Anomalies In Embryos Research Articles

Assessing the toxicity of aegerolysin-based bioinsecticidal complexes using the sea urchin Paracentrotus lividus as model organism

The use of alternative solutions for pest management to replace pesticides in agriculture is of great interest. Proteinaceous complexes deriving from edible oyster mushrooms were recently proposed as environmentally friendly bioinsecticides. Such complexes, composed of ostreolysin A6 (OlyA6) and pleurotolysin B (PlyB), target invertebrate-specific membrane sphingolipids in insect's midgut, causing death through the formation of transmembrane pores. In this work, the potential impact of OlyA6/PlyB complexes was tested in the Mediterranean sea urchin Paracentrotus lividus, as an indicator of environmental quality. The ability of the fluorescently tagged OlyA6 to bind sea urchin gametes (sperm, eggs), the lipidome of sea urchin gametes, and the potential toxic effects and developmental anomalies caused by OlyA6/PlyB complexes on P. lividus early development (embryo, larvae) were investigated. The binding of the fluorescently tagged OlyA6 could be observed only in sea urchin eggs, which harbor OlyA6 sphingolipid membrane receptors, conversely to sperm. High protein concentrations affected sea urchin fertilization (>750 µg/L) and early development (> 375 µg/L in embryos; >100 µg/L in larvae), by causing toxicity and morphological anomalies in embryos and larvae. The main anomalies consisted in delayed embryos and incorrect migration of the primary mesenchyme cells that caused larval skeletal anomalies. The classification of these anomalies indicated a slight environmental impact of OlyA6/PlyB complexes at concentrations higher than 750 µg/L. Such impact should not persist in the marine environment, due to the reversible anomalies observed in sea urchin embryos and larvae that may promote defense strategies. However, before promoting the use of OlyA6/PlyB complexes as bio-pesticides at low concentrations, further studies on other marine coastal species are needed.

O-302 Triploid conceptions are predominantly caused by female meiosis II errors and their risk increases with advancing maternal age

Abstract Study question What are the incidence and origin of ploidy abnormalities in embryos derived from 2 pronuclei (2PN) zygotes, and is their risk linked to maternal age? Summary answer While embryo haploidy is usually due to male meiosis errors, triploidy is mainly caused by female meiosis II errors, whose incidence increases with maternal age. What is known already Normal fertilization is denoted by the appearance of 2PN 16-18 hours after insemination. Deviation from 2PN is considered evidence of abnormal fertilization and ploidy anomalies. Nonetheless, even 2PN embryos can be diagnosed with ploidy abnormalities during preimplantation genetic testing (PGT). The incidence of 3PN has already been linked to advanced maternal age. However, no conclusive and well-powered studies have yet investigated the incidence, origin, and maternal age correlation of genetically-diagnosed ploidy abnormalities. A targeted NGS-based approach that simultaneously analyzes copy number and genotyping data provides a comprehensive ploidy status assessment and helps address numerous basic and clinical research questions. Study design, size, duration Retrospective study including 96,660 trophectoderm biopsies analyzed between 2020 and 2022 using a targetedNGS-based PGT platform. A total of 1,063 embryos carrying haploidy or triploidy were used to investigate maternal age correlation, and parental/meiotic origin of the anomaly. Of these, 57 embryos were from concomitant PGT-M (PGT for monogenic disorders) cycles where also parental DNA was available. These trios (embryo-parents) were used to independently estimate parental/meiotic contribution to the ploidy abnormality and genome-wide recombination events. Participants/materials, setting, methods Targeted-NGS’s accuracy in genotyping and copy number (CN) detection were previously validated on triploid cell lines and multifocal blastocyst biopsies with known ploidy status derived from abnormally fertilized oocytes. Parental and meiotic origins were estimated using two independent approaches. First, they were inferred using sex chromosomes CN from 1,063 embryos with altered ploidy status. Secondly, they were directly calculated using genotyping data from 57 trios (embryo and parents) from PGT-M cycles. Main results and the role of chance The prevalence of ploidy abnormalities in 2PN-derived embryos was 1.1% (n = 1,063/96,660), with triploids accounting for 83.0% (n = 882/1,063) and haploids for 17.0% (n = 181/1,063). Remarkably, the incidence of ploidy anomalies is positively correlated with maternal age (OR = 1.046 per year; p < 0.001). Triploidy showed strong correlation with age (OR = 1.059 per year; p < 0.001), while haploidy did not (OR = 0.96 per year; p = 0.1). Based on sex chromosomes CN analysis, the extra haploid set of triploid embryos was almost completely of maternal origin (94.6%; 95%CI:93.0-96.1), with male errors accounting for only 5.4% (95%CI:4.0-7.1). On the other hand, haploid embryos were the result of paternal errors in 97.8% of cases (95%CI:94.4-99.4), with the missing haploid set being of maternal origin in the remaining 2.2% (95%CI:0.6-5.6). In terms of triploidy’s meiotic origin, two-thirds of the errors occurred during MII (95%CI:63.4-69.8), while one-third occurred during MI (95%CI:30.2-36.5). An independent method using genotyping data of 57 trios confirmed the predominance of paternal error in haploidy (n = 12/14) and the exclusively maternal origin of all embryonic triploidies (n = 43/43). The extra haploid set resulted from an error during MI in 27.9% (n = 12/43) and during MII in 72.1% (n = 31/43) of cases. Interestingly, 16.3% of triploids (n = 7/43) showed no genome-wide recombination events. Limitations, reasons for caution Despite the uniquely large sample size, the inference based on sex chromosomes CN suffers the limitations of the modeling assumptions (independence between parental and meiotic errors), which require further validation. Due to the low prevalence of paternal errors, clinical correlation with male factor could not be investigated with sufficient power. Wider implications of the findings Thanks to the exceedingly high sample size, this is the first study to reveal an increased risk of triploid conception with advancing female age (76% higher at age 40 than at age 30), providing meaningful insights for patients counseling. Importantly, the meiotic origin of ploidy anomalies in embryos were unveiled. Trial registration number not applicable

Association between hereditary predisposition to common cancers and congenital multimalformations.

In a previous article we reported that mutations favoring cancer at adulthood seemed to improve fertility and limit miscarriages. Because spontaneous abortion may result from anomalies in embryo, we questioned if an increased frequency of congenital malformation could be evidenced among cancer‐prone families. Oncogenetics database (≈193 000 members) of the comprehensive cancer center Jean Perrin was crossed with regional registry of congenital malformations (≈10 000). Among children born between 1986 and 2011, 176 children with malformation matched in both databases. In breast/ovaries cancer‐prone families, the risk for malformations was multiplied by 2.4 [1.2‐4.5] in case of a BRCA1 mutation. Frequencies of malformation in BRCA2 and MMR mutated families were similar to families without a cancer syndrome. In comparison to malformations concerning a unique anatomical system, multimalformations were significantly more frequent in case of BRCA or MMR mutations: compared to families without cancer syndrome, the risk of multimalformations was multiplied by 4.1 [0.8‐21.7] for cancer‐prone families but with no known deleterious mutation, by 6.9 [1.2‐38.6] in families with a known mutation but an unknown parental mutational status and by 10.4 [2.3‐46.0] when one parent carried the familial mutation. No association with the type of anatomical system was found, nor with multiple births. These results suggest that BRCA and MMR genes play an important role in human embryogenesis and that if their function is lowered because of heterozygote mutations, congenital malformations are either more likely (BRCA1 mutations) and/or more susceptible to concern several anatomical systems.

Неслучайное распределение кариотипов эмбрионов у женщин с привычным невынашиванием беременности

Привычное невынашивание беременности (ПНБ) - это потеря двух и более беременностей подряд, затрагивающая до 5% супружеских пар в популяции. Средняя частота хромосомных аномалий у спонтанных абортусов человека составляет около 50%, и если считать возникновение хромосомных аномалий случайным событием, то, независимо от кариотипа первого выкидыша, последующий в половине случаев должен быть цитогенетически нормальным. Целью данной работы было определить, существует ли закономерная повторяемость хромосомной конституции у спонтанных абортусов от одной супружеской пары. Прокариотипировано 108 случаев повторной гибели эмбриона в 51 семье. Кариотип абортусов был получен с использованием комбинации нескольких методов: стандартного цитогенетического анализа, сравнительной геномной гибридизации (CGH), флуоресцентной гибридизации in situ (FISH) и сравнительной геномной гибридизации на микрочипах (aCGH) (73, 29, 3 и 3 образца соответственно). Среди обследованных женщин 35% (18/51) были здоровы, а 59% (30/51) имели заболевания женской половой сферы, ассоциированные с ПНБ. Отношение шансов (OR) потери второго эмбриона с таким же кариотипом (нормальным или аномальным), как и у первого абортуса, составило 6,98 (95% CI: 2,04-23,88; p = 0,0013). У 19 женщин из 51 (37%) все погибшие зародыши были с нормальным кариотипом, причем у 7 из этих женщин не было выявлено патологии, обусловливающей невынашивание беременности. Оба выкидыша с аномалиями кариотипа имели 35% (18/51) женщин, из них у 6 женщин аномалии эмбрионов представляли собой повторные трисомии по различным хромосомам (гетеротрисомии), еще в одной семье оба абортуса имели трисомию 16. Одиннадцать случаев повторной гибели эмбрионов оказались сочетанием различных типов аномалий. Средний возраст женщин с двумя трисомными выкидышами оказался выше, чем у женщин с двумя выкидышами с различными типами аномалий (33,1 ± 3,45 и 28,7 ± 5,78 года соответственно, p = 0,025). Вероятность того, что последующий абортус будет иметь такой же кариотип (нормальный или аномальный), что и предыдущий, повышена. Повторные выкидыши с нормальным кариотипом могут быть обусловлены наличием у женщин этиологических факторов невынашивания, не диагностируемых стандартными методами обследования. Некоторые пациентки с повторными трисомиями абортусов, возможно, имеют более высокий риск хромосомного нерасхождения, чем другие женщины в том же возрасте. Сочетание у абортусов от одной женщины аберраций различных типов скорее всего случайно: маловероятно, чтобы наличие конкретного типа аномалии могло быть связано с повышенным риском возникновения другого типа аномалий, так как их формирование обусловлено различными механизмами. Introduction. Recurrent pregnancy losses is the loss of two or more consecutive pregnancies, it affects up to 5% of couples in the population. The average frequency of chromosomal abnormalities in human spontaneous abortions is about 50%, and if the chromosomal abnormalities are random events, the subsequent abortion should be cytogenetically normal in half of the cases, regardless of the karyotype of the previous miscarriage. The aim of this study was to determine is there a regular occurrence of the chromosome constitution in spontaneous abortions from the same woman. Materials and methods. A total of 108 cases of recurrent embryonic death in 51 families were studied. The karyotype of abortion was obtained using a combination of methods: standard cytogenetic analysis (73), CGH (29), FISH and aCGH (3 samples each). 35% (18/51) of women were healthy, 59% (30/51) had diseases associated with miscarriage. Results. The odds ratio (OR) of the loss of the second embryo with the same karyotype (normal or abnormal), as in the first abortion, was 6.98 (95% CI: 2.04-23.88, p = 0.0013). In 19 women from 51 (37%), all the dead embryos had normal karyotypes, and 7 of these women did not have a pathology that caused miscarriage. Among women with RM 35% (18/51) had both miscarriages with abnormal karyotypes. Of these, in 6 women, embryo anomalies were repeated trisomy of different chromosomes (heterotrisomies), in one family both abortions had trisomy 16. Eleven cases of repeated death of embryos proved to be a combination of different types of anomalies. The average age of women with two trisomic miscarriages was higher than in women with miscarriages with different types of anomalies (33.1 ± 3.45 and 28.7 ± 5.78 years, respectively, p = 0.025). Conclusions. The likelihood for subsequent abortion to be of the same karyotype (normal or abnormal), as the previous one, is increased. Repeated miscarriages with normal karyotypes may be due to the women’s etiological factors of miscarriage that were not diagnosed by standard survey methods. Some patients with recurrent trisomic abortions may have an increased risk of chromosomal nondisjunction than other women at the same age. The combination of abortions with different types of abnormalities from one woman is most likely random: it is unlikely that the presence of a specific type of anomaly could be associated with an increased risk of another type of anomaly, because different mechanisms are involved in their formation.

ВЛИЯНИЕ ТЕМПЕРАТУРЫ В ЭМБРИОГЕНЕЗЕ НА РЫБОВОДНО-БИОЛОГИЧЕСКИЕ ПОКАЗАТЕЛИ МОЛОДИ ГИБРИДА БЕСТЕРА

The article focuses on the influence of different temperature parameters during incubation of bester hybrid Acipenser ruthenus (Linnaeus, 1758) × ( Huso huso (Linnaeus, 1758) × × Acipenser ruthenus (Linnaeus, 1758)) of the Aksay breed of the fourth generation (F4) on the abnormalities in embryogenesis and on the rate of growth and survival of the hybrid juveniles in recirculating aquaculture systems. As a result of experimental work, it has been proved that the optimum incubation temperature for bester roe is 16°C, and the fastest rate of weight growth is observed in bester juveniles bred from eggs incubated at a water temperature 18-20°C. A distinctive feature of the incubation of eggs at 18-20°C is elimination of a large number of fertilized eggs (25%) even at the early stages of development. Also, at lower water temperature (8 and 12°C), the highest percentage of embryo anomalies at the hatching stage has been observed. The lowest survival rate had juveniles from eggs, incubation of which took place at a temperature of 8°C (14-32%). The highest survival rate from one-day prelarva to fry with average weight of 1 g was observed in bester species grown from eggs which were incubated at a temperature of 12-13°C (38-90%). It is shown that the temperature difference of 1-2°C during cultivation affects juveniles' weight. Data are given on the effect of growing density on the survival rates and weight of juveniles. The stocking density of bester at the stage of larvae that switched to active feeding to the juvenile with a mass of 1 g should not exceed 2-3 kg/m3 in order to guarantee optimal growth rates, survival rate, and to maximize the use of tanks volumes. It has been stated that the lower the temperature of water during the incubation of eggs, the slower the fish grows in the future. However, this effect is leveled in species by the age of 250-300 days, if the optimal growing densities are non-compliance.

Histological analysis of hatchlings of the Australian lungfish, Neoceratodus forsteri, from water impoundments reveals fundamental flaws in development

Anomalies in embryos and hatchlings of the Australian lungfish are now found in many of the environments inhabited by lungfish, such as reservoirs (Lakes) created over natural rivers, and affect many tissues and organs in the body, most obviously the epidermis and related sense organs, but extending to blood vessels and skeletal structures as well. Development of muscular tissues is affected, but only by the lack of nutrition in the eggs as laid by the parents, and by failure of the hatchlings to feed. They are found in embryos collected from the Lakes and reared in the laboratory by standard methods, and are also present in embryos and hatchlings collected directly from the Lakes. They are not found in fish collected from unaltered parts of Rivers below the Lakes. Recently, in the Lakes, the anomalies are so widespread, and so serious, that they affect all the young of one season. The most probable cause of abnormal development is a lack of volatile fatty acids in the diet of adults, resulting in the production of poor quality eggs, as has been found in other species of fish. The results of this study have serious implications for survival of the species, since almost every habitat where lungfish are found, and are still spawning, has now been altered by the building of water impoundments. Restoration of freshwater environments in south-east Queensland should be a priority for the State and Federal Governments and for water authorities.

4‐O‐methylhonokiol Inhibits Serious Embryo Anomalies Caused by Nicotine via Modulations of Oxidative Stress, Apoptosis, and Inflammation

Since the increasing smoking rate among women has resulted in higher rates of embryonic malformations, it is important to search for an efficient and inexpensive agent that can help reduce the rate of serious fetal anomalies caused by maternal cigarette smoking. In this study, the bioavailability of 4-O-methylhonokiol isolated from Magnolia officinalis was first demonstrated in the mouse embryos exposed to nicotine using a whole embryo culture system. Mouse embryos on embryonic day 8.5 were cultured with 1 mM nicotine and/or 4-O-methylhonokiol (1 × 10(-4) or 1 × 10(-3) μM) for 48 hr and were analyzed on the viewpoints of embryo developmental changes, oxidative damages, and apoptotic and inflammatory changes. Embryos exposed to 1 mM nicotine developed not only severe morphological anomalies, increased expressions of tumor necrosis factor-α, interleukin-1β, and caspase 3 mRNAs; and elevated levels of lipid peroxidation, but also decreased levels of cytoplasmic superoxide dismutase, cytosolic glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase, hypoxia inducible factor-1α, and B-cell lymphoma-extra large mRNAs, and reduced superoxide dismutase activity. However, these parameters were significantly improved when embryos exposed to the nicotine were concurrently treated with 4-O-methylhonokiol (1 × 10(-4) or 1 × 10(-3) μM). These findings indicate that 4-O-methylhonokiol reduces serious embryo anomalies caused by nicotine in mouse embryos via the modulations of oxidative stress, apoptosis, and inflammation, suggesting that 4-O-methylhonokiol may be a preventive and therapeutic agent against the dysmorphology induced by maternal smoking during pregnancy.

Sperm DNA fragmentation index does not correlate with the sperm or embryo aneuploidy rate in recurrent miscarriage or implantation failure patients

The aneuploidy rate is higher in poor-quality sperm samples, which also have higher DNA fragmentation index values. The aim of this study was to assess the relationship between sperm DNA fragmentation in samples from infertile men belonging to couples with recurrent miscarriage or implantation failure and the aneuploidy rate in spermatozoa as well as in embryos from patients. This prospective study evaluated DNA damage and the aneuploidy rate in fresh and processed (density gradient centrifugation) ejaculated sperm as well as the aneuploidy rate in biopsied embryos from fertility cycles. Fluorescence in situ hybridization was used for the aneuploidy analysis. Results were compared using linear regression and analysis of variance. A total of 154 embryos were evaluated from 38 patients undergoing PGD cycles; 35.2% of the embryos were chromosomally normal. Analysis of the same sperm samples showed an increased DNA fragmentation after sperm preparation in 76% of the patients. There was no correlation between DNA fragmentation and the aneuploidy rate in embryos or in fresh or processed sperm samples. Sperm DNA fragmentation is not related to chromosomal anomalies in embryos from patients with recurrent miscarriage or implantation failure. However, we cannot rule out the possibility that a relationship between DNA fragmentation and aneuploidy exists for other causes of infertility. Furthermore, the different methods used to evaluate DNA fragmentation may produce different results.

Aneuploidy in the Human Blastocyst

Studies of human cleavage stage embryos, 3 days after fertilization of the oocyte, have revealed remarkably high levels of chromosome abnormality. In addition to meiotic errors derived from the gametes, principally the oocyte, mitotic errors occurring after fertilization are also common, leading to widespread chromosomal mosaicism. The prevalence of chromosome anomalies in embryos may explain the relatively poor fertility and fecundity in humans and the low success rates of assisted reproductive treatments (e.g., IVF). While much is known concerning the incidence of aneuploidy during the first 3 days following fertilization, it is only in the last couple of years that large numbers of embryos at the final stage of preimplantation development, the blastocyst stage, 5 days after fertilization, have been subjected to detailed analysis. Here we discuss the latest data from the comprehensive cytogenetic analysis of blastocysts. These findings indicate that the majority of selection against chromosome abnormalities does not occur until the time of implantation or shortly after, with aneuploidy typically affecting more than 50% of blastocysts. Additionally, clinical results presented suggest that screening of blastocyst stage embryos for chromosome abnormality, with preferential transfer to the uterus of those found to be euploid, may help to improve the success rates of assisted reproductive treatments.

Vascular defects in a mouse model of hypotrichosis-lymphedema-telangiectasia syndrome indicate a role for SOX18 in blood vessel maturation

Mutations in the transcription factor gene SOX18 cause vascular, lymphatic and hair follicle defects in humans with dominant and recessive forms of hypotrichosis-lymphedema-telangiectasia (HLT) syndrome. Here, we clarify the role of SOX18 in the vascular dysfunction in HLT by ultrastructural, immunofluorescence, molecular and functional analysis of vascular anomalies in embryos of the naturally occurring Sox18-mutant mouse strain ragged-opossum (Ra(Op)). Early genesis and patterning of vasculature was unimpaired in Ra(Op) embryos, but surface capillaries became enlarged from 12.5 dpc and embryos developed massive surface hemorrhage by 14.5 dpc. Large focal breaches in the endothelial barrier were observed, in addition to endothelial hyperplasia associated with impaired pericyte recruitment to the microvasculature. Expression of the genes encoding the endothelial factors MMP7, IL7R and N-cadherin was reduced in Ra(Op) embryos, suggesting that these are downstream targets of SOX18. Together, our results indicate that vascular anomalies in HLT arise from defects in regulation of genes required for the acquisition of structural integrity during microvascular maturation.

Implications for Providers and Patients: A Comment on the Regulatory Framework for Preimplantation Genetic Diagnosis in New Zealand

Preimplantation Genetic Diagnosis (PGD) is a reproductive genetic technology that was first introduced into clinical medicine over a decade and a half ago. 2 PGD enables the detection of genetic anomalies in embryos created via IVF prior to implantation. On the basis of the genetic information derived from PGD, choices may be made as to which embryo, or embryos, are transferred to a woman's uterus. This technology was undertaken for the first time in New Zealand in late 2005. The provision of PGD by fertility services in New Zealand was preceded by the formulation of Guidelines on Preimplantation Genetic Diagnosis by the National Ethics Committee on Assisted Human Reproduction. Since the introduction of the Human Assisted Reproductive Technology Act 2004, these have been designated as interim Guidelines of the statutory policy-making body created by the Act, the Advisory Committee on Assisted Reproductive Technology. An Order in Council has since authorised a category of PGD, in almost identical terms to those specified in the Guidelines, which may be carried out as a routine clinical procedure without external oversight. This article examines the scope of these regulatory initiatives. It argues that there are inherent uncertainties in both the Guidelines and the Order in Council which may pose difficulties for providers and patients in the clinical context.

Teratogenic effects of bis‐diamine on the developing cardiac conduction system

Congenital heart defects, including conotruncal anomalies, are often associated with arrhythmias. Bis-diamine induces conotruncal anomalies in embryos when administered to pregnant female rats. To investigate the mechanism of arrhythmia in conotruncal anomalies, we histologically examined the development of the cardiac conduction system in this animal model. A single dose of 200 mg of bis-diamine was administered to pregnant Wistar rats on ED 10.5 of pregnancy. The embryos were removed on each day from ED 11.5 to 15.5. Immunoexpression of HNK-1, connexin40, and connexin43 were examined in serial sections. The distribution pattern of TUNEL-positive cells around the conduction system was also examined. HNK-1 immunoreactivity was evident in interventricular septum, in both the control and the bis-diamine-treated embryos from ED 12.5. Although a chain of connexin40-immunoreactive cells from interventricular septum to trabeculae, corresponding to the His bundle and its branches, was demonstrated at ED 13.5 in the control embryos, this chain was first detected at ED 14.5 in the bis-diamine-treated embryos. Immunoexpression of connexin43 in the working myocardium was also less in the bis-diamine-treated embryos than in the control at ED 13.5. The number of TUNEL-positive cells in the interventricular septum was highest at ED 12.5 in the control and at ED 13.5 in the bis-diamine-treated embryos. Furthermore, these TUNEL-positive cells were HNK-1 negative, vimentin-positive, and alpha smooth muscle actin-positive. Bis-diamine disturbed the normal development of gap junctions and apoptosis of myofibroblasts around the HNK-1-positive conduction tissue through overall poor myocardial proliferation and growth.

Embryopathy in experimental diabetic gestation:assessment of PGE2 level, gene expression of cyclooxygenases and apoptosis

The causes of, and predisposing conditions for, increased congenital anomalies in embryos of experimental diabetic gestation are not fully identified. In the present study, some possible factors involved in diabetes-induced embryopathy are explored. The concentration of PGE2, the gene expression of cyclooxygenases (COX-1 and COX-2) and level of apoptosis (measured by caspase-3 activity) are assessed during organogenesis in the embryos of streptozotocin-induced diabetic rats. The concentrations of PGE2 in the embryos of diabetic rats were lower than controls, with the lowest values in malformed embryos and their associated membranes (yolk sacs). The pattern of change in PGE2 was similar in the embryos of the control and diabetic groups, which showed a steady decline between days 9 and 11 of gestation. These changes in PGE2 were accompanied by a small decrease in COX-1 expression in all embryos and associated membranes during the same gestational period. Expression of COX-2, which was below normal in diabetic embryos, decreased between days 9 and 11 of gestation in all groups. In the membranes of non-malformed embryos, COX-2 expression peaked on day 10 of gestation. It was found that there was little or no detectable COX-2 expression in the membranes of malformed embryos on day 9 of gestation and although its expression was detectable on the following days it was much lower than in the other groups. Caspase-3 activity increased substantially between days 9 and 11 of gestation. Embryos from the experimentally diabetic group showed higher activity than did controls, with the largest increases in the malformed embryos. It would appear that COX-2 expression and PGE2 concentration (in both embryo and associated membranes) play a significant role in organ formation. The data presented here suggest that an unhealthy placenta may be instrumental in the development of malformed embryos.

Germ cell aneuploidy in zebrafish with mutations in the mitotic checkpoint gene mps1.

Aneuploidy, resulting from chromosome missegregation during meiosis, is a major cause of human infertility and birth defects. However, its molecular basis remains incompletely understood. Here we have identified a spectrum of chromosome anomalies in embryos of zebrafish homozygous for a hypomorphic mutation in Mps1, a kinase required for the mitotic checkpoint. These aneuploidies are caused by meiotic error and result in severe developmental defects. Our results reveal Mps1 as a critical regulator of chromosome number in zebrafish, and demonstrate how slight genetic perturbation of a mitotic checkpoint factor can dramatically reduce the fidelity of chromosome segregation during vertebrate meiosis.

Maternal autoimmune diseases and immunologically induced embryonic and fetoplacental damage

Autoimmune diseases are a group of illnesses in which autoantibodies are produced against various organs, presenting with a variety of clinical symptoms. In this review, we discuss the different aspects of autoimmune diseases in pregnancy. We also describe experimental models that help to understand the etiology and pathogenesis of the effects of this maternal disease on the developing embryo, fetus and placenta. The possible direct effects of sera or IgG obtained from women with systemic lupus erythematosus/antiphospholipid syndrome (SLE/APS) and recurrent pregnancy loss (RPL) were examined on cultured 10.5- and 11.5-day-old rat embryos and on cultured human placental explants, as compared to sera from healthy women or synthetic medium that were used as controls. In addition, we examined the effects of the sera obtained from these women after successful treatment that allowed the birth of normal infants. We observed increased embryonic death and anomalies in embryos cultured on sera and IgG from SLE/APS women. Similarly, when human placental explants were cultured on these sera, trophoblastic cell growth was reduced and apoptotic rate was increased. Successful treatment also reduced the damage caused by the sera from these women in the cultured embryos and placentas. Our results, and the cited studies, point to the important role of the placental damage in the etiology of RPL associated with SLE/APS. Animal models, both in vivo and in vitro, as well as cultured early human placental explants can be used successfully to understand some of the pathogenic aspects of SLE/APS and RPL.

Synthetic matrix metalloproteinase inhibitor decreases early cardiac neural crest migration in chicken embryos.

During early embryonic development, cardiac neural crest (NC) cells emerge from the forming neural tube, migrate beneath the ectoderm, enter the pharyngeal arches, and subsequently participate in the septation of the heart. Like tumor cells, NC cells penetrate through basement membranes and invade extracellular matrix during their emigration and migration and, therefore, are liable to use similar invasive mechanisms. Matrix metalloproteinases (MMPs) are a family of zinc proteolytic enzymes known to be important in cell migration and invasion of normal and metastatic cells. In an earlier study, we found that the spatial and temporal distribution pattern of MMP-2 positively correlates with cardiac NC migration, suggesting MMP enzymatic activity may be important in mediating cardiac cell NC migration. To test this hypothesis, a synthetic MMP inhibitor, KB8301, was used to block MMP enzymatic activity during in vitro and in vivo cardiac NC cell migration in chick embryos. Injection of KB8301 into the cell-free space adjacent to the neural tube at the level of the second somite before the NC cells emigrated caused major morphologic anomalies in embryos and disrupted cardiac NC morphogenesis. Unilateral injection of KB8301 at lower concentrations, significantly decreased cardiac NC migration on the injected side compared with the noninjected side and compared with that of the injected controls. This decrease correlated with a decrease in MMP activity in the embryos and was not attributable to differences in embryo size or rate of embryonic development after injection. KB8301 also significantly decreased the rate of NC cell motility and distance NC cells migrated from explanted neural tubes and increased cell area and perimeter. These data suggest that MMP enzymatic activity is an important mediator of early cardiac NC migration and that perturbation of endogenous MMP activity may lead to NC-related congenital defects.

The use of microangiography in detecting aberrant vasculature in zebrafish embryos exposed to cadmium

Embryonic vascular patterns in zebrafish ( Danio rerio) could be visualised by confocal microscopy coupled with microinjected fluorescent microbeads. This microangiographic technique was adopted here, for the first time, to study the effects of cadmium on cardiovascular development in zebrafish embryos. Zebrafish embryos were incubated in culture medium containing 100 μM cadmium from 5 h post fertilisation (hpf) to 48 hpf. At 48 hpf, embryos were examined for viability and occurrence of malformations. The 100 μM cadmium caused 32.21±3.65% mortality and 20.33±4.04% visible malformations in surviving embryos. In the remaining embryos with no visible signs of malformations, further assessments for less obvious abnormalities were performed. Assessments on craniofacial development were made by digital measurements on areas of brains and eyes. Cardiac development was assessed by immunostaining the heart with the antibody MF20 specific for myosin heavy chain. Body lengths of the embryos were also measured. Embryonic development of brains, eyes, hearts and body lengths of visibly healthy embryos in the cadmium treatment group showed no significant difference from the controls. Embryonic vasculature of these visibly healthy embryos was then studied by microinjecting fluorescent microbeads of diameter 0.02 μm into the circulation. All the cadmium treated embryos showed localised vascular defects in the dorsal aortae, segmental and cranial vessels while none of the control embryos showed any aberrant patterns in the networking of the vasculature. Improved image analyses on the anterior regions revealed that cadmium treated embryos had markedly less complex networks of cranial vessels with fewer vessels perfusing the craniofacial regions. The number of branch points in the vascular network was counted. In untreated embryos, there were 135.6±51 branches in the vasculature in entire body. In the cadmium treated embryos, there were 64.5±31 branches. The difference was significant when assessed with Student's t-test. It appeared that although cadmium did not cause any signs of external malformations in these visibly healthy embryos, nonetheless induced impaired branching and anastomsis of the cranial vessels. This study revealed, for the first time, that vital vascular structures in fish embryos could be affected by exposure to cadmium. This technique allowed visualisation of vascular anomalies in embryos showing no external signs of malformations. The impairment of anatomical features during embryonic development might serve as meaningful health endpoints in ecotoxicological studies and in risk assessment.

Relationship between esophageal atresia with tracheoesophageal fistula and vertebral anomalies in mammalian embryos

Background/Purpose: The association of esophageal atresia with tracheoesophageal fistula and vertebral anomalies is well known, although the embryology of the combined defects has not yet been analysed. The present study describes the origin and development of esophageal atresia with tracheoesophageal fistula and vertebral anomalies in embryos using a rat model of VATER association produced by Adriamycin administration. Results: The lung buds were seen to develop from the laryngotracheal groove but the trachea failed to grow normally and the foregut overgrowth compensated for this failure. The trachea developed by trachealization of the foregut, which continued as a fistula to the lower esophageal segment. The notochord did not separate from the foregut at the correct time (before day 11 in rat embryos, Carnegie stage 11 in human embryos). Overgrowth of the foregut ventrally and caudally carried with it the attached notochord in the same direction leading to abnormal bending of this structure. Conclusion: This irregularity of the notochord may be responsible for abnormal development of the vertebrae.

Relationship between associations of NOR and chromosomal anomalies in the abnormal embryos of nonobese diabetic and STZ-diabetic mouse.

Associations of nucleolar organizing regions (NORs) in the postimplantation stage embryos of nonobese diabetic (NOD) mice, and diabetic ICR mice induced by streptozotocin (ST), were studied to investigate the possible cause of the numerical anomalies of the chromosomes in their abnormal embryos. The incidence of NOR associations in abnormal embryos from diabetic NOD (NOD-DM) and STZ-diabetic mice was 11.7 and 7.7%, respectively. This incidence was significantly higher than that (1.2%, p < 0.05) of normal embryos from ICR mice which were used as control. From the results analyzed cytogenetically it was suggested that the higher incidence of chromosomal numerical anomalies in the embryos from NOD-DM and STZ-diabetic mice were caused by the chromosomal nondisjunction induced by associations of NORs. Furthermore, it was suggested that NOD-DM embryos have a tendency to increase the associations of NOR in a diabetic condition together with other factors such as autoimmune disease, however a diabetic condition alone induced chromosomal anomalies. Regarding relationships between the incidence of associations of NOR and the types of malformed embryos, it was also clear that all of the abnormal embryos from NOD-DM and STZ-diabetic mice had a high incidence of associations of NOR, and that the incidence was not related to the types of congenital anomalies. Furthermore, in the mal-developed tissue of embryos from STZ-diabetic mice, many chromosomal anomalies were found (26.6%), and the incidence was similar to that of whole embryos (22.6%).(ABSTRACT TRUNCATED AT 250 WORDS)

IgG exchange as a means of partial correction of anomalies in rat embryos in vitro, induced by sera from women with recurrent abortion

Many recurrent abortions have an unknown aetiology. A significant proportion of sera from women with spontaneous abortions induced 50% or more anomalies (‘high risk’ sera) in cultures of 10.5-day-old rat embryos. Because it is assumed that the teratogenic factor(s) in recurrent abortion is often an immunoglobulin G (IgG) antibody, it was attempted to reduce the rate of anomalies induced by high risk sera by removing the IgG fractions and adding IgG fractions from control sera (sera from women in a normal second trimester of pregnancy and after delivery). Concomitantly, an attempt was made to induce anomalies in embryos cultured in control sera by adding IgG fractions from high risk sera. It was found that among the high risk sera there were three groups, according to the response to the IgG exchange: (1) a group in which IgG exchange ‘corrected’ the anomalies in high risk sera, whereas it induced anomalies in control sera; (2) a group in which anomalies were found both in embryos cultured in high risk sera and those cultured in control sera; (3) a group in which the IgG exchange did not affect the results in the ‘experimental’ or ‘control’ embryos. Examination by transmission electron microscopy, of yolk sacs cultured in sera with IgG from women belonging to the third group did not reveal ultrastructural changes. In conclusion, in some high risk sera the teratogenic factor appears to be an IgG only; in others, teratogenicity is brought about not only by an IgG, but also by additional teratogenic factor(s); in the last group, the teratogenic factor(s) does not appear to be an IgG at all.