Incidence Of Mosaicism Research Articles

Effect of freeze-thawing, cell collection, and laser irradiation cycles on mosaicism occurrence in preimplantation genetic testing for aneuploidy

ObjectiveIn preimplantation genetic testing for aneuploidy, opinions regarding the handling of mosaic embryos vary. In this study, we aimed to investigate the effects of freeze-thawing, the number of cells obtained, and the number of laser irradiation cycles on the degree of embryonic mosaicism. Study designThis study was conducted in three parts. First, we classified specimens into the normal biopsy (control) (119 patients, 304 blastocysts) and thawed-biopsy (TB group) (26 patients, 72 blastocysts)) groups. The control and TB groups were then classified into three categories (euploidy, mosaic and aneuploidy) according to next-generation sequencing (NGS) results, and the number of cells collected and laser irradiation cycles were compared for each category. Subsequently, the effects of differences in the number of cells collected and laser irradiation cycles on NGS results were investigated in the control and TB groups. Finally, data on cell collection and laser irradiation cycles and NGS analysis results for the groups were compared. ResultsThe TB group had a significantly higher incidence of chromosomal mosaicism than the control group. Neither the number of cells collected nor the laser irradiation cycles affected the percentage of chromosomal mosaicism. However, the freeze–thaw process increased the occurrence of mosaicism. ConclusionsThis study showed that repeated freeze–thaw cycles increase the incidence of mosaicism, but the embryos are not aneuploid and are therefore suitable for transfer.

O-155 Limited utility of mosaicism reporting in PGT-A: independent confirmation from a European multisite, double-blinded analysis including 4293 embryo transfers following the first US experience

Abstract Study question Are results showing limited clinical utility of mosaicism reporting in PGT from a blinded mosaic embryo transfer American study, confirmed in a European setting? Summary answer Even between different patient populations and clinical settings, putative mosaicism reporting has limited clinical utility for embryo selection when co-evaluated with other clinical/embryological factors. What is known already Intermediate chromosomal copy number (ICN) results in NGS-based PGT-A are commonly interpreted as embryo mosaicism. A recent prospective, non-selection study in America showed that reporting mosaicism would not significantly improve embryo selection. At odds with this, results suggesting that embryo selection based on putative mosaicism can improve clinical outcomes continue to be reported, possibly reflecting limitations in accurate mosaicism identification or retrospective analysis biases. This study replicates the design of previously reported American study, using the same PGT-A assay, examining the predictive value of whole-chromosome ICN (wICN) and segmental ICN (sICN) following concurrent copy-number and genotyping-PGT in a European setting. Study design, size, duration A multisite double-blinded study involving 14 European IVF clinics was conducted from April 2022 to July 2023, including 3365 patients and 4293 single embryo transfers. Embryos, reported as negative for uniform aneuploidies, including those with wICN or sICN, were chosen for transfer based solely on standard developmental and morphological evaluation. Primary outcome measure was live birth rate defined as any pregnancy reaching the 24th week of gestation (LBR). Participants/materials, setting, methods Trophectoderm biopsies were analysed using a validated targeted-NGS assay with approximately 5000 loci across the genome, providing both quantitative and genotyping information to support aneuploidy classification. Putative mosaicism was inferred from ICN deviations from the expected two copies for autosomes and partly supported by corresponding SNP B-allele frequency patterns. Related clinical and embryological variables were analysed in a multivariate analysis and a Random Forrest regression used to model the clinical utility of putative mosaicism. Main results and the role of chance Of the embryos transferred, 6.8% (293/4293) were wICN only; 6.2% (269/4293) were sICN only and 0.5% (23/4293) were a combination of wICN and sICN. The detected ICN ranged from 23%-84% with no overrepresentation of any individual chromosomes. The LBR of embryos in the control group (without ICN), sICN and wICN were 46.8% (1736/3708; 95%CI:45.2-48.4%), 36.4% (98/269; 95%CI:30.9-42.3%) and 39.2% (115/293; 95%CI:33.8-44.9%) respectively. The miscarriage rate for control, sICN and wICN were 12.9% (257/1993; 95%CI:11.5-14-4%), 19.7% (24/122; 95%CI:13.6-27-6%) and 16.7% (23/138; 95%CI:11.4-23.8%), respectively. Logistic regression (P = 0.05) showed that the presence of sICN (OR: 0.7; 95%CI: 0.54-0.9) and wICN (OR: 0.62; 95%CI: 0.36-1.07) were modestly but significantly associated with LBR along with other embryological and clinical factors including embryo morphology (OR: 0.89; 95%CI: 0.84-0.96), day of biopsy (OR: 0.65;95%CI: 0.56-0.75), previous ET failures (OR: 0.79; 95%CI: 0.71-0.88) that were more strongly associated. No significant effect was observed of any specific intermediate CN cut-off on the association with LBR. Random Forrest regression, considering all correlating variables, showed a LBR prediction (AUC) equal to 0.578 without putative mosaicism and AUC of 0.583 with putative mosaicism included, a negligible increase considering the low incidence of mosaicism in our clinical setting along with its modest predictive value. Limitations, reasons for caution Follow-up prenatal and post-natal data at this time was not available, hence conclusions about these outcomes wasn’t possible. Despite the overall large sample size, the analysis of specific effects at different ICN values lacked sufficient power. Furthermore, this study’s data is platform-specific and cannot be translated to other PGT-A assays. Wider implications of the findings Although putative mosaicism was associated with modestly impaired pregnancy rate, two independent studies now confirm that reporting mosaicism has limited impact when prospectively selecting embryos for transfer based on standard embryological parameters. Considering the established downsides, including potential embryo disposal, the practice of mosaicism reporting in PGT is questionable. Trial registration number Not applicable

A retrospective cohort study and review of the literature about germline mosaicism in Duchenne/Becker muscular dystrophy prenatal counseling: How to estimate the recurrence risk in clinical settings?

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are the most common inherited neuromuscular diseases. Following the identification of a pathogenic causative variant in the DMD gene of a proband, potential carriers can be informed of their risk of having offspring with the disease. Germline mosaicism is a variant that is confined to the gonads that can be transmitted to offspring and is usually reported when a non-carrier of a DMD pathogenic variant has two or more offspring carrying the variant in question. On average, one third of cases are the result of a de novo variant, and as DMD and BMD are prone to germline mosaicism, its inclusion in genetic counseling is mandatory. In this retrospective cohort study, we presented clinical data from an unpublished DMD/BMD cohort of 332 families with incidence of germline mosaicism in families with de novo transmission of 8.1%. This is also the first systematic literature review searching PubMed to provide an accurate assessment of the current literature on germline mosaicism in DMD and BMD, including 17 case reports and 20 original studies. The incidence of documented germline mosaicism in de novo event families ranged from 6.0 to 40%, with a mean of 8.3%. The estimated recurrence risk for mothers of a patient with a proven de novo causal variant ranged from 4.3 to 11%, with a mean of 5.8% for a male fetus. By providing an up-to-date and comprehensive overview of the literature, this review aims to improve our understanding of germline mosaicism in DMD and to promote the development of effective strategies and reliable data for occurrence risk assessment in genetic counseling of de novo event families.

Tackling mosaicism in gene edited livestock

The farming of livestock has a critical role to play in global nutritional security and poverty alleviation. To meet these goals through more efficient, environmentally sustainable and animal welfare focused means, gene editing technologies could be integrated into current breeding programs. A common issue with gene editing in livestock zygotes is the high incidence of genetic mosaicism. Genetic mosaicism, characterised by a single individual carrying distinct genotypes in different cell lineages, can lead to inconsistent presentation of a desired trait phenotypically, or the absence of the intended genotype in the animal’s germline. This review explores the present status of genetic mosaicism associated with CRISPR-Cas9 gene editing in cattle, sheep, and pigs, and identifies four areas for refinement; (1) the type of CRISPR-Cas9 genome editor used; (2) the CRISPR-Cas9 formats and timing of gene editing during embryonic development; (3) the method used to deliver the genome editor and (4) the genetic screening strategies applied. We also discuss alternatives to direct zygote gene editing, including surrogate sire technology and blastomere separation, which circumvent the production of mosaic offspring. By exploring these avenues for reducing mosaicism, gene editing protocols in livestock could become more efficient and effective, which will ultimately pave the way for traits to be introduced that improve animal welfare standards and help address gaps in the security of global nutrition access.

#347 : Factors Related to Mosaicism of Human Embryo: Conventional Statistics and Machine Learning Analysis

Background and Aims: Mosaicism arises from errors in the cell division process and can lead to cells containing a combination of normal and mutated genes. Mosaic embryos can produce healthy babies. However, it may be associated with a lower implantation and a higher miscarriage rate. Despite extensive research, the scientific understanding of the underlying causes of embryonic mosaicism remains limited, and conclusions remain subject to ongoing debate. Machine learning (ML) can capture complex relationships and automatically learn the most informative features, while conventional statistics (CS) are simple to understand. We aimed to apply both ML and CS to determine the critical factors that may be associated with mosaicism. Method: A retrospective analysis was conducted at the Infertility Department of Hung Vuong Hospital between 2018 and 2022 using medical data from 51 IVF couples. The study utilized both machine learning and conventional statistical methods to identify possible factors associated with mosaicism in human embryos. Results: Out of the 246 embryos studied, 84 (32%) were found to be mosaic. The performance of the classifiers in the machine learning analysis was relatively poor when using all features, with F1-scores ranging from 0.08 to 0.26. While feature selection helped to improve performance, the F1-scores remained low. On the other hand, the conventional statistical analysis revealed that patients with cryptozoospermia showed a positive association with mosaicism (OR 2.68, 95% CI 1.08 to 6.66, p=0.033). Conclusion: According to the machine learning algorithms, the current set of clinical features has no relation to the incidence of mosaicism. However, the conventional statistical analysis suggests that patients with cryptozoospermia may contribute to a higher risk of mosaicism. Further research is needed to fully understand the mechanisms underlying mosaicism in human embryos.

O-060 Single-cell DNA sequencing reveals that current bulk DNA sequencing methods lead to an underestimation of chromosomal mosaicism in human blastocysts

Abstract Study question What is the incidence of chromosomal mosaicism in trophectoderm and inner cell mass from good quality human blastocysts after NGS-based single-cell molecular karyotyping? Summary answer Single-cell analysis showed that almost all human blastocysts are mosaic, involving low level mosaicism and/or abnormal cells resulting from reciprocal error events. What is known already Chromosome segregation in early human embryos is considered to be error prone. While meiotic errors affect all cells within embryos, mitotic errors lead to chromosomal mosaicism, the presence of cytogenetically different cells within an embryo. The reported incidence of mosaicism in blastocysts after bulk DNA sequencing of multicellular trophectoderm (TE) biopsies performed for preimplantation genetic testing for aneuploidy (PGT-A) varies between 2-19%. However, bulk DNA analysis can only assess net average chromosome gains or losses with a detection limit of 20-30%, and it makes distinguishing true mosaicism from technical artefact problematic. Study design, size, duration Observational study in human good quality surplus embryos which were donated for research purposes (CCMO, NL82597.000.22). The embryos were thawed and cultured until the blastocyst stage. From the blastocysts with at least a morphology grade 3BB, the ICM was dissociated from the TE. From 55 embryos, both samples were successfully disaggregated into single cells and manually placed in 384 well plates. For the cytogenetic analysis, a validated method for single-cell molecular karyotyping was used (scKaryo-seq). Participants/materials, setting, methods Embryos with at least two cytogenetically different cells were considered mosaic. To investigate if bulk DNA sequencing would have detected this mosaicism, we performed an in-silico reanalysis on our single-cell data. For each embryo, TE cells with the same mitotic abnormality were quantified. Next, we determined the number of mosaic embryos in which these abnormalities were present in at least 20% of TE cells, as a cut-off for what PGT-A methods are expected to detect. Main results and the role of chance On average 42% of the total number of cells that could be isolated per embryo were successfully karyotyped, giving a total of 1057 karyotyped cells (522 normal, 535 abnormal) from 55 embryos. Six embryos (11%) were normal, four (7%) were uniformly abnormal and 45 (82%) were mosaic. From these, 14 (26%) embryos were aneuploid mosaic with cytogenetically different abnormal cells and 31 (56%) were diploid-aneuploid mosaic with normal and abnormal cells. Here cytogenetically different abnormal cells were also frequently observed, with products of reciprocal events detected in 20 embryos. The in-silico reanalysis was performed on 38 embryos with a mosaic TE. It predicted that only 11% of the mitotic abnormalities observed in TE cells through scKaryo-seq would have been detected by bulk DNA sequencing. In 19 out of 38 embryos at least one mitotic abnormality affected more than 20% of the TE cells, meaning that only 50% of the embryos would have been recognized as mosaic with bulk DNA analysis. Bulk DNA sequencing methods used for PGT-A lead to an underestimation of mosaicism, since low-level mosaicism that affects a few cells within an embryo and products of reciprocal events without a net gain or loss remain undetected. Limitations, reasons for caution Isolation and cytogenetic analysis of viable single cells at the blastocyst stage is technically challenging and only 42% of the cells per embryo could be successfully karyotyped. Furthermore, it is unknown to what extent in vitro culture conditions and the freezing-thawing process influenced our findings. Wider implications of the findings These single cell observations support the notion that mosaicism is a common biological phenomenon in human blastocysts. Any selective mechanism eliminating embryos or cells with chromosomal abnormalities is still not fully active at this stage. The underestimation of mosaicism by bulk DNA analysis has potential implications for current PGT-A practices Trial registration number not applicable

The correlation between morphological parameters and the incidence of de novo chromosomal abnormalities in 3238 biopsied blastocysts

PurposeThe aim of this study was to determine the relationship between morphological parameters and the incidence of de novo chromosomal abnormalities.MethodsThis was a retrospective cohort study of 652 patients who underwent 921 cycles with 3238 blastocysts biopsied. The embryo grades were evaluated according to Gardner and Schoolcraft’s system. The incidence of euploidy, whole chromosomal aneuploidy (W-aneuploidy), segmental chromosomal aneuploidy (S-aneuploidy), and mosaicism in trophectoderm (TE) cell biopsies was analyzed.ResultsThe euploidy decreased significantly with maternal age and was positively correlated biopsy day and morphological parameters. The W-aneuploidy increased significantly with maternal age and was negatively correlated biopsy day and morphological parameters. Parental age, TE biopsy day, and morphological parameters were not associated with S-aneuploidy and mosaicism, except that TE grade C blastocysts had significantly higher mosaicism than TE grade A blastocysts. Subanalysis in different female age groups showed that euploidy and W-aneuploidy had a significant correlation with TE biopsy day among women aged ≤ 30 y and 31–35 y, with expansion degree among women aged ≥ 36 y, with ICM grade among women aged ≥ 31 y, and with TE grade among all female age ranges.ConclusionFemale age, embryo developmental speed and blastocyst morphological parameters are associated with euploidy and whole chromosomal aneuploidy. The predictive value of these factors varies across female age groups. Parental age, embryo developmental speed, expansion degree, and ICM grade are not associated with the incidence of segmental aneuploidy or mosaicism, but TE grade seemingly has a weak correlation with segmental aneuploidy and mosaicism in embryos.

The embryo mosaicism profile of next-generation sequencing PGT-A in different clinical conditions and their associations.

Uniform chromosome abnormalities are commonly seen in early pregnancy loss, with analyses of the product of conception suggesting the presence of mosaic autosomal trisomy in ∼10% of cases. Although chromosomal mosaicism occurs in a minority of embryos, their relative commonality and uncertainty regarding associated transfer outcomes have created discussion at both the clinical and research levels, highlighting the need to understand the clinical conditions associated with the incidence of embryo mosaicism. We took advantage of a preimplantation genetic testing for aneuploidy (PGT-A) database created from 2019 to 2022 in more than 160 in vitro fertilization (IVF) clinics in Brazil, the second-largest world market for IVF. We carried out descriptive statistical and associative analyses to assess the proportions of mosaicism associated with clinical conditions and reported incidence by chromosome, clinic origin, and biopsy operator. Chromosomal analysis revealed that most mosaic aneuploidies occurred in the last three chromosomes, with 78.06% of cases having only one chromosome affected. Low mosaicism in trisomy represented the most ordinary form, followed by low mosaicism in monosomy. We identified associations between low (negatively-associated) and high mosaicism (positively-associated) and maternal age, indication (male factor and uterus/ovarian factor negatively associated with low and high mosaic, respectively), day of blastocyst development (day five has an overall better outcome), morphology grade (lower quality increased the chances of low and high mosaicism), origin (vitrified oocyte and embryo increased the rates of low and high mosaicism, respectively), and embryo sex (male embryos negatively associated with low mosaic). With these results, we hope to foster an improved understanding of the chromosomal mosaicism linked with distinct clinical conditions and their associations in Brazil.

UTILIZING MULTI-ETHNIC DISEASE MODELING TO UNDERSTAND THE BIOLOGICAL PROCESSES OF EMBRYONIC MOSAICISM

While disparities in Assisted Reproductive Technology outcomes have been correlated with race and ethnicity, it has not been determined whether this disparity extends to the incidence of embryo mosaicism. The aim of this study is to analyze the mosaic status of embryos using preimplantation genetic testing for aneuploidy (PGT-A) among various racial groups.

Application of machine learning to predict aneuploidy and mosaicism in embryos from in vitro fertilization cycles

BACKGROUNDThe factors associated with embryo aneuploidy have been extensively studied. Mostly maternal age and to a lesser extent male factor and ovarian stimulation have been related to the occurrence of chromosomal alterations in the embryo. On the other hand, the main factors that may increase the incidence of embryo mosaicism have not yet been established.OBJECTIVEThis study aimed to establish a machine learning model that would allow prediction of aneuploidies and mosaicism in embryos conceived via in vitro fertilization, and thus help to determine which variables are associated with these chromosomal alterations.STUDY DESIGNThe study design was observational and retrospective. A total of 6989 embryos from 2476 cycles of preimplantation genetic testing for aneuploidies were included (January 2013 to December 2020). The trophoectoderm biopsies on day-5, -6, or -7 blastocysts were analyzed by preimplantation genetic testing for aneuploidies (PGT-A). The different maternal, paternal, couple, embryo, and in vitro fertilization cycle characteristics were recorded in a database (22 predictor variables) from which predictive models of embryo aneuploidy and mosaicism were developed; 16 different unsupervised classification machine learning algorithms were used to establish the predictive models.RESULTSTwo different predictive models were performed: one for aneuploidy and the other for mosaicism. The predictor variable was of multiclass type because it included the segmental- and whole-chromosome alteration categories. The best predicting models for both aneuploidies and mosaicism were those obtained from the Random Forest algorithm. The area under ROC curve (AUC) value was 0.792 for the aneuploidy explanatory model and 0.776 for mosaicism. The most important variable in the final aneuploidy model was maternal age, followed by paternal and maternal karyotype and embryo quality. In the predictive model of mosaicism, the most important variable was the technique used in preimplantation genetic testing for aneuploidies and embryo quality, followed by maternal age and day of biopsy.CONCLUSIONIt is possible to predict embryo aneuploidy and mosaicism from certain characteristics of the patients and their embryos.

ESHRE survey results and good practice recommendations on managing chromosomal mosaicism.

How should ART/preimplantation genetic testing (PGT) centres manage the detection of chromosomal mosaicism following PGT? Thirty good practice recommendations were formulated that can be used by ART/PGT centres as a basis for their own policy with regards to the management of 'mosaic' embryos. The use of comprehensive chromosome screening technologies has provided a variety of data on the incidence of chromosomal mosaicism at the preimplantation stage of development and evidence is accumulating that clarifies the clinical outcomes after transfer of embryos with putative mosaic results, with regards to implantation, miscarriage and live birth rates, and neonatal outcomes. This document was developed according to a predefined methodology for ESHRE good practice recommendations. Recommendations are supported by data from the literature, a large survey evaluating current practice and published guidance documents. The literature search was performed using PubMed and focused on studies published between 2010 and 2022. The survey was performed through a web-based questionnaire distributed to members of the ESHRE special interest groups (SIG) Reproductive Genetics and Embryology, and the ESHRE PGT Consortium members. It included questions on ART and PGT, reporting, embryo transfer policy and follow-up of transfers. The final dataset represents 239 centres. The working group (WG) included 16 members with expertise on the ART/PGT process and chromosomal mosaicism. The recommendations for clinical practice were formulated based on the expert opinion of the WG, while taking into consideration the published data and results of the survey. Eighty percent of centres that biopsy three or more cells report mosaicism, even though only 66.9% of all centres have validated their technology and only 61.8% of these have validated specifically for the calling of chromosomal mosaicism. The criteria for designating mosaicism, reporting and transfer policies vary significantly across the centres replying to the survey. The WG formulated recommendations on how to manage the detection of chromosomal mosaicism in clinical practice, considering validation, risk assessment, designating and reporting mosaicism, embryo transfer policies, prenatal testing and follow-up. Guidance is also provided on the essential elements that should constitute the consent forms and the genetic report, and that should be covered in genetic counselling. As there are several unknowns in chromosomal mosaicism, it is recommended that PGT centres monitor emerging data on the topic and adapt or refine their policy whenever new insights are available from evidence. Rather than providing instant standardized advice, the recommendations should help ART/PGT centres in developing their own policy towards the management of putative mosaic embryos in clinical practice. This document will help facilitate a more knowledge-based approach for dealing with chromosomal mosaicism in different centres. In addition to recommendations for clinical practice, recommendations for future research were formulated. Following up on these will direct research towards existing research gaps with direct translation to clinical practice. Emerging data will help in improving guidance, and a more evidence-based approach of managing chromosomal mosaicism. The WG received technical support from ESHRE. M.D.R. participated in the EQA special advisory group, outside the submitted work, and is the chair of the PGT WG of the Belgian society for human genetics. D.W. declared receiving salary from Juno Genetics, UK. A.C. is an employee of Igenomix, Italy and C.R. is an employee of Igenomix, Spain. C.S. received a research grant from FWO, Belgium, not related to the submitted work. I.S. declared being a Co-founder of IVFvision Ltd, UK. J.R.V. declared patents related to 'Methods for haplotyping single-cells' and 'Haplotyping and copy number typing using polymorphic variant allelic frequencies', and being a board member of Preimplantation Genetic Diagnosis International Society (PGDIS) and International Society for Prenatal Diagnosis (ISPD). K.S. reported being Chair-elect of ESHRE. The other authors had nothing to disclose. This Good Practice Recommendations (GPR) document represents the views of ESHRE, which are the result of consensus between the relevant ESHRE stakeholders and are based on the scientific evidence available at the time of preparation. ESHRE GPRs should be used for information and educational purposes. They should not be interpreted as setting a standard of care or be deemed inclusive of all proper methods of care, or be exclusive of other methods of care reasonably directed to obtaining the same results. They do not replace the need for application of clinical judgement to each individual presentation, or variations based on locality and facility type. Furthermore, ESHRE GPRs do not constitute or imply the endorsement, or favouring, of any of the included technologies by ESHRE.

Regional and developmental characteristics of human embryo mosaicism revealed by single cell sequencing.

Chromosomal mosaicism is common throughout human pre- and post-implantation development. However, the incidence and characteristics of mosaicism in human blastocyst remain unclear. Concerns and confusions still exist regarding the interpretation of chromosomal mosaicism on preimplantation genetic testing for aneuploidy (PGT-A) results and embryo development. Here, we aimed to estimate the genetic concordance between trophectoderm (TE), inner cell mass (ICM) and the corresponding human embryonic stem cells (hESCs), and to explore the characteristics of mosaicism in human blastocyst and hESCs on a single cell level. The single cell sequencing results of TE cells indicated that 65.71% of the blastocysts were mosaic (23 in 35 embryos), while the ICM sequencing results suggested that 60.00% of the blastocysts were mosaic (9 in 15 embryos). The incidence of mosaicism for the corresponding hESCs was 33.33% (2 in 6 embryos). No significant difference was observed between the mosaic rate of TE and that of ICM. However, the mosaic rate of the corresponding hESCs was significantly lower than that of TE and ICM cells, suggesting that the incidence of mosaicism may decline during embryonic development. Upon single cell sequencing, we found several “complementary” copy number variations (CNVs) that were usually not revealed in clinical PGT-A which used multi-cell DNA sequencing (or array analysis). This indicates the potential diagnostic risk of PGT-A based multi-cell analysis routinely in clinical practice. This study provided new insights into the characteristics, and considerable influences, of mosaicism on human embryo development, as well as the clinical risks of PGT-A based on multi-cell biopsies and bulk DNA assays.

Chromosomal mosaicism in human blastocysts: a cytogenetic comparison of trophectoderm and inner cell mass after next-generation sequencing

Research questionWhat is the incidence of chromosomal mosaicism in human blastocysts and can a single trophectoderm (TE) biopsy accurately predict the chromosomal constitution of the inner cell mass (ICM)? DesignObservational study in 46 surplus cryopreserved preimplantation embryos of unknown chromosomal constitution. For each embryo, a TE biopsy was performed and the ICM was collected separately. Both samples underwent next-generation sequencing (NGS) for cytogenetic analysis and were classified as chromosomally normal, abnormal or mosaic. Mosaic samples were classified as low or high mosaic, based on the majority dominance of either normal or abnormal cells in the biopsied sample. Findings within each embryo were compared. ResultsChromosomal mosaicism was detected in 59% (n = 27/46) of the embryos, with a cytogenetic concordance rate between TE and corresponding ICM of 48% (n = 22/46). Concordance was higher from a clinical perspective: in 86% of embryos with a high-mosaic or abnormal TE, the ICM was also high-mosaic or abnormal. In 88% of the blastocysts with a normal or low-mosaic TE biopsy, a normal or low-mosaic ICM was observed. ConclusionDespite the low cytogenetic concordance rate due to chromosomal mosaicism present in blastocysts, it was found that a single TE biopsy could correctly predict whether the ICM consists of mostly normal or abnormal cells in the majority of cases.

SELECTION OF POTATO APICAL LEAF CURL VIRUS DISEASE-RESISTANT GENOTYPES, PEST SURVEILLANCE, SEED DEGENERATION, AND MANAGEMENT OF APHID IN FIELD CONDITIONS

Objectives: Under All India Co-ordinated Research Project on Potato, studies on the selection of potato apical leaf curl virus disease-resistant genotypes, pest surveillance, seed degeneration, and management of aphid in field conditions were conducted at Hisar during 2017–2018. Methods: Sowing of seed tubers was done at recommended time and spacing in every experiment followed by spraying of required insecticides. Observations on different aspects were recorded and statistically analyzed to present results in tabular format. Results and Discussion: Out of 100 germplasm, seven were resistant and one was moderately resistant to potato apical leaf curl New Delhi virus, all the local genotypes were susceptible to disease. There was infestation of aphids, jassids, thrips, and whitefly irrespective of cultivars. Severe mosaic incidence ranged from 63.6 to 93.2%. The breeder seed was superior as it resulted in the highest seed tuber emergence (98.94%) and the least incidence of leafroll disease (0.80%) followed by seed tuber of seed plot technique (SPT). The conventional seed performed the poorest in terms of emergence and exhibited the highest disease incidence. Aphid appeared in the 47th meteorological week and it approached the ET level in the 52nd week. Whitefly population was the highest in the 47th week which continuously declined. Jassid population was very mild and thrips appeared in the 52nd week. Flonicamid 50 WG caused higher aphid mortality than imidacloprid after 24 h of spray. The maximum and minimum temperatures varied from 16.9 to 35.7 and 2.6 to 16.2° C, respectively. The morning and evening relative humidity was in the range of 82–100 and 24–75%, respectively. The wind speed ranged from 1.0 to 3.6 KMPH and the sun was shined from 0.3 to 7.6 h. The results of this study are close conformity of earlier researchers. Conclusion: It is concluded that resistant genotypes, breeder seed, and seed produced through SPT are promising in managing the disease successfully. Flonicamid is a good alternative of imidacloprid. Aphid is a major pest that plays a key role in transmitting viral disease of this crop, its population touched ET level in the 52nd week.

Risk factors related to chromosomal mosaicism in human blastocysts

Research questionDo factors relating to patients, ART, or both, affect the incidence of chromosomal mosaicism in human blastocysts? DesignBlastocysts (n = 5718) from 1198 PGT-A cycles were biopsied between 2015 and 2021. All samples were amplified with multiple displacement amplification, and MiSeq system was used for next-generation sequencing. Mosaicism prevalence, and ART and patient factors potentially affecting mosaicism, were analysed. ResultsAmong 5718 blastocysts detected, 1245 were mosaic (21.8%). Day-6 biopsied blastocysts yielded a statistically significantly higher mosaicism rate than day-5 blastocysts (24.5% versus 19.1%; OR 1.174; 95% CI 1.017 to 1.355, P = 0.028). Mosaicism rate increased as morphological score declined (excellent quality [13.2%], good quality [19.0%], average quality [22.0%] and poor quality [26.4%], P < 0.001). Compared with excellent-quality embryos, the OR of mosaicism was 1.490 (95% CI 1.069 to 2.078, P = 0.019) for good-quality, 1.751 (95% CI 1.274 to 2.407, P = 0.001) for average-quality, and 2.113 (95% CI 1.512 to 2.952, P < 0.001) for poor-quality embryos. Biopsy technicians were related to the incidence of mosaicism. One of the four technicians had a significantly higher mosaicism rate than the others (27.9%; 20.9%; 20.5%; 20.5%, respectively; P < 0.001). Parental ages, female BMI, history of infertility, sperm quality, antral follicular counts, ovarian stimulation protocols, stimulation length, total gonadotrophin dosage and number of retrieved oocytes, were not related to the incidence of mosaicism. ConclusionSlow developing, poor-quality blastocysts are at higher risk of mosaicism. Biopsy technician may contribute to artificial mosaicism as an extrinsic factor.

Identification of Newer Insecticides and Plant Extracts against Whitefly for Integration in Disease Management Modules in Brinjal

Efficacy of eight insecticides along with five plant extracts were tested during rabi 2017–18 and kharif 2018–19 at Agricultural Research Station, Virinjipuram, Vellore, Tamil Nadu. Four applications of treatments viz., acetamiprid 20 SP @ 30 g ai ha−1, imidacloprid 17.8 SL @ 44.5 g ai ha−1, spiromesifen 240 SC @ 120 g ai ha−1, thiacloprid 240 SC @ 120 g ai ha−1, thiamethoxam 25 WG @ 50 g ai ha−1, diafenthiuron 50 WP @ 250 g ai ha−1, dimethoate 30 EC @ 300 g ai ha−1 and profenophos 50 EC @ 300 g ai ha−1, azadirachtin 10000 ppm @ 3.0 mL L−1, neem seed kernel extract 5%, the leaf extracts of neem, Bougainvillea and sorghum/coconut at 10% along with an untreated check at one month interval on 30 day old plants were given. The results showed that spraying of spiromesifen (3.0 Nos. per 3 leaves) was found to record the lowest population of whitefly throughout the crop period followed by thiacloprid (3.3 Nos. per 3 leaves) and thiamethoxam (3.4 Nos. per 3 leaves). The highest population reduction of 79.2, 77.6 and 76.8% was achieved in plots treated with spiromesifen, thiacloprid and thiamethoxam, respectively, over untreated check. With respect to viral disease, spraying of spiromesifen recorded the least incidence of mosaic (4.4%) followed by thiacloprid (5.2%), thiamethoxam (5.9%) and azadirachtin (6.2%). The plots treated with spiromesifen, thiacloprid and thiamethoxam recorded the highest fruit yield of 28.8, 28.0 and 27.2 t ha−1, respectively, with 36.6, 32.9 and 29.3% increase over untreated check and also the highest cost: benefit ratio.

Prenatal Diagnosis of a Lethal Skeletal Dysplasia.

Prenatal Diagnosis of a Lethal Skeletal Dysplasia.

Effect of Different Planting Material on Physiological Parameters and Cassava Mosaic Incidence in Cassava (Manihot esculenta Crantz.)

An investigation was carried out to assess the effect of planting materials on physiological parameters, cassava mosaic incidence and yield of Cassava varieties viz. H-226 and CO.2 at the Department of Vegetable crops, Horticultural College and Research Institute, TNAU, Coimbatore during September 2010 to June 2011. The experiments were laid out in Factorial Randomized Block Design with four treatments and three replications in two varieties. The results revealed that the tissue culture plants were found be the best in virtue of its high leaf area index, net assimilation rate, dry matter content, tuber yield (26.33 t ha-1) and least incidence of cassava mosaic virus. Among the varieties, CO.2 showed less incidence of CMD. The tissue cultured plants may be recommended to farmers to reduce mosaic disease incidence and get maximum yield in cassava.

SINGLE CELL DNA SEQUENCING OF WHOLE HUMAN BLASTOCYSTS: NOVEL INSIGHTS INTO THE PHENOMENON OF EMBRYONIC MOSAICISM

Conventional wisdom suggests that viable human embryos are the result of faithful cloning of euploid zygotes. However, study of blastocysts grown in vitro often identifies mosaicism that is likely the result of post-fertilization mitotic errors1. Yet, the true incidence of embryonic mosaicism remains poorly understood. Although prior work has studied cleavage stage embryos or blastocyst biopsies, to our knowledge, whole embryonic analysis via single cell sequencing of every cell from a human blastocyst has yet to be performed.

THE EFFECT OF INSEMINATION METHODS ON EMBRYO MOSAICISM IN PREIMPLANTATION GENETIC TESTING FOR ANEUPLOIDY (PGT-A) CYCLES

The objective of this study was to determine if conventional insemination vs. intracytoplasmic sperm injection (ICSI) results in a higher incidence of embryo mosaicism in IVF cycles with PGT-A. This exempt retrospective chart review was evaluated and approved by the Eastern Virginia Medical School Institutional Review Board. Cycle data were collected for 545 IVF cycles with PGT-A between July 2018 to October 2020 with one of the following methods of insemination: all ICSI (N = 369), all conventional insemination (IVF; N = 103), or a combination of both ICSI and IVF (COMBO; N = 73). A total of 2,672 embryos were biopsied from these cycles, and samples were sent to a single genetics facility for testing with next generation sequencing. Rates of euploid, aneuploid and mosaic embryos were compared between insemination methods by analyzing the aggregate data by two proportional z-tests. Secondary outcomes – including embryos fertilized, good day 3 development, total blastocyst development, mosaic embryo anomalies, clinical uterine gestation (CIG), and implantation rate – were analyzed by two proportional z-tests, and the Pearson chi-square test was used to compare outcomes for frozen embryo transfers (FET). Statistical analysis was performed using Intellectus Statistics software, and a p-value of less than 0.05 was defined as statistically significant, with a 95% confidence interval. Rates of mosaicism were similar between ICSI and conventional insemination when examining total aggregate data (11% vs. 11%), COMBO cycles, intra-cohort comparison, oocyte age groups, and normal and oligospermic semen samples. A significant difference was only found between embryos from testicular cycles and oligospermic cycles (17% vs. 9%, p=0.019). Occurrences of increased aneuploidy with ICSI, when compared to conventional insemination, were found from aggregate data (p=0.006), COMBO cycles (p<0.001), all IVF vs. IVF embryos from COMBO cycles (p=0.007), maternal age ≥38 (p=0.033), and in cycles with normal sperm concentration (p=0.001). However, FET outcomes, including pregnancy loss and implantation, did not differ between insemination methods. Within mosaic embryos, significantly higher percentages were seen of low-level compared to high-level (p=<0.001) and whole chromosome vs. segmental (p<0.001) mosaicism, but there were no differences found for the same comparisons by insemination type. Conventional insemination did not have a significant impact on levels of embryo mosaicism, and the only significant increase in mosaicism resulted from testicular samples. Although more research is needed, this study did observe a significant increase in aneuploidy from ICSI-derived embryos, which may suggest the use of ICSI preferably for cycles with male infertility only.