Aneuploidy Status Research Articles

P-568 Correlation of ploidy analysis in two types of zygotes with 3 pronuclei (3PN) observed: same size 3PNs and one micronucleus 3PNs

Abstract Study question Do all zygotes with three observed pronuclei have an actual triploid chromosomal complement? Summary answer Not all zygotes with 3 pronuclei observed are triploid, most of embryos with two even pronuclei and one smaller micronucleus are diploid. What is known already In the in vitro fertilization (IVF) setting, normal oocyte fertilisation is commonly confirmed 16–19 hours after conventional insemination or intracytoplasmic sperm injection (ICSI) with the appearance of two pronuclei (2PN). The presence three pronuclei (3PN) shows an abnormal oocyte fertilization. The incidence of 3PN zygotes in IVF is between 5.0%- 8.1% in conventional insemination and between 2.0%- 6.2% in ICSI. 3PN fertilized embryos are commonly discarded due to the suspected increased risk of a triploid chromosomal complement. However, no definitive genetic evidence has been obtained to show that all 3PN zygotes are chromosomally abnormal. Study design, size, duration An Ethics Committee for Investigation accredited this prospective observational study performed in a private centre between June 2023 and January 2024. 120 3PN zygotes from 113 cycles were cultivated until blastocyst stage. 46 were biopsied to clarify ploidy and euploidy status. 3PNs were divided into 2 groups based on the size of their pronuclei: Micro Triploid (MT) (smallest pronucleus <70% of the diameter of the largest pronucleus) and Pure Triploid (PT) (smallest >70% of the largest). Participants/materials, setting, methods Patients with 1 or more 3PN embryo were offered to participate, culturing and assessing their 3PNs. If 3PNs reached blastocyst stage, they were biopsied and analysed using Next-Generation Sequencing strategy with Single Nucleotide Polymorphisms technology without the need for parental samples, to assess ploidy and aneuploidy status. Measures of the pronuclei size were taken to sort the 3PNs in both groups. Comparisons between these groups regarding blastocyst rates, ploidy status and aneuploidy rates were assessed. Main results and the role of chance 120 3PNs zygotes were identified in 1054 cycles, representing 1.9% of all fertilized embryos. The blastocyst rate (BR) for overall 3PNs was 38.3%, while the BR for 2PNs on those 113 cycles was 58.3%, showing significant differences (p < 0.001). 3PN zygotes were classified into the MT and PT groups described above. The MT group had 40.4% BR, and the PT group had 37.0% BR. No significant differences were found. Of the 46 biopsied embryos, 37 were informative (80.4%), 17 for the MT group and 20 for the PT group. The diploidy rate for the overall 3PNs was 37.8% (n = 14). The MT group had 82.4% (n = 14) of diploidy rate, while the PT group had 0% diploid rate (n = 0), showing significant differences between them. (p < 0.001). The overall euploidy rate for 3PN zygotes was 28.6% (n = 12) (36.2 mean age), which was similar to the euploidy rate (30.3%) for our overall 2PNs biopsied during 2023 (38.2 mean age). No differences found. Out of the 14 diploid 3PNs, 28.6% (n = 4) were euploid, showing no differences with our 2023 euploidy rate. Two diploid 3PNs were mosaic, resulting in a total of 6 embryos that could potentially be transferred. Limitations, reasons for caution This study is limited by the number of triploid embryos recruited. As blastocyst rate for 3PNs population is lower than observed in the 2PN population the number of biopsied embryos is reduced. Euploidy rates for 3PNs and 2PNs are very similar, but the mean age differs by 2 years. Wider implications of the findings It is very important to distinguish between both types of 3PNs observed: PT are not worthy to keep and analyse, while MT embryos are very likely to be diploid, and it is worthwhile to culture and analyse them. Patients with few embryos available in particular could benefit from this study. Trial registration number not applicable

The severity of meiotic aneuploidy is associated with altered morphokinetic variables of mouse oocyte maturation.

Is there an association between morphokinetic variables of meiotic maturation and the severity of aneuploidy following in vitro maturation (IVM) in the mouse? The severity of meiotic aneuploidy correlates with an extended time to first polar body extrusion (tPB1) and duration of meiosis I (dMI). Morphokinetic variables measured using time-lapse technology allow for the non-invasive evaluation of preimplantation embryo development within clinical assisted reproductive technology (ART). We recently applied this technology to monitor meiotic progression during IVM of mouse gametes. Whether there is a relationship between morphokinetic variables of meiotic progression and aneuploidy in the resulting egg has not been systematically examined at the resolution of specific chromosomes. Next-generation sequencing (NGS) is a robust clinical tool for determining aneuploidy status and has been reverse-translated in mouse blastocysts and oocytes. Therefore, we harnessed the technologies of time-lapse imaging and NGS to determine the relationship between the morphokinetics of meiotic progression and egg aneuploidy. Cumulus-oocyte complexes were collected from large antral follicles from hyperstimulated CD-1 mice. Cumulus cells were removed, and spontaneous IVM was performed in the absence or presence of two doses of Nocodazole (25 or 50 nM) to induce a spectrum of spindle abnormalities and chromosome segregation errors during oocyte meiosis. Comprehensive chromosome screening was then performed in the resulting eggs, and morphokinetic variables and ploidy status were compared across experimental groups (control, n = 11; 25 nM Nocodazole, n = 13; 50 nM Nocodazole, n = 23). We monitored IVM in mouse oocytes using time-lapse microscopy for 16 h, and time to germinal vesicle breakdown (tGVBD), tPB1, and dMI were analyzed. Following IVM, comprehensive chromosome screening was performed on the eggs and their matched first polar bodies via adaptation of an NGS-based preimplantation genetic testing for aneuploidy (PGT-A) assay. Bioinformatics analysis was performed to align reads to the mouse genome and determine copy number-based predictions of aneuploidy. The concordance of each polar body-egg pair (reciprocal errors) was used to validate the results. Ploidy status was categorized as euploid, 1-3 chromosomal segregation errors, or ≥4 chromosomal segregation errors. Additionally, aneuploidy due to premature separation of sister chromatids (PSSC) versus non-disjunction (NDJ) was distinguished. We applied and validated state-of-the-art NGS technology to screen aneuploidy in individual mouse eggs and matched polar bodies at the chromosome-specific level. By performing IVM in the presence of different doses of Nocodazole, we induced a range of aneuploidy. No aneuploidy was observed in the absence of Nocodazole (0/11), whereas IVM in the presence of 25 and 50 nM Nocodazole resulted in an aneuploidy incidence of 7.69% (1/13) and 82.61% (19/23), respectively. Of the aneuploid eggs, 5% (1/20) was due to PSSC, 65% (13/20) to NDJ, and the remainder to a combination of both. There was no relationship between ploidy status and tGVBD, but tPB1 and the dMI were both significantly prolonged in eggs with reciprocal aneuploidy events compared to the euploid eggs, and this scaled with the severity of aneuploidy. Eggs with ≥4 aneuploid chromosomes had the longest tPB1 and dMI (P < 0.0001), whereas eggs with one to three aneuploid chromosomes exhibited intermediate lengths of time (P < 0.0001). N/A. We used Nocodazole in this study to disrupt the meiotic spindle and induce aneuploidy in mouse oocytes. Whether the association between morphokinetic variables of meiotic progression and the severity of aneuploidy occurs with other compounds that induce chromosome segregation errors remain to be investigated. In addition, unlike mouse oocytes, human IVM requires the presence of cumulus cells, which precludes visualization of morphokinetic variables of meiotic progression. Thus, our study may have limited direct clinical translatability. We validated NGS in mouse eggs to detect aneuploidy at a chromosome-specific resolution which greatly improves the utility of the mouse model. With a tractable and validated model system for characterizing meiotic aneuploidy, investigations into the molecular mechanisms and factors which may influence aneuploidy can be further elaborated. Time-lapse analyses of morphokinetic variables of meiotic progression may be a useful non-invasive predictor of aneuploidy severity. This work was supported by the Bill & Melinda Gates Foundation (INV-003385). Under the grant conditions of the Foundation, a Creative Commons Attribution 4.0 Generic License has already been assigned to the Author Accepted Manuscript version that might arise from this submission. The authors have no conflict of interest to disclose.

Expansion Grade and Morphokinetic Parameters Associated with Aneuploidy Status of Embryo Fifth Day

Objective: To determine the potential of examining embryo morphology, and morphokinetic parameters in predicting the chromosomal status of embryos.&#x0D; Methods: This study is a cross-sectional that requires patients undergoing IVF followed by chromosome examination with NGS that was conducted at the IVF Center at Pondok Indah Hospital and Morula IVF Center at Bunda Hospital from December 2021 to December 2022. Each embryo that reaches the blastocyst stage on day 5 or 6 will be washed and put into a PCR tube for a week; then, embryologists annotate them to determine morphological assessment and morphokinetic parameters using Time-Lapse Microscopy. The chi-square test was used to analyse bivariate variables.&#x0D; Results: One hundred twenty-four samples were collected on day 5 of patients undergoing the IVF procedure. 50.8% of the samples were aneuploid chromosomes, and 49.2% were euploid. The morphokinetic characteristics median was 3.86 fold. It was found that expansion grade, time to pro-nuclear fading, and time to the synchrony of the third cell cycle were significantly associated with euploid status (p = 0.000; 0.041 and 0.036).&#x0D; Conclusion: The expansion grade has been proven as the most influential component for accurately predicting the ploidy status of embryos.&#x0D; Keyword: blastocyst, embryo, euploid status, expansion grade, morphokinetics&#x0D; &#x0D; Abstrak&#x0D; Tujuan: Untuk mengetahui potensi pemeriksaan morfologi embrio, dan parameter morfokinetik dalam memprediksi status kromosom embrio.&#x0D; Metode: Penelitian ini merupakan penelitian potong lintang yang memerlukan pasien yang sedang menjalani program bayi tabung yang dilanjutkan dengan pemeriksaan kromosom dengan NGS yang dilakukan di Pusat IVF RS Pondok Indah dan Pusat IVF Morula RS Bunda pada bulan Desember 2021 hingga Desember 2022. Setiap embrio yang mencapai tahap blastokista pada hari ke 5 atau 6 akan dicuci dan dimasukkan ke dalam tabung PCR selama seminggu; kemudian, ahli embriologi membuat anotasi untuk menentukan penilaian morfologi dan parameter morfokinetik menggunakan Mikroskop Time-Lapse. Uji chi-square digunakan untuk menganalisis variabel bivariat.&#x0D; Hasil: Seratus dua puluh empat sampel dikumpulkan pada hari ke 5 dari pasien yang menjalani prosedur IVF. 50,8% sampel adalah kromosom aneuploid, dan 49,2% adalah euploid. Median karakteristik morfokinetik sebesar 3,86 kali lipat. Ditemukan bahwa tingkat ekspansi, waktu menuju pemudaran pro-nuklir, dan waktu hingga sinkronisasi siklus sel ketiga berhubungan secara signifikan dengan status euploid (p = 0,000; 0,041 dan 0,036).&#x0D; Kesimpulan: Tingkat ekspansi telah terbukti sebagai komponen yang paling berpengaruh dalam memprediksi status ploidi embrio secara akurat.&#x0D; Kata kunci: blastosist, embrio, status euploid, tingkat ekspansi, morfokinetik

P-171 Identification of aneuploid embryos using cell free DNA in embryo culture medium

Abstract Study question Can noninvasive preimplantation genetic testing (PGT) using cell free DNA from embryo culture medium discriminate embryonic aneuploidy status? Summary answer Aneuploid embryos in older patients can be identified by cell free DNA in embryo culture medium. What is known already There have been reports of non-invasive PGT by analysing cell free DNA (cfDNA) released from embryos into culture medium, and found to be highly correlated with the analysis results of Trophectoderm (TE)-biopsy (Carmen.Rubio, 2020).We have initiated in 2021, a preliminary research program for the clinical application of niPGT. In aneuploid embryos, we have found a tendency for a high concordance rate between cfDNA analysis and blastocyst analysis results, suggesting the usefulness of cfDNA analysis in terms of identifying aneuploid embryos.Subsequently, we examined the usefulness of cfDNA analysis by comparing the concordance rate with cfDNA analysis and TE biopsy of blastocysts. Study design, size, duration After thawing pronuclear-stage frozen embryos for which consent was obtained for research use, they were co-cultured until Day 4, and then individually cultured on in new dishes.40 embryos that reached morula on day 4 or day 5 and blastocyst on day 6 or day 7 were included. Participants/materials, setting, methods The biopsied TE, the biopsied blastocyst, and the cfDNA obtained after individual culture were all sent to an external provider (Igenomix: Valencia, Spain) for chromosomal aneuploidy analysis by NGS. The concordance rate of the analysis for blastocysts in cfDNA and TE was calculated by dividing them into euploidy and aneuploidy, and by age. Concordance between the results of the blastocyst analysis and the culture medium were assessed according to the standards of the inspection company. Main results and the role of chance For the blastocyst analysis results, the concordance rate with cfDNA was 76.5% (26/34) and the concordance rate with TE was 85.3% (29/34), showing no difference. For the euploid blastocysts analyzed, the matching rate with cfDNA was 61.1% (11/18) and with TE was 77.8% (14/18), showing no difference. Similarly, for aneuploid blastocysts, the concordance rate with cfDNA was 93.8% (15/16) and with TE was 93.8% (15/16), showing no difference. Detailed analysis of aneuploid blastocysts revealed that the concordance rate with cfDNA was 88.9% (8/9) and the concordance rate with TE was 88.9% (8/9) in women under 39 years of age. In addition, the concordance rate with cfDNA was 100.0% (7/7) with TE was 100.0% (7/7) in those aged 40 and over. (Fisher's exact test) Limitations, reasons for caution Concordance between cfDNA and blastocyst analyzes in euploid embryos was low, continued follow-up is considered necessary. Wider implications of the findings In a detailed analysis of aneuploid blastocysts aged 40 years or older, Similar to TE, cfDNA showed a high concordance rate with the results of blastocyst analysis, demonstrating that cfDNA provides sufficient accuracy for identifying aneuploid embryos in women over 40 years of age. Trial registration number not applicable

MATERNAL FIBROID IMPACT ON CELL FREE DNA (cfDNA) SCREENING

In cell free DNA (cfDNA) screening, a proportion of the circulating cfDNA is placental and serves as a proxy for fetal aneuploidy status. As the remaining cfDNA originates from maternal tissue, identification of unexpected maternal findings is possible. Like other tissues, fibroids can contribute cfDNA fragments to maternal plasma. ∼50% of fibroids have abnormal chromosome complements which may confound fetal aneuploidy screening.1-3 Here we investigate cfDNA data and results from 57 patients with known fibroids.

P-239 Embryonic quality assessment from microRNA profiling

Abstract Study question Does the microRNA expression profile in blastocysts change with different embryonic quality parameters? Summary answer MicroRNA expression showed significant changes when aneuploidy was present, in day-6 compared to day-5 blastocysts and with different trophectoderm (TE) morphology grades. What is known already MicroRNA (miRNA) is a gene expression regulatory mechanism used as a diagnostic marker for some conditions. Despite the great magnitude of studies investigating the miRNA role in implantation and pregnancy complications, limited work has been done to evaluate it as a potential marker for preimplantation embryo quality. With the accelerated changes in gene expression at early embryos, the miRNA profile is a potential indicator of embryo quality. This study investigates the blastocyst miRNA profile in relation to three quality parameters: aneuploidy, the day of blastocyst formation and morphology. Study design, size, duration We collected 122 surplus embryos donated for research by 46 consenting couples from Centre for Reproductive and Genetic Health, London. All samples were ICSI derived blastocysts (day5/6) with PGT-A results. For aneuploidy status comparison, samples were categorized according to the number of chromosomes affected (single/multiple) and type of aneuploidy (gain/loss/partial), compared to euploid. The day of blastocyst development has (day5/day6) groups, and finally samples were compared based on TE morphology (excellent/good/fair). Participants/materials, setting, methods Frozen human embryos were individually thawed and transferred to a lysis buffer. The miRNAs were extracted from each sample then sequenced by next generation sequencing (NGS) using miRNA Library Kit (QIAGEN) with NextSeqTM 500 (illumina). The differential expression analysis was conducted using DESeq2 via GeneGlobe (QIAGEN). We set the significance cut-offs to (-2&amp;gt; foldchange &amp;gt;2) and (&amp;lt;0.05 FDR p-value). For gene ontology and pathway annotation, we used miRTargetLink 2.0 and miRPathDB 2.0 platforms. Main results and the role of chance Compared to the euploid blastocysts, all aneuploid categories, except the single chromosome aneuploidy, had significant differences in miRNA expression. Nine miRNAs were altered in the group with multiple aneuploidies, and one was highly upregulated in embryos with partial losses or gains. The majority of differentially expressed (DE) miRNAs in samples with whole chromosomal gain or loss were downregulated. Conversely, four miRNAs showed a significant increase in day-6 blastocysts compared to day-5. Morphology grade comparisons showed no differences between excellent and good TE; however, six miRNAs were significantly changed in fair TE compared to good. The frequently altered miRNAs in these comparisons were ( hsa-let-7c-5p, hsa-miR-206, hsa-miR-184 and hsa-miR-203a-3p ). Unsurprisingly, pathway annotation of the DE miRNAs showed an involvement of key biological processes, like apoptosis, cell-cycle and differentiation, signalling and gene expression regulation, in all groups. The pathways of note in aneuploid blastocysts included DNA damage response, intrinsic and extrinsic apoptotic signalling, and regulation of cell-cycle G1/S associated events. In blastocyst day5/day6 comparison, embryonic and organ development were the most involved pathways, while in TE morphology, kinase activity and phosphorylation along with apoptosis and DNA damage response were significant. Limitations, reasons for caution Differential expression analysis based on inner cell mass (ICM) morphology and blastocyst expansion were not applicable due to the inadequate number of embryos per group; as all the samples collected were in excellent or good quality. Wider implications of the findings Gene expression profiling of miRNAs in human blastocysts will potentially identify biomarkers predictive of implantation success. Trial registration number not applicable

Comprehensive analysis of aneuploidy status and its effect on the efficacy of EGFR-TKIs in lung cancer.

BackgroundLung cancer has the highest mortality rate among cancers worldwide, and most patients are diagnosed with non-small-cell lung cancer (NSCLC), and evaluating the clinical efficacy of molecularly targeted cancer therapy remains a major challenge.MethodsThis paper retrospectively investigated the outcome information of 291 lung cancer patients detected by next-generation sequencing (NGS) analysis and fluorescence in situ hybridization (FISH), including 63 patients with lung cancer who were followed up. We analyzed epidermal growth factor receptor (EGFR) mutation abundance and aneuploidy status to evaluate clinical efficacy.ResultsThe progress free survival (PFS) of patients diagnosed as euploidy was actually higher than that of patients diagnosed with aneuploidy, and was related to both the objective response rate (ORR) and disease control rate (DCR). Patients with an epidermal growth factor receptor (EGFR) mutation abundance ≥28.86% had slightly higher ORR and similar DCR. Two-way analysis of variance was used to assess the effects of EGFR mutation abundance and tumor aneuploidy status on patients’ PFS. The results indicated a strong correlation between aneuploidy status and clinical efficacy, with euploid patients having a higher ORR and DCR.ConclusionsAneuploidy status could effectively evaluate the clinical efficacy of patients with lung cancer. However, EGFR mutations abundance could not predict the extent of benefit from tyrosine kinase inhibitors (EGFR-TKI) treatment.

Chromosomally normal miscarriage is associated with vaginal dysbiosis and local inflammation

BackgroundEmerging evidence supports an association between vaginal microbiota composition and risk of miscarriage; however, the underlying mechanisms are poorly understood. We aim to investigate the vaginal microbial composition and the local immune response in chromosomally normal and abnormal miscarriages and compare this to uncomplicated pregnancies delivering at term.MethodsWe used 16S rRNA gene based metataxonomics to interrogate the vaginal microbiota in a cohort of 167 women, 93 miscarriages (54 euploid and 39 aneuploid using molecular cytogenetics) and 74 women who delivered at term and correlate this with the aneuploidy status of the miscarriages. We also measured the concentrations of IL-2, IL-4, IL-6, IL-8, TNF-α, IFN-γ, IL-1β, IL-18 and IL-10 in cervical vaginal fluid.ResultsWe show that euploid miscarriage is associated with a significantly higher prevalence of Lactobacillus spp. deplete vaginal microbial communities compared to aneuploid miscarriage (P = 0.01). Integration of matched cervicovaginal fluid immune-profiles showed that Lactobacillus spp. depleted vaginal microbiota associated with pro-inflammatory cytokine levels most strongly in euploid miscarriage compared to viable term pregnancy (IL-1β; P < 0.001, IL-8; P = 0.01, IL-6; P < 0.001).ConclusionsOur data suggest the vaginal microbiota plays an important aetiological role in euploid miscarriage and may represent a target to modify risk of pregnancy loss.

Sperm DNA fragmentation and reproductive outcomes

In the last few years, male infertility is increasingly catching the attention of researchers. The impact of spermatozoal factors on the fertilization and early embryo development is being deciphered. An association of sperm aneuploidy status in unexplained recurrent pregnancy loss has been observed. Sperm DNA damage or the sperm DNA fragmentation (sDF) is now measurable as DNA fragmentation index. The methods of evaluating sDF are many and they differ in their technique and paraphernalia needed to perform them. The most common methods are SCSA, TUNEL, SCD, COMET. TUNEL utilizes tagging of DNA in places where there is a break and then quantifying them. SCD is based on the principal of susceptibility of fragmented DNA to acid denaturation. COMET assay involves variable movement of intact and fragmented DNA under the influence of electric field. There are different methods to perform each of the previous mentioned tests though the principle remains the same. Each laboratory performing these tests needs to standardize them and should have their own cutoffs, which should relate to the procedure [intrauterine insemination/in vitro fertilization (IVF)/intracytoplasmic sperm injection] being performed. The role of sDF is slowly getting defined in recurrent implantation failures, unexplained infertility, varicocele, and IVF failures. Yet the jury is still out.

CLINICAL BENEFITS OF CULTURING INTRACYTOPLASMIC SPERM INJECTION-DERIVED NONPRONUCLEATED (0PN) AND MONOPRONUCLEATED (1PN) ZYGOTES, AND THE IMPORTANCE OF VERIFYING BIPARENTAL CHROMOSOME INHERITANCE IN 1PN-DERIVED BLASTOCYSTS

Evaluate the developmental competence and aneuploidy status of blastocysts derived from 0PN and 1PN zygotes following intracytoplasmic sperm injection (ICSI) and preimplantation genetic testing for aneuploidy (PGT-A). Elucidate biparental chromosome inheritance in female 1PN-derived blastocysts.

P–262 Comparison of embryo aneuploidy rate and reproductive outcomes of ART cycles using fresh and vitrified donor oocytes

Abstract Study question Is there any difference in blastocyst morphology, embryo aneuploidy rate and ART clinical outcomes when using fresh or vitrified donor oocytes? Summary answer Frequency of good quality blastocyst obtained from fresh oocytes is significantly higher compared to vitrified.No difference in embryo aneuploidy rates nor clinical outcomes were found. What is known already Oocytes vitrification is an efficient method that allows non only fertility preservation but also the creation of donor oocytes banks, optimizing clinical resources for patients undergoing Assisted Reproductive Technology. Although the benefits of donor oocytes vitrification are well known, some studies have shown that this cryopreservation process can induce spindle abnormalities and chromosomal changes, leading to aneuploidy. Comparative studies between fresh and vitrified oocytes to evaluate embryo developmental competence, aneuploidy and clinical pregnancy rate (CPR) are needed. Study design, size, duration This retrospective study includes ICSI cycles with fresh donor oocytes(N = 2795) and vitrified donor oocytes (N = 1225) between January 2019 and September 2020. Pre-implantation Genetic Testing for Aneuploidy (PGT-A) was performed on Day 5 and Day 6 blastocysts. Fertilization rate, blastocyst morphology, aneuploidy status and CPR were analysed and compared between the groups. Recipients were equally distributed in terms of maternal age (40.86 years) and previous history, sperm samples were also similar in profile and origin (fresh-frozen). Participants/materials, setting, methods A total of 266 subfertile couples participated in the study, ICSI was carried out in all cycles. Vitrification and warming protocols were performed with a commercial kit. All embryos were cultured to blastocyst stage in a Time-Lapse incubator and assessed by Gardner’s blastocyst grading scale. PGT-A testing was performed on trophectoderm biopsies by Next Generation Sequencing (NGS). Single/double embryo transfers were performed in all cases. Odd-ratios were calculated,and Chi-square was performed for the statistical analysis. Main results and the role of chance A total of 266 patients underwent 289 donor oocyte cycles yielding an overall of 4557 oocytes. ICSI was performed on 2795 fresh and 1225 vitrified mature oocytes. Similar fertilization rates were achieved with fresh and vitrified oocytes (75.9% (2122/2795) and 75.2% (921/1225), respectively (P = 0.6)) yielding a significant difference in blastocyst rate of 71.7% (1522/2122) and 62.5% (576/921) (OR 1,519; 95% CI 1,290–1,789; p &amp;lt; 0.001). In addition, when blastocysts morphology was analysed, a significant difference was shown in the frequency of good quality embryos that decreases from 56.6% (861/1522) with fresh oocytes to 51% (294/576) with vitrified oocytes (OR 1,249; 95% CI 1,031–1,514; P &amp;lt; 0.02). PGT-A testing of blastocysts revealed not significant differences in euploidy rates (73.6% in fresh oocytes vs 76.8% vitrified oocytes, P = 0.2). With regards to clinical outcomes, similar results were found between the groups. A total of 322 embryo transfers were performed (237 from fresh and 85 vitrified) achieving a CPR of 48.9% (116/237) with fresh oocytes and 54% (46/85) with vitrified (P = 0.7) and a pregnancy loss of 6.7%(16/237) in fresh oocytes and 11.7%(10/85) vitrified oocytes (P = 0.1). Limitations, reasons for caution The study was conducted on a small number of cases. Further studies are needed to confirm our findings. Moreover, although the same stimulation protocol was used, donors from different background were included. Wider implications of the findings: This study supports the use of vitrified oocytes in the laboratory routine without compromising clinical outcomes. Although oocyte vitrification may have an influence on embryo morphology, blastocyst rate, no impact of this cryopreservation process is seen on embryo aneuploidy, developmental competence and CPR. Trial registration number Not applicable

O-129 Lactobacillus deplete vaginal microbial composition is associated with chromosomally normal miscarriage and local inflammation

Abstract Study question To investigate the vaginal microbial composition and the local immune response in chromosomally normal and abnormal miscarriages and compare this to uncomplicated pregnancies delivering at term. Summary answer We show that euploid miscarriage is associated with a significantly higher prevalence of Lactobacillus spp. deplete vaginal microbial communities compared to aneuploid miscarriage. What is known already Emerging evidence supports the role of the vaginal microbiota in adverse pregnancy outcome, but the underlying mechanisms are poorly understood. A dominance of Lactobacillus spp. in pregnancy provides protection against pathogenic bacteria by producing lactic acid and antimicrobial compounds. A depletion in Lactobacillus spp. is often linked to adverse pregnancy outcomes.Current work also implicates the reproductive tract microbiota as a key modulator of local inflammatory and immune pathways. We have previously shown that miscarriage is associated with vaginal dysbiosis but without knowledge of the cytogenetic status of those miscarriages or the local immune profile. Study design, size, duration This study was a prospective observational cohort study based at Queen Charlotte’s &amp; Chelsea Hospital, Early Pregnancy Unit, London between March 2014-February 2019. Vaginal swabs were collected from the posterior vaginal fornix of 167 patients. Participants/materials, setting, methods We used 16S rRNA gene based metataxonomics to interrogate the vaginal microbiota in a cohort of 167 women, 93 miscarriage patients (54 euploid and 39 aneuploid using molecular cytogenetics) and 74 women who delivered at term and correlate this with the aneuploidy status of the miscarriages. We also measured the concentrations of IL-2, IL-4, IL-6, IL-8, TNF-α, IFN-γ, IL-1β, IL-18 and IL-10 in cervical vaginal fluid using Human Magnetic Luminex Screening Assay (8-plex). Main results and the role of chance We show that euploid miscarriage is associated with a significantly higher prevalence of Lactobacillus spp. deplete vaginal microbial communities compared to aneuploid miscarriage (P=0.008). In women having Lactobacillus spp. deplete vaginal microbial communities, euploid miscarriage associates with higher concentrations of pro-inflammatory cytokines IL-1β, IL-8, IL-6 (P&amp;lt;0.001, P=0.01 and P&amp;lt;0.001 respectively) and lower concentrations of anti-inflammatory cytokines IL10 (P&amp;lt;0.001) when compared to viable term pregnancy. We identified Prevotella bivia and Streptococcus as particularly common in euploid miscarriage and as drivers of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α). Co-occurrence network analyses revealed low levels of co-occurrence between Lactobacillus crispatus and other organisms and strong co-occurrence between Streptococcal species. Our data show a combination of both an adverse vaginal microbiota and a cytokine response to it influences early pregnancy outcome. Although this may be a reflection of intrinsic maternal immune response, it appears that the cytokine response is largely driven by the bacterial taxa present in the vagina, which presents an opportunity for specific, directed intervention. The negative co-occurrence between L.crispatus and all other organisms suggests a possible therapeutic role for probiotics containing this organism. The influence of Streptococci also suggests a potential benefit of targeted antibiotics with probiotics for some patients. Limitations, reasons for caution There were no longitudinal samples in this cohort and our results are based on the assumption that the vaginal microbial composition is stable throughout the first trimester.Future longitudinal studies with larger sample sizes are needed to corroborate these findings and provide insights to the mechanisms that trigger the inflammatory response. Wider implications of the findings These findings support the hypothesis that the vaginal microbiota plays an important aetiological role in euploid miscarriage and may represent a target to modify the risk of pregnancy loss. Trial registration number n/a

Detection of Aneuploidy in Cerebrospinal Fluid from Patients with Breast Cancer Can Improve Diagnosis of Leptomeningeal Metastases.

Detection of leptomeningeal metastasis is hampered by limited sensitivities of currently used techniques: MRI and cytology of cerebrospinal fluid (CSF). Detection of cell-free tumor DNA in CSF has been proposed as a tumor-specific candidate to detect leptomeningeal metastasis at an earlier stage. The aim of this study was to investigate mutation and aneuploidy status in CSF-derived cell-free DNA (cfDNA) of patients with breast cancer with a clinical suspicion of leptomeningeal metastasis. cfDNA was isolated from stored remnant CSF and analyzed by targeted next-generation sequencing (NGS; n = 30) and the modified fast aneuploidy screening test-sequencing system (mFAST-SeqS; n = 121). The latter method employs selective amplification of long interspaced nuclear elements sequences that are present throughout the genome and allow for fast and cheap detection of aneuploidy. We compared these results with the gold standard to diagnose leptomeningeal metastasis: cytology. Leptomeningeal metastasis was cytology proven in 13 of 121 patients. Low DNA yields resulted in insufficient molecular coverage of NGS for the majority of samples (success rate, 8/30). The mFAST-SeqS method, successful in 112 of 121 (93%) samples, detected genome-wide aneuploidy in 24 patients. Ten of these patients had cytology-proven leptomeningeal metastasis; 8 additional patients were either concurrently diagnosed with central nervous system metastases by radiological means or developed these soon after the lumbar puncture. The remaining six cases were suspected of leptomeningeal metastasis, but could not be confirmed by cytology or imaging. Aneuploidy was associated with development of leptomeningeal metastasis and significantly worse overall survival. Aneuploidy in CSF-derived cfDNA may provide a promising biomarker to improve timely detection of leptomeningeal metastasis.

Clinical benefits of culturing monopronucleated zygotes derived from intracytoplasmic injection cycles

Objective To evaluate the developmental competence and aneuploidy status of blastocysts derived from monopronucleated (1PN) zygotes following intracytoplasmic injection (ICSI) and preimplantation genetic testing for aneuploidy (PGT-A). Design Prospective, cohort study using chi-square tests to assess differences (p Materials and methods Fertilization was assessed ∼16-18 hrs post-ICSI for the presence of 2PN. All zygotes were cultured in LG medium + additives and subdivided by PN status. Blastocyst formation was assessed on days 5, 6 and 7 of development, and blastocysts of suitable quality were biopsied and/or cryopreserved. The trophectoderm samples were analyzed at Ovation Genetics for aneuploidy using NGS. Results We obtained 75 1PN zygotes from 54 fresh ICSI cycles, representing 26.60% of the total fresh ICSI cycles. Twenty-six (34.67%) developed to the blastocyst stage, with 14 (18.67%) being biopsied/cryopreserved. From the same fresh ICSI cycles we also obtained 626 2PN zygotes of which 456 (72.84%) developed to the blastocyst stage, and 372 (59.43%) were biopsied and/or cryopreserved. Both the 1PN blastocyst rate and the 1PN biopsy/cryopreservation rate were found to be significantly lower than that of 2PN-derived embryos. Interestingly 85.71% of the biopsied 1PN blastocysts reported a euploid result, compared to 53.03% for 2PN blastocysts. The latter euploidy rates were determined to be higher (P Conclusions Previously, 1PN zygotes were routinely considered abnormal and thus discarded. Our data confirms that 1PN zygotes can develop into euploid blastocysts. This coincides with previous work showing the potential of 1PN zygotes to develop into genetically normal blastocysts capable of producing healthy live births. A standard protocol for the selection of normal 1PN embryos derived from ICSI cycles has however not yet been established. Our data suggests that in fresh ICSI cycles blastocyst stage growth followed by biopsy and PGT-A testing can select for euploid 1PN embryos. However, while PGT-A using NGS technology is designed to detect numerical abnormalities of whole chromosomes, it does not have the ability to screen for uniparental disomy or haploidy. Therefore, to consider female 1PN-derived embryos for transfer additional genetic testing is suggested to discern if the embryo is diploid with a bi-parental genome. These tests and additional data recovery are ongoing as we seek to discover the clinical value of 1PN, ICSI-derived zygotes, especially in cycles where few blastocysts are produced or biopsied. Disclosures None Funding None

MLPA and DNA index improve the molecular diagnosis of childhood B-cell acute lymphoblastic leukemia

Aneuploidy occurs within a significant proportion of childhood B-cell acute lymphoblastic leukemia (B-ALL). Some copy number variations (CNV), associated with novel subtypes of childhood B-ALL, have prognostic significance. A total of 233 childhood B-ALL patients were enrolled into this study. Focal copy number alterations of ERG, IKZF1, PAX5, ETV6, RB1, BTG1, EBF1, CDKN2A/2B, and the Xp22.33/Yp11.31 region were assessed by Multiplex Ligation-dependent Probe Amplification (MLPA). The MLPA telomere kit was used to identify aneuploidy through detection of whole chromosome loss or gain. We carried out these procedures alongside measurement of DNA index in order to identify, aneuploidy status in our cohort. MLPA telomere data and DNA index correlated well with aneuploidy status at higher sensitivity than cytogenetic analysis. Three masked hypodiploid patients, undetected by cytogenetics, and their associated copy number neutral loss of heterozygosity (CN-LOH) were identified by STR and SNP arrays. Rearrangements of TCF3, located to 19p, were frequently associated with 19p deletions. Other genetic alterations including iAMP21, IKZF1 deletions, ERG deletions, PAX5AMP, which have clinical significance or are associated with novel subtypes of ALL, were identified. In conclusion, appropriate application of MLPA aids the identifications of CNV and aneuploidy in childhood B-ALL.

Identification of a rare tri-allelic inheritance at the D8S1179 locus in a case of paternity testing

Short Tandem Repeat (STR) typing is routinely used in forensic casework, paternity disputes and in the detection of aneuploidies. Anomalous peak patterns are occasionally encountered in STR typing due to genetic variations, mutations and, other abnormalities. We report here a rare tri-allelic variant at the D8S1179 locus in a 6-month-old female child, observed during routine paternity testing in the Indian Tamil population.DNA eluted from the blood samples collected on FTA elute microcards were subjected to STR typing for autosomal and sex STR loci using the commercial AmpFlSTR Identifiler Plus kit and the Promega PowerPlex® Fusion kit. Capillary electrophoresis was carried out in ABI 3130xl Genetic Analyzer and genotyped using GeneMapper ID-X Software v1.5. Copy number variations and aneuploidy status of chromosome 8 were analyzed using Multiplex ligation-dependent probe amplification (MLPA) and Prenatal BoBs assays. In this case of paternity exclusion, the child displayed a Type II tri-allelic pattern (10,13,14) at the D8S1179 locus. In addition, the mother displayed an abnormal 10,14 genotype (with heterozygous peak imbalance - 2:1) at the D8S1179 locus suggesting 10,10,14 pattern. MLPA and prenatal BoBs analyses did not detect any abnormalities on chromosome 8. These results suggest an intrachromosomal segmental duplication event at the D8S1179 locus (8q24.13) as the reason behind tri-allelic pattern and its inheritance. Understanding anomalous cases such as these can help in appropriate interpretation of the results in STR typing.

13. CORRELATION BETWEEN ANEUPLOIDY, MOSAICISM AND MORPHOKINETIC DEVELOPMENT IN 1550 BIOPSIED BLASTOCYSTS

Introduction Despite the high number of recent studies, conflicting data are reported, and there is still considerable disagreement regarding which morphokinetic parameters are useful to predict blastocyst formation, implantation potential and ploidy status of the embryo. The first attempt to develop a model predicting embryo implantation used the time of division to 5 cells, the time between division from 3 to 4 cells and the time between division from 2 to 3 cells (Meseguer et al., 2011). More recently, the aneuploidy status of embryos was related to the start of blastulation and the formation of a full blastocyst. The aneuploidy risk classification built proved beneficial in a correlation with live birth when applied to non-biopsied embryos (Campbell et al., 2013). Material and Methods This retrospective study was based on 1550 blastocysts which were subjected to preimplantation genetic testing for aneuploidy (PGT-A) between August 2011 and December 2018 in Istanbul Memorial Hospital (n=659 euploids; n=831 aneuploids; n=60 mosaics). Incubation was performed in time-lapse incubators (EmbryoScope™). All relevant events (fertilization, cleavages, morula and blastocyst formation) were checked daily, and time of cleavage to two-cell embryo (t2) and subsequent divisions t3, t4, t5, t6, t7, t8, t9+, tM, tSB, tB and tEB were recorded in the EmbryoViewer® workstation. PGT-A was done by NGS ReproSeq on Ion Torrent S5 (Thermo Fisher Scientific) or aCGH (Illumina) following trophectoderm biopsy. Mann-Whitney U and Kruskal Wallis tests were used in this study. Results When euploid embryos were compared with aneuploids, the time to reach the blastocyst stage was 3 hours earlier in euploids (tB=104.60h vs. 107.64h; p Conclusions The mosaic embryos’ morphokinetic parameters were similar with the euploid blastocysts. Observed time-lapse differences between euploid and aneuploid blastocysts suggests that the embryos are being affected by the chromosomal constitution throughout the embryo development. Even though the variances at the initial cleavages are as short as half an hour, this increases to a three hour at the blastocyst stage. The study presented above can be used to prioritize the embryos according to the morphokinetic variables to biopsy and diagnose the embryos with highest chance of being euploid thus reducing the costs especially for couples who have more than one blastocyst.

Abstract 974: The molecular and genomic drivers of immunologically cold tumors: Novel discoveries from pan-cancer analysis

Abstract Immune-checkpoint blockade (ICB) is able to elicit remarkable immune response but currently the durable response is limited to a minority of cancer patients. Recent evidence suggests that a prerequisite for the efficacy of ICB, for example the PD-1 inhibitor, is T-cells infiltration in the tumor microenvironment (TME). The immunologically ‘quite’ and T-cell depleted (immune ‘desert’) tumors are less likely respond to ICB. However, we still lack a detailed understanding of the genomic and molecular determinants that drive the ‘cold’ immune phenotypes. We here performed a comprehensive pan-cancer analysis to define and characterize the immunologically ‘hot’ (inflamed) and ‘cold’ (quite or T-cell depleted) tumors by high-dimensional, multi-platform data integration of 9,887 tumors from The Cancer Genome Atlas (TCGA). This study provided an unprecedented catalog of the molecular and genomic drivers of immune ‘cold’ tumors by analyzing the immune cell composition, patterns of their spatial organization within the TME, the dysregulated chemokines networks, somatic mutations, DNA copy-number alterations, aneuploidy status, differentially expressed immune regulatory genes, and enriched signaling pathways. We showed that the immunologic status is highly heterogeneous within and across TCGA tumor types and subtypes. We integrated the information of T-cell spatial organization with our immune phenotypes and found that the ‘band-like’ spatial organization (T cells clustered at the invasive margin of the tumor and didn’t get into the tumor body) pattern is strongly associated with the ‘cold’ phenotype, particularly in colon and stomach cancers. Our analysis revealed a number of other factors that are associated with the ‘cold’ phenotype by either preventing T-cell infiltration or promoting T-cell dysfunction, and we performed functional validation on several targets of interest. This study provided an in-depth understanding of the genomic and molecular determinants of immunologically ‘cold’ tumors and suggested potential biomarkers that can be targeted for the development of novel combination therapy strategies to convert the ‘cold’ tumors into ‘hot’ ones and improve the odds of cancer immunotherapy success. Citation Format: Guangchun Han, Ruiping Wang, Jianfeng Shen, Pankaj Singh, Shaojun Zhang, Arvind Rao, Jianjun Gao, Alexander Lazar, Andrew Futreal, Guang Peng, Kunal Rai, Linghua Wang. The molecular and genomic drivers of immunologically cold tumors: Novel discoveries from pan-cancer analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 974.

Uncertainty of fetal fraction determination in Non-Invasive Prenatal Screening by highly polymorphic SNPs

Fetal fraction and the chromosome representation are the two key quantities used in Non-Invasive Prenatal Screening (NIPS) to determine the aneuploidy status of a fetus. Several methods for fetal fraction determination have been proposed in the literature, including a class of the methods, denoted snpFF, based on high-coverage targeted sequencing of highly polymorphic Single Nucleotide Polymorphisms (SNPs). The variant of snpFF, investigated here, has similar properties as the other variants of snpFF. We point out that the variability of the individual informative SNPs-based estimates of fetal fraction increases with the increase of fetal fraction. At 4% fetal fraction the Inter-Quartile Range (IQR) of the individual estimates of fetal fraction is around 3% and it increases to 6% at 15% fetal fraction. snpFF cannot detect fetal fraction below 2.5% because the number of informative SNPs becomes too small, even zero.

Ensuring high standards for the delivery of NIPT world-wide: Development of an international external quality assessment scheme.

ObjectiveTo ensure accurate and appropriate reporting of non‐invasive prenatal testing (NIPT) results, the standard of testing should be measured and monitored by participation in external quality assessment (EQA) schemes. The findings from international pilot EQAs for NIPT for the common trisomies are presented.MethodsIn the first pilot, three EQA providers used artificially manufactured reference materials to deliver an EQA for NIPT. The second pilot used clinically collected maternal plasma samples. The testing and reporting for aneuploidy status was performed by participating laboratories using routine procedures. Reports were assessed against peer ratified criteria and EQA scores were returned to participants.ResultsForty laboratories participated in the first. Genotyping accuracy was high; four laboratories reported a critical genotyping error (10%) and two reported partial results. Eighty seven laboratories participated in the second pilot using maternal plasma, two reporting a critical genotyping error (2.3%). For both rounds, report content was variable with key information frequently omitted or difficult to identify within the report.ConclusionsWe have successfully delivered an international pilot EQA for NIPT. When compared with currently available manufactured materials, EQA for NIPT was best performed using clinically collected maternal plasma. Work is required to define and improve the standard of reporting.