Day-3 Biopsy Research Articles

P-742 Universal genome-wide haplotyping-based preimplantation genetic testing: a systematic seven-year experience at a single center

Abstract Study question To record the outcome and clinical benefit of universal genome-wide haplotyping-based preimplantation genetic testing (PGT). Summary answer Universal PGT allows for standardization of protocols and reduces turnaround time. Apart from accurate genetic diagnosis, it also provides chromosome information, improving embryo selection strategies. What is known already Developments in genome-wide technologies leveraged the implementation of universal PGT that combines haplotyping and copy number typing across the whole genome. Universal PGT provides diagnosis for any familial monogenic disorder (PGT-M) or structural rearrangement (PGT-SR), and allows for concurrent aneuploidy screening (PGT-A) in PGT-M/SR cycles, all using the same generic protocol and the same biopsy. For this reason, various universal PGT approaches have been developed in the last 2-3 years and are increasingly making their way to the clinic worldwide. Study design, size, duration Universal PGT as a first-tier test was implemented at UZ Leuven mid-2014, using in-house developed algorithm (siCHILD/haplarithmisis). This is a retrospective analysis of universal PGT data, collected between 1 January 2015 and 31 December 2022. Data on PGT indication, PGT cycles and biopsy results was extracted. Implantation rate (IR; with IU/EU fetal sac) per embryo transfer was calculated from frozen embryo transfer (FET) cycles for patients with known result by the end of 2021. Participants/materials, setting, methods By the end of 2022, 718 couples have successfully finalized preclinical PGT workup. At the time of writing, 573 couples (80%) have further proceeded with PGT cycles. In total, 1264 PGT cycles were performed with day-3 (D3) cleavage-stage (n = 3344) or trophectoderm (TE) biopsies (n = 1524). For 403 patients with known outcome from 954 FET cycles, IR per embryo transfer was calculated per biopsy type and embryo ranking, considering genome-wide chromosome information. Main results and the role of chance Couples were referred to PGT due to various autosomal dominant (AD, 65%), autosomal recessive (AR, 14%) and X-linked disorders (XL, 10%). Various PGT-M indications encompassed >200 genes in total, but almost 20% of couples enrolled due to inherited BRCA1/BRCA2 mutations. PGT-SR was indication in 7% of cases, mainly for familial microduplications/deletions (64%). For all PGT cycles, approximately 15% and 18% of all performed D3 and TE biopsies, respectively, were scored as abnormal largely due to meiotic errors in the embryo. All unaffected embryos were further ranked as R1 (euploid) or R2 (mosaic), and the number of R2 embryos was expectedly higher after D3 biopsies, compared to TE biopsies (250/1063, 23.6% vs 37/510, 7.3%, p<0.001). In accordance with inheritance mode, the number of unaffected embryos suitable for transfer was significantly lower in AD PGT cycles, compared to AR (32.3% (1116/3455) vs 44.0% (262/596) ; p<0.0001)). For 403 patients with FET cycles, IR was higher in R1 vs R2 embryos after D3 biopsy (231/590, 39.2% vs 30/159, 18.9 %, p <0.0001), whereas no difference was observed between R1 and R2 embryos after TE biopsy, although low number of R2 embryos after TE biopsy was transferred (85/142, 59.9% vs 7/12, 58.3%, p=ns). Limitations, reasons for caution The analyzed data is derived from a single center and the clinical outcome may not reflect the experience of other IVF/PGT centers due to confounding factors, such as PGT indication, used PGT technology and IVF lab practice. Wider implications of the findings The current dataset provides a comprehensive overview on general trends and outcome of universal PGT practice. Apart from accurate genetic diagnosis, universal PGT provides information on chromosomes, which improves embryo selection for transfer. This in turn impacts clinical counselling, interpretation and reporting of the results, and embryo transfer policies. Trial registration number Not applicable

O-288 Implantation, ultrastructure and metabolic profiling of embryos from PGT-M and PGT-A Cycles

Abstract Study question Can metabolic profiling predict embryos at risk of single gene defects and chromosomal abnormalities and how are these reflected in their ultrustructure? Summary answer Different metabolic profiles are observed between embryos with chromosomal abnormalities and monogenic disorders and normal embryos, linked to altered mitochondrial and other organelles’ structure/function What is known already Down’s syndrome embryos and Monosomy 21 embryos have previously been shown to have differential expression of metabolites compared to normal embryos, but limited studies, have investigated in detail the metabolic profiling of embryos with other abnormalities or those with monogenic disorders in comparison to chromosomally normal embryos or their reflection in their ultrustructure. Study design, size, duration 520 embryos were biopsied on day5 for PGT-A (n = 178-47 cycles) or on day3 for PGT-A (n = 178-33 cycles) and PGT-M (n = 164-24 cycles). Following transfer of normal embryos, spare embryos, rejected for transfer following day5 or day3 biopsy were processed for TEM (n = 60 day3 biopsied, n = 60 day5 biopsied/vitrified), 60 unbiopsied/vitrified embryos from egg donation cycles used as control. Culture media were collected from the embryos and analysed by hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS). Participants/materials, setting, methods PGT-A and PGT-M were performed in 2 IVF Units. Metabolic profiling was conducted in a Forensic Toxicology Laboratory by HILIC-MS/MS (100 metabolites). Ultrastructure analysis by TEM was carried out in an Academic Hospital and Histology/Embryology Laboratory following embryo fixation in 3% glutaraldehyde, 1% osmium tetroxide, washes in PBS and staining with 1% aqueous uranyl acetate. Main results and the role of chance The implantation rates (+ve hCG/ET) for the PGT-M cycles was 68.2% and the for the PGT-A (blastocyst and cleavage biopsy cycles) 64.5% and 65% respectively. Characteristic patient specific metabolic profiles after screening for >100 primary metabolites were observed which differed between normal embryos that had resulted in a viable pregnancy and aneuploid and chaotic embryos although it was more difficult to find clear patterns in embryos with monogenic disorders. Logistic regression analysis revealed a number of metabolites with high predictive value which in combination with embryo score could serve in the future as non-invasive markers for the detection of chromosomal abnormalities before embryo transfer. TEM analysis revealed differences in the quality of cells and organelle activity which were reflected in the embryo metabolic profiles. Abnormal but well developed hatching blastocysts had mainly cells with good mitochondrial morphology/ activity, nice Golgi apparatus and well developed rough and smooth endoplasmic reticulum but depending on the aneuploidy or gene mutation involved, inner cell mass cells with limited organelles and occasionally lipofuscin droplets in the trophectoderm were evident. Chaotic poor quality embryos showed a lower number of mitochondria, often with no cisternae, increased number of vacuoles, and frequently problems in junctions between cells. Limitations, reasons for caution Although metabolic profiles were compared between normal and abnormal embryos, all the normal embryos were transferred to the uterus or remain vitrified for clinical purposes. Therefore the ultrastructure analysis is based only on biopsied abnormal embryos and as control unbiopsied embryos from egg-donation cycles with high chance of being normal. Wider implications of the findings This study shows high implantation rates after PGT—M and PGT-A and identified distinct differences in the metabolic profiles of normal and abnormal embryos providing unique metabolites which in the future could serve as non-invasive biomarkers for the detection of abnormalities before embryo transfer. Trial registration number Not applicable

P–261 The human embryo following biopsy on day5 vs day3: Implantation, cytoskeleton, ultrastructure and effects of endometrial damage/inflammation on receptivity as revealed by scanning electron microscopy

Abstract Study question Are there any differences in implantation, cytoskeleton and ultrastructure of embryos biopsied on day5 vs day3 and how endometrial damage/inflammation may affect receptivity and implantation? Summary answer No differences are observed in implantation rates but vitrification following day5 biopsy led to more cytoskeletal/ultrastructural anomalies. Infections and epithelial damage severely affected endometrial receptivity. What is known already Successful implantation is dependent on the correct synchronization of the window of implantantation with the transfer of chromosomally/genetically normal embryos, in a well prepared receptive endometrium. This is the first study to examine the effects of day5 vs day3 embryo biopsy by comparing implantation/pregnancy rates and by analysing cytoskeleton using Confocal Laser Scanning Microscopy (CLSM), and ultrastructure by Transmission Electron Microscopy (TEM). In addition, Scanning Electron Microscopy (SEM) was used on endometrial biopsies to assess possible uterine pathologies/inflammation that may be responsible for the failed implantation after PGT-A/M and if subsequent treatment can increase implantation/pregnancy rates in succeeding PGT-A/M cycles. Study design, size, duration 470 embryos were biopsied on day5 for PGT-A (n = 152–37 cycles) or on day3 for PGT-A (n = 162–29 cycles) and PGT-M (n = 156–22 cycles). Following transfer of normal embryos, spare embryos, rejected for transfer following day5 or day3 biopsy were processed for Cytoskeletal analysis (n = 30 fresh day3 biopsied, n = 30 day5 biopsied/vitrified) or TEM (n = 20 fresh day3 biopsied, n = 20 day5 biopsied/vitrified). Also, patients with a –ve hCG test, underwent endometrial biopsy to detect infection/inflammation and assess receptivity. Participants/materials, setting, methods Cytoskeletal analysis was performed by embryo immunostaining with α-tubulin, γ-tubulin, acetylated-tubulin antibodies and DAPI or/ PI to visualise DNA. TEM analysis was carried out following embryo fixation in gluteraldehyde, incubation in osmium, aqueous uranyl acetate, dehydration through ethanol series, and immersion in Epon. Endometrial biopsies were fixed in gluteraldehyde solution and processed for SEM using standard methods. The study was conducted in an academic hospital with an IVF/PGD laboratory and 3 private IVF Units. Main results and the role of chance 162 embryos were biopsied on day 3 for PGT-A (29 cycles), 10 cycles had no normal embryos for transfer, 30 normal embryos were transferred in 19 cycles leading to 12/19 (63.2%) +vehCG/ET and 11/19 (57.9%) Ongoing pregnancy rate/ET. 156 embryos were biopsied on day 3 for PGT-M (22 cycles), 2 cycles had no normal embryos for transfer, 34 normal or carrier embryos were transferred in 20 cycles leading to 15/20 (75.0%) +vehCG/ET and 13/20 Ongoing pregnancy rate/ET (65.0%). 152 embryos were biopsied on day 5 for PGT-A (37 cycles), 8 cycles had no normal embryos for transfer, 34 normal embryos were transferred in 29 cycles leading to 18/29 (62.1%) +vehCG/ET and 16/29 (55.2%) Ongoing pregnancy rate /ET. Analysis of endometrial biopsies with SEM revealed bacterial infections, inflammation and epithelial damage. So far, 33.3% of patients who received intracavitary infusions-antibiotic treatment per os achieved a +vehCG/ET in their next PGT-A/M cycle. Cytoskeletal analysis showed that the majority of spindles examined in both day3 and day5 biopsied embryos were normal (85/114(74.6%) and 87/137(63.5). However vitrification following day5 biopsy led to more cytoskeletal/ultrastructural anomalies which included multipolar/abnormally shaped spindles, chromosome-bridging, chromosome-lagging and more vacuoles, lipofuscins. and distension of mitochondria. Limitations, reasons for caution Patients undergoing PGT-A have various aetiologies (heterogeneous group). The embryos used for cytoskeletal and ultrastructural analysis in this study were all diagnosed with either chromosomal abnormalities or single gene defects following PGT-A or PGT-M. Wider implications of the findings: This is the first study to compare implantation/pregnancy rates, cytoskeleton and ultrastructure of day5 vs day3 biopsied embryos. The similarities observed in implantation/pregnancy rates, and the limited ultrastuctural and cytoskeletal anomalies identified confirm the procedures’ safety and indicate in certain cases endometrial factors/inflammation responsible for failed implantation following PGT-A/M. Trial registration number Not applicable

44. CAN PGT-A USING ARRAY CGH IMPROVE IVF CLINICAL RESULTS? (THE EGYPTIAN IVF-ET CENTER EXPERIENCE)

Introduction Chromosomal aneuploidies are considered the main cause of failed development of embryos, implantation failures, and early reproductive losses. Preimplantation genetic testing (PGT) using array comparative genomic hybridization (array-CGH) allows selection of embryos with normal karyotype, before embryo transfer. This should increase the implantation rate and reduce the frequency of early pregnancy loss. The aim of this study is to assess if PGT using array-CGH improve IVF clinical results. Material & methods During 2016 - 2018, a total of 97 cycles for 94 patients underwent blastomere biopsy by laser on day 3, and 90 fresh transfer cycles were performed. The indications were repeated IVF failure (30), repeated pregnancy loss (26), previous history of aneuploid offspring (10) and for aneuploidy screening and sex selection (28). The maternal ages ranged from 25 to 42yrs. 732 embryos from 97 cycles were subjected to day-3 biopsies, the DNA was amplified using modified whole genome amplification (WGA) protocol. The amplified DNA from 710 embryos was labeled and analyzed using array-CGH software (IBSA Genetics., Switzerland). Results 710 embryos was tested, 69.44% (493/710) of the embryos were aneuploid267, and 30.46% (217/710) were euploid.70 euploid embryos were cryopreserved, and 147 euploid embryos were transferred to 90 patients resulting in 50% (45/90) pregnancies, 22 deliveries with healthy babies and 20 ongoing pregnancies and 3 miscarriages. Conclusion: Our results show that array CGH has the potential to provide high pregnancy and delivery rates after transfer of euploid embryos.

PGT-A and RCT proof in AMA and SMF couples

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

Embryonic Aneuploidy after Preimplantation Genetic Screening: Age- and Indication-Matched Comparative Study between Indian and Spanish Population.

Background:Recent studies show that there are differences in female fertility in different ethnic groups with ovarian aging and IVF treatment outcomes. Advanced maternal age is a known risk factor for miscarriage, accounting largely due to genetically abnormal fetus.Aims and Objectives:This study investigates if there are any differences in rates of embryo aneuploidy based on age and indications for preimplantation genetic screening (PGS) between Indian and Spanish women.Materials and Methods:This multicenter study was carried out at fertility centers in India and Spain. Data from autologous IVF cycles of women <45 years age (Spanish: 39.4 ± 3.8 years; Indian: 35.3 ± 4.6 years) were included. A total of 37,962 embryos from 7009 IVF cycles from Spain and 1894 embryos from 308 IVF cycles from India, having similar clinical indications, underwent similar IVF treatment protocol. The embryos were analyzed by PGS using either a day-3 or day-5/6 embryo biopsy.Results:Both Indian and Spanish ethnic population showed a reduction in aneuploidy rate in day-5/6 biopsy compared with day-3 biopsy (Spanish: 53.3% vs. 81.1%, P < 0.01; Indian: 50% vs. 75%, P < 0.02). There was a significant decrease in highly abnormal or chaotic embryos in trophectoderm biopsies compared with day-3 biopsies (Spanish: 2% vs. 16.1%, P < 0.01; Indian: 2.5% vs. 17.7%, P < 0.01). Both the populations showed similar trend in aneuploidy rate with maternal age. The results showed no significance between aneuploidy rate compared within different age groups and indications. However, there was a significant reduction in the miscarriage rate in Spanish population in day-3 biopsy compared with Indian population (10.7% vs. 19.8%; P < 0.05; 95% confidence interval [0.0044–0.0712]). There were no differences in the clinical outcomes compared between the two populations.Conclusion:This study shows that the aneuploidy rates between Indian and Spanish women of the same age group undergoing IVF treatment do not differ. An in-depth analysis to compare the types of anomalies reported with PGS in both the population will be of much interest.

Is more better? A higher oocyte yield is independently associated with more day-3 euploid embryos after ICSI.

Is the number of oocytes retrieved after ovarian stimulation for ICSI independently associated with the number of day-3 euploid embryos (EE)? A larger oocyte yield is independently associated with more day-3 EE, although the expected benefit decreases significantly with advancing age. Although traditionally ovarian stimulation aims at collecting more than one oocyte in order to increase the chance of pregnancy, there is evidence suggesting that excessive ovarian response leads to lower live birth rates. Whether a larger oocyte yield after ovarian stimulation is associated with the genetic composition of the resulting embryos and therefore with their reproductive potential is still largely unknown. This is a multi-centered retrospective cohort study analyzing 724 cycles of preimplantation genetic testing for aneuploidy (PGT-A) cycles using day-3 biopsy and array-comparative genomic hybridization between March 2011 and December 2016 in three laboratories. The primary outcome measure was the number of EE on day-3. Statistical analysis was performed using the generalized estimating equations (GEE) framework and multivariate regression models to control for the clustered nature of the data while adjusting for potential confounders. A multivariate regression GEE model including all significant population and stimulation characteristics as covariates as well as an interaction term between female age and number of oocytes revealed that the number of oocytes retrieved was still positively associated with the number of EE (coeff: +0.40, 95% CI: 0.24-0.56). The interaction term was highly significant (coeff: -0.01, P < 0.001) indicating an effect modifying role of female age on the association of oocytes retrieved with the number of EE. The number of oocytes retrieved was also positively associated with cumulative live birth rates (odds ratio: 1.07, 95% CI: 1.03-1.12). This study is retrospective and the presence of residual unknown bias cannot be excluded. Furthermore, the population analyzed in this study might not be completely representative of the general population undergoing ICSI. These results provide an explanatory mechanism for the recently published positive association between the number of oocytes retrieved and cumulative live birth rates. CAV is supported by a NHMRC Early Career Fellowship (GNT1147154)/ No competing interests to declare.

Clinical reassessment of human embryo ploidy status between cleavage and blastocyst stage by Next Generation Sequencing.

One of the most important limitations of genetic testing in preimplantation embryos is embryonic mosaicism, especially when performed on D3 with only a single blastomere evaluated. Previous publications, using Array-Comparative Genomic Hybridization (a-CGH) to compare day 3 (D3) biopsies versus trophectoderm biopsies for the analysis of aneuploid embryos, showed similar high concordance rates per embryo diagnosis for D3 biopsies and trophectoderm biopsies. Next generation sequencing (NGS) was introduced lately as a new technique for preimplantation genetic testing for aneuploidies (PGT-A). Using this technique, this retrospective descriptive study evaluated the degree of the concordance of the diagnosis between preimplantation human cleavage stage (D3) and blastocyst stage (D5) embryos. Double biopsies on D3 and D5 were performed on 118 embryos, reaching blastocyst stage on D5 and had not been selected for transfer. As the fertilization law of the United Arab Emirates does not allow embryo freezing, also surplus euploid embryos after D 3 biopsy were included.Analysis of the NGS results from D3 and D5 embryo biopsies showed a total concordance rate per embryo diagnosis of 85.6% for euploid and aneuploid embryos. The concordance rates per embryo chromosomal pattern for embryo diagnosed as aneuploid at both biopsy stages was 82.2%. However, the status regarding the affected chromosomes was not identical on D3 and D5. Hence, the total concordance rate between D3 biopsy and D5 biopsy was limited to 67.8%.This current study clearly demonstrated that the concordance rates between D3 and D5 biopsies in aneuploid and euploid embryos are lower than previously reported.

PGD-A in advanced maternal age: final results of a day-3 biopsy RCT

PGD-A in advanced maternal age: final results of a day-3 biopsy RCT

Chromosomal Status Assessed by CGH after a Day-3 Biopsy, in Embryos Classified by Standard Scoring Methodology at Pronuclear, Cleavage and Blastocyst Stage

Chromosomal Status Assessed by CGH after a Day-3 Biopsy, in Embryos Classified by Standard Scoring Methodology at Pronuclear, Cleavage and Blastocyst Stage

Effects of maternal age on euploidy rates in a large cohort of embryos analyzed with 24-chromosome single-nucleotide polymorphism–based preimplantation genetic screening

To determine the effect of maternal age on the average number of euploid embryos retrieved during oocyte harvest as part of an invitro fertilization (IVF) cycle, including the probability of retrieving at least one euploid embryo in a cohort (PrE). Retrospective study. Preimplantation genetic screening (PGS) laboratory. Women aged 18 to 48years undergoing IVF treatment. Use of 24-chromosome single-nucleotide polymorphism (SNP)-based PGS of day-3 and day-5 embryo biopsies. Relationships between maternal age and the rate of embryos that tested as euploid (hereafter referred to as "euploid embryos"), the average number and proportion of euploid embryos per IVF cycle, and PrE. We analyzed 22,599 day-3 embryos and 15,112 day-5 embryos. In women aged 27 to 35years, the median proportion of euploid embryos in each cycle remained constant at ∼35% in day-3 biopsies and ∼55% in day-5 biopsies, but it decreased rapidly after age 35. On average, women in their late 20s had four euploid embryos (day 3 or day 5) per cycle, but this number decreased linearly (R(2) ≥ 0.983) after 35years of age. The effect of maternal age on PrE was similar, with a rapid exponential decline (R(2) = 0.986). Across all maternal ages, the euploid proportion and number of embryos per cycle were counterbalanced, so the number of euploid embryos per cycle was the same for day-3 and day-5 biopsies. This suggests that the loss of embryos from day 3 to day 5 was primarily due to aneuploidy. Our results confirm the known inverse relationship between advanced maternal age (>35years) and embryo euploidy, demonstrating that equal numbers of euploid embryos are available at day 3 and day5.

Confirmation rates of array-CGH in day-3 embryo and blastocyst biopsies for preimplantation genetic screening.

The purpose of this study was to compare the confirmation rate of day-3 embryo biopsy (blastomere) and trophectoderm biopsy using array-comparative genomic hybridization (array-CGH) technology. A blinded study was conducted to re-analyse 109 embryos previously diagnosed as chromosomally abnormal by array-CGH. Preimplantation genetic screening (PGS) was performed using array-CGH on day 3 (n = 50) or day 5 (n = 59). Partial chromosome gains or losses were excluded (n=6), and only whole chromosome aneuploidies were considered. Re-analysis of whole blastocysts was carried out following the same array-CGH protocol used for PGS. The PGS result was confirmed in the whole blastocyst in (a) 49/50 (98 %) abnormal embryos after day-3 biopsy and (b) 57/59 (96.6 %) abnormal embryos after trophectoderm biopsy. One embryo (1/50; 2 %) was diagnosed as abnormal, with monosomy 18, on day 3, and software analysis of the whole blastocyst gave a euploid result; however, a mosaic pattern was observed for monosomy 18 in the whole blastocyst. Two trophectoderm biopsy cases (3.4 %) did not have the abnormalities (trisomy 7, and trisomy 1 and 4, respectively) verified in the whole embryo. Concordance rates for both biopsy strategies and for individual chromosomes were evaluated by Fisher's exact test and showed no significant differences. Both types of biopsies showed similar high concordance rates with whole blastocyst results. Therefore, regarding the confirmation rates shown in this work, day-3 embryo biopsies can be representative of the whole embryo and both types of biopsy can be used for clinical analysis in PGS following the described array-CGH protocol.

Direct detection of FMR1 CGG repeats causative for fragile X syndrome coupled with 24-chromosome aneuploidy screening in single cells

Fragile X syndrome (FXS) is caused by the expansion of CGG sequences in the 5’ untranslated region of the Fragile X Mental Retardation 1 (FMR1) gene. Most current pre-implantation genetic diagnosis (PGD) protocols are based on indirect analysis of linkage markers because amplification of trinucleotide repeats is difficult. The aim of our study is to develop a dual test that can achieve direct diagnosis of FXS and screen for 24-chromosome aneuploidy from the same sample while being sensitive at the single cell level and applicable to PGD for IVF. Experimental study. Four single-cell and five-cell samples of (1) a lymphocyte cell line carrying an intermediate mutation for FXS, (2) a fibroblast cell line carrying a full mutation for the FXS, (3) a trisomic cell line for chromosome 13, (4) a cell line monosomic for chromosome 21 and (5 and 6) two normal cell lines (46 XX and 46 XY) were subjected to whole genome amplification (WGA). All cell lines were established and commercially available. After WGA, a first aliquot was processed through the AmplideX™ protocol (Asuragen) for CGG repeats assessment of the FMR1 gene. A second aliquot was used for 24-chromosome aneuploidy screening on array-CGH from BlueGnome (Illumina). Normal alleles (27 and 30), intermediate mutation (52) and full mutation of FMR1 (>200) were respectively detected in control cells (8/8), lymphocyte (8/8) and fibroblast (8/8) cell lines affected with FXS, in a very sensitive and reproducible manner. Concurrent aneuploidy screening was effectively achieved from the same WGA sample, as shown by the confirmation diagnosis of known trisomic 13, monosomic 21 or normal cell lines. This study is the first report of direct diagnosis for FXS coupled with 24-chromosome aneuploidy screening. The advantages of this new test are (1) it provides accurate sizing of FMR1 alleles up to 200 CGG repeats, allowing to define a pre-, intermediate or full Fragile X mutation, (2) it can be achieved from a unique biopsy specimen, offering the possibility to diagnose for multiple conditions on limited starting material, (3) it is reliable on a single cell, therefore applicable to pre-implantation genetic diagnosis on D3 blastomere as well as on D5/6 trophectoderm biopsy, (4) it can be completed within 24 to 48 hours, hence allowing for Day3 biopsy with Day5 fresh transfer.

Blastocyst culture selects for euploid embryos: comparison of blastomere and trophectoderm biopsies

Preimplantation genetic diagnosis and screening improves the chances of achieving a viable pregnancy, not only free of undesired single-gene defects but also aneuploidy. In addition, improvements in vitrification provide an efficient means of preserving embryos (blastocysts). By combining trophectoderm biopsy with recent improvements in vitrification methods, only those embryos that have proved themselves viable and potentially more competent are tested. Using array comparative genomic hybridization (aCGH) to assess all 24 chromosomes, aneuploidy rates were compared between day-3 blastomere biopsy and day-5 trophectoderm biopsy. Of those 1603 embryos, 31% were euploid, 62% were aneuploid and 7% not analysable. A significantly larger proportion of embryos were euploid on day-5 biopsy (42%) compared with day-3 biopsy (24%, P<0.0001). The number of euploid embryos per patient was not significantly different. Combining extended culture, trophectoderm biopsy and aneuploidy assessment by aCGH and subsequent vitrification can provide a more efficient means of achieving euploid pregnancies in IVF.Preimplantation genetic diagnosis and screening improves the chances of achieving a viable pregnancy, not only free of undesired single-gene defects but also aneuploidy. In addition, improvements in vitrification provide an efficient means of preserving embryos (blastocysts). By combining trophectoderm biopsy with recent improvements in vitrification methods, only those embryos that have proved themselves viable and potentially more competent are tested. Using array comparative genomic hybridization to assess all 24 chromosomes, aneuploidy rates were compared between day-3 blastomere biopsy and trophectoderm biopsy. Of those 1603 embryos, 31% were euploid, 62% were aneuploid and 7% not analysable. A significantly larger proportion of embryos were euploid with trophectoderm biopsy (42%) compared with blastomere biopsy (24%, P<0.0001). The number of euploid embryos per patient was not significantly different. Combining extended culture, trophectoderm biopsy and aneuploidy assessment by array comparative genomic hybridization and subsequent vitrification can provide a more efficient means of achieving euploid pregnancies in IVF.

Efficiency of preimplantation genetic screening (PGS) using array-CGH compared to matched control IVF patient populations with and without day-3 PGS fish

Efficiency of preimplantation genetic screening (PGS) using array-CGH compared to matched control IVF patient populations with and without day-3 PGS fish

Significant decrease in miscarriages after preimplantation genetic diagnosis (PGD) for recurrent pregnancy loss using array comparative genome hybridization (Array CGH)

Array CGH can analyze all chromosomes for aneuploidy and may further reduce the risk of miscarriage in patients suffering from recurrent pregnancy loss (RPL). The objective of this study was to determine any beneficial effects of PGD by array CGH for RPL patients in comparison to their expected loss rate and to PGD by FISH results. Multicenter retrospective study. The study included 177 cycles of couples with idiopathic RPL (defined as 3 or more miscarriages), average maternal age of 36.3 years, undergoing ART at multiple fertility centers. PGD was done using day-3 biopsy (n = 143) or day-5 biopsy (n = 34), followed by analysis by aCGH. Each PGD patient was matched with their expected loss rate for RPL patients (Brigham et al. 1999). An ongoing pregnancy was defined as past second trimester. In total 1517 embryos were analyzed, of which 33% were normal, 63% were abnormal and 4% none analyzable (no nucleus or no DNA amplification). Of the 176 cycles, pregnancy data was available for 154, of which 66 (43%) became pregnant with an implantation rate of 45% (95 sacs / 212 replaced embryos) and 62 cycles (40%) are ongoing past second trimester or delivered. We would expect a 34.9% miscarriage rate in this specific group of patients, but the miscarriage rate found was only 6% (4/66) (P<0.001). Previous PGD results using FISH showed that the miscarriage rate in idiopathic RPL patients was significantly reduced from 26% to 10% in patients younger than 35, and from 39% to13% in older patients. Current PGD results with aCGH indicate a significant decrease in the miscarriage rate of idiopathic RPL patients and high pregnancy rates for the current average maternal age of 36. These data suggest that aCGH further improves the results previously obtained by FISH. Furthermore, this confirms that idiopathic recurrent miscarriage is mostly caused by embryonic chromosome abnormalities.

Aneuploidy Patterns in 3143 day-3 Embryos Analyzed by Array CGH

BACKGROUND: Previous FISH studies with day-3 biopsy showed an excess of monosomies, a correlation between maternal age and percentage of normal embryos (lower maternal age = higher percentage of normal embryos), and no correlation between maternal age and mosaicism. These studies were performed with 9-12 chromosome probes and thus did not include more than half of the chromosome complement. OBJECTIVE(S): To assess the types of chromosome abnormalities observed by array CGH and their relationship to maternal age. MATERIAL AND METHOD(S): Single blastomeres from day-3 embryos were analyzed by aCGH (24Sure, BlueGnome, UK). This technique can analyze all chromosomes with a very low error rate, including errors caused by mosaicism, (2%, Gutierrez-Mateo et al. 2010) and thus we assume that day-3 PGD results are mostly representative of the rest of the embryo. An embryo with 3 or more chromosomes out of the 24 being abnormal was considered “complex abnormal”. RESULT(S): Complex abnormal embryos and double aneuploidies significantly increased with advancing maternal age, while trisomies and monosomies did not. Normal embryos significantly decreased with advancing maternal age (48% in the <35 years of age group to 21% in the >39 years). We observed an excess of monosomy events (n=1006) compared to trisomy events (n=695) with one third of that excess being caused by monosomy X. There was a slightly higher average maternal age for aneuploidy events on chromosomes 13 to 22 (av. Age 38.4) than 1 to 12 (37.7), while monosomy X results showed an average maternal age of 36.4 years. Normal embryo results had an average maternal age of 35.9 years, compared to complex abnormal embryo results of 38.7 years. The most common aneuploidies were for chromosomes 16 (n=204), 22 (157), X (125), 15 (119), 21 (107), and 19 (91). CONCLUSION(S): Large studies using FISH (Munne et al. 2007) considered complex abnormalities akin to chaotic mosaics. However, by analyzing all chromosomes with aCGH, many embryos classified by aCGH as complex abnormal must be true aneuploid, as seen by their increased maternal age. The percentage of abnormal embryos does increase significantly with maternal age, as does the percentage of double aneuploids and complex abnormals. The excess of monosomies observed is not an artifact, as described in a separate abstract by Colls et al. SUPPORT: Departmental funds

Clinical results on single cells from 470 embryos using 23-chromosome single nucleotide polymorphism (SNP) microarray preimplantation genetic screening (PGS) from 45 patients

OBJECTIVE: We amplified DNA from a single blastomere or trophectoderm cells and performed complex SNP microarray genetic analyses. DESIGN: Prospective study. MATERIALS AND METHODS: PGS patients between 12/1/09 and 4/25/10 were analyzed. 45 patients underwent embryo biopsy by laser and SNP microarray PGS primarily due to > 2 spontaneous miscarriages. 3 of these patients were tested for translocation abnormalities and aneuploidy. The maternal ages ranged from 34 to 44yrs. 398 embryos from 37 patients underwent day-3 biopsies, whereas 72 embryos from 8 patients underwent blastocyst trophectoderm biopsies. We amplified the DNA using a modified whole genome amplification (WGA) protocol. We used the Illumina Cyto-12 microarray to determine chromosome aberrations and to obtain genotype data for ∼300K SNPs. A high-resolution copy-number profile was used to identify copy number variations (CNVs). Parental DNA was simultaneously analyzed with embryo DNA for CNVs. Data was analyzed with Illumina GenomeStudio software. RESULTS: 45 patients and 470 embryos were tested. 37 patients undergoing a day-3 biopsy were considered for embryo transfer along with 1 patient undergoing a blastocyst biopsy. All embryos from an additional 7 patients undergoing blastocyst biopsies and PGS were frozen. 59% (277/470) of the embryos were abnormal. Many embryos with CNVs were considered normal because of parental CNV analysis. 3.1 ± 2 euploid embryos were identified per patient/cycle. The overall clinical pregnancy rate was 62%. The blastocyst biopsy transferred patient is pregnant.Tabled 1Clinical Pregnancy by Age GroupAgeClinical PregnancyMiscarriageNo TransferFrozen〈3556% (5/9)4% (1/23)0235-3763% (5/8)01038-4075% (9/12)004〉4044% (4/9)041 Open table in a new tab CONCLUSION: 23-chromosome SNP microarray PGS is a valuable technology in an effort to optimize the transfer of euploid embryos and to increase the delivery rates of couples undergoing in vitro fertilization (IVF) due to repeat miscarriages.

P40 Short-CGH in a PGD for a reciprocal translocation: first clinical application

Introduction: An increase of embryonic chromosome abnormalities with maternal age contributes to low implantation rates and high miscarriage rates. PGS may reduce this trend by selecting chromosomally normal embryos for replacement, provided that the biopsy is done with minimal damage to the embryo and the analysis is accurate. Unfortunately, different studies using day-3 biopsy and FISH have used diverse methodologies yielding conflicting results. Although day 5 biopsy, CGH and vitrification combined seem to produce outstanding results, patients and doctors still prefer fresh over frozen cycles. An improvement over CGH is array CGH (aCGH), which provides complete chromosome analysis in one day. The Here we present our preliminary clinical data on the use of aCGH. Methods: aCGH was performed on 87 cycles from 19 fertility centers. Average maternal age was 37.6. The indications were advanced maternal age ( 38, n = 46), recurrent pregnancy loss (RPL, n = 28) or other (n = 13). A single cell was biopsied per embryo on day 3 of development. Normal embryos were replaced on day 5. Some abnormal embryos were fully fixed and reanalyzed by FISH to determine the error rate of the technique. Pregnancy rates and miscarriage rates from each center were compared to SART data from the same center controlling for age. Results: A total of 747 embryos were analyzed. 2.4% of cells did not show DNA amplification. Of the rest 38% were euploid and the rest abnormal. Euploidy decreased with maternal age from 45.7% in Conclusions: aCGH provides results in one day with very low no result frequency (2%) and error rate (5%). Pregnancy and miscarriage rates seem to indicate an improvement over historic controls. While comparing pregnancy rates in this manner may be subject to confounding factors, miscarriage rates are probably even higher in an ideal control that should include >25% RPL patients.