gestational age, defined by an absent heartbeat by ultrasound for a previously reported live pregnancy. PARTICIPANTS/MATERIALS, SETTING, METHODS: Pregnant women with live singleton pregnancies were included and serial three-dimensional transvaginal ultrasound scans were performed. Embryonic morphological development was assessed by the Carnegie developmental stages and evaluated using virtual reality techniques. The embryonic morphology was compared to clinically used growth parameters (i.e. crown-rump length (CRL) and embryonic volume (EV)). Linear mixed models were used to evaluate the association between miscarriage and the Carnegie stages. Logistic regression with generalized estimating equations was used to calculate the odds of a miscarriage after a delay in Carnegie stages. Adjustments were made for potential confounders or covariates and include age, parity, and smoking status. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 611 ongoing pregnancies and 33 pregnancies ending in a miscarriage were included between 7 + 0 and 10 + 3 weeks gestational age, resulting in 1127 assigned Carnegie stages for evaluation. Compared to an ongoing pregnancy, a pregnancy ending in a miscarriage is associated with a lower Carnegie stage (ßCarnegie = -0.824, 95% CI -1.190; -0.458, P < 0.001). A live embryo of a pregnancy ending in a miscarriage will reach the final Carnegie stage with a delay of 4.0 days compared to an ongoing pregnancy. A pregnancy ending in a miscarriage is associated with a smaller CRL (ßCRL = -0.120, 95% CI -0.240; -0.001, P = 0.049) and EV (ßEV = -0.060, 95% CI -0.112; -0.007, P = 0.027). The delay in Carnegie stage increases the odds of a miscarriage by 1.5% per delayed Carnegie stage (ORCarnegie = 1.015, 95% CI 1.002; 1.028, P = 0.028). LIMITATIONS, REASONS FOR CAUTION: We included a relatively small number of pregnancies ending in a miscarriage from a study population that is recruited from a tertiary referral centre. Furthermore, results of genetic testing on the products of the miscarriages or information on the karyotype of the parents were not available. WIDER IMPLICATIONS OF THE FINDINGS: Embryonic morphological development, assessed by the Carnegie stages, is delayed in live pregnancies ending in a miscarriage. In the future, embryonic morphology may be used to estimate the likelihood of a pregnancy continuing to the delivery of a healthy baby. This is of crucial importance for all women but in particular for those at risk of a recurrent pregnancy loss. As part of supportive care, both women and their partners may benefit from information on the prospective outcome of the pregnancy and the timely identification of a miscarriage. STUDY FUNDING/COMPETING INTEREST(S): The work was funded by the Department of Obstetrics and Gynaecology, Erasmus MC, University Medical Centre, Rotterdam, The Netherlands. The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

The impact of maternal smoking on embryonic morphological development: the Rotterdam Periconception Cohort.

STUDY QUESTION: Is periconceptional maternal smoking associated with embryonic morphological development in ongoing pregnancies? SUMMARY ANSWER: Smoking during the periconceptional period is associated with a delayed embryonic morphological development which is not fully recuperated beyond the first trimester of pregnancy. WHAT IS KNOWN ALREADY: Smoking during pregnancy decreases prenatal growth, increasing the risk of preterm birth, small for gestational age (GA) and childhood obesity. STUDY DESIGN, SIZE, DURATION: Between 2010 and 2018, 689 women with ongoing singleton pregnancies were periconceptionally enrolled in a prospective cohort study with follow-up until 1 year after delivery. PARTICIPANTS/MATERIALS, SETTING, METHODS: Between 7 + 0 and 10 + 3 weeks, GA serial three-dimensional transvaginal ultrasound scans were performed. Embryonic morphological development as assessed by the Carnegie developmental stages was evaluated using Virtual Reality techniques. In the absence of fetal morphology classification methods beyond the embryonic period, fetal ultrasound measurements at around 20 weeks' GA, and birth weight were used to assess fetal growth. Linear mixed models were used to evaluate the association between smoking and the Carnegie stages. Regarding first-trimester morphological development, we additionally stratified our findings for mode of conception. Multiple linear regression models were used to study the association between smoking, fetal growth and birth weight. To investigate to which extent delayed embryonic morphological development mediated the effect of smoking, contemporary mediation analysis was used. Adjustments were made for potential confounders and other covariates. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 689 singleton ongoing pregnancies were included and 1210 Carnegie stages were determined. Maternal periconceptional smoking represented by the number of cigarettes/day was associated with a slight non-significant delay of the Carnegie stages (ßcigarettes/day = -0.058, 95% CI -0.122; 0.007, P = 0.080). Smoking of ≥10 cigarettes/day showed the strongest association (ß≥10 cigarettes/day = -0.352, 95% CI -0.648; -0.057, P = 0.019), as reflected by a 0.9-day delay in reaching the final Carnegie stage. Stratification for mode of conception showed a stronger negative association between the number of cigarettes/day in the IVF/ICSI group (ßcigarettes/day = -0.126, 95% CI -0.200; -0.051, P = 0.001) compared to naturally conceived pregnancies (ßcigarettes/day = 0.009, 95% CI -0.093; 0.111, P = 0.867). In the IVF/ICSI group, periconceptional smoking of ≥10 cigarettes/day was associated with in a 1.6 day delay in reaching the final Carnegie stage (ß≥10 cigarettes/day = -0.510, 95% CI -0.834; -0.186, P = 0.002). In the second trimester, periconceptional smoking was associated with a smaller femur length (ßcigarettes/day = -0.077, 95% CI -0.147; -0.008, P = 0.029) and a larger head circumference (ß1-9 cigarettes/day = 0.290, 95% CI 0.065; 0.514, P = 0.012). Smoking was associated with a lower birth weight, with a dose-response effect (ßcigarettes/day = -0.150, 95% CI -0.233; -0.068, P < 0.001). Furthermore, using the unadjusted model, 40-60% of the association between smoking and fetal ultrasound parameters and 6.3% of the association between smoking and birth weight can be explained by a delayed embryonic morphology. LIMITATIONS, REASONS FOR CAUTION: The study population was recruited from a tertiary referral center. Smoking habits were explored using self-reported questionnaires and checked for consistency by trained researchers. WIDER IMPLICATIONS OF THE FINDINGS: This study shows that the association of periconceptional maternal smoking and human morphological development can already be detected early in the first trimester of pregnancy using embryonic morphology as outcome. One of the key messages of this study is that the delay, or dysregulation, in embryonic morphology is associated with allometric growth reflected by smaller fetal measurements at 20 weeks gestation and lower weight at birth. The delay in embryonic morphology, measured in early pregnancy, cannot be recuperated during the pregnancy. The results of this study emphasize the importance of smoking intervention programs prior to conception. More research is warranted to assess the association between periconceptional smoking cessation and embryonic development. STUDY FUNDING/COMPETING INTEREST(S): The work was funded by the Department of Obstetrics and Gynaecology, Erasmus MC, University Medical Centre, Rotterdam, The Netherlands. The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

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.

Periconceptional maternal one-carbon biomarkers are associated with embryonic development according to the Carnegie stages.

STUDY QUESTION: Is periconceptional maternal one-carbon (I-C) metabolism associated with embryonic morphological development in non-malformed ongoing pregnancies? SUMMARY ANSWER: Serum vitamin B12, red blood cell (RBC) folate and plasma total homocysteine (tHcy) are associated with embryonic development according to the Carnegie stages. WHAT IS KNOWN ALREADY: Derangements in maternal I-C metabolism affect reproductive and pregnancy outcomes, as well as future health of the offspring. STUDY DESIGN, SIZE, DURATION: Between 2010 and 2014, women with singleton ongoing pregnancies were enrolled in a prospective periconceptional cohort study. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 234 pregnancies, including 138 spontaneous or IUI pregnancies with strict pregnancy dating and 96 pregnancies derived from IVF, ICSI or cryopreserved embryo transfer (IVF/ICSI pregnancies), underwent longitudinal transvaginal three-dimensional ultrasound (3D US) scans from 6+0 up to 10+2 weeks of gestation. Carnegie stages were defined using internal and external morphologic criteria in a virtual reality system. Maternal venous blood samples were collected at enrollment for serum vitamin B12, RBC folate and plasma tHcy assessment. Associations between biomarker concentrations and longitudinal Carnegie stages were investigated using linear mixed models. MAIN RESULTS AND THE ROLE OF CHANCE: We performed a median of three 3D US scans per pregnancy (range 1-5) resulting in 600 good quality data sets for the Carnegie stage annotation (80.5%). Vitamin B12 was positively associated with embryonic development in the total study population (ß = 0.001 (95% CI: 0.000; 0.002), P < 0.05) and in the subgroup of strictly dated spontaneous pregnancies (ß = 0.002 (95% CI: 0.001; 0.003), P < 0.05). Low vitamin B12 concentrations (-2SD, 73.4 pmol/l) were associated with delayed embryonic development by 1.4 days (95% CI: 1.3-1.4) compared with high concentrations (+2SD, 563.1 pmol/l). RBC folate was positively associated with Carnegie stages only in IVF/ICSI pregnancies (ß = 0.001 (95% CI: 0.0005; 0.0015), P < 0.05). In this group, low RBC folate concentrations (-2SD, 875.4 nmol/l) were associated with a 1.8-day delay (95% CI: 1.7-1.8) in development compared with high concentrations (+2SD, 2119.9 nmol/l). tHcy was negatively associated with embryonic development in the total study population (ß = -0.08 (95% CI: -0.14; -0.02), P < 0.01), as well as in the IVF/ICSI subgroup (ß = -0.08 (95% CI: -0.15; -0.01), P < 0.05). High tHcy concentrations (+2SD, 10.4 µmol/l) were associated with a delay of 1.6 days (95% CI: 1.5-1.7) in embryonic development compared with low concentrations (-2SD, 3.0 µmol/l). LIMITATIONS, REASONS FOR CAUTION: The study was performed in a tertiary care center, resulting in high rates of folic acid supplement use and comorbidity that may reduce the external validity of our findings. WIDER IMPLICATIONS OF THE FINDINGS: In periconceptional care, maternal I-C biomarkers should be taken into account as predictors of embryonic morphological development. Combining embryonic size measurements with morphological assessment could better define normal embryonic development. STUDY FUNDING/COMPETING INTEREST(S): The work was funded by the Department of Obstetrics and Gynaecology, Erasmus MC, University Medical Centre, Rotterdam, The Netherlands. RPMST is CSO of the startup company Slimmere Zorg and CEO of eHealth Care Solutions. The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: Not applicable.

Upper arm posture during human embryonic and fetal development.

The upper extremity posture is characteristic of each Carnegie stage (CS), particularly between CS18 and CS23. Morphogenesis of the shoulder joint complex largely contributes to posture, although the exact position of the shoulder joints has not been described. In the present study, the position of the upper arm was first quantitatively measured, and the contribution of the position of the shoulder girdle, including the scapula and glenohumeral (GH) joint, was then evaluated. Twenty-nine human fetal specimens from the Kyoto Collection were used in this study. The morphogenesis and three-dimensional position of the shoulder girdle and humerus were analyzed using phase-contrast X-ray computed tomography and magnetic resonance imaging. Both abduction and flexion of the upper arm displayed a local maximum at CS20. Abduction gradually decreased until the middle fetal period, which was a prominent feature. Flexion was less than 90° at the local maximum, which was discrepant between appearance and measurement value in our study. The scapular body exhibited a unique position, being oriented internally and in the upward direction, with the glenoid cavity oriented cranially and ventrally. However, this unique scapular position had little effect on the upper arm posture because the angle of the scapula on the thorax was canceled as the angle of the GH joint had changed to a mirror image of that angle. Our present study suggested that measuring the angle of the scapula on the thorax and that of the GH joint using sonography leads to improved staging of the human embryo.

Dynamic transcriptome profiling toward understanding the development of the human embryonic heart during different Carnegie stages.

Transcriptional regulation participates in heart development. However, the transcriptomes of human embryonic hearts during Carnegie stage (CS)10-CS16 have not been elucidated. Here, we found marked changes in the morphology and transcriptome of the human embryonic heart from CS10 to CS11. At CS12-CS14, the embryonic heart undergoes hypoxia-to-aerobic transformation. At CS14-CS16, transcriptome functions were related to energy metabolism, regulation of cholesterol, and processes related to inorganic substances. Moreover, the transcriptomes of cardiac progenitor cells derived from human embryonic stem cells (hESCs) most overlapped with those of human embryonic hearts at CS10. Cardiomyocytes derived from hESCs considerably overlapped with embryonic hearts at CS14-CS16. Overall, these results provide a new perspective into the characteristics of human embryonic heart development.

Vesicular swelling in the cervical region with lymph sac formation in human embryos.

Vesicular swelling in the cervical region (VSC) is occasionally observed among human embryos around Carnegie stage (CS) 21. However, its mechanism and significance in fetal development are unclear. The present study aimed to analyze the relation of development of VSC with jugular lymph sac (JLS) formation. Serial histological sections that were digitalized from 14 embryos at CS20 and CS21 stored at the Kyoto Collection were used for the analysis. Subcutaneous edema and enlargement of the subarachnoid space were found to cause VSC. No obvious abnormalities in cranial regions that may be related to the VSC were detected on histological sections. Three-dimensional reconstructions revealed the following: (a) the JLS was located bilaterally at the levels between the first and fourth cervical vertebrae; (b) the JLS was pyramidal in shape; and (c) no severe deformity and/or malformation was found in all samples. The JLS was not connected to the subcutaneous tissue and subarachnoid space in all samples. The mean volume of the JLS increased nine-times from CS20 (0.02 mm3 in VSC [-] group) to CS21 (0.18 mm3 in VSC [-] group). The mean volume of the JLS was comparable between the VSC [-] and VSC (+) groups at both CS20 and CS21. A moderate correlation was observed between VSCd and the mean volume of the JLS in both groups at CS20 (R2 = 0.75) and CS21 (R2 = 0.56). In conclusion, the dynamics of the lymphatic system at the cervical region may contribute to VSC observed around CS21. © 2019 Japanese Teratology Society.

Spatial Relationship Between the Metanephros and Adjacent Organs According to the Carnegie Stage of Development.

The morphological changes in the metanephros and its spatial relationship to the adjacent organs was evaluated based on the Carnegie stages (CSs) from 14 through 23. The imaging modalities used included magnetic resonance imaging (N = 4), phase-contrast X-ray computed tomography (N = 11), and serial histological sections (N = 40), supplemented by three-dimensional image reconstruction. The orientation of the hilus of the metanephros changed significantly between CS17 (34.4 ± 13.7 degrees) and 18 (122.3 ± 38.1 degrees), with an increase in the number of branches of the urinary collecting system, from 1.61 ± 0.42 at CS17 to 3.20 ± 0.35 at CS18. This increase in the number of branches influenced the growth of the metanephros and the orientation of its hilus. The right and left metanephroses were in proximity throughout the embryonic period. The local maximum interpole distances were observed at CS18 (0.87 ± 0.11 mm for the upper and 0.50 ± 0.25 mm for the lower pole). Mesenchymal tissue was observed between the metanephros and iliac arteries, as well as between the right and left metanephros. Throughout development, the position of the lower pole of the metanephros remained adjacent to the aortic bifurcation. The position of the upper pole, referenced with respect to the aortic bifurcation, increased by >2.0 mm, reflecting the longitudinal growth of the metanephros. Our findings provide a detailed description of the morphogenesis of the metanephros and of its hilus, which might contribute to our understanding of congenital malformations and malpositions of the kidneys. Anat Rec, 302:1901-1915, 2019. © 2019 American Association for Anatomy.

Correlation of external ear auricle formation with staging of human embryos.

The formation of auricles in human embryos was evaluated between Carnegie stage (CS)19 and CS23, and the findings were correlated across the stages. The auricle was categorized into 11 steps according to Streeter's criteria with modifications. Mesenchyme cell condensation was observed at Step 7, and two layers of cartilage consisting of the auricle were recognized at Step 11. The representative steps at each CS shifted from Step 3 to Step 11 during CS16 and CS23, although several steps overlapped between adjacent CSs. These results indicate that observations of the auricle between CS19 and CS23 may be utilized for determining embryo staging as convincing supportive evidence of external features reflecting the internal histological structure, although other findings should also be taken into account.