Normal Human Embryos Research Articles

Transcriptome analysis reveals that defects in cell cycle regulation contribute to preimplantation embryo arrest

Patients with preimplantation embryo arrest (PREMBA) often experience assisted reproductive failure primarily due to the lack of transferable embryos, and the molecular mechanisms underlying PREMBA remain unclear. In our study, the embryos from five women with recurrent preimplantation embryo arrest and three women with tubal factor infertility were used for single-embryo transcriptome sequencing. Meanwhile, the transcriptomes of normal human preimplantation embryos obtained from GSE36552 were utilized to perform a comparative analysis with the transcriptomes of PREMBA embryos. Our results showed dysregulation of the cell cycle phase transition might be a potential pathogenic factor contributing to PREMBA. Through integrated analysis of the differentially expressed genes (DEGs) and weighted gene co-expression network analysis (WGCNA), we identified a number of hub genes using the protein-protein interaction network. The top 5 hub genes were as follows: CCNB2, BUB1B, CDC25A, CCNB3, and PLK3. The expression of hub genes was validated in PREMBA embryos and donated embryos using RT-qPCR. The knockdown of Ccnb2 in mouse zygotes led to an increase in embryo fragmentation, a rise in apoptosis, and a reduction in blastocyst formation. Furthermore, silencing the expression of CDC25A in HEK293T cells resulted in a decrease in cell proliferation and an increase in apoptosis, providing further support for our findings. Our findings could predict the development outcomes of preimplantation embryos and be used as potential therapeutic targets to prevent recurrent failures of IVF/ICSI attempts.

P-252 A roadmap to normal human embryo development – a timelapse study of embryos resulting in the live birth

Abstract Study question What are the morphokinetic parameters of preimplantation development in normal human embryos? Summary answer We provide a timeline of early developmental events and the incidence of embryo morphological features compatible with childbirth. What is known already Timelapse embryo monitoring provides unique insight into early human development. Implementation of this technique in clinical practice allows for assessing developmental dynamics for improved embryo selection. Several markers and ranking algorithms have been proposed to predict blastulation or implantation outcomes, but only a few studies assessed time-lapse data with live birth as the primary endpoint. Therefore, the comprehensive morphokinetic profile of embryos that gave rise to live birth has not been defined. Study design, size, duration Retrospective observational analysis of 300 time-lapse records of IVF embryos known to develop into healthy newborns. Out of total 73 quantitative/qualitative signs annotated, we focused on 9 developmental events and 14 morphological features and evaluated their timing, duration, and incidence. The influence of potential confounding factors (i.e., egg age, donor egg, donor sperm, sperm quality, and sex of the child) was evaluated by advanced statistical methods. Participants/materials, setting, methods Embryo monitoring was performed by a GERI-timelapse incubator. Image acquisition of individual embryo development lasted 5-6 days, starting 10 minutes after ICSI, which was used as reference time point. Transmitted light images were automatically captured in 5-minute intervals and 7-11 focal planes. The embryo selection for transfer was based solely on its morphology. The annotation data represent the consensus of minimum 3 experienced embryologists. Generalized linear models were used to explore relationships between dataset variables. Main results and the role of chance The average age of female participants was 27.5 years; 71.67% of embryos were derived from young donor eggs without fertility disorders. The severe andrological factor was present in 8% of male patients; donor sperm was used in 23% of cases; 16% of embryos were derived from dysmorphic oocytes. Both sexes were equally represented among analyzed embryos. Our analysis showed that early development was characterized by remarkably short first mitosis (2.68 ± 0.73h) and the interval between the second and third division (t4-t3 = 0.65 ± 0.66h), whereas the third cleavage was less synchronous (t8-t5 = 5.46 ± 4.9h) No association between quantitative signs and confounding factors was found. Interestingly, 2-cell stage embryos often contained multiple nuclei in at least one blastomere (51%), but multinucleation was reduced at the 4-cell stage (8%). Multipolar divisions were absent during zygotic division and rare (2.3%) during the second cleavage. Preterm and partial compaction was observed in 26.6% and 34% of embryos, respectively. Importantly, we noticed that 11.6% of embryos in our dataset deviated from conventional scenario of cleavage dynamics. These “atypical” embryos were characterized by a significantly higher incidence of multipolar divisions during the second cleavage compared to “typical,” regularly dividing embryos. Additionally, the odds of partial compaction increase 10 times in “atypical” embryos. Limitations, reasons for caution The presented analysis only involved embryos confirmed to result in a live birth after transfer. The comparison with data from embryos that did not produce pregnancy was not performed because it would be impossible to discriminate between the role of the embryo and endometrial factors in implantation failure. Wider implications of the findings Human embryos may develop into healthy children despite irregular cell division. A high incidence of partial compaction in “atypical” embryos suggests a self-correction mechanism rescuing their developmental potential. A larger-scale analysis of features compatible with live birth is needed to improve embryo selection criteria for evidence-based decision-making. Trial registration number not applicable

Expanding the HPSE2 Genotypic Spectrum in Urofacial Syndrome, A Disease Featuring a Peripheral Neuropathy of the Urinary Bladder.

Urofacial (also called Ochoa) syndrome (UFS) is an autosomal recessive congenital disorder of the urinary bladder featuring voiding dysfunction and a grimace upon smiling. Biallelic variants in HPSE2, coding for the secreted protein heparanase-2, are described in around half of families genetically studied. Hpse2 mutant mice have aberrant bladder nerves. We sought to expand the genotypic spectrum of UFS and make insights into its pathobiology. Sanger sequencing, next generation sequencing and microarray analysis were performed in four previously unreported families with urinary tract disease and grimacing. In one, the proband had kidney failure and was homozygous for the previously described pathogenic variant c.429T>A, p.(Tyr143*). Three other families each carried a different novel HPSE2 variant. One had homozygous triplication of exons 8 and 9; another had homozygous deletion of exon 4; and another carried a novel c.419C>G variant encoding the missense p.Pro140Arg in trans with c.1099-1G>A, a previously reported pathogenic splice variant. Expressing the missense heparanase-2 variant in vitro showed that it was secreted as normal, suggesting that 140Arg has aberrant functionality after secretion. Bladder autonomic neurons emanate from pelvic ganglia where resident neural cell bodies derive from migrating neural crest cells. We demonstrated that, in normal human embryos, neuronal precursors near the developing hindgut and lower urinary tract were positive for both heparanase-2 and leucine rich repeats and immunoglobulin like domains 2 (LRIG2). Indeed, biallelic variants of LRIG2 have been implicated in rare UFS families. The study expands the genotypic spectrum in HPSE2 in UFS and supports a developmental neuronal pathobiology.

Metabolic state of human blastocysts measured by fluorescence lifetime imaging microscopy.

Can non-invasive metabolic imaging via fluorescence lifetime imaging microscopy (FLIM) detect variations in metabolic profiles between discarded human blastocysts? FLIM revealed extensive variations in the metabolic state of discarded human blastocysts associated with blastocyst development over 36 h, the day after fertilization and blastocyst developmental stage, as well as metabolic heterogeneity within individual blastocysts. Mammalian embryos undergo large changes in metabolism over the course of preimplantation development. Embryo metabolism has long been linked to embryo viability, suggesting its potential utility in ART to aid in selecting high quality embryos. However, the metabolism of human embryos remains poorly characterized due to a lack of non-invasive methods to measure their metabolic state. We conducted a prospective observational study. We used 215 morphologically normal human embryos from 137 patients that were discarded and donated for research under an approved institutional review board protocol. These embryos were imaged using metabolic imaging via FLIM to measure the autofluorescence of two central coenzymes, nicotinamide adenine (phosphate) dinucleotide (NAD(P)H) and flavine adenine dinucleotide (FAD+), which are essential for cellular respiration and glycolysis. Here, we used non-invasive FLIM to measure the metabolic state of human blastocysts. We first studied spatial patterns in the metabolic state within human blastocysts and the association of the metabolic state of the whole blastocysts with stage of expansion, day of development since fertilization and morphology. We explored the sensitivity of this technique in detecting metabolic variations between blastocysts from the same patient and between patients. Next, we explored whether FLIM can quantitatively measure metabolic changes through human blastocyst expansion and hatching via time-lapse imaging. For all test conditions, the level of significance was set at P < 0.05 after correction for multiple comparisons using Benjamini-Hochberg's false discovery rate. We found that FLIM is sensitive enough to detect significant metabolic differences between blastocysts. We found that metabolic variations between blastocyst are partially explained by both the time since fertilization and their developmental expansion stage (P < 0.05), but not their morphological grade. Substantial metabolic variations between blastocysts from the same patients remain, even after controlling for these factors. We also observe significant metabolic heterogeneity within individual blastocysts, including between the inner cell mass and the trophectoderm, and between the portions of hatching blastocysts within and without the zona pellucida (P < 0.05). And finally, we observed that the metabolic state of human blastocysts continuously varies over time. Although we observed significant variations in metabolic parameters, our data are taken from human blastocysts that were discarded and donated for research and we do not know their clinical outcome. Moreover, the embryos used in this study are a mixture of aneuploid, euploid and embryos of unknown ploidy. This work reveals novel aspects of the metabolism of human blastocysts and suggests that FLIM is a promising approach to assess embryo viability through non-invasive, quantitative measurements of their metabolism. These results further demonstrate that FLIM can provide biologically relevant information that may be valuable for the assessment of embryo quality. Supported by the Blavatnik Biomedical Accelerator Grant at Harvard University. Becker and Hickl GmbH and Boston Electronics sponsored research with the loaning of equipment for FLIM. D.J.N. is an inventor on patent US20170039415A1. N/A.

Reprint of: Embryo morphology, developmental rates, and maternal age are correlated with chromosome abnormalities

To determine some of the unresolved questions related to chromosome anomalies in early human embryos, such are the detection of any advanced maternal age effect; the complete assessment of mosaicism, which requires analysis of all cells; and the relationship with embryonic dysmorphism. Fluorescence in situ hybridization has been used in this study to answer these issues.Fluorescence in situ hybridization analysis of human embryos using simultaneously probes for three or five chromosomes. Five hundred twenty-four cleavage-stage human embryos obtained by IVF were analyzed by fluorescence in situ hybridization. Embryos were allocated into three groups according to morphological and developmental characteristics (arrested; slow and/or fragmented; morphologically and developmentally normal). The embryos also were analyzed according to maternal age.Dysmorphic embryos had higher rates of polyploidy and diploid mosaicism. Aneuploidy increased with maternal age in nonarrested embryos. Preimplantation genetic diagnosis successfully detected these abnormalities.This study demonstrates that, in morphologically and developmentally normal human embryos, cleavage-stage aneuploidy significantly increases with maternal age. The results suggest that implantation failure in older women largely could be due to aneuploidy.

Genetic predictors of human blastocyst hatching success in the IVF cycles

Introduction: According to in vitro experiments, up to 75% of morphologically normal human embryos cannot spontaneously leave the zona pellucida (ZP). Molecular mechanisms of hatching have still not been completely studied.

Understanding the Development, Variations, and Defects of the Muscular System in Normal Human Embryos, Fetuses, and Newborns

There has been, until now, a limited knowledge on the development of the human muscular system, as most textbooks and atlases on human development described in detail the development of the skeletal system and other structures such as the internal organs without mentioning ‐ or doing it in a very brief way ‐ the muscular system. Moreover, within the few published works that did focus on the ontogeny of human muscles, most of them were restricted to a single muscle, a muscle group, a region, or a specific period of time (e.g. before 7 weeks of gestation). This makes the study of muscle anomalies, as well as of variations, very difficult due to the lack of good comparative models about normal development. We recently dissected 17 phenotypically ‘normal’ human newborns, fetuses and embryos between 7 and 39 weeks of development, and compared our results with the scarce literature available on the normal development of the muscular system in humans. Importantly, in the individuals dissected by us there was an indirect relationship between the ages of the fetuses and the number of seemingly fused (i.e. undifferentiated) muscles. That is, muscle anlagen in the early embryonic development often give rise to several muscles even after 7 weeks of gestation. This would contradict what is often described in the literature that by end of week 7 of gestation, all muscles resemble those of the adults (i.e. are differentiated). In addition, the number of the variations (i.e. phenotypes that deviate from the norm, within the ‘normal’ human population) per upper limbs dissected is significantly higher than the number of variations per lower limb studied, a pattern that is strikingly similar to that often reported in the literature about birth defects. Therefore, these and other results support Alberch's (1989) ill‐named “logic of monsters” theory that predicts a parallel between the variant phenotypes of the ‘normal’ populations and the defective phenotypes of genetically ‘abnormal’ individual due to the strong internal constraints that limit the number of possible outcomes. Furthermore, we present, for the first time, detailed data about the development of each head and limb human muscle from late embryonic stages to newborn configuration, and discuss the broader implications of these new data within a broad comparative and developmental framework.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The embryological basis of subclinical hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is caused by mutations in sarcomeric proteins, the commonest being MYBPC3 encoding myosin-binding protein C. It is characterised by left ventricular hypertrophy but there is an important pre-hypertrophic phenotype with features including crypts, abnormal mitral leaflets and trabeculae. We investigated these during mouse cardiac development using high-resolution episcopic microscopy. In embryonic hearts from wildtype, homozygous (HO) and heterozygous (HET) Mybpc3-targeted knock-out (KO) mice we show that crypts (one or two) are a normal part of wildtype development but they almost all resolve by birth. By contrast, HO and HET embryos had increased crypt presence, abnormal mitral valve formation and alterations in the compaction process. In scarce normal human embryos, crypts were sometimes present. This study shows that features of the human pre-hypertrophic HCM phenotype occur in the mouse. In an animal model we demonstrate that there is an embryological HCM phenotype. Crypts are a normal part of cardiac development but, along with the mitral valve and trabeculae, their developmental trajectory is altered by the presence of HCM truncating Mybpc3 gene mutation.

Morphogenesis of the spleen during the human embryonic period.

We aimed to observe morphological changes in the spleen from the emergence of the primordium to the end of the embryonic period using histological serial sections of 228 samples. Between Carnegie stages (CSs) 14 and 17, the spleen was usually recognized as a bulge in the dorsal mesogastrium (DM), and after CS 20, the spleen became apparent. Intrasplenic folds were observed later. A high-density area was first recognized in 6 of the 58 cases at CS 16 and in all cases examined after CS 18. The spleen was recognized neither as a bulge nor as a high-density area at CS 13. The mesothelium was pseudostratified until CS 16 and was replaced with high columnar cells and then with low columnar cells. The basement membrane was obvious after CS 17. The mesenchymal cells differentiated from cells in the DM, and sinus formation started at CS 20. Hematopoietic cells were detected after CS 18. The vessels were observed at CS 14 in the DM. Hilus formation was observed after CS 20. The parallel entries of the arteries and veins were observed at CS 23. The rate of increase in spleen length in relation to that of stomach length along the cranial-caudal direction was 0.51 ± 0.11, which remained constant during CSs 19 and 23, indicating that their growths were similar. These data may help to better understand the development of normal human embryos and to detect abnormal embryos in the early stages of development.

GJB6, of which mutations underlie Clouston syndrome, is a potential direct target gene of p63

Clouston syndrome is a rare autosomal dominant condition characterized by hypotrichosis, nail dystrophy, and occasionally palmoplantar keratoderma. The disease is caused by mutations in GJB6 gene, which encodes a gap junction protein connexin 30 (Cx30). To disclose the molecular basis of Clouston syndrome in a Lebanese-German family, and also to determine precise expression of Cx30 in normal skin of humans and mice, as well as transcriptional regulation for the GJB6 expression. We searched for mutations in the GJB6 gene using DNA of the family members with Clouston syndrome. We performed immunostaining to localize the Cx30 expression in normal human skin and mouse embryos. In addition, we did a series of in vitro studies to investigate if the GJB6 could be a direct transcriptional target gene of p63. We identified a recurrent heterozygous mutation c.31G>C (p.Gly11Arg) in the GJB6 gene in the Lebanese-German family with Clouston syndrome. Immunostaining in normal human skin sections demonstrated predominant expression of Cx30 in hair follicles, nails, and palmoplantar epidermis, which partially overlapped with p63 expression. We also showed co-expression of Cx30 and p63 in developing mouse hair follicles and nail units. In cultured cells, the GJB6 expression was significantly upregulated by ΔNp63α isoform. Further in vitro analyses suggested that ΔNp63α was potentially involved in the GJB6 expression via binding to the sequences in intron 1 of the GJB6 gene. Our data further underscore the crucial roles of Cx30 in morphogenesis and development of skin and its appendages.

Transcriptome profiling of genes involved in neural tube closure during human embryonic development using long serial analysis of gene expression (long‐SAGE)

Neural tube defects (NTDs) are common human birth defects with a complex etiology. To develop a comprehensive knowledge of the genes expressed during normal neurulation, we established transcriptomes from human neural tube fragments during and after neurulation using long Serial Analysis of Gene Expression (long-SAGE). Rostral and caudal neural tubes were dissected from normal human embryos aged between 26 and 32 days of gestation. Tissues from the same region and Carnegie stage were pooled (n ≥ 4) and total RNA extracted to construct four long-SAGE libraries. Tags were mapped using the UniGene Homo sapiens 17 bp tag-to-gene best mapping set. Differentially expressed genes were identified by chi-square or Fisher's exact test, and validation was performed for a subset of those transcripts using in situ hybridization. In silico analyses were performed with BinGO and EXPANDER. We observed most genes to be similarly regulated in rostral and caudal regions, but expression profiles differed during and after closure. In silico analysis found similar enrichments in both regions for biologic process terms, transcription factor binding and miRNA target motifs. Twelve genes potentially expressing alternate isoforms by region or developmental stage, and the microRNAs miR-339-5p, miR-141/200a, miR-23ab, and miR-129/129-5p are among several potential candidates identified here for future research. Time appears to influence gene expression in the developing central nervous system more than location. These data provide a novel complement to traditional strategies of identifying genes associated with human NTDs and offer unique insight into the genes associated with normal human neurulation.

Syndromic craniosynostosis, fibroblast growth factor receptor 2 (FGFR2) mutations, and sacrococcygeal eversion presenting as human tails

There have been 23 previously published cases of patients with syndromic craniosynostosis and human tails. In many of these, the tail was composed of prominent coccygeal and sacral vertebrae, curved in a retroverted instead of in the normal anterograde fashion. This has been termed sacrococcygeal eversion. In those cases in which genetic testing results are reported, there were fibroblast growth factor receptor 2 (FGFR2) mutations. We present three patients with Pfeiffer syndrome and sacrococcygeal eversion. Two had genetic testing and both had FGFR2 mutations, one of them a novel mutation in patients with syndromic craniosynostosis and sacrococcygeal eversion. We also briefly review the literature on craniosynostosis and human tails. All but one reported patient has had either Pfeiffer, Crouzon, or Beare-Stevenson syndrome. Most patients, including ours, have had severe manifestations of their syndrome. Although the pathogenesis of sacrococcygeal eversion is unknown, a similarly posteriorly curved tail bud develops in normal human embryos during the second month of gestation. Perhaps increased FGFR2 activation during this embryonic period leads to abnormal differentiation or regression of the tail bud and, in turn, sacrococcygeal eversion, in certain patients with severe syndromic craniosynostosis.

Altered Methylation of the DNA Repair Gene MGMT Is Associated with Neural Tube Defects

DNA repair is critical for proper embryogenesis, and altered expression of DNA repair genes has been associated with neural tube defects (NTDs). The expression of DNA repair enzymes may be controlled, in part, by methylation of the promoter region. To assess whether disturbed promoter methylation pattern increases the incidence of NTDs, we employed methylation specific-multiplex ligation-dependent probe amplification (MS-MLPA) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to quantify the methylation levels of multiple promoter CpG sites in normal human embryos and embryos with NTDs. Of the total seven DNA repair genes, two genes (MGMT, MSH6) were examined as having disturbed methylation levels by MS-MLPA kit, while only one gene was confirmed with a significant alternated methylation pattern by MALDI-TOF MS. In our research, methylation profiling of the DNA repair gene O (6)-methylguanine-DNA methyltransferase (MGMT) showed gender difference in embryogenesis. Comparison of MGMT promoter methylation revealed that hypomethylation was associated with an increased risk for cephalic malformations, especially with female embryos (Adjusted Odds Ratio = 8.250). The majority of individual CpG units within the promoter demonstrated hypomethylation. Meanwhile, the expression of MGMT was proven to increase significant in female cases. These results underscore the role of stable promoter methylation in the DNA repair enzymes MGMT for proper embryogenesis.

Gene expression analysis of a new source of human oocytes and embryos for research and human embryonic stem cell derivation

To create developmentally competent embryos from failed-to-fertilize oocytes for use in infertility research and human embryonic stem cell derivation. Attempts to recover developmental potential of failed-to-fertilize oocytes were made by using either parthenogenetic activation or reinsemination by intracytoplasmic sperm injection. Resulting embryos were cultured to various stages up to and including blastocyst, and single embryos exhibiting normal development were analyzed for gene expression by quantitatively profiling representative transcripts. Hospital-based assisted reproductive technology laboratory and University academic laboratories. One hundred sixty-five couples undergoing assisted fertility treatment. Metaphase II stage oocytes were either parthenogenetically activated or reinseminated with donor sperm, then allowed to develop up to and including the blastocyst stage. Gene expression analysis was performed on oocytes and embryos by quantitative reverse transcriptase-polymerase chain reaction for markers of developmental competence. Fertilization occurred in 65% of the activated or reinseminated oocytes, which resulted in a blastocyst formation rate of 8%. Evaluation of a number of developmentally important genes in those embryos exhibiting normal development revealed profile and levels of expression similar to control embryos. One blastocyst from an activated oocyte yielded a novel pluripotent stem cell line indistinguishable from those derived from embryos surplus to infertility treatment. Clinically unusable oocytes represent a valuable alternative source of normal human embryos for human infertility and stem cell research without conflicting with patient treatment.

Genome-wide microarray evidence that 8-cell human blastomeres over-express cell cycle drivers and under-express checkpoints

PurposeTo understand cell cycle controls in the 8-Cell human blastomere.MethodsData from whole human genome (43,377 elements) microarray analyses of RNAs from normal 8-Cell human embryos were compiled with published microarrays of RNAs from human fibroblasts, before and after induced pluripotency, and embryonic stem cells. A sub database of 3,803 genes identified by high throughput RNA knock-down studies, plus genes that oscillate in human cells, was analyzed.ResultsThirty-five genes over-detected at least 7-fold specifically on the 8-Cell arrays were enriched for cell cycle drivers and for proteins that stabilize chromosome cohesion and spindle attachment and limit DNA and centrosome replication to once per cycle.ConclusionsThese results indicate that 8-cell human blastomere cleavage is guided by cyclic over-expression of key proteins, rather than canonical checkpoints, leading to rapidly increasing gene copy number and a susceptibility to chromosome and cytokinesis mishaps, well-noted characteristics of preimplantation human embryos.Electronic supplementary materialThe online version of this article (doi:10.1007/s10815-010-9407-6) contains supplementary material, which is available to authorized users.

Morphometric study on the characteristic external features of normal and abnormal human embryos

The embryonic period is characterized by organogenesis and accompanying dynamic changes in external features. The measurement of human embryos has been limited to whole body dimensions, such as crown-rump length. More detailed measurements would add quantitative information about these characteristic events and provide a better understanding of normal and abnormal embryonic development. In the present study, we defined axes, landmarks, and measurements for human embryos, and measured 250 externally normal human embryos at Carnegie stages 14-23 (6.5-29.3 mm in crown-rump length, approximately 5-8 weeks of estimated ovulation age) that were fixed in Bouin's solution and preserved in 10% formalin solution. The axes, landmarks, and measurements defined for human embryos are corresponding to those in human and primate fetuses. The whole body, head, face, and extremities were measured using a scale attached to a dissecting microscope. Axial length, head height plus ear-shoulder length plus trunk height, was designated as a new measurement of the whole body, which is comparable with crown-rump length. Approximate standards of these measurements were obtained. The ratios of some measurements to trunk height and between the different parts were also obtained, and several different developmental patterns were recognized. The reproducibility of each measurement was evaluated by measuring 50 specimens three times each at intervals of one or two months. As a pilot study for the application of the proposed measurements, 84 human embryos with external anomalies, including holoprosencephaly, anomalies of extremities, and pharyngeal arch anomalies, were measured using the same method, and a few tendencies characteristic to holoprosencephaly were noticed.

Allometric growth ratios are independent of Igf2 gene dosage during development

In the mouse, allelic dosage of the paternally expressed gene coding for insulin-like growth factor II (Igf2), from null to bi-allelic, results in dose-dependent growth, an effect which appears to be fully established during a discrete period of embryogenesis that then persists throughout life. Here, we specifically quantify the influence of Igf2 allelic dosage on the proportionality of regional embryonic growth rather than overall growth. Remarkably, preservation of allometric growth ratios between head and body regions were observed throughout development, irrespective of the range of overall growth phenotype (60-130% of wild type). Evaluation of log-log plots suggests that each allele of Igf2 expressed corresponds to the equivalent of 2-4 days of relative growth. Igf2 is predominantly expressed in extra-embryonic mesoderm (E7.5-E8.25), 24 h before alterations in cell number are known to occur in embryos with disruption of the paternally expressed allele. We hypothesized that the preservation of proportionality may result from modification of extra-embryonic development and subsequent alteration of systemic nutritional supply. Morphological analyses of chorio-allantoic and placental development between E9 and E9.5 appeared Igf2 independent. This suggests either an intrinsic but systemic Igf2-dependent activity within the embryo or a more complex developmental mechanism accounts for the proportional phenotype. Allelic IGF2 expression is subject to stochastic variation in humans, with 10% of the population estimated to be functionally bi-allelic. Evaluation of allometric growth of normal and pathological human embryos, suggest intra-uterine growth phenotypes associated with altered IGF2 imprinting are also likely to be proportionate.

Computerized three-dimensional analysis of the heart and great vessels in normal and holoprosencephalic human embryos

The developing heart and great vessels undergo drastic morphogenetic changes during the embryonic period. To analyze the normal and abnormal development of these organs, it is essential to visualize their structures in three and four dimensions, including the changes occurring with time. We have reconstructed the luminal structure of the hearts and great vessels of staged human embryos from serial histological sections to demonstrate their sequential morphological changes in three dimensions. The detailed structures of the embryonic heart and major arteries in normal and holoprosencephalic (HPE) human embryos could be reconstructed and visualized, and anatomical structures were analyzed using 3D images. By 3D analysis, cardiac anomalies such as double-outlet right ventricle and malrotation of the heart tube were identified in HPE embryos, which were not easily diagnosed by histological observation. Reconstruction and analysis of 3D images are useful for the study of anatomical structures of developing embryos and for identifying their abnormalities.

Hypospadias and associated penile anomalies: a histopathological study and a reconstruction of the pathogenesis

Hypospadias is considered to be the result of inadequate fusion of urethral folds and, possibly, of canalization of a glandar epithelial cord during the formation of the spongy urethra. This theory had to be reconsidered because a recent study in normal human embryos has exposed such fusion and invagination as misconceptions. Autopsy specimens of five penises with hypospadias from foetuses and neonates were studied histologically. The findings complemented with data from the literature were correlated to the normal developmental process to reconstruct the pathogenesis of the disorder. Histopathological analysis revealed that the hypospadic orifice was the proximal part of a mucosal delta which revealed the structure of the roof and meatus of a flattened distal urethra. Branches of the raphe bordering the delta and terminating in prominent 'dog ears' had the characteristics of the transient urethral labia (folds). Associated curvature and torsion could be related to structural abnormalities of vascular structures, notably the distal corpus spongiosum, and fasciae predominantly proximal to the hypospadic orifice. Correlation with normal development indicated that hypospadias and associated anomalies are not caused by disturbed fusion or glandar invagination but by maldevelopment of a complex of primordial fascial and vascular tissue proximal to the urethral orifice which normally form the venter side of the penis by disproportionately strong proliferation and make the urethral orifice shift distalward. Insufficient growth may disturb that shift with the degree of deficiency determining the precise position of the urethral orifice, size of the urethral delta and defect of the prepuce. Shortage and/or poor organisation of these tissues explain curvature and, if asymmetrical, torsion, both of which can occur also with minimal urethral deformity or as congenital ventral curvature and torsion without hypospadias.

Origin of the Deciduous Upper Lateral Incisor and its Clinical Aspects

The upper lateral incisor in humans is often affected by dental anomalies that might be explained developmentally. To address this question, we investigated the origin of the deciduous upper lateral incisor (i2) in normal human embryos at prenatal weeks 6-8. We used serial frontal histological sections and computer-aided 3D reconstructions. At embryonic days 40-42, two thickenings of the dental epithelia in an "end-to-end" orientation were separated by a groove at the former fusion site of the medial nasal and maxillary processes. Later, these dental epithelia fused, forming a continuous dental lamina. At the fusion site, i2 started to develop. The fusion line was detectable on the i2 germ until the 8th prenatal week. The composite origin of the i2 may be associated with its developmental vulnerability. From a clinical aspect, a supernumerary i2 might be a form of cleft caused by a non-fusion of the dental epithelia.