Rate Of Mosaicism Research Articles

Digital droplet PCR (ddPCR) to investigate parental mosaicism in New Zealand families with apparently de novo syndromic disorders

A recurrence risk of under 1% is usually quoted for parents of children who have apparently de novo genetic conditions. Since 2018, our service has offered digital droplet PCR (ddPCR) testing to determine parental mosaicism rates for apparently de novo variants identified in children with syndromic disorders Aim: To determine the rate of parental mosaicism in a cohort of New Zealand families with apparently de novo pathogenic variants. Method: Specific assays were designed for each pathogenic variant. DNA derived from varying combinations of leukocytes, urothelial buccal and semen was tested by ddPCR for the pathogenic variants identified in the affected children. Results: 102 trio cases have been analysed. Seven parents were identified as being mosaic (6.8%). There is a preponderance of paternal mosaicism (6 of 7 being paternally derived) and two couples had a recurrence of the condition in a second child. Conclusion: This small cohort demonstrates a higher rate of parental mosaicism compared to the typically quoted recurrence risk of <1% and has implications for genetic counselling and genetic testing provision.

Utilization of standardized preimplantation genetic testing for aneuploidy (PGT-A) via artificial intelligence (AI) technology is correlated with improved pregnancy outcomes in single thawed euploid embryo transfer (STEET) cycles

PurposeTo investigate the role of standardized preimplantation genetic testing for aneuploidy (PGT-A) using artificial intelligence (AI) in patients undergoing single thawed euploid embryo transfer (STEET) cycles.MethodsRetrospective cohort study at a single, large university-based fertility center with patients undergoing in vitro fertilization (IVF) utilizing PGT-A from February 2015 to April 2020. Controls included embryos tested using subjective NGS. The first experimental group included embryos analyzed by NGS utilizing AI and machine learning (PGTaiSM Technology Platform, AI 1.0). The second group included embryos analyzed by AI 1.0 and SNP analysis (PGTai2.0, AI 2.0). Primary outcomes included rates of euploidy, aneuploidy and simple mosaicism. Secondary outcomes included rates of implantation (IR), clinical pregnancy (CPR), biochemical pregnancy (BPR), spontaneous abortion (SABR) and ongoing pregnancy and/or live birth (OP/LBR).ResultsA total of 24,908 embryos were analyzed, and classification rates using AI platforms were compared to subjective NGS. Overall, those tested via AI 1.0 showed a significantly increased euploidy rate (36.6% vs. 28.9%), decreased simple mosaicism rate (11.3% vs. 14.0%) and decreased aneuploidy rate (52.1% vs. 57.0%). Overall, those tested via AI 2.0 showed a significantly increased euploidy rate (35.0% vs. 28.9%) and decreased simple mosaicism rate (10.1% vs. 14.0%). Aneuploidy rate was insignificantly decreased when comparing AI 2.0 to NGS (54.8% vs. 57.0%). A total of 1,174 euploid embryos were transferred. The OP/LBR was significantly higher in the AI 2.0 group (70.3% vs. 61.7%). The BPR was significantly lower in the AI 2.0 group (4.6% vs. 11.8%).ConclusionStandardized PGT-A via AI significantly increases euploidy classification rates and OP/LBR, and decreases BPR when compared to standard NGS.

The importance of standardizing criteria for PGT-A interpretation of blastocysts analyzed by next-generation sequencing.

To compare the preimplantation genetic testing for aneuploidy (PGT-A) results using the three most frequent criteria employed by preimplantation genetic laboratories and evaluate its impact on the number of euploid embryos available for transfer. Retrospective and descriptive study including patients who underwent PGT-A between January 2018 and December 2020. Five hundred and nine PGT-A cycles and 2,079 blastocysts were analyzed by next-generation sequencing (NGS). We re-assigned the diagnosis of all blastocysts using three different criteria: strict (mosaicism thresholds from 20% to 80%), standard (from 30% to 70%) and excluding (mosaicism is not reported). We compared the euploid, aneuploid and mosaic embryos obtained in each criteria used. We observed PGT-A results discrepancies in 32.5% (165/509) of the cycles when the three different criteria were applied. The standard and excluding criteria showed 92 more euploid embryos (875/2,079) compared to the strict criteria (783/2,079). Evaluating the PGT-A results per cycle with the strict, standard and excluding criteria, the euploidy rates were 34.0%, 38.4% and 38.4% (p<0.001); aneuploidy rates were 59.0%, 55.8% and 61.6% (p<0.001) and mosaic rates were 7.0% and 5.8% (p<0.047), respectively. The mean number of euploid blastocysts available for transfer was 1.54±1.67 with the strict criteria, while the possibility to obtain an euploid embryo was higher if the standard or the excluding criteria were used 1.72±1.78 (p<0.001). This study highlights the importance of standardizing the criteria used for the interpretation of PGT-A blastocysts. We observed significant differences on PGT-A results associated solely to the criteria used.

Quantitative assessment of low-level parental mosaicism of SNVs and CNVs in Waardenburg syndrome.

Waardenburg syndrome (WS) is a rare inherited autosomal dominant disorder caused by SOX10, PAX3, MITF, EDNRB, EDN3, and SNAI2. A large burden of pathogenic de novo variants is present in patients with WS, which may be derived from parental mosaicism. Previously, we retrospectively analyzed 90 WS probands with family information. And the frequency of de novo events and parental mosaicism was preliminary investigated in our previous study. In this study, we further explored the occurrence of low-level parental mosaicism in 33 WS families with de novo variants and introduced our procedure of quantifying low-level mosaicism. Mosaic single nucleotide polymorphisms (SNPs) were validated by amplicon-based next-generation sequencing (NGS); copy-number variants (CNVs) were validated by droplet-digital polymerase chain reaction (ddPCR). Molecular validation of low-level mosaicism of WS-causing variants was performed in four families (12.1%, 4/33). These four mosaic variants, comprising three SNVs and one CNV, were identified in SOX10. The rate of parental mosaicism was 25% (4/16) in WS families with de novo SOX10 variants. The lowest allele ratio of a mosaic variant was 2.0% in parental saliva. These de novo WS cases were explained by parental mosaicism conferring an elevated recurrence risk in subsequent pregnancies of parents. Considering its importance in genetic counseling, low-level parental mosaicism should be systematically investigated by personalized sensitive testing. Amplicon-based NGS and ddPCR are recommended to detect and precisely quantify the mosaicism for SNPs and CNVs.

Introduction of the FecGF mutation in GDF9 gene via CRISPR/Cas9 system with single-stranded oligodeoxynucleotide

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9 system has been a recent focus of breeders owing to its potential to improve economically significant traits of livestock. The introduction of defined point mutations into the ovine genome via CRISPR/Cas9-mediated homology-directed repair has been reported; however, indel and mosaic events observed in genetically modified animals limit the practical application of this system in sheep breeding. The FecGF mutation (g. G1111A, p. V371 M) in the growth differentiation factor 9 (GDF9) gene is strongly associated with litter size in Belclare and Norwegian White Sheep. In the present study, we introduced the FecGF mutation in GDF9 by co-injecting the CRISPR/Cas9 system, single-stranded oligodeoxynucleotide (ssODN), and Scr7 into ovine zygotes. Scr7 at various concentrations (0 μM, 1 μM, and 2 μM) had no adverse effects on embryonic development in vitro. No significant differences in total mutation, point mutation, and indel rates in embryos were observed among groups treated with different concentrations of Scr7. However, the mosaicism rates of embryos from zygotes microinjected with 1 and 2 μM Scr7 were significantly lower than that for 0 μM Scr7 (7.7% and 7.5% vs. 19.7%). We successfully obtained lambs with defined nucleotide substitutions by the coinjection of Cas9 mRNA, sgRNA, ssODN, and 1 μM Scr7 into Altay sheep zygotes. The single nucleotide mutation efficiency was 7.69% (3/39) in newborn lambs, with one mosaic. Our findings provide evidence that Scr7 could improve the specificity of the CRISPR/Cas9 system for the introduction of a defined point mutation in livestock to some extent.

Reproductive genetics laboratory may impact euploid blastocyst and live birth rates: a comparison of 4 national laboratories' PGT-A results from vitrified donor oocytes

To evaluate potential variation in the euploid blastocyst rate and live birth rate (LBR) per single frozen euploid blastocyst transfer, among 4 unique United States reproductive genetics laboratories. Analyses were limited to blastocysts derived from vitrified donor oocytes, to minimize variation in oocyte quality. Retrospective cohort study from 2016 to2020. Donor Egg Bank Database. Patients undergoing invitro fertilization with donor oocytes. We excluded patients with uterine factor, male factor, or surgically extracted sperm. Only healthy women <34 years old were accepted as oocyte donors. Next generation sequencing platforms for chromosomal analysis MAIN OUTCOME MEASURE(S): Euploid blastocyst rate and LBR per euploid transfer. Secondary outcomes included the rate of aneuploidy, mosaic calls, biochemical pregnancy loss, and miscarriage rate. A total of 2,633 embryos were included. Four laboratories had >200 embryos tested. Euploid blastocyst rate was significantly higher in laboratory A (73.6%) vs. B (63.3%), C (60.9%), and D (52.3%). Mosaic rate was significantly lower for Laboratories B (2.8%) and C (5.5%) vs. Laboratories A (9.9%) and D (11.5). The LBR was significantly higher in laboratory A (58.73%) vs. laboratory D (47.3%). There were no significant differences in the implantation or miscarriage rate among laboratories. In this large study, controlling for oocyte quality, some preimplantation genetic testing for aneuploidy (PGT-A) laboratories report a significantly higher euploid blastocyst rate with concurrent higher LBR. This type of comparison is important as it provides insight into the role of the PGT-A process in outcomes. Further research is needed to evaluate the differences in laboratory techniques and bioinformatic algorithms accounting for variable outcomes across PGT-A laboratories.

Do chromosomal inversion carriers really need preimplantation genetic testing?

This study aimed to evaluate the rates of euploidy, aneuploidy, and mosaicism in preimplantation genetic testing for structural rearrangements (PGT-SR) cycles from chromosomal inversion carriers. In addition, this work also focused on assessing the impact of some contributors on the incidence of parental originating aneuploidy and mosaicism. This retrospective review enrolled chromosomal inversion carrier couples of whom the females were under 38years old undergoing PGT-SR at a single academic reproductive center. Subgroups were divided according to the gender of carriers, the inversion type, and the semen parameters of male carriers (male factor infertility (MF) or non-MF). Patient demographics, cycle characteristics, and PGT-SR outcomes were compared among subgroups. A total of 71 PGT-SR cycles from 57 inversion carrier couples were included for analysis. Among the 283 blastocysts, 48.4% were identified as euploidy, 27.9% as aneuploidy, and the remaining 23.7% as mosaicism. Only 32.9% of aneuploid embryos and 1.5% of mosaic embryos involved the parental inversion chromosomes. Notably, the female inversion carriers seemed to produce more parental originating aneuploid embryos than male inversion carriers (45.5% vs 23.9%, p = 0.044). The type of inversion and sperm parameters of male chromosomal inversion carriers did not affect the ploidy status of embryos. The incidence of parental originating aneuploidy in inversion carrier couples is lower than expected. For male chromosomal inversion carriers with normal sperm condition whose female partners are under 38years old, natural conception combined with prenatal diagnosis could be provided as an option during fertility counseling.

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.

Systematic detection of mosaicism by using digital NGS reveals three new MEN1 mosaicisms.

PurposeMosaicism is a feature of several inherited tumor syndromes. Only a few cases of mosaicism have been described in multiple endocrine neoplasia type 1 (MEN1). Next-generation sequencing (NGS) offers new possibilities for detecting mosaicism. Here, we report the first study to systematically look for MEN1 mosaicism, using blood DNA, in MEN1-suspected patients but without MEN1 pathogenic variants (PV) in a heterozygous state.MethodsDigital targeted NGS, including unique molecular identifiers (UMIs), was performed in routine practice, and the analytic performance of this method was verified.ResultsAmong a cohort of 119 patients harboring from 2 to 5 MEN1 lesions, we identified 3 patients with MEN1 mosaic PVs. The allele frequencies ranged from 2.3 to 9.5%. The detection rate of MEN1 mosaicism in patients bearing at least 3 MEN1 lesions was 17% (3/18). No cases were detected in patients with two lesions.ConclusionWe report here three new cases with MEN1 mosaicism. This study examined the performance of UMI in the diagnosis of MEN1 mosaicism in routine practice, and our results underline that the frequency of mosaicism is probably underestimated in patients with suspected MEN1.

Correlation between number of laser pulses and rate of mosaicism in human blastocysts trophectoderm biopsy

Correlation between number of laser pulses and rate of mosaicism in human blastocysts trophectoderm biopsy

MOSAICISM INCIDENCE IS INDEPENDENT OF THE TYPE OF CULTURE MEDIUM – A COMPARISON BETWEEN CONTINUOUS CULTURE MEDIUM (CCM) AND SEQUENTIAL CULTURE MEDIUM (SCM)

To evaluate whether the type of culture medium affects mosaicism rates when sibling oocytes were cultured using CCM or SCM.

No laser use in “flicking” embryo biopsy does not reduce the mosaicism rate

No laser use in “flicking” embryo biopsy does not reduce the mosaicism rate

Oocytes With Smooth Endoplasmic Reticulum Aggregates May Not Impact Blastocyst Euploidy Rate.

ObjectiveTo investigate whether the euploidy rate of blastocysts derived from smooth endoplasmic reticulum aggregates (SERa) positive cycles and oocytes are impacted.DesignRetrospective cohort study.Method(s)A total of 601 preimplantation genetic testing (PGT) cycles with at least one oocyte retrieved in our center between April 2017 and May 2021 were initially included in the study. Women>35 years and PGT cycles with chromosomal structural rearrangements (PGT-SR) were excluded. Embryological and blastocyst ploidy outcomes were compared among SERa+ oocyte, sibling SERa- oocytes and oocytes in SERa- cycles.ResultsNo significant difference was observed among the SERa+ oocyte group, sibling SERa- oocyte group, and SERa- cycle group in the normal fertilization rate (82.1% vs. 77.8% vs. 83.1%, respectively, P=0.061), blastocyst formation rate (71.0% vs. 72.5% vs. 68.4%, respectively, P=0.393), good quality blastocyst formation rate (46.4% vs. 48.3% vs. 42.6%, respectively, P=0.198). No significant difference was observed in the euploidy rate (50.0% vs. 62.5% vs. 63.3%, respectively, P=0.324), mosaic rate (12.5% vs. 9.7% vs. 13.4%, respectively, P=0.506), and aneuploidy rate (37.5% vs. 27.8% vs. 23.2%, respectively, P=0.137) among the three groups.ConclusionOur results suggest that the euploidy rate of blastocysts derived from SERa+ cycles and oocytes may not be impacted.

Bio-responsive composite liposomes against Campylobacter jejuni in vitro and its application in chicken preservation

The infection of Campylobacter jejuni (C. jejuni) in food can cause huge economic and health costs, and liposomes based on biological switch is a promising antibacterial strategy. In this study, a bio-responsive composite liposome was designed with silk fibroin and L-fucose (FLPs) based on the chemotaxis and protease of C. jejuni, and Litsea cubeba essential oil as antibacterial agent. The oil encapsulation and protein mosaic rates of the composite liposome were 70.6 ± 0.5% and 47.9 ± 1.0%, respectively. The in vitro release experiment verified that the release rates of essential oil from the liposome exceeded 95% at 5 days. Furthermore, FLPs possessed excellent antibacterial activity, the inhibition zone was 23.5 mm and 20% (v/v) of the composite liposomes could inactivate 99% C. jejuni. Moreover, laser scanning confocal microscopy and scanning electron microscopy images confirmed the antimicrobial activity of FLPs was better than that of other liposomes, and it could attach to bacteria well. Sprayed liposomes revealed sustained ability of retarding bacterial reproduction within 5 days with the refrigerated chicken meat inoculated with C. jejuni, and had no impact on the color and texture of the food. Hence, the bio-responsive composite liposome has a promising application in inhibiting C. jejuni growth in food. Industrial relevanceIn this study, the composite liposome with high encapsulation efficiency shows significant and sustained antibacterial activity, and has no negative effects on the color and texture of chicken meat. The findings of this study will facilitate the development and application of liposomes as active antimicrobial packaging in food industry.

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.

P-556 Younger maternal age as a factor associated with embryonic mosaicism. Analysis of 3222 blastocyst by Next Generation Sequencing

Abstract Study question Is there an association between healthy younger women under 30 years old and blastocyst mosaicism rate evaluated by NGS analysis? Summary answer Embryo mosaicism is independent to younger maternal age. However, morphological embryo quality is associated with embryo mosaicism. What is known already Embryo aneuploidy rate is strongly associated with maternal age, increasing in women older than 36y. On the contrary, the aneuploidy rate remains lower in women between 22 to 35y. However, the mosaicism rate in younger women seems to be slightly higher compared to older women. Although, there is still controversy on the relationship with maternal age and others factors. Study design, size, duration This retrospective study include 786 oocytes donation cycles were performed from 2016 to 2020. These IVF cycles generated 3222 blastocyst that were analyzed with PGT-A(NGS. Veryseq-Illumina). Mosaic embryos were call when variation was between 20-80%. Mosaic embryos were also sub-classify as a whole or segmental chromosome mosaicism. Mosaic embryos were according to the impact level as a: high Mosaic level (&amp;gt;50%) and low mosaic level (≤50%). Data was classified by donor age: group-1(18-22y)(n = 288) and group-2(23-30y)(n = 498). Participants/materials, setting, methods All donors had a normal ovarian reserve and without any pathological or chromosome translocation. Donors had conventional oocyte stimulation with antagonist protocol trigger with agonist GnRH. Embryos were cultured with Lifeglobal media family under 7% CO2 and 5%O2. A multilevel model was made and associations between variables by logistic regression were adjusted according to paternal age, morphological blastocyst quality, fertilization method, biopsy operator, day of embryo biopsy, number of chromosomes impacted per embryo and sperm quality. Main results and the role of chance Oocyte donation cycles generated 3222 blastocyst that were analyzed by NGS. The euploidy rate was 60.4% (n = 723) and 58.6% (n = 1186). Aneuploidy rate was 22.6% (n = 271) and 26.2% (n = 531) (p = 0.04). Mosaicism rate was 17% (n = 204) and 15.1% (n = 309) in group 1 and 2 respectively (p = 0.4) Mosaic embryos were also further studied and classified according to the impact level. Interestingly, a vast majority of mosaic embryos have low mosaicism level (86.1%) compared to high mosaic level (13.9%) (p &amp;lt; 0.001). The multilevel model shows that embryo quality correlated with embryo mosaicism where good quality embryos have a lower mosaicism rate compared to fair and poor quality blastocyst. Finally, the chromosomes 21, 22 and 14 were the most frequent chromosome affect in whole chromosome mosaicism while the chromosomes 1, 2 and 5 were affected by segmental chromosome mosaicism and it was independent of maternal age. Limitations, reasons for caution The analysis was limited only for healthy women under 30 year old and it should not be extrapolate to patients with other pathologies. Wider implications of the findings The present study revealed that embryo mosaicism remains at similar percentage in younger healthy women. The mosaic embryos generated from young women have a lower chromosome impact, which according to other studies has a good implantation potential. Trial registration number N.A

P-519 Relationship between the quality control parameters in PGT-A results by NGS with the chromosomal mosaicism diagnosis in trophoectoderm biopsies

Abstract Study question Are quality control (QC) parameters in NGS results related to mosaicism rate in trophoectoderm samples? Summary answer A high number of filtered reads in PGT-A results correlates with a higher mosaicism rate. But the overall noise does not influence the mosaicism rate. What is known already The implementation of NGS have led to increased reporting of mosaicism. There is intense research on the biological mechanisms behind mosaicism, but also on the technical factors that might be influencing in its diagnosis. Low quality samples and DNA amplification artefacts lead to an increase in overall noise after sample analysis. The Derivative Log Ratio (DLR) quality control parameter in sequencing results measures the overall noise present in a sample. High DLR values indicate noisy profiles. Additionally, insufficient numbers of sequencing reads will result in noisy profiles and false copy number assignments. Study design, size, duration A retrospective analysis of PGT-A results, corresponding to trophoectoderm biopsies from embryos on day 5, 6 or 7 of development, was performed (February 2017- November 2021). After the quality analysis, 5245 samples that reached the optimum QC measures were included (samples with DLR &amp;lt; 0.4 and number of filtered reads &amp;gt; 250.000). Participants/materials, setting, methods PGT-A was carried out with the VeriSeq kit (Illumina), after genome amplification with the Picoplex technique. Sequencing data was analyzed with BlueFuse Multi software (Illumina). Data analysis was performed using SPSSv20.0. An embryo was designated as mosaic when the percentage of aneuploidy for at least one chromosome was between 25 and 50%. For this study, we considered euploid-aneuploid and aneuploid-aneuploid mosaic embryos. Main results and the role of chance Considering the 5245 samples included in the analysis, the number of embryos carrying chromosomal mosaicism was 960, which corresponds to 18.3%. To analyze whether the percentage of mosaic embryos varied according to the DRL, we used a logistic regression analysis, introducing maternal and paternal age, embryo quality, and biopsy day as confounding variables, and categorized the DLR using as a point cut the median (0.19). There were no significant differences in the percentage of embryos carrying mosaicism between the group of higher DLR (≥0.19) compared to the group of lower DLR (&amp;lt;0.19) (18.7% vs 18.0%; p = 0.278). On the other hand, to study the relationship between mosaicism and the number of filtered reads obtained in the sequencing analysis, we performed the same analysis using the median of filtered reads (556.688) as cut point. The samples with a number of filtered reads higher than the median had a higher rate of mosaicism (19.3% vs 17.4%, p = 0.041). Moreover, we analyse the data according to the type of mosaicism (segmental or whole chromosome) and we did not observe any difference neither for the DLR nor for the reads. Limitations, reasons for caution The main limitation of this study compared with other studies is the different criteria in the mosaicism diagnosis between the different laboratories. Moreover, we are not able to identify if worse QC are due to issues with the biopsy procedure, sample quality, DNA amplification or library preparation. Wider implications of the findings The overall noise in PGT-A results doesn't seem to influence the mosaicism rate. However, increased mosaicism rate is observed in samples with high number of filtered reads. This finding suggests that too many cells may lead to amplification saturation and underestimation of the relative quantity of DNA. Trial registration number Not applicable

P-520 Factors associated with embryonic mosaicism: a systematic review and meta-analysis

Abstract Study question What factors are involved in chromosomal mosaicism in human blastocysts? Summary answer Progenitors’ age and embryo biopsy day are associated with mosaicism rate. What is known already Chromosomal abnormalities are common in embryos analyzed in preimplantation genetic testing for aneuploidy (PGT-A) cycles. Mosaicism is a usual event in embryos derived from IVF cycles. With the development of Next-Generation Sequencing (NGS) techniques, the ability to detect mosaicism in embryos has been improved. Several studies show that mosaic embryos have reduced potential to reach term compared to euploid. Furthermore, the existing scientific evidence regarding the cause of embryonic mosaicism is scarce with conclusions generating considerable controversy. Study design, size, duration A systematic review and meta-analysis were conducted following the PRISMA guidelines on Medline and Google Scholar (January 2016 to December 2021). Studies addressing factors associated with embryo mosaicism were comprehensively analyzed. As inclusion criteria: mosaicism should have been detected from a blastocyst trophectoderm biopsy on day 5,6 o 7 and analyzed by NGS. Embryo quality, maternal and paternal age, day of biopsy and seminal quality were the outcomes variables included for analysis. Participants/materials, setting, methods A comprehensive database search resulted in 195 articles, 18 of which were included for abstract reading. Search, screen, and data extraction were performed by two reviewers independently based on inclusion criteria. The study by meta-analysis of the effect of the different factors associated with embryonic mosaicism was carried out using the inverse variance method. Depending on the existence of heterogeneity between studies, the fixed effects method or the random effects method were used. Main results and the role of chance After critically and thoroughly reading the selected papers, 10 of them were included in the meta-analysis. Data from our reproductive clinic were also enclosed in the analysis (2513 cycles, 7242 embryos). The results of the meta-analysis show that neither embryo quality nor seminal quality (male factor) were related to mosaic embryo rate (OR: 1.15; 95%CI: 0.98-1.35 and OR: 1.01; 95%CI: 0.80-1.27, respectively). In contrast, a positive association with embryo mosaicism was observed for the variable “biopsy day” (OR: 1.06; 95% CI: 1.01-1.11): in embryos biopsied on day 6 or 7 of embryonic development, a small increase in the mosaicism rate was observed, in comparison with day 5. A significative positive association was also observed when we studied “paternal age” factor (OR: 1.13; 95% CI: 1.02-1.26): embryo mosaicism increases with paternal age. On the other hand, maternal age showed a negative association with mosaicism (OR: 0.84; 95% CI: 0.74-0.95). Interestingly, in opposite to what happens with the aneuploidy rate, embryo mosaicism is higher in young women. Limitations, reasons for caution The main limitation is the low number of papers analyzed in the meta-analysis. Limitations also include the retrospective design and heterogeneity of studies, limiting comparison and pooling of data. Nonetheless, our conclusions were based on studies with low risk of bias. Wider implications of the findings Our result, combined with the body of medical evidence available suggest that embryo mosaicism rate is influenced by trophectoderm biopsy day and maternal and paternal age. This information will add in the knowledge for elucidating the uncertainties surrounding the factors by which mosaicism is generated in embryos. Trial registration number Not applicable

P-564 Advanced paternal age is associated with higher rate of segmental chromosomal aberrations in preimplantation embryos

Abstract Study question Is there a correlation between paternal age and the frequency of chromosomal aberrations in embryos created by in vitro fertilization (IVF)? Summary answer There is an association between paternal age and segmental aberration rates in embryos derived from young oocyte providers after adjusting for age,sperm,and IVF-cycle characteristics. What is known already Paternal reproductive aging(APA) is less understood than maternal reproductive aging(AMA). Embryo aneuploidy presents one of the most important determinants of transfer cycle success. The association of AMA with increased rate of aneuploidy is well established. The systematic review and meta-analysis published by our group did not identify an association of APA with aneuploidy, based on ∼10,000 pooled embryos, however, the power to detect subchromosomal aberrations was limited by the sensitivity of the PGT-A platform used by each center. Our aim was to study the impact of APA on chromosomal aberrations in a large sample size analyzed by high resolution PGT-A. Study design, size, duration This was a retrospective cohort study performed at the CReATe Fertility Centre Genetics Laboratory, Toronto, Canada, between January 2019-December 2021. A total of 17731 blastocysts from 3506 cycles (2502 patients) were analyzed using high resolution NGS PGT-A (10Mb resolution; 30%-70% reported as mosaicism). Clinical, laboratory and demographic data were obtained for statistical analysis. Participants/materials, setting, methods Embryos were allocated into two groups by oocyte-provider age: ≤33years and ≥34years. Each group was divided into four sub-groups by paternal age: Group A 20-29(n = 484); GroupB 30-39(n = 9704); GroupC 40-49(n = 6341); GroupD≥50(n = 1202). Multiple logistic/ordinal regressions were conducted to evaluate euploidy, mosaicism and aneuploidy rates between age groups, as appropriate. Each was further sub-analyzed by type of abnormality A-single-whole-chromosome, S-segmental, C-complex (≥2 full-chr-aberrations). Segmental aberrations were divided into subgroups of S + (segmental gain), S-(segmental loss) and CS(complex segmental). Main results and the role of chance High resolution NGS PGT-A analysis of 17731 embryos showed 54.9% were euploid, 31.5% were aneuploid and 13.6% mosaic. In the oocyte provider’s age of ≤ 33 group, significant differences between paternal age group A vs. B, C and D were found in euploidy rates (68.8%, 62.5%, 61.7%, 59.0% respectively; p = 0.00559). There were no significant differences in aneuploidy rates (19.1%, 22.7%, 22.5%, 24.6% respectively; p = 0.07184) or mosaicism rates (12.1%, 14.8%, 15.8%, 16.3%; respectively, p = 0.23754). In the oocyte provider’s age of ≥ 34 group, decreasing euploidy rates and increasing aneuploidy rates were observed between all paternal age groups (p = 0.00001 and p &amp;lt; 0.00001,respectively), and no significant differences in mosaicism rates (14%, 12%, 10.2%, 11.6% respectively) reflecting the maternal age effect on euploidy/aneuploidy. In the oocyte provider’s age of ≤ 33 group, paternal group A (2.3%) had significantly lower segmental (S) rates compared to groups B (4.3%, p = 0.027599), C (3.3%, p = 0.039924) and D (4.6%, p = 0.018478). In addition, segmental losses (S-) were lower in group A (1.3%) compared to groups B (3.4%, p = 0.020431), C (3.1%, p = 0.043166) and D (3.7%, p = 0.016385). Limitations, reasons for caution The main limitation was the retrospective nature of this study; however, multiple regression analyses were conducted to limit potential bias by adjusting for sperm and oocyte characteristics. In addition, PGT-A analysis cannot provide information on origin of the aneuploidy and all conclusions are deduced from indirect associations to paternal age. Wider implications of the findings The results of this study provide important information to assist fertility practitioners when providing pre-conception counseling to patients, particularly for couples in which the male partner belongs to an older age group. Trial registration number na

O-075 Implicit bias in diagnosing mosaicism amongst preimplantation genetic testing providers: results from a large multicenter analysis of 36395 blastocysts

Abstract Study question Does the diagnosis of mosaicism affect ploidy rates across different providers offering preimplantation genetic testing for aneuploidies (PGT-A)? Summary answer Our analysis of 36395 blastocyst biopsies across 8 genetic testing laboratories revealed that euploidy rates were significantly higher in providers reporting low rates of mosaicism. What is known already Diagnoses consistent with chromosomal mosaicism have emerged as a third category of possible options for embryo ploidy outcomes in PGT-A. However, diagnosing mosaicism using current PGT-A platforms remains hindered by several biological and technical factors. This has led to substantial variability in mosaicism rates amongst genetic testing laboratories. Furthermore, reservations regarding the clinical value of diagnosing mosaicism have led to varying practices in reporting mosaic calls amongst providers. Critically, it remains unknown whether these differences impact the number of euploid embryos available for transfer. Ultimately, this may significantly affect clinical outcomes, with important implications for PGT-A patients. Study design, size, duration Retrospective, international, multicenter cohort study of 10875 PGT-A cycles conducted between October 2015 and October 2021. A total of 18 IVF centers associated with 8 PGT-A providers, across 5 countries and 3 continents participated in the study, which included 36395 blastocysts, tested using trophectoderm biopsy and next generation sequencing (NGS). Both autologous and donation cycles were assessed. Preimplantation genetic testing for structural rearrangements (PGT-SR) cycles were excluded from the analysis. Participants/materials, setting, methods Ploidy rates were analyzed using multilevel mixed linear regression. Providers were categorized (A to H), with the most frequent provider used as the reference for statistical analysis. Analyses were adjusted for maternal age, paternal age, donor status, number of embryo biopsied and day of biopsy, as appropriate. The overall significance of categorical variables in the regression models was tested using a Chi-squared test. P-values &amp;lt;0.05 were considered significant. Data analysis was performed using STATA, v.15.0. Main results and the role of chance The mean maternal age(+SD) across all providers was 36.9(±5.1). As expected, maternal age and day of biopsy had a significant impact on euploidy rates (p &amp;lt; 0.0001). Mosaicism rates were associated with PGT-A provider and independent of all other parameters (maternal age, paternal age, donor status, number of embryos biopsied and day of biopsy). Out of the 8 providers, 7 reported chromosomal mosaicism. Amongst these 7 providers, the rate of mosaic calls varied from 2.9% to 23.9%. After adjusting for confounders, two providers reported significantly higher mosaicism rates compared to the reference (4.2%): Provider-C 10.4% (OR = 2.43, 95%CI: 1.84-4.25) and Provider-F 23.9% (OR = 4.47, 95%CI: 2.92-6.86), while euploidy and aneuploidy rates did not differ. Conversely, the chance of diagnosing mosaicism was lower in Provider-B (OR = 0.34, 95%CI: 0.22-0.54) and Provider-E (OR = 0.59, 95%CI: 0.38-0.90). Here, aneuploidy rates were comparable to the reference, yet the chance of diagnosing a euploid embryo was significantly higher: Provider-B (OR = 2.38, 95%CI: 1.87-3.03) and Provider-E (OR = 1.62, 95%CI: 1.28-2.05). Compared to the reference, euploidy rates were also higher when mosaicism was not reported: 53.5% vs. 44.2% (OR = 2.04, 95% CI: 1.60-2.59). Moreover, the chance of having at least one euploid blastocyst available for transfer significantly increased when mosaicism was not diagnosed (OR = 1.30, 95%CI: 1.13-1.50). Limitations, reasons for caution Due to the retrospective nature of the study, associations can be ascertained, however causality cannot be established. Certain parameters were not available in the dataset, therefore full elucidation of all potential confounders accounting for the variability may not be possible. Wider implications of the findings Our findings highlight the significant impact of the genetic testing provider on PGT-A results. We demonstrate that reporting mosaicism primarily comes at the expense of euploid diagnoses, raising concerns regarding the accuracy of mosaicism predictions and their impact on clinical outcomes. Moving forward, greater standardization amongst providers will be essential. Trial registration number NA