Low Mosaicism Research Articles

P-532 Preimplantation genetic testing versus morphology as selection criteria for single embryo transfer in women aged 35-42: results of a pilot randomized controlled trial

Abstract Study question To assess the feasibility of running a large randomised controlled trial (RCT) evaluating the benefit of preimplantation genetic testing for aneuploidy (PGT-A) for embryo selection. Summary answer Running a PGT-A RCT is feasible. Considerations include participant recruitment, seven-day biopsy-embryologist availability and logistical complexities. What is known already Critics question the effectiveness of PGT-A leading to concerns about false positives and negatives, unnecessary embryo discarding and clinical effectiveness. Past studies have been criticised for only performing biopsy on top quality embryos, their criteria for randomization and the age of the population being assessed. Given these uncertainties there is a need for a rigorous RCT evaluating the role of PGT-A using current methodologies and biopsy techniques in women most affected by aneuploidy. Our pilot study is the first study where all embryos generated will undergo biopsy and ranked in terms in implantation potential prior to transfer. Study design, size, duration In this prospective, concealed allocation two-arm parallel RCT, our goal was to randomise 100 women with ≥3 good quality embryos on day 3 post egg collection into two groups: embryos screened using next-generation sequencing (NGS)-based PGT-A or selection-based on morphology alone. PGT-A patients underwent a trophectoderm biopsy on day 5/6/7 following egg collection. Biopsied embryos were frozen and subsequently transferred. Euploid and embryos described as being low mosaic (<30% abnormal cells) were prioritised for transfer. Participants/materials, setting, methods Women aged between 35–42 years undergoing IVF±ICSI were included, with those undergoing PGT for inherited genetic disorders, gamete donation cycles, untreated hydrosalpinges or untreated uterine abnormalities excluded. Primary objective during the pilot phase was to assess the ability to recruit, randomise, adherence to the study protocol and plan for a full study. Secondary outcomes were clinical pregnancy rate per embryo transferred, clinical pregnancy rate per couple randomised, miscarriage rate and time to pregnancy. Main results and the role of chance Out of 247 eligible women,100 were randomised, and 94 received the assigned treatment. The recruitment rate was 70.18%(173/247, 95%CI 61.65%-78.70%). An average of 6 participants were randomised each month. All randomised participants completed the follow-up. Our main recruitment challenges included 19% of eligible patients choosing to have PGT-A privately and 6% withdrawing their consent choosing to have a fresh transfer. Lack of biopsy-embryologist also meant patients whose potential biopsy would have been on a weekend had to be excluded in the first 4 months of our recruitment process. Baseline characteristics including age, BMI and cause of infertility were comparable in both arms and a total of 56 euploid, 53 aneuploid and 3 low mosaic embryos were identified. There was no statistically difference in CPR (23/50 vs 21/50, RR 1.09 95%CI:0.83-1.15, p-value>0.23) and miscarriage rate (3/50 vs 2/50, RR 1.5 95%CI:0.84-1.75, p-value: 0.15) per single embryo transfer or per woman randomised (23/46 vs 21/48, RR 1.14 95%CI 0.78-1.27, p-value:0.24 and 3/46 vs 2/48, RR 0.72 95%CI 0.58-1.17 p-value: 0.17) in either arm of the study. We cannot yet confirm the clinical benefit of PGT-A owing to the insufficient sample size in this pilot study. No low mosaic embryos were transferred. Limitations, reasons for caution The study was conducted in a single centre. If future definitive trials are conducted in multiple centres, the criteria for recruitment, randomisation, intervention, and follow-up still need to be unified. Many women aged above 40 failed to reach randomization due to challenges in producing three good-quality embryos. Wider implications of the findings The pilot study found that the eligibility rate is adequate however researchers should consider reviewing the conditions for randomisation in main study such as reducing the number of good embryos required to two. In addition, stratified randomization should be used in order to better balance female age within each group. Trial registration number NCT05009745

P-451 Improvement and validation of a cost effective PGT-A test for the detection of aneuploidy, triploidy and mosaicism

Abstract Study question This study aims to validate a PGT-A platform to simultaneously sequence and genotype embryos to detect triploidy as well as high and low frequency mosaicism. Summary answer Previda Plus platform validation establishes the capability to concurrently identify aneuploidy, triploidy and mosaicism with high and low frequencies at a significantly reduced cost. What is known already Low pass sequencing platforms can identify with great accuracy monosomy and trisomy karyotypes from embryo biopsies by comparing DNA quantities across chromosomes, however, these platforms exhibit limitations in identifying triploidy since all chromosomes exhibit uniform concentration. Triploidy detection necessitates genotyping involving the detection of highly variable SNPs to distinguish different haplotypes. Intermediate copy number may represent putative mosaicism. Recent publications show that embryos with low-level mosaicism demonstrate comparable implantation rates to euploid embryos, therefore validating cut offs to differentiate between low-level and high-level mosaics from euploid and aneuploid embryos is necessary. Study design, size, duration To validate triploidy detection, we measured allele frequencies (AF) using a high frequency SNP panel. The SNP frequency profile of a diploid exhibits a mix of AF of 100% and 50%, while a triploid exhibits AF of 100%, 66%, and 33%. To validate mosaicism, cell lines that were diploid, trisomic, or monosomic for a specific chromosome were mixed in pairs in increments of 10% (From 0%:100% line-1:line-2, to 100%:0%) with multiple iterations of each combination. Participants/materials, setting, methods Fifteen cell lines (triploid or diploid) underwent amplification using a 384 highly variable SNP panel. Pooled SNP and PGT-A products underwent processing in a single sequencing workflow. Ploidy Scores (PS) were derived from the total number of SNPs, 66% and 33%, and cutoff values were established to discriminate between diploid and triploid. For mosaicism validation, thirteen different trisomic cell lines were mixed in 10% ratio increments with 15 different monosomic cell lines. Main results and the role of chance Amplified samples exhibited distinct separation in ploidy scores between diploidy (PS of 0.91 to 1.85), and triploidy samples (PS of 0.28 to 0.61) with no overlap of boxplots. Specificity, sensitivity, and accuracy were 100%. For mosaicism validation, mixing different ratios of cell lines featuring monosomy and cell lines for a trisomy for different chromosomes resulted in ratios of monosomy: disomy for one chromosome and trisomy: disomy for the other. We assessed 32 combinations of cell lines, each one in 10% increments of each cell line from 0:100% to 100%:0%. Mixes with 40% trisomy mosaicism exhibited a 2.35 median ploidy, while 50%, 60%, 70% and 80% mixes show 2.45, 2.50, 2.60, and 2.70, respectively. Mixes with 40% monosomy mosaicism exhibited a median ploidy of 1.6, while 50%, 60%, 70% and 80% mixes showed 1.50, 1.45, 1.35, and 1.25, respectively. The literature recommends considering <40% mosaicism as euploid and 80-100% as aneuploid, therefore, ploidy scores below 2.35 are deemed euploid, ploidy scores at or above 2.35, but below 2.6 (40%-60%), are classified as low level mosaic, and ploidy scores equal to or above 2.6, but below 2.7, are classified as high mosaic. Embryos equal to or above 2.8 (80%+) are considered aneuploid. Limitations, reasons for caution This test is not designed to screen for tetraploidy (92 XXYY) and has not undergone validation for it nor haploidy. Validation procedures solely utilized cell lines. Reanalysis of the remainder of embryos deemed triploid or high-rate mosaic using this validation results are currently underway. Wider implications of the findings This Previda Plus platform combined with proprietary chemistry provides a rapid, cost effective, and a streamlined workflow for aneuploidy, triploidy, and mosaicism for use in PGT-A of blastocysts biopsies. Trial registration number not applicable

O-154 Chromosomal, gestational, and neonatal outcomes of mosaic embryos: analysis of 3074 cases from the international registry of mosaic embryo

Abstract Study question Which are the clinical outcomes and post-natal results of mosaic embryos with low and high-level of mosaicism? Summary answer The level of mosaicism negatively influences implantation and ongoing pregnancy rate. 6/7 cases in which mosaicism persisted in the foetuses were from low-level mosaic. What is known already Chromosomal mosaic embryos are characterized by the presence of chromosomally different cell lines within the same embryo. While the transfer of these embryos is now offered as an option for women who undergo in vitro fertilization (IVF), several concerns remain. For instance, the limited data on pregnancy outcome and the possibility that intra-biopsy mosaicism in the TE is a poor predictor of the ploidy status of the ICM. Therefore, some argue that mosaicism should be not reported until a clear classification of such embryos in relation to their reproductive potential has been defined. Study design, size, duration We collected the clinical outcomes of 3074 mosaic embryos transferred in women who underwent IVF between May 2019-May 2023. All embryos were cultured to blastocyst stage; trophectoderm (TE) biopsy was performed on Day-5 of development or Day6/7 for slow-growing embryos. The clinical outcome obtained after the transfer of mosaic embryos with the different chromosomal constitutions was compared with each other. Prenatal and post-natal outcomes were collected for available cases. Participants/materials, setting, methods Preimplantation genetic testing (PGT) was performed using high-resolution next-generation sequencing (NGS) methodology. TE biopsies were classified as mosaic if they had 20%-80% abnormal cells. For statistical analysis, mosaic embryos were divided into groups based on mosaic levels and chromosomal constitution detected in TE: single mosaic aneuploidy (monosomy/trisomy; SM), double mosaic chromosomes (monosomy/trisomy or combination, DM), complex mosaic aneuploidy (>2 different aneuploidies; CM) and mosaic segmental aneuploidy (single and double deletion/insertion >5Mb, MS). Main results and the role of chance Embryos classified as ‘low-mosaic’ by NGS-based PGT-A have a higher likelihood of achieving implantation compared to ‘high-mosaic’ embryos (48% vs. 39.%; p < 0.05 ), as well as ongoing pregnancy/live birth (40% vs. 29%; p < 0.05). Chromosomal composition of mosaicism abnormalities dictates the success rate of mosaic embryo transfers, with low and high segmental mosaics being preferable over low- and high-mosaics involving whole chromosomes. For 621/670 pregnancies, parental tests and post-natal data were available. The majority (99.75%) of the babies were largely healthy by routine physical inspection by neonatologists (no gross abnormalities in babies from mosaic embryos, n = 495). A combination of NIPT, CVS, and Amniocentesis prenatal testing results were collected for 552 pregnancies of mosaic embryo transfers, with predominantly normal findings. The mosaicism detected at the embryonic stage by PGT-A was reflected in prenatal testing in only 7 out of 552 pregnancies (0.9%), in which the mosaicism identified with PGT-A at the blastocyst stage was reflected in gestation by prenatal chromosomal testing as true fetal mosaicism. Limitations, reasons for caution Additional clinical data must be obtained to evaluate the contribution of each different chromosome before this approach can be evaluated as an additional tool to choose mosaic embryos for transfer. Wider implications of the findings The international registry of mosaic embryo transfers continues to grow in sample size, in turn increasing the power of analysis. The findings of the mosaic embryo transfer registry can help educate the management and selection of embryos in the clinic. Trial registration number no

Prenatal diagnosis of fetal microdeletion and microduplication syndromes among pregnant women with advanced maternal ages

To assess the value of combined chromosomal karyotyping and chromosomal microarray analysis (CMA) and/or copy number variation sequencing (CNV-seq) for the prenatal diagnosis for women with advanced maternal ages, and to explore the challenges of prenatal genetic counseling brought by the types of fetal CNVs and uncertainty of related phenotypes. A retrospective analysis was carried out on 1 841 women with advanced maternal age who underwent interventional prenatal diagnosis at the Prenatal Diagnosis Center of Xiamen University Affiliated Women and Children's Hospital from January 2017 to December 2020. Routine chromosomal karyotyping analysis and CMA/CNV-seq detection were carried out. CMA/CNV-seq had detected pathogenic variants in 2 cases which had failed karyotyping analysis. Two hundred and twenty one fetal chromosomal abnormalities were detected by karyotyping analysis, among which 187 were detected by CMA/CNV-seq. CMA/CNV-seq analysis of 23 cases with balanced chromosome structural aberrations and 10 cases with low proportion mosaicisms (including a marker chromosome) had yielded a negative result. In addition, 26 cases (26/1 841, 1.4%) with pathogenic CNVs were discovered among those with a normal karyotype, of which 13 (50.0%) were recurrent CNVs associated with neurocognitive impairment, with 22q11.21 microdeletions and microduplications being the most common types (26.92%). The combination of karyotyping analysis and CMA/CNV-seq not only increased the rate of prenatal diagnosis, but also complemented with each other, which has facilitated genetic counseling and formulation of prenatal diagnosis strategy for the affected families.

Fraccaro Syndrome (49,XXXXY): Case study in antenatal and postnatal from South Indian patients, an awareness to the human society

Aim: 49, XXXXY syndrome is a rare chromosomal abnormality with an approximate incidence of 1:85000 – 1:100000. Early diagnosis is important to improve the quality of life of patient. This study was carried out to assess the chromosomal abberations in prenatal and postnatal sample of patients through cytogenetics study. Methodology: Sex chromosomal aneuplodies were performed in both amniotic fluid and peripheral venous blood samples. Cytogenetic studies such as Karyotyping was done in in-vitro culture cells by GTG banded metaphase slides. FISH test was conducted in raw sample to rule out low grade mosaicism and DNA isolated from both the samples to rule out the gain and loss of genes by Chromosomal microarray technique. Results: The two cases performed showed aneuploidy with pentasomy sex chromosomes in Karyotyping in all the metaphase analysed and FISH test resulted with five signals in sex chromosome with four copies of X chromosomes and one copy of Y chromosome in all the cells. There was no loss of genes and only gain of genes in X chromosomes as identified in Chromosomal microarray. Interpretation: Based on the findings, antenatal screening is essential and should not be avoided for any USG findings and biochemical screening. The test should not be limited only to trisomy studies. Extensive studies should be performed to rule out the genetic disorders. Chromosomal aneuploidies can be identified by cytogenetic studies like Karyotyping, FISH and Chromosomal microarray technique. Key words: Aneuploidy, Chromosomal microarray analysis, Cytogenetics, Fraccaro syndrome, Karyotyping, Pentasomy sex chromosome

Efficient Optimization of High‐Quality Epitaxial Lithium Niobate Thin Films by Chemical Beam Vapor Deposition: Impact of Cationic Stoichiometry

AbstractLithium niobate is a material of special interest for its challenging functional properties, which can suit various applications. However, high quality 200‐mm LixNb1‐xO3 thin film grown on sapphire substrate have never been reported so far which limits these potential applications. This paper reports the efficient optimization of high quality LiNbO3 thin film deposition on sapphire (001) substrate through chemical beam vapor deposition in a combinatorial configuration. With this technique, flow ratio of Li/Nb can be tuned from ≈0.25 to ≈2.45 on a single wafer. Various complementary characterizations (by means of diffraction, microscopy and spectroscopy techniques) have been performed at different areas of the film (different cationic ratios) in order to investigate the impact of the cationic stoichiometry deviation on the film properties. Close to cationic stoichiometry (LiNbO3), the epitaxial films are of high quality (single phase in spite of two in‐plane domains, low mosaicity of 0.04°, low surface roughness, refractive index and band gap close to bulk values). Deviating from the stoichiometry conditions, secondary phases are detected (LiNb3O8 for Nb‐rich flow ratios, and Li3NbO4 with partial amorphization for Li‐rich flow ratios). LiNbO3 films are of high interest for various key applications in data communications among others.

An Integral Approach to the Molecular Diagnosis of Tuberous Sclerosis Complex: The Role of Mosaicism and Splicing Variants

Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder characterized by the presence of hamartomas in multiple organs. At the molecular level, the disease is caused by pathogenic variants in the TSC1 and TSC2 genes, and only 10% to 25% of clinically diagnosed patients remain negative after multiplex ligation-dependent probe amplification and exon sequencing of both genes. Here, to improve the molecular diagnosis of TSC, we developed an integral approach that includes multiplex ligation-dependent probe amplification and deep-coverage next-generation sequencing of the entire TSC1 and TSC2 genes, along with an adapted bioinformatic pipeline to detect variants at low allele frequencies (>1%). Using this workflow, the molecular cause was identified in 29 of 42 patients with TSC, describing here, for the first time, 12 novel pathogenic variants in TSC genes. These variants included seven splicing variants, five of which were studied at the cDNA level, determining their effect on splicing. In addition, 8 of the 29 pathogenic variants were detected in mosaicism, including four patients with previous negative study results who presented extremely low mosaic variants (allele frequency, <16%). We demonstrate that this integral approach allows the molecular diagnosis of patients with TSC and improves the conventional one by adapting the technology to the detection of low-frequency mosaics.

P-711 Are there any differences among blastocyst euploid and mosaicism rate (high or low) using fresh or frozen ejaculate sperm in donated oocytes?

Abstract Study question To compare euploid and mosaicism rate (high or low) among blastocysts derived from fresh and frozen ejaculate sperm in donated oocytes. Summary answer No statistically significant differences were found on blastocyst euploid and mosaicism rate between blastocysts derived from fresh versus frozen. What is known already Even though 5%-10% autosomal aneuploidies and 5%-100% sex chromosomes aneuploidies are paternally derived, the clinical relevance of paternal contribution to chromosome aneuploidy is frequently overlooked. Besides, male infertility could contribute to the generation of chromosomal abnormalities in the resulting embryos. Sperm cryopreservation is a widely used technique in assisted reproduction for different reasons. However, little is known regarding the potential effect of sperm freezing on embryo aneuploidies. Study design, size, duration Retrospective, observational and multicenter study of patients undergoing Preimplantation Genetic Testing for Aneuploidies (PGT-A) from January 2017 to December 2022 in IVIRMA Madrid, Valencia and Barcelona. To elucidate the effect of advance maternal age, 1,780 biopsied blastocysts coming from the egg donation program were analyzed. Autologous sperm samples with less than 2 million spermatozoa per milliliter and male partners with altered karyotype, abnormal spermatozoa fish or any genetic disease were excluded from the study. Participants/materials, setting, methods Embryos were cultured in regular incubators, with an atmospheric concentration of 5% O2, 6.5% CO2 and a temperature of 37ºC. Blastocyst biopsy was performed either on day 5 or day 6 of embryo development and analyzed through Next Generation Sequencing (NGS). Sperm freezing was done following manufacturer’s instructions (Sperm freeze, Origio). Continuous variables were compared with a student´s t-test and categorical variables with a Chi-square using SPSS statistical program. Main results and the role of chance Mean paternal age (44.3±5.20 years old vs 43.2±5.20 years old), sperm concentration (46.67±33.05 mill/ml vs 35.1±32.92 mill/ml) were not significantly different among fresh versus frozen sperm group (p &amp;gt; 0.05). A total 1780 blastocysts tested for PGT-A were included: 1267 blastocysts coming from fresh ejaculate sperm (n = 194 patients) and 513 blastocysts from frozen sperm (n = 81 patients). No statistically significant differences were found in blastocyst euploid rate between fresh versus frozen ejaculate sperm (64.5% versus 67.2%, p = 0.278 ;) respectively. Blastocyst mosaicism rate: High mosaicism (&amp;gt;50% of aneuploidy trophectoderm cells) and low mosaicism (30-50% of aneuploidy trophectoderm cells) was comparable between fresh versus frozen sperm (2.7% versus 4.5%; p = 0.90) and (3.9% versus 3.7%), respectively. Limitations, reasons for caution This study is based on a retrospective data. Wider implications of the findings According to our findings, there are no statistically significant differences in the blastocyst euploid and mosaicism rate between fresh and frozen ejaculate sperm. Trial registration number Not Applicable

P-726 The impact of reporting low-grade mosaicism in PGT cycles according to the number of cycles with at least one embryo for transfer

Abstract Study question How did the unmasking of low-grade mosaic embryos affect the number of cycles with an embryo for transfer for patients undergoing preimplantation genetic testing? Summary answer 5.4% of PGT-A cycles that would have resulted in no embryos for transfer, had one low-grade mosaic embryo that could be considered for transfer. What is known already Before the publication of the last practice recommendations for managing mosaic embryos (2022), many fertility clinics requested masking mosaic results in PGT-A reports (mosaic embryos were reported as aneuploid). Recent publications revealed that low-range mosaic embryos had pregnancy rates, live birth rates and miscarriage rates comparable to euploid embryos (Capalbo et al, 2022). New recommendations suggested that it is not appropriate to discard low mosaic embryos, as they had a high chance of resulting in a healthy baby. Therefore, fertility clinics are now asking for the report of mosaic embryos and are developing their own procedures for mosaic embryo transfer. Study design, size, duration A cohort of 1696 blastocysts from 553 PGT-A cycles were retrospectively included. The data was collected from PGT-A cases analyzed in the same genetic laboratory between July and December 2022. PGT-A cycles were performed in 17 different fertility clinics from Argentina and all of them, since July 2022, began to request the report of mosaic embryos. Participants/materials, setting, methods Trophectoderm biopsies from 1696 day 5, 6 or 7 blastocysts were analyzed by NGS using Ion ReproSeq PGS kit, Ion Cheftm, and Ion-S5 System (ThermoFisher Scientific). A customized algorithm was applied for the interpretation of PGT-A results. Mosaic embryos were reported as low mosaic (one or two chromosomes between 30-50% of copy number variation) or high mosaic (one or two chromosomes between 50-70%). Maternal age ranges from 22-44 years. Egg donor cycles were also included. Main results and the role of chance From the 1696 blastocysts analyzed, 1630 were informative (96.1%). 49.4% (805/1630) of embryos were euploid. This corresponded to 366 PGT-A cycles (66.2%) for which at least one euploid embryo was available for transfer. From the cycles with no euploid embryos (187/553, 33.8%), 30 patients (5.4%) have at least one low-mosaic embryo for transfer. The new recommendations for mosaic embryo management made it possible to increase the number of PGT-A cycles with at least one embryo for transfer by 5.4% during this period of time. According to maternal age, these 30 cycles correspond to: 33.3% (10/30) women &amp;lt; 38 y/o and egg donation cycles, 46.7% (14/30) for women between 38-40, and 20% (6/30) for &amp;gt; 40 y/o. The impact was observed mainly in advanced maternal age. For this group of patients, where it could be more difficult to repeat an IVF cycle and obtain euploid embryos, the possibility of transferring a low mosaic embryo could be their only chance. These 30 PGT-A cycles would have resulted in no embryos for transfer if previous policies for masking mosaic embryos (requested by the clinic) would be applied. The possibility for low-mosaic embryo transfer must be then reviewed by the patient and the physician. Limitations, reasons for caution Molecular laboratories performing PGT must execute an in-house validation. Our validated algorithm reports low-grade mosaic embryos between 30-50%. However, other platforms could have been validated for different ranges and these results may not be comparable. Five affected pregnancies were reported after transferring mosaic embryos (Viotti, 2021), so caution is required. Wider implications of the findings Results from a short period of time (six months) were considered for this analysis and clinical data was not possible to assess. Further analysis is required to evaluate clinical benefits in patients that proceed to mosaic embryo transfers. Trial registration number Not applicable

O-076 Update on the International Registry of Mosaic Embryo Transfers

Abstract Study question Which are the results obtained by the International Registry of Mosaic Embryo Transfers? Summary answer An update of clinical outcomes and post-natal results of mosaic embryos with low and high-level of mosaicism is provided. What is known already Chromosomal mosaic embryos are characterized by the presence of chromosomally different cell lines within the same embryo. While the transfer of these embryos is now offered as an option for women who undergo in vitro fertilization (IVF), several concerns remain. For instance, the limited data on pregnancy outcome and the possibility that intra-biopsy mosaicism in the TE is a poor predictor of the ploidy status of the ICM. Therefore, some argue that mosaicism should be not reported until a clear classification of such embryos in relation to their reproductive potential has been defined. Study design, size, duration We collected the clinical outcomes of 2045 mosaic embryos transferred in women who undergoing IVF between May 2019-May 2022. All embryos were cultured to blastocyst stage; trophectoderm (TE) biopsy was performed on Day-5 of development or Day6/7 for slow-growing embryos. The clinical outcomes obtained after the transfer of mosaic embryos with the different chromosomal constitutions were compared. Prenatal and post-natal outcome was collected for available cases. Participants/materials, setting, methods Preimplantation genetic testing for aneuploidies (PGT-A) was performed using high-resolution next-generation sequencing (NGS) methodology. TE biopsies were classified as mosaic if they had 20%-80% abnormal cells. For statistical analysis, mosaic embryos were divided into groups based on mosaic levels and chromosomal constitution detected in TE: single mosaic aneuploidy (monosomy/trisomy; SM), double mosaic chromosomes (monosomy/trisomy or combination, DM), complex mosaic aneuploidy (&amp;gt;2 different aneuploidies; CM) and mosaic segmental aneuploidy (single and double deletion/insertion &amp;gt;5Mb, MS). Main results and the role of chance Embryos classified as ‘low-mosaic’ by NGS-based PGT-A have a higher likelihood of achieving implantation compared to ‘high-mosaic’ embryos (48% vs. 39.%; p &amp;lt; 0.05 ), as well as ongoing pregnancy/live birth (40% vs. 29%; p &amp;lt; 0.05). Chromosomal composition of mosaicism abnormalities dictates the success rate of mosaic embryo transfers, with low and high segmental mosaics being preferable over low- and high-mosaics involving whole chromosomes. For 550/670 pregnancies, parental tests and post-natal data were available. The majority (99.75%) of the babies were largely healthy by routine physical inspection by neonatologists (no gross abnormalities in babies from mosaic embryos, n = 495). Prenatal testing performed on pregnancies from mosaic embryo transfers were generally normal. The mosaicism detected at the embryonic stage by PGT-A was reflected in prenatal testing in only 5 out of 550 pregnancies (0.9%) in which the mosaicism identified with PGT-A at the blastocyst stage was reflected in gestation by prenatal chromosomal testing as true fetal mosaicism. Limitations, reasons for caution Additional clinical data must be obtained to evaluate the contribution of each different chromosome before this approach can be evaluated as an additional tool to choose mosaic embryos for transfer. Wider implications of the findings The International Registry of Mosaic Embryo transfers continues to grow in sample size, in turn increasing the power of analysis. The findings of the mosaic embryo transfer registry can help educate the management and selection of embryos in the clinic. Trial registration number Not applicable

Genetic analysis of a child with Pitt-Hopkins syndrome due to a novel variant of TCF4 gene derived from low percentage maternal mosaicism

To explore the genetic etiology of a child with Pitt-Hopkins syndrome. A child who had presented at the Medical Genetics Center of Gansu Provincial Maternal and Child Health Care Hospital on February 24, 2021 and his parents were selected as the study subjects. Clinical data of the child was collected. Genomic DNA was extracted from peripheral blood samples of the child and his parents and subjected to trio-whole exome sequencing (trio-WES). Candidate variant was verified by Sanger sequencing. Karyotype analysis was also carried out for the child, and her mother was subjected to ultra-deep sequencing and prenatal diagnosis upon her subsequent pregnancy. The clinical manifestations of the proband included facial dysmorphism, Simian crease, and mental retardation. Genetic testing revealed that he has carried a heterozygous c.1762C>T (p.Arg588Cys) variant of the TCF4 gene, for which both parents had a wild-type. The variant was unreported previously and was rated as likely pathogenic based on the guidelines of the American College of Medical Genetics and Genomics (ACMG). Ultra-deep sequencing indicated that the variant has a proportion of 2.63% in the mother, suggesting the presence of low percentage mosaicism. Prenatal diagnosis of amniotic fluid sample suggested that the fetus did not carry the same variant. The heterozygous c.1762C>T variant of the TCF4 gene probably underlay the disease in this child and has derived from the low percentage mosaicism in his mother.

The value of chromosomal microarray analysis and fluorescence in situ hybridization for the prenatal diagnosis of chromosomal mosaicisms

To assess the value of chromosomal microarray analysis (CMA) and fluorescence in situ hybridization (FISH) for the prenatal diagnosis of chromosomal mosaicisms. A total of 775 pregnant women who had visited the Prenatal Diagnosis Center of Yancheng Maternal and Child Health Care Hospital from January 2018 to December 2020 were selected as study subjects. Chromosome karyotyping analysis and CMA were carried out for all women, and FISH was used to validate the suspected mosaicism cases. Among the 775 amniotic fluid samples, karyotyping has identified 13 mosaicism cases, which yielded a detection rate of 1.55%. Respectively, there were 4, 3, 4 and 2 cases for sex chromosome number mosaicisms, abnormal sex chromosome structure mosaicisms, abnormal autosomal number mosaicisms and abnormal autosomal structure mosaicisms. CMA has only detected only 6 of the 13 cases. Among 3 cases verified by FISH, 2 cases were consistent with the karyotyping and CMA results, and clearly showed low proportion mosaicism, and 1 case was consistent with the result of karyotyping but with a normal result by CMA. Eight pregnant women had chosen to terminate the pregnancy (5 with sex chromosome mosaicisms and 3 with autosomal mosaicisms). For fetuses suspected for chromosomal mosaicisms, CMA, FISH and G-banding karyotyping should be combined to determine the type and proportion of mosaicisms more precisely in order to provide more information for genetic counseling.

Epitaxial GaSb films directly grown on on-axis Si(001) with low defect density by MBE

In recent years, GaSb-on-Si direct heteroepitaxy has been highly desirable to extend the operating wavelength range into mid-infrared and high-mobility applications, such as free-space communications, gas sensing, and hyperspectral imaging. High-quality GaSb films on Si remain challenging due to the high density of defects generated during the growth. For this purpose, epitaxial GaSb films were grown by molecular beam epitaxy on on-axis Si(001). Due to the large lattice mismatch (12.2%) between GaSb and Si, here, we proposed a radical design and growth strategy with the primary objective of achieving the annihilation of antiphase boundaries (APBs) and the reduction of threading dislocation density (TDD). Benefitting from a V-grooved Si hollow structure, we demonstrated the growth of emerging-APB-free GaSb film on Si(001) with low mosaicity. Moreover, by introducing InGaSb/GaSb dislocation filtering layers, the atomically flat surface root mean square roughness is improved to 0.34 (on Si) and 0.14 nm (on GaAs/Si). Moreover, the corresponding TDD can be reduced to 3.5 × 107 and 2 × 107 cm−2, respectively, one order of magnitude lower than the minimum value found in the literature. These reported results are a powerful lever to improve the overall quality of epitaxial Si-based antimonide, which is of high interest for various devices and critical applications, such as laser diodes, photo-detectors, and solar cells.

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.

AmpSeqR: an R package for amplicon deep sequencing data analysis

Amplicon sequencing (AmpSeq) is a methodology that targets specific genomic regions of interest for polymerase chain reaction (PCR) amplification so that they can be sequenced to a high depth of coverage. Amplicons are typically chosen to be highly polymorphic, usually with several highly informative, high frequency single nucleotide polymorphisms (SNPs) segregating in an amplicon of 100–200 base pair (bp). This allows high sensitivity detection and quantification of the frequency of each sequence within each sample making it suitable for applications such as low frequency somatic mosaicism detection or minor clone detection in mixed samples. AmpSeq is being increasingly applied to both biological and medical studies, in applications such as cancer, infectious diseases and brain mosaicism studies. Current bioinformatics pipelines for AmpSeq data processing lack downstream analysis, have difficulty distinguishing between true sequences and PCR sequencing errors and artifacts, and often require bioinformatic expertise. We present a new R package: AmpSeqR, designed for the processing of deep short-read amplicon sequencing data, with a focus on infectious diseases. The pipeline integrates several existing R packages combining them with newly developed functions to perform optimal filtering of reads to remove noise and improve the accuracy of the detected sequences data, permitting detection of very low frequency clones in mixed samples. The package provides useful functions including data pre-processing, amplicon sequence variants (ASVs) estimation, data post-processing, data visualization, and automatically generates a comprehensive Rmarkdown report that contains all essential results facilitating easy inclusion into reports and publications. AmpSeqR is publicly available at https://github.com/bahlolab/AmpSeqR.

What proportion of embryos should be considered for transfer following a mosaic diagnosis? A study of 115 clinics from a central diagnostic laboratory.

The aim of this study is to identify what proportion of mosaic embryo diagnoses should be considered for transfer, and thereby assess the impact on patient cases. We categorised mosaic embryos into 3 groups; high, medium and low priority for transfer based on the percentage of biopsy sample being aneuploid and the chromosomes involved. The categories were applied to those patients that had no euploid embryo diagnoses but 1 or more mosaic embryos identified as mosaic available after PGT-A. In total, 6614 PGT-A cases from 115 clinics and a single diagnostic laboratory were reviewed. Further, 1384 [20.9%] cases only had aneuploid embryos, 4538 [68.6%] cases had one or more euploid embryos and 692 [10.5%] cases had no euploid and one or more mosaic embryo. The mosaic embryos in the no euploid, one or more mosaic group, when reviewed using priorities, resulted in: 111 [1.7%] of cases having at least one high priority mosaic available. 184 [2.8%] of cases having no high priority but at least one medium priority mosaic available. 397 [6.0%] of cases only having low priority mosaic embryos available. Based on this data, embryos identified as mosaic will only be considered for transfer in the first instance for around 4.5% (when taking high and medium priority and excluding low priority cases) of all PGT-A cases.

What is the Impact of Mosaicism for Embryo Selection: Experience Obtained Subsequent to the 1000 Mosaic Embryo Transfer- A Mini Review

Chromosomal mosaicism, implying the co-occurrence of cells having possession of separate Chromosomal amount, has got illustrated in human embryos for the past3 decades. The initial versions that were generated with regards to preimplantation genetic testing for aneuploidy (PGT-A) did not estimate mosaicism, in view of classically just the evaluation of a single cell was carried out or the approach did not possess the capacity of precise Identification. Despite, a straight forward diagnosis was yielded (an embryo got the label of normal or abnormal, thus just evading the topic, with now as hindsight might have been resulting in umpteen mistakes in the diagnosis that caused negative clinical outcomes .The modern PGT-A estimates a multicellular trophectoderm biopsy sample with the methodology possessing the capacity of recognition of intermediate copy number signals for the chromosomes or the subchromosomal areas. Thus we get faced with the topic of mosaicism in addition to thehurdles with regards to the management of the embryos that generate such outcomes in the clinical scenario. Here we conducted a systematic mini review utilizing search engine pubmed, google scholar; web of science; embase; Cochrane review library utilizing the MeSH terms for this mini review like PGT-A; aneuploidy; euploidy; mosaicism; implantation; ongoing pregnancy; spontaneous abortions; 100-1000mosaic embryo transfer study/studies; self-correction of mosaic embryos; multipolar mitosis; tripolar mitosis; low mosaic; high mosaic; final outcomes from 1990 to 2021 till date. We found a total of 500 articles out of which we selected 38 articles for this mini review. No meta-analysis was done. Here we have tried to detail the that demonstrated that mosaicism besides in general, however particular characteristics picked up with PGT-A are correlated with differing clinical outcomes. Thus requirements are there regarding mosaicism to get considered in the context of greater informed in addition to embryo selection in the clinical scenario

Natural Regeneration; 4 Years after the Ekiugbo Oil Spill, at Ughelli, Delta State, Nigeria: Implication for Phytoremediation Potential

This study assessed the floristic composition, structural classification and phytosociology of life form regeneration, mode of regeneration and demographic status of regeneration in vegetation forest, 4 years after pollution impact. It was aimed at evaluating the phytoremediation potential of some hydrocarbon tolerant macrophytes (HTM). Conventional ecological approach involving stratified simple random design method and phytosociological indices were used. Result has classified the study site flora as low land secondary mosaic vegetation with heterogenous continuum in spatial and closed horizontal structural arrangement. Phytosociological dynamics of 51 herbaceous and 12 shrubby life forms of 63 representative species under 21 families and 49 genera of angiosperms recorded changes. Four prevalently dominant families very abundant with highest species diversity richness and three families in abundance were recorded. Shrubby recruit was lower than Herbaceous recruits with the Herbaceous sedge (HS) recording highest recruits among regenerating life forms (HS&gt;HG&gt;HH&gt;HCl). The herbaceous life form had Chamaephytes 33(64.71%) and Hemicryptophytes 18(35.29%). The shrubby life form recorded 2(11.11%) Nanophanerophytes and Mesophanerophytes respectively and 8(66.67%) Microphanerophytes. The herbaceous life form mode of regeneration had 28 recruits with multiplier mode, and 23 recruits with single mode of regeneration. Four recruits exhibited multiplier mode and eight with single mode of regeneration across shrubby life form. Demographic status of regeneration revealed greater seedling than sapling density devoid of adult tree recruits, thus implies “successful and new regeneration” Keywords: Coppice, Recruits, Rhizome, Sapling, and Seedlings. DOI: 10.7176/JEES/12-11-04 Publication date: November 30 th 2022

Vacuolization in embryos on days 3 and 4 of in vitro development: Association with stimulation protocols, embryo development, chromosomal status, pregnancy and neonatal outcomes.

Is vacuolization in embryos on Days 3 and 4 associated with parent-related factors, stimulation protocols, embryo development, embryo ploidy, pregnancy and neonatal outcomes? This is a retrospective cohort study that comprised 5,703 embryos from 611 patients who underwent preimplantation genetic testing and time-lapse monitoring of their embryos from August 2017 to September 2021. Embryo vacuolization on Days 3 and 4 is associated with the LH level on the day of the hCG trigger and the number of retrieved oocytes. Compared to vacuole-negative embryos, the rates of blastocyst formation and good-blastocyst formation was significantly lower in vacuole-positive embryos. We observed no significant difference in the rates of euploidy, implantation, ongoing pregnancy, and live birth between vacuole-positive and vacuole-negative embryos. In vacuole-positive embryos, the embryos of which the vacuole-positive blastomeres were involved in embryo compaction exhibited significantly higher mosaicism rate compared with those of which the vacuole-positive blastomeres were not involved in embryo compaction. Vacuolization in embryos on Days 3 and 4 is associated with reduced blastocyst formation rate and high-quality blastocyst rate. Blastocysts had a low mosaicism rate if the vacuole-containing cells were rejected in compaction process, which supports the hypothesis that exclusion of abnormal blastomeres from compaction is a self-correction mechanism.

THE INFLUENCE OF MATERNAL AGE ON THE PREVALENCE OF FOUR DISTINCT MOSAIC RESULT CLASSIFICATIONS

To determine whether maternal age influences the prevalence of four distinct mosaic result classifications observed in preimplantation genetic testing for aneuploidy (PGT-A): isolated low mosaic, isolated high mosaic, complex low mosaic, and complex high mosaic.