Trisomic Embryos Research Articles

Embryonic statistical analyses reveal 2 growth phenotypes in mouse models of Down syndrome

Down syndrome is associated with several comorbidities, including intellectual disability, growth restriction, and congenital heart defects. The prevalence of Down syndrome-associated comorbidities is highly variable, and intellectual disability, although fully penetrant, ranges from mild to severe. Understanding the basis of this interindividual variability might identify predictive biomarkers of in utero and postnatal outcomes that could be used as endpoints to test the efficacy of future therapeutic interventions. The main objective of this study was to examine if antenatal interindividual variability exists in mouse models of Down syndrome and whether applying statistical approaches to clinically relevant measurements (ie, the weights of the embryo, placenta, and brain) could define cutoffs that discriminate between subgroups of trisomic embryos. Three commonly used mouse models of Down syndrome (Dp(16)1/Yey, Ts65Dn, and Ts1Cje) and a new model (Ts66Yah) were used in this study. Trisomic and euploid littermate embryos were used from each model with total numbers of 102 for Ts66Yah, 118 for Dp(16)1/Yey, 92 for Ts65Dn, and 126 for Ts1Cje. Placental, embryonic, and brain weights and volumes at embryonic day 18.5 were compared between genotypes in each model. K-mean clustering analysis was applied to embryonic and brain weights to identify severity classes in trisomic embryos, and brain and placental volumetric measurements were compared between genotypes and classes for each strain. In addition, Ts66Yah embryos were examined for malformations because embryonic phenotypes have never been examined in this model. Reduced body and brain weights were present in Ts66Yah, Dp(16)1/Yey, and Ts65Dn embyos. Cluster analysis identified 2 severity classes in trisomic embryos-mild and severe-in all 4 models that were distinguishable using a putative embryonic weight cutoff of <0.5 standard deviation below the mean. Ts66Yah trisomic embryos develop congenital anomalies that are also found in humans with Down syndrome, including congenital heart defects and renal pelvis dilation. Statistical approaches applied to clinically relevant measurements revealed 2 classes of phenotypic severity in trisomic mouse models of Down syndrome. Analysis of severely affected trisomic animals may facilitate the identification of biomarkers and endpoints that can be used to prenatally predict outcomes and the efficacy of treatments.

P-702 Secretion of beta-human chorionic gonadotropin (b-hCG) by post-implantation embryos differs according to different chromosomal status.

Abstract Study question To evaluate secretion of b-hCG hormone by human post-implantation embryos cultured in-vitro up to day 10 according to their chromosomal status. Summary answer Secreted b-hCG levels differs among embryos with different chromosomal load, being severely diminished in monosomic embryos when compared either to euploid or trisomic embryos. What is known already Exploring early post-implantation embryo development is subject of growing interest because errors in correct embryo development at this period might cause early pregnancy losses or development of pathologies affecting postnatal health. Down's Syndrome (DS) is one of the most common examples of chromosomal supernumerary (trisomy 21; T21) aneuploidy that leads to live birth but associated to pathology in postnatal life while monosomy of chromosome 21 (M21) is more likely to cause embryo developmental arrest. Furthermore, conclusions extracted from our previous studies put forward the idea of different gene and metabolite expression according to different chromosomal load (Sanchez-Ribas et al., 2019). Study design, size, duration This is an exploratory comparative study which includes a total of 33 supernumerary science-donated human blastocysts on day 6 from in-vitro fertilization cycles followed by preimplantation genetic test for aneuploidies. Euploid (n = 10), pure T21 (n = 13) and pure M21 (n = 10) were included in the study. All day 6 blastocyst included were good-quality embryos and were initiating hatching at the time of vitrification. Embryos degenerated during culture were not used for b-hCG quantifications. Participants/materials, setting, methods Euploid, T21 and M21 day blastocysts were thawed and cultured in-vitro until day 10. Embryos were cultured in culture medium specifically designed to support post-implantation embryo development. Each day of culture, half volume of culture media was replaced by fresh media. Levels of b-hCG in conditioned embryo media were quantified by ELISA immunoassay at day 7, 9, and 10. Results are expressed as mean ± standard deviation. Embryo morphology was tracked by bright-field images. Main results and the role of chance All euploid and T21 embryos thawed managed to hatch completely and expand the blastocoel cavity, while 40% of M21 embryos failed to hatch being arrested inside the zona pellucida on day 7. On day 7, mean concentration of b-hCG secreted did not differ among euploid, T21 and M21 embryos (37,29±6,27; 36,05±6,61 and 27,16±9,95 ng/mL respectively). Interestingly, euploid and T21 embryos reported a progressive time-dependent increase in b-hCG levels along post-implantation in-vitro culture while secreted levels of b-hCG by M21 embryos showed a decreasing trend, reporting diminished b-hCG secretion at the end of the culture (day 10) compared to day 7 (14,86±2,57 vs 27,16±9,95 ng/mL; p &amp;lt; 0.05). No significant differences in b-hCG secretions were observed between euploid and T21 embryos neither on day 9 d.p.f. (61,05±8,47 and 56,08±8,10 ng/mL respectively) nor on day 10 (74,88±6,36 and 71,75±7,01 ng/mL respectively). However, b-hCG secreted by M21 embryos on day 9. and day 10 (27,11±11,45 and 14,86±2,57 ng/mL respectively) were significantly reduced (p &amp;lt; 0.01) compared with euploid or T21 embryos. Besides, while all euploid and T21 embryos developed until day 10, 16,67% of expanded M21 embryos degenerated before day 9. Limitations, reasons for caution This research focused only on embryo development, no maternal tissues were considered. Further proteins should be investigated to reveal other pathways implicated in healthy embryo development and not only trophoblast proliferation. Wider implications of the findings Monosomic embryos, associated to implantation failures, secrete lower b-hCG when compared to either euploid or trisomic embryos, which show similar behavior. Besides, b-hCG in culture media could become a non-invasive marker to track embryo progression along post-implantation stages and detect embryos with increased likelihood of developmental arrest or early miscarriage. Trial registration number Not Applicable

O-085 In-depth analysis of embryo development: Differences among monosomic, trisomic and chromosomally chaotic embryos compared to euploid embryos

Abstract Study question Is there any visible variation in the development of aneuploid embryos depending on the type of chromosome abnormality? Summary answer There were significant visible differences in the development of euploid, monosomic, trisomic and, especially, chaotic embryos. What is known already Aneuploidy rates are remarkably high in in vitro fertilized human embryos, with up to 50% of embryos diagnosed as aneuploid based on preimplantation genetic testing for aneuploidies (PGT-A). However, very little is known about the impact of specific aneuploidies during the early human embryo development. A recent publication showed that embryos with single chromosomal gain or loss reached the blastocyst stage later or earlier depending on the chromosome affected (Shahbazi et al., 2020). In our study, we wanted to detect observable differences in embryo behavior between embryos with different chromosomal abnormalities during the entire in vitro development. Study design, size, duration This was a retrospective study including 2,500 blastocysts with PGT-A results. Embryos were cultured in EmbryoScope systems until the fifth/sixth day of development (up to the time of trophectoderm biopsy). Automatic-annotations for division times and quality gradings were supervised routinely by senior embryologists using Guided Annotations Tool. Out of the total, 1,000 were euploid embryos used for reference and 1,500 were aneuploid embryos with one or more defects, including monosomic, trisomic and chromosomally chaotic embryos. Participants/materials, setting, methods Chromosome analysis was performed using next-generation sequence technology. Then, an in-depth analysis of time-lapse videos and supervised-automatic annotations was performed. We calculated the proportion of embryos, in each aneuploid category, that reached one specific event later than the expected value for euploid embryos plus one standard deviation. Later, we calculated the “relative risk” of an embryo of reaching the milestone late. We did the same for the time between milestones and for pairs of milestones. Main results and the role of chance Every aneuploid category was more likely to reach each specific embryo developmental event later than euploid embryos and the time gaps between developmental milestones were also statistically longer in aneuploid embryos (p &amp;lt; 0.0001). The following results were the most interesting relative risks (RR) when we compared the proportion of embryos (in each aneuploid category) to the proportion of euploid embryos (RR for euploid = 1). For reaching the division time to two cells (t2): 1.31 in monosomic embryos, 1.50 in trisomic embryos and 2.43 in chaotic embryos. For the division time to four cells (t4): 1.42 in monosomic embryos, 1.54 in trisomic embryos and 3.07 in chaotic embryos. For the division time to eight cells (t8) and the time of starting blastulation: 1.45 in monosomic embryos, 1.22 in trisomic embryos and 2.74 in chaotic embryos. Combined milestones were stronger indicators than each milestone by itself, the RR were: 1.63 in monosomic embryos, 1.81 in trisomic embryos and 3.35 in chaotic embryos for t2 and t4; 1.50 in monosomic embryos, 1.80 in trisomic embryos and 2.84 in chaotic embryos for t2 and t8; 1.46 in monosomic embryos, 1.90 in trisomic embryos and 3.43 in chaotic embryos for t4 and t8. Limitations, reasons for caution At this stage, we did not go down to specific chromosome abnormality as there were very few cases in each fully detailed category. Also, not all the embryos reached every developmental milestone. Wider implications of the findings Aneuploid embryos were significantly different from euploid embryos in the first five days of development. A large proportion of aneuploid embryos could be rejected because their developmental milestones falling outside the normal range. This could form part of an automated system for determining euploidy/aneuploidy from observation of embryos in vitro. Trial registration number 1902-VLC-018-MM

Novel insights from fetal and placental phenotyping in 3 mouse models of Down syndrome

In human fetuses with Down syndrome, placental pathology, structural anomalies and growth restriction are present. There is currently a significant lack of information regarding the early life span in mouse models of Down syndrome. The objective of this study was to examine embryonic day 18.5 and placental phenotype in the 3 most common mouse models of Down syndrome (Ts65Dn, Dp(16)1/Yey, Ts1Cje). Based on prenatal and placental phenotyping in 3 mouse models of Down syndrome, we hypothesized that one or more of them would have a similar phenotype to human fetuses with trisomy 21, which would make it the most suitable for in utero treatment studies. Here, C57BL6J/6 female mice were mated to Dp(16)1/Yey and Ts1Cje male mice and Ts65Dn female mice to C57BL/B6Eic3Sn.BLiAF1/J male mice. At embryonic day 18.5, dams were euthanized. Embryos and placentas were examined blindly for weight and size. Embryos were characterized as euploid or trisomic, male or female by polymerase chain reaction. A subset of embryos (34 euploid and 34 trisomic) were examined for malformations. The Ts65Dn mouse model showed the largest differences in fetal growth, brain development, and placental development when comparing euploid and trisomic embryos. For the Dp(16)1/Yey mouse model, genotype did not impact fetal growth, but there were differences in brain and placental development. For the Ts1Cje mouse model, no significant association was found between genotype and fetal growth, brain development, or placental development. Euploid mouse embryos had no congenital anomalies; however, 1 mouse embryo died. Hepatic necrosis was seen in 6 of 12 Dp(16)1/Yey (50%) and 1 of 12 Ts1Cje (8%) mouse embryos; hepatic congestion or inflammation was observed in 3 of 10 Ts65Dn mouse embryos (30%). Renal pelvis dilation was seen in 5 of 12 Dp(16)1/Yey (42%), 5 of 10 Ts65Dn (50%), and 3 of 12 Ts1Cje (25%) mouse embryos. In addition, 1 Ts65Dn mouse embryo and 1 Dp(16)1/Yey mouse embryo had an aortic outflow abnormality. Furthermore, 2 Ts1Cje mouse embryos had ventricular septal defects. Ts65Dn mouse placentas had increased spongiotrophoblast necrosis. Fetal and placental growth showed varying trends across strains. Congenital anomalies were primarily seen in trisomic embryos. The presence of liver abnormalities in all 3 mouse models of Down syndrome (10 of 34 cases) is a novel finding. Renal pelvis dilation was also common (13 of 34 cases). Future research will examine human autopsy material to determine if these findings are relevant to infants with Down syndrome. Differences in placental histology were also observed among strains.

Centriolar defects, centrin 1 alterations, and FISH studies in human spermatozoa of a male partner of a couple that produces aneuploid embryos in natural and artificial fertilization

PurposeTo study the potential paternal contribution to aneuploidies in the man of a couple who obtained trisomic embryos with natural and assisted fertilization.MethodsSemen analysis, immunofluorescence for localization of tubulin and centrin 1, transmission electron microscopy (TEM), and fluorescence in situ hybridization (FISH) analysis for chromosomes 18 and 9 were performed. Sperm of fertile men were used as controls.ResultsThe percentages of sperm motility and normal forms were decreased. The percentages of sperm with tail reduced in dimension, headless tails, coiled tails, and altered head-tail junction were significantly higher (P < 0.01) in the patient than in controls, whereas the percentage of sperm with a normal centrin 1 localization (two spots in the centriolar area) was significantly reduced (P < 0.01) in the patient. Immunofluorescence with anti-tubulin antibody showed that in most of the patient’s sperm connecting pieces (83.00 ± 1.78%), two spots were present, indicating prominent proximal centriole/centriolar adjunct and evident distal centriole, whereas controls’ sperm displayed a single spot, indicating the proximal centriole. The percentage of sperm with two spots was significantly higher (P < 0.01) in the patient than in controls. TEM analysis showed that centriolar adjuncts of the patient’s sperm were significantly longer (721.80 ± 122.26 nm) than in controls’ sperm (310.00 ± 64.11 nm; P < 0.001). The aneuploidy frequencies of the patient’s sperm, detected by FISH analysis, were increased with respect to controls.ConclusionA paternal contribution to sperm aneuploidies cannot be excluded since the patient’s sperm showed altered morphology, immature centriolar adjunct, presence of evident distal centriole, scarce presence of centrin 1, and high aneuploidy frequency.

Failure to recombine is a common feature of human oogenesis

Failure of homologous chromosomes to recombine is arguably the most important cause of human meiotic nondisjunction, having been linked to numerous autosomal and sex chromosome trisomies of maternal origin. However, almost all information on these "exchangeless" homologs has come from genetic mapping studies of trisomic conceptuses, so the incidence of this defect and its impact on gametogenesis are not clear. If oocytes containing exchangeless homologs are selected against during meiosis, the incidence may be much higher in developing germ cells than in zygotes. To address this, we initiated studies of exchangeless chromosomes in fetal ovarian samples from elective terminations of pregnancy. In total, we examined more than 7,000 oocytes from 160 tissue samples, scoring for the number of foci per cell of the crossover-associated protein MLH1. We identified a surprisingly high level of recombination failure, with more than 7% of oocytes containing at least one chromosome pair that lacked an MLH1 focus. Detailed analyses indicate striking chromosome-specific differences, with a preponderance of MLH1-less homologs involving chromosomes 21 or 22. Further, the effect was linked to the overall level of recombination in the cell, with the presence of one or two exchangeless chromosomes in a cell associated with a 10%-20% reduction in the total number of crossovers. This suggests individuals with lower rates of meiotic recombination are at an increased risk of producing aneuploid offspring.

Abstracts of the 24th Annual Congress of the SBRA, 2020 (Virtual Meeting).

Objective:Human embryos are affected by a high rate of aneuploidy, around 35% in natural conceptions and this prevalence is expected to be higher in pre-implantation embryos derived from in vitro fertilization (IVF). One of the most common alteration is the trisomy and preimplantation genetic testing for aneuploidies (PGT-A) is currently used in IVF to minimize the chances of transferring genetically abnormal embryos. The introduction of time-lapse monitoring (TLM) allowed additional noninvasive criteria to best embryo selection. Markers of embryonic kinetic from the first-cleavage onwards has been studied and applied in algorithms for best embryo choice. The aim of this study was to analyze the association of trisomy with the embryo morphokinetics in embryos derived from IVF and analyzed by PGT-A.Methods:This retrospective study included 509 embryos from 109 patients undergoing IVF between April/2018-September/2019. Mature oocytes were fertilized by ICSI and cultured in a TLM system (Embryoscope®). Blastocysts were biopsied for PGT-A and analyzed at Igenomix Brasil through Next Generation Sequencing (NGS). Twenty-seven embryos presenting trisomy of chromosomes 13, 15, 16, 18, 21 and 22 were selected and a matched analysis was performed, since a euploid embryo from the same patient was selected as a control for each trisomic embryo.Results:The women age was 38.0±3.0 years and euploid blastocyst rate was 36.3%. Two-way repeated measured ANOVA was performed based on times for achieving pronucleous-fade, two-cells, three-cells, four-cells, five-cells, eight-cells, nine-cells and blastocyst. Embryo morphokinetics significantly differed between euploid and trisomic embryos (p=0.052). The pairwise comparisons demonstrated significant difference on times between euploid and trisomic embryos from eight cells until blastocyst stage. The mean difference time between euploid and trisomic embryos to get the eight-cells stage was 5.1 hours (61.0±10.8x55.9±7.1, p=0.033) and to attain blastocyst stage was 6.2 hours (112.3±11.7x106.1±10.4, p=0.013).Conclusion:In this study, trisomic embryos showed a faster development than their paired euploid ones. Despite of this study have included a small number of embryos, the inclusion criteria was for each trisomic embryo there is an euploid embryo of the same patient, which exclude a number of confounders. The literature describes the association of accelerated embryonic development with lower success potential; however, the reason for that relationship is not clear. Our findings allow us to hypothesize that the decreased implantation potential of faster embryos could be due to chromosomal alteration, specifically trisomy, and to suggest that accelerated embryonic development may be an indication for genetic evaluation. These findings must be confirmed in a higher number of embryos and further studies are being conducted to investigate the association of genetic embryo alterations and embryo development.

Chromosomal segregation in sperm of the Robertsonian translocation (21;22) carrier and its impact on IVF outcome.

To assess the variability of meiotic segregation patterns in sperm of Robertsonian translocation (RobT) carrier t(21;22) and present effect on reproductive outcome. Infertile couple enrolled in IVF/ICSI program. Sperm chromosomal segregation analysis was done using FISH; preimplantation genetic testing for aneuploids (PGT-A) was performed by NGS. Patients had a low fertilization rate and a negative outcome after the first IVF/ICSI cycle, so they were advised to do chromosomal aberration analysis before their next attempt. The second IVF/ICSI procedure resulted in pregnancy, and two blastocysts were cryopreserved. The NIFTY test has shown low risk for all tested trisomies, sex chromosomes aneuploidis, and deletion syndromes, so a healthy female child was born. During pregnancy, karyotypisation results revealed that the male partner is a RobT carrier t(21;22). Sperm segregation analysis of chromosomes 21 and 22 has shown six types of sperm chromosome sets. The majority of sperm cells had a normal/balanced RobT form of a haploid set of chromosomes (68.5-76%) called an "alternate." Sperm cells that had additional chromosome 21 or 22, or lack of chromosome 21 or 22, were present in 4-12%. PGT-A performed on two cryopreserved blastocysts revealed one embryo euploid and the other with the mosaic aneuploidy of chromosome 7 present in 50% of the cells. Infertile couples with a RobT male carrier who have semen comprising of normal/alternate form in the majority have a good prognosis of IVF/ICSI outcome. PGT is recommended because of the possible occurrence of viable trisomic embryos and potential interchromosomal effect.

PGT-A and RCT proof in AMA and SMF couples

Aneuploidy is the most common genetic abnormality in human embryos. Preimplantation genetic diagnosis for aneuploidy (PGT-A) can increase pregnancy rates in infertile couples requiring assisted reproductive technology (ART). Large datasets from testing preimplantation embryos demonstrate that over half of embryos produced by in vitro fertilization (IVF) are aneuploid. Conventional embryo morphology evaluation as well as morphokinetics can not properly discriminate euploid from aneuploid embryos. Particularly, trisomic embryos that behave very similarly to euploid embryos. For these reasons, PGT-A started to be applied for several indications. In the present talk the results of two randomized trials (RCTs) in advanced maternal age (AMA) and severe male factor (SMF) will be discussed. In both studies, livebirths/ongoing pregnancy rates were compared among patients with blastocyst transfer as a control group and a study group in which day-3 biopsy was performed, and array CGH was applied for 24-chromosome analysis, with the transfer of euploid blastocysts. The final results of the AMA study conducted in women between 38 and 41 years of age included a total of 205 cycles. The PGT-A group exhibited significantly fewer embryo transfers (68.0%, vs. 90.5% for control; p=0.0001), and lower miscarriage rates (2.7%, vs. 39.0% for control; p=0.0007). Delivery rate after the first transfer attempt was significantly higher in the PGT-A group per transfer (52.9% vs. 24.2%; p=0.0002), and per patient (36.0% vs. 21.9%; p=0.0309). No significant differences were observed in the cumulative delivery rates per patient six months after closing the study. However, the mean number of embryo transfers needed per live birth was lower in the PGD-A group compared to control group (1.8 vs. 3.7), as was the time to pregnancy (7.7 vs. 14.9 weeks). The interim analysis of SMF study was performed when 101 cycles were completed and included women We can conclude that the benefits of testing embryos for common chromosomal abnormalities, include: increased ongoing implantation and pregnancy rates per transfer, decreased miscarriage rates per patient, and faster time to pregnancy when compared to conventional embryo scoring by morphology alone. Also, we concluded that PGT-A with the new approaches with throphectoderm biopsy and Next Generation Sequencing (NGS) the cost per treatment can decrease, being like a conventional cycle if we consider the endpoint of a healthy livebirth per cycle.

Risk factors for Down syndrome.

Down syndrome (DS) originates, in most of the cases (95%), from a full trisomy of chromosome 21. The remaining cases are due to either mosaicism for chromosome 21 or the inheritance of a structural rearrangement leading to partial trisomy of the majority of its content. Full trisomy 21 and mosaicism are not inherited, but originate from errors in cell divisions during the development of the egg, sperm or embryo. In addition, full trisomy for chromosome 21 should be further divided into cases of maternal origin, the majority, and cases of paternal origin, less than 10%. Among cases of maternal origin, a further stratification should be performed into errors that have occurred or originated during the first meiotic division in the maternal grandmother's body and errors that occurred later in life during the second maternal meiotic division. This complex scenario suggests that our understanding of the risk factors for trisomy 21 should take into account the above stratification as it reflects different individuals and generations in which the first error has occurred. Unfortunately, most of the available literature is focused on maternal risk factors, and the only certain risk factors for the birth of a child with DS are advanced maternal age at conception and recombination errors, even though the molecular mechanisms leading to chromosome 21 nondisjunction are still a matter of debate. This article critically reviews the hypotheses and the risk factors which have been suggested to contribute to the birth of a child with DS, including folate metabolism, dietary, lifestyle, environmental, occupational, genetic and epigenetic factors, with focus on maternal and paternal risk factors, and taking into account the possible contribution of the maternal grandmother and that of the developing trisomic embryo, in a complex scenario depicting the birth of a child with DS as the result of complex gene-environment interactions and selection processes involving different generations.

Transmission of trisomy decreases with maternal age in mouse models of Down syndrome, mirroring a phenomenon in human Down syndrome mothers.

BackgroundDown syndrome incidence in humans increases dramatically with maternal age. This is mainly the result of increased meiotic errors, but factors such as differences in abortion rate may play a role as well. Since the meiotic error rate increases almost exponentially after a certain age, its contribution to the overall incidence aneuploidy may mask the contribution of other processes.ResultsTo focus on such selection mechanisms we investigated transmission in trisomic females, using data from mouse models and from Down syndrome humans. In trisomic females the a-priori probability for trisomy is independent of meiotic errors and thus approximately constant in the early embryo. Despite this, the rate of transmission of the extra chromosome decreases with age in females of the Ts65Dn and, as we show, for the Tc1 mouse models for Down syndrome. Evaluating progeny of 73 Tc1 births and 112 Ts65Dn births from females aged 130 days to 250 days old showed that both models exhibit a 3-fold reduction of the probability to transmit the trisomy with increased maternal ageing. This is concurrent with a 2-fold reduction of litter size with maternal ageing. Furthermore, analysis of previously reported 30 births in Down syndrome women shows a similar tendency with an almost three fold reduction in the probability to have a Down syndrome child between a 20 and 30 years old Down syndrome woman.ConclusionsIn the two types of mice models for Down syndrome that were used for this study, and in human Down syndrome, older females have significantly lower probability to transmit the trisomy to the offspring. Our findings, taken together with previous reports of decreased supportive environment of the older uterus, add support to the notion that an older uterus negatively selects the less fit trisomic embryos.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-016-0412-3) contains supplementary material, which is available to authorized users.

Abnormal mineralization of the Ts65Dn Down syndrome mouse appendicular skeleton begins during embryonic development in a Dyrk1a-independent manner

The relationship between gene dosage imbalance and phenotypes associated with Trisomy 21, including the etiology of abnormal bone phenotypes linked to Down syndrome (DS), is not well understood. The Ts65Dn mouse model for DS exhibits appendicular skeletal defects during adolescence and adulthood but the developmental and genetic origin of these phenotypes remains unclear. It is hypothesized that the postnatal Ts65Dn skeletal phenotype originates during embryonic development and results from an increased Dyrk1a gene copy number, a gene hypothesized to play a critical role in many DS phenotypes. Ts65Dn embryos exhibit a lower percent bone volume in the E17.5 femur when compared to euploid embryos. Concomitant with gene copy number, qPCR analysis revealed a ~1.5 fold increase in Dyrk1a transcript levels in the Ts65Dn E17.5 embryonic femur as compared to euploid. Returning Dyrk1a copy number to euploid levels in Ts65Dn, Dyrk1a+/− embryos did not correct the trisomic skeletal phenotype but did return Dyrk1a gene transcript levels to normal. The size and protein expression patterns of the cartilage template during embryonic bone development appear to be unaffected at E14.5 and E17.5 in trisomic embryos. Taken together, these data suggest that the dosage imbalance of genes other than Dyrk1a is involved in the development of the prenatal bone phenotype in Ts65Dn embryos.

Non‐trisomic homeobox gene expression during craniofacial development in the Ts65Dn mouse model of Down syndrome

Trisomy 21 in humans causes cognitive impairment, craniofacial dysmorphology, and heart defects collectively referred to as Down syndrome. Yet, the pathophysiology of these phenotypes is not well understood. Craniofacial alterations may lead to complications in breathing, eating, and communication. Ts65Dn mice exhibit craniofacial alterations that model Down syndrome including a small mandible. We show that Ts65Dn embryos at 13.5 days gestation (E13.5) have a smaller mandibular precursor but a normal sized tongue as compared to euploid embryos, suggesting a relative instead of actual macroglossia originates during development. Neurological tissues were also altered in E13.5 trisomic embryos. Our array analysis found 155 differentially expressed non-trisomic genes in the trisomic E13.5 mandible, including 20 genes containing a homeobox DNA binding domain. Additionally, Sox9, important in skeletal formation and cell proliferation, was upregulated in Ts65Dn mandible precursors. Our results suggest trisomy causes altered expression of non-trisomic genes in development leading to structural changes associated with DS. Identification of genetic pathways disrupted by trisomy is an important step in proposing rational therapies at relevant time points to ameliorate craniofacial abnormalities in DS and other congenital disorders.

Analysis of overlapping phenotypes from trisomic and euploid embryos of a Down syndrome mouse model (Ts65Dn): implications for human medicine

Down syndrome (DS), i.e. trisomy of Hsa21, exhibits nearly 80 clinically defined traits with varying phenotypic severity. Recent understanding of the genetic basis of DS phenotype has come through the use of mouse models of DS. The trisomic Ts65Dn chromosome seems to be inherited in a Mendelian ration but both trisomic and euploid offspring are lost in pre and postnatal stages and at weaning the frequency of trisomy is significantly lower than Mendelian ratio. The phenotytpic variability of euploid and trisomic embryos has not been analyzed in detail. Our current study suggests that euploid and trisomic mice embryos from a trisomic mother share some common adverse phenotypes in mouse: we investigated phenotypic variability in embryonic day 13.5 offspring from Ts65Dn mice and the euploid embryos reveal a milder version of the trisomic phenotype, but we detected anomalies in cardiac, pharyngeal as well as in neural features along with some specific malformations in embryonic liver as well as developing pituitary in trisomic as well as euploid embryos. Unfortunately there are no detailed published accounts about fine‐tuned analyses of developmental delay, intellectual impairment, muscle hypotonia and other phenotypic anomalies in human euploids born from trisomic mothers; our study therefore provides new insight in this arena and paves the way for future studies on this complex, crucial, and understudied aspect of DS.Grant Funding Source: N/A

Fish reanalysis of inner cell mass and trophectoderm samples of previously array-CGH screened blastocysts reveals high accuracy of diagnosis and no sign of mosaicism or preferential allocation

To study the blastocyst genetic constitution (possible chromosomal mosaicism between ICM and TE and within TE), after isolating ICM by a novel method.and to evaluate array-CGH accuracy against FISH testing Cross-sectional study. 32 blastocysts diagnosed as genetically abnormal by array-CGH were donated to research. ICM was isolated from the trophectoderm (TE) using a novel mechanical separation which was confirmed by KRT18 immunostaining. 24 of the 32 embryo had single or double aneuploidy and 8 aneusomic for unbalanced product of translocation or for long single arm deletions/duplications but with normal ploidy (by aCGH). After biopsy, individual samples were fixed by Carnoy method and analyzed with conventional 9-chromosomes FISH and locus specific probes. We reanalyzed 32 ICM and 96 TE samples (3 separate groups of cells from each TE of the same embryo). Looking at the single chromosome level, 28 out of 28 aneuploidies diagnosed by a-CGH were confirmed by FISH reanalysis both in ICM and TE samples resulting in a analytical sensitivity of 100%(95%CI 90.2-100). For almost all embryos, the reported aneuploidies were constitutive. As regard to chromosomes diagnosed as disomic, only 2 out of 248 (specificity 99.1 95%CI 97.4-99.8) were misdiagnosed by a-CGH, a trisomy 13 in an unbalanced embryo from reciprocal translocation carrier observed in a constitutive state and a mosaic nullisomy/monosomy 16 in a double aneuploid embryo. Consequently, only one out of 32 embryos was misdiagnosed as false negative. Only in one case we observed a confined trisomy 17 in all nuclei of a TE sample from a trisomic embryo for chromosome 13. In all other embryos ICM and TE samples were concordant and confirmed the original a-CGH diagnosis. Our data show a very high analytical accuracy of a-CGH to diagnose embryo chromosomal constitution. Concordance between ICM and TE was reported for all embryos analyzed, showing no sign of mosaicism or preferential allocation at blastocyst stage.

Sonographic markers of aneuploidies at 6–10 weeks of gestation

Objectives To determine if sonographic features of live embryos at 6–10 weeks' gestation are altered in aneuploidies. Methods Embryonic crown rump length (CRL), embryonic heart rate, gestational sac diameter (GSD) and yolk sac diameter (YSD) were measured by transvaginal sonography in 5603 live embryos from singleton pregnancies at 6–10 weeks' gestation. The measurements were expressed as differences from the expected normal mean for CRL (delta values) and median delta values in the aneuploid cases were compared to the euploid group. Results 5393 pregnancies resulted in the live birth of phenotypically normal neonates and these cases constituted the euploid group. In 55 cases there was subsequent prenatal diagnosis of aneuploidies (trisomy 21, n = 28; trisomy 18, n = 10; trisomy 13, n = 10; triploidy, n = 4; Turner syndrome, n = 3). The median and interquartile range (IQR) of delta embryonic heart rate in trisomy 18 was − 19.44 (− 23.77 to − 7.20) bpm and in trisomy 13 it was 11.12 (7.25 to 20.39) bpm, which were significantly lower and higher, respectively, than in euploid embryos (median − 0.05, IQR − 6.18 to 6.21 bpm). The median delta YSD in trisomy 21 was higher than in euploid fetuses (median 0.56, IQR 0.23 to 0.79 and median − 0.17, IQR − 3.11 to 2.82 mm). There were no other significant differences in measurements between the groups. Conclusion At 6–10 weeks' gestation there are sonographically detectable differences between euploid and trisomic embryos.

Cardiovascular Development and Survival During Gestation in the Ts65Dn Mouse Model for Down Syndrome

The Ts65Dn mouse model for Down syndrome (DS) exhibits many phenotypes seen in human DS. Previous research has revealed a reduced rate of transmission of the T65Dn marker chromosome in neonates. To analyze potential fetal loss, litters from trisomic females at 10.5dpc through 14.5dpc were genotyped. No significant differences from the expected Mendelian ratio were found in transmission of T65Dn at any stage. Cardiovascular defects found in trisomic neonates are associated with formation of pharyngeal arch arteries. Vessel tracing was used to identify anomalies in 10.5dpc, 11.5dpc, and 13.5dpc embryos. Comparison of trisomic versus euploid embryos injected with India ink revealed delay and abnormality in cardiovascular development in trisomic embryos at each stage. Through the analysis of transmission rate and cardiovascular development in embryonic mice, we learn more about prenatal mortality and the origins of cardiac abnormality in the Ts65Dn mice to assist in understanding cardiovascular malformation associated with DS.

Preimplantation aneuploid embryos undergo self-correction in correlation with their developmental potential

To investigate the incidence of embryos' self-correction during preimplantation development in terms of mosaicism and in correlation with its developmental stage. Prospective study to compare the chromosome status of embryos on day 3 with that of day 5, in correlation with their developmental stage. In vitro fertilization unit of a university-affiliated hospital. Eighty-three aneuploid embryos. Fluorescence in situ hybridization (FISH) reanalysis. Day 3 embryos classified as mosaic or chromosomally abnormal by preimplantation genetic screening (PGS) were reanalyzed on day 5. The results were evaluated in correlation with the embryos' developmental stage. Out of 83 day 3 aneuploid embryos, 15 (18.1%) were diagnosed with mosaicism. The FISH reanalysis on day 5 demonstrated that 27 embryos (32.6%) were partly or entirely normal disomic. Of these 83 aneuploid embryos, 8 (9.7%) underwent complete self-correction. The PGS results demonstrated that 26.5% of the embryos were trisomic, of which 41.0% underwent trisomic rescue by day 5. Self-correction was in correlation with the embryo's developmental stage, i.e., 38.1% of aneuploid embryos that developed to the blastocyst stage underwent self-correction compared with only 12.5% of embryos that only cleaved after biopsy. Our results demonstrate that self-correction of aneuploid and mosaic embryos occurs probably more significantly during development toward the blastocyst stage than in delayed embryos. In addition, trisomic embryos correct themselves more than other aneuploidies. These findings suggest that PGS results must be interpreted with caution.

Preimplantation aneuploid embryos undergo self-correction in correlation to their developmental potential

OBJECTIVE: Morphological assessment of the preimplantation embryo is used to select top-quality embryos with the best implantation potential. Yet, only 60-70% of day 3 good-quality embryos are euploid as demonstrated by FISH analysis. It is not clear, however, whether the chromosomal constitution of the day 3 embryo, as diagnosed by PGS, truly represents the chromosomal constitution of the implanted fetus. The objective of this study is to investigate the incidence of self-correction of embryos during preimplantation development in terms of mosaicism and in correlation with its developmental stage. DESIGN: A prospective study to compare the chromosome status of embryos on day 3 with that of day 5, in correlation to their developmental stage. MATERIALS AND METHODS: A total of 83 embryos classified by PGS as chromosomally abnormal were re-analyzed on day 5 in two rounds of hybridization (probes for chromosomes 13, 15, 16, 17, 18, 21, 22, X, Y). Correction and mosaicism rate were defined for each embryo in correlation to its developmental stage at day 5. RESULTS: Two blastomeres were subjected to PGS in 80% of the embryos. Of these, 18.1% were diagnosed with mosaics between blastomeres. FISH reanalysis on day 5 was performed on an average of 8.9±6.4 blastomeres per embryo and demonstrated that 32.6% of the embryos were partly or entirely normal disomic. PGS results demonstrated that 26.5% of the embryos were trisomic, of which 41.0% showed disomic cells by day 5, presumably by trisomic rescue, a mechanism of self correction. The relatively high percent of self-correction was in correlation with the embryo's developmental stage at day 5; i.e.38.1% of aneuploid embryos that developed to the blastocyst stage underwent self-correction as compared to only 12.5% of embryos that merely cleaved following biopsy. CONCLUSIONS: Our results demonstrate that self-correction of aneuploid and mosaic preimplantation embryos, occurs probably more significantly during development towards the blastocyst stage than in delayed embryos. In addition, trisomic embryos correct themselves more than other aneuploidies. These findings suggest that PGS results must be interpreted with caution.

Determination of the genetic status of cleavage‐stage human embryos by microsatellite marker analysis following multiple displacement amplification

To analyse genotype information from cleavage-stage human embryos and assess the chromosomal status and feasibility of performing aneuploidy screening by microsatellite analysis. DNA from 49 blastomeres from eight cleavage-stage human embryos was amplified using multiple displacement amplification, then tested for panels of 64 polymorphic microsatellite markers on seven different chromosomes, and for two non-polymorphic sequences on the X and Y chromosomes. There was an overall allele drop out (ADO) rate of 28%. Novel alleles in single cells were seen in 0.3% of amplifications, interpreted as either somatic microsatellite mutation events or 'slippage' of the MDA phi 29 polymerase. Three-allele results for a single marker in a single cell were found in 0.07% of amplifications, interpreted as 'slippage' of the MDA phi 29 polymerase. One apparent segmental duplication was found. Only one embryo with no normal cells was found, probably arising from the chaotic cleavage division following a triploid conception. Six embryos were mosaic, of which four had only one abnormal cell. Abnormalities in human embryos may be present in only a single cell, leading to potentially false abnormal results at pre-implantation genetic diagnosis. ADO associated with MDA reduces the efficacy of this approach for detection of aneuploidy. Statistical analysis showed that, for ADO of 28%, seven informative markers would be required to give 95% confidence of detecting trisomic embryos.