High concordance in preimplantation genetic testing for aneuploidy between automatic identification via Ion S5 and manual identification via Miseq.

The Ion S5 (Thermo Fisher Scientific) and Miseq (Illumina) NGS systems are both widely used in the clinical laboratories conducting PGT-A. Each system employs discrepant library preparation steps, sequencing principles, and data processing algorithms. The automatic interpretation via Ion Reporter software (Thermo Fisher Scientific) and the manual interpretation via BlueFuse Multi software (Illumina) for chromosomal copy number variation (CNV) represent very different reporting approaches. Thus, it is intriguing to compare their ability of ploidy detection as PGT-A/NGS system. In the present study, four aneuploid cell lines were individually mixed with a diploid cell line at different aneuploid ratios of 0% (0:5), 10% (1:9), 20% (1:4), 40% (2:3), 50% (3:3), 60% (3:2), 80% (4:1) and 100% (5:0) to assess the sensitivity and specificity for whole chromosomal and segmental aneuploidy detection. The clinical biopsies of 107 blastocysts from 46 IVF/PGT-A cycles recruited between December 2019 and February 2020 were used to calculate the concordance. Initially, the pre-amplified products were divided into two aliquots for different library preparation procedures of each system. Applying the same calling criteria, automatic identification was achieved through the Ion Reporter, while well-trained technicians manually identified each sample through the BlueFuse Multi. The results displayed that both systems reliably distinguished chromosomal CNV of the mixtures with at least 10% aneuploidy from karyotypically normal samples ([Ion S5] whole-chromosomal duplication: 2.14 vs. 2.05, p value = 0.009, segmental deletion: 1.88 vs. 2.05, p value = 0.003; [Miseq] whole-chromosomal duplication: 2.12 vs. 2.03, p value = 0.047, segmental deletion: 1.82 vs. 2.03, p value = 0.002). The sensitivity and specificity were comparable between the Ion S5 and Miseq ([sensitivity] 93% vs. 90%, p = 0.78; [specificity] 100% vs. 100%, p value = 1.0). In the 107 clinical biopsies, three displayed chaotic patterns (2.8%), which could not be interpreted for the ploidy. The ploidy concordance was 99.04% (103/104) per embryo and 99.47% (2265/2277) per chromosome pair. Since their ability of detection were proven to be similar, the automatic identification in Ion S5 system presents comparatively faster and more standardized performance.

Rapid preimplantation genetic screening using a handheld, nanopore-based DNA sequencer.

OBJECTIVE: To determine if a handheld, nanopore-based DNA sequencer can be used for rapid preimplantation genetic screening (PGS). DESIGN: Laboratory study. SETTING: Academic medical center. PATIENT(S): Amplified genomic DNA from euploid and aneuploid trophectoderm biopsy samples (n=9) that was also tested using traditional next generation sequencing (NGS). INTERVENTION(S): Short-read DNA library preparation and nanopore-based sequencing using a hand-held MinION sequencer. MAIN OUTCOME MEASURE(S): Comparison of cytogenetic testing result from NGS and nanopore-based sequencing and the time required for library preparation and sequencing. RESULT(S): Multiplexed short-read DNA library preparation was completed in 45 minutes. Sequencing on a single sample was completed within 20 minutes and 5 samples were simultaneously sequenced in under 2 hours. Whole-chromosome aneuploidy screening results obtained from nanopore-based sequencing were identical to those obtained using NGS. CONCLUSION(S): Here we report the first application of nanopore-based sequencing for PGS on trophectoderm biopsy samples using a novel rapid multiplxed short-read nanopore sequencing library preparation protocol. Sequencing for aneuploidy screening could be performed on a single sample in 20 minutes and on 5 samples, simultaneously, within 2 hours. Overall, nanopore sequencing is a promising tool to perform rapid PGS onsite, enabling same day testing and embryo transfer, thus obviating the need for complex, large and expensive DNA sequencers or embryo freezing.

Novel technologies emerging for preimplantation genetic diagnosis and preimplantation genetic testing for aneuploidy.

INTRODUCTION: Preimplantation genetic diagnosis (PGD) was introduced as an alternative to prenatal diagnosis: embryos cultured in vitro were analysed for a monogenic disease and only disease-free embryos were transferred to the mother, to avoid the termination of pregnancy with an affected foetus. It soon transpired that human embryos show a great deal of acquired chromosomal abnormalities, thought to explain the low success rate of IVF - hence preimplantation genetic testing for aneuploidy (PGT-A) was developed to select euploid embryos for transfer. Areas covered: PGD has followed the tremendous evolution in genetic analysis, with only a slight delay due to adaptations for diagnosis on small samples. Currently, next generation sequencing combining chromosome with single-base pair analysis is on the verge of becoming the golden standard in PGD and PGT-A. Papers highlighting the different steps in the evolution of PGD/PGT-A were selected. Expert commentary: Different methodologies used in PGD/PGT-A with their pros and cons are discussed.

Next generation sequencing for preimplantation genetic testing of blastocysts aneuploidies in women of different ages.

Most of the current preimplantation genetic screening of aneuploidies tests are based on the low quality and low density comparative genomic hybridization arrays. The results are based on fewer than 2,700 probes. Our main outcome was the association of aneuploidy rates and the women's age. Between August-December 2013, 198 blastocysts from women (mean age 36.3+-4.6) undergoing in vitro fertilization underwent routine trophectoderm biopsy. NGS was performed on Ion Torrent PGM (Life Technologies). The results were analyzed in five age groups (<31, 31-35, 36-38, 39-40 and >40). 85 blastocysts were normal according to NGS results. The results in the investigated groups were (% of normal blastocyst in each group): <31 (41.9%), 31-35 (47.6%), 36-38 (47.8%), 39-40 (37.7%) and >40 (38.5%). Our study suggests that NGS PGD is applicable for routine preimplantation genetic testing. It allows also for easy customization of the procedure for each individual patient making personalized diagnostics a reality.

Simultaneous assessment of aneuploidy, polymorphisms, and mitochondrial DNA content in human polar bodies and embryos with the use of a novel microarray platform.

OBJECTIVE: To develop a microarray platform that allows simultaneous assessment of aneuploidy and quantification of mitochondrial DNA (mtDNA) in human polar bodies and embryos. DESIGN: Optimization and validation applied to cell lines and clinical samples (polar bodies, blastomeres, and trophectoderm biopsies). SETTING: University research laboratory and a preimplantation genetic diagnosis (PGD) reference laboratory. PATIENT(S): Samples from 65 couples who underwent PGD for aneuploidy and/or a single-gene disorder. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): 1) Comparison of aneuploidy screening results obtained with the use of the new microarray with those derived from two well established cytogenetic techniques. 2) mtDNA quantification. 3) Analysis of single-nucleotide polymorphisms. RESULT(S): The fully optimized microarray was estimated to have an accuracy of ≥97% for the detection of individual aneuploidies and to detect 99% of chromosomally abnormal embryos. The microarray was shown to accurately determine relative quantities of mtDNA. Information provided from polymorphic loci was sufficient to allow confirmation that an embryo was derived from specific parents. CONCLUSION(S): It is hoped that methods such as those reported here, which provide information on several aspects of oocyte/embryo genetics, could lead to improved strategies for identifying viable embryos, thereby increasing the likelihood of successful implantation. Additionally, the provision of genotyping information has the potential to reveal DNA contaminants and confirm parental origin of embryos.

New perspectives on embryo biopsy: not how, but when and why?

During the past 2 decades, biopsy for preimplantation genetic diagnosis at three stages, that is, before conception (the first polar body), after fertilization (the second polar body), and early cleavage (D3 blastomeres) or blastocyst stage (D5/D6 trophectoderm biopsy), have been optimized and performed clinically in hundreds of in vitro fertilization centers around the world. Although opening the zona pellucida by mechanical or chemical means is still effectively in use, noncontact laser has become the indispensable instrument. Overall, the invasive nature of biopsy at any given stage is recognized. It is believed that removal of the polar bodies from M-II oocytes and fertilized zygotes may have the least detrimental effects on subsequent embryonic development; hence increasing applications of polar body biopsy are anticipated. Although D3 biopsy is currently the most frequently used method, the effectiveness of D3 cleavage-stage biopsy is unsettling because of the mosaicism in early cleavage human embryos. Controversies exist in several areas; particularly, the efficacy of preimplantation genetic screening based on one cell removed from a D3 embryo remains to be confirmed. With new genetic testing technology, there may be no need to biopsy two cells because accuracy from one cell is high and the risk of misdiagnosis is very low when sufficient markers are used for chromosome copy number assessment or for mutation detection of single-gene disorders. And finally, it appears that limited harm is caused by biopsy at the blastocyst stage and mosaicism seems to be less common as compared with earlier stages. Therefore, use of the blastocyst-stage biopsy combined with cryopreservation protocol can be effectively used for several indications. Furthermore, faster genetic analytical methods that can be completed within several hours will make this strategy more practical and attractive as fresh embryo transfer is possible.

Effects of laser polar-body biopsy on embryo quality.

OBJECTIVE: To evaluate the effect of laser polar-body biopsy (PBB) for preimplantation genetic diagnosis on embryo quality. STUDY DESIGN: Retrospective case-control analysis. The quality of 145 embryos after PBB was compared to 276 embryos of the same group of women without biopsy. SETTING: University-based tertiary-care medical center. PATIENT(S): Women with inherited genetics disease. INTERVENTION(S): Laser PBB of IVF embryos for genetic diagnosis. MAIN OUTCOME MEASURE(S): The study and control embryos were compared for fertilization rate, pronuclear grading, and cleavage-stage parameters on days 1, 2, and 3 after oocyte retrieval. RESULT(S): The study embryos demonstrated higher rates of cleavage arrest (3.6% vs. 0.7%), higher rate of significant fragmentation on day 2 (9.5% vs. 3.0%), and lower rate of good cleavage embryos on day 2 (69.1% vs. 78.4%) compared with control embryos. On day 3, the study embryos had lower cleavage rates (six or more blastomeres; 56.5% vs. 74.5%), higher fragmentation (11.7% vs. 3.9%), higher rate of embryos presenting inferior cleavage pattern (57.2% vs. 38.5%), and lower mean blastomere number (5.8 ± 2.1 vs. 6.6 ± 1.9) compared with control embryos. CONCLUSION(S): Polar-body biopsy may have a negative effect on embryo quality.

ESHRE PGD consortium best practice guidelines for amplification-based PGD.

In 2005, the European Society for Human Reproduction and Embryology (ESHRE) PGD Consortium published a set of Guidelines for Best Practice PGD to give information, support and guidance to potential, existing and fledgling PGD programmes. The subsequent years have seen the introduction of a number of new technologies as well as the evolution of current techniques. Additionally, in light of recent advice from ESHRE on how practice guidelines should be written and formulated, the Consortium believed it was timely to revise and update the PGD guidelines. Rather than one document that covers all of PGD, as in the original publication, these guidelines are separated into four new documents that apply to different aspects of a PGD programme, i.e. Organization of a PGD centre, fluorescence in situ hybridization-based testing, Amplification-based testing and Polar Body and Embryo Biopsy for PGD/preimplantation genetic screening. Here, we have updated the sections that pertain to amplification-based PGD. Topics covered in this guideline include inclusion/exclusion criteria for amplification-based PGD testing, preclinical validation of tests, amplification-based testing methods, tubing of cells for analysis, set-up of local IVF centre and Transport PGD centres, quality control/quality assurance and diagnostic confirmation of untransferred embryos.

Cell technology employing femtosecond laser pulses.

Practical advantages of using femtosecond laser pulses for manipulations in cell surgery were demonstrated. The use of femtosecond laser pulses enables precision punching of the zona pellucida of the embryo without damaging its cells. With the help of femtosecond laser tweezers/scalpel, auxillary laser hatching was performed and a technique of optical biopsy of mammalian embryo was developed, which enabled non-contact sampling of embryonic material for preimplantation diagnostics. Our findings suggest that about 90% embryos retained the ability to develop at least to the blastula stage after this manipulation.

One-step multiplex polymerase chain reaction for preimplantation genetic diagnosis of Huntington disease.

OBJECTIVE: To develop a multiplex polymerase chain reaction (PCR) method for Huntington disease (HD) preimplantation genetic diagnosis (PGD) based on the coamplification of CAG repeats and three different polymorphic microsatellites in a single step of PCR. DESIGN: Techniques and instrumentation. SETTING: Tertiary clinical and academic medical center. PATIENT(S): Thirty-six embryos from seven clinical PGD cycles. INTERVENTION(S): Patients underwent a PGD cycle with transfer of two unaffected embryos on day 5. MAIN OUTCOME MEASURE(S): PGD based on mutation identification or exclusion testing for at-risk HD carriers. RESULT(S): Thirty-six embryos from seven clinical PGD cycles were analyzed with the new method here developed, and results were obtained for 34 of them. Two embryos were transferred on day 5, resulting in two singleton pregnancies. CONCLUSION(S): An interesting application of this approach can be considered for PGD cycles in which numerous markers must be used. We have also used this one-step multiplex method for PGD for other pathological conditions.

Symposium: innovative techniques in human embryo viability assessment. Human oocyte respiration-rate measurement--potential to improve oocyte and embryo selection?

Oocyte and embryo selection are not highly successful, with fewer than 10% of oocytes in assisted reproduction resulting in a delivery. Techniques for oocyte and embryo selection rely on highly subjective morphology assessment, with few true quantitative techniques available. One aspect of oocyte health that could be considered is the ability to produce ATP through respiration. Using a non-invasive technology, the respiration rates of individual human oocytes were recorded in an attempt to correlate respiration and oocyte health with probable subsequent development. Oocytes used were either immature or mature, non-fertilized oocytes from a clinical assisted reproduction programme. Differences in respiration rates between oocytes within a cohort and between cohorts of oocytes were recorded. The differences between cohorts reflected many of the currently known differences in oocyte health, related to age and FSH concentrations. However, within a cohort, differences between oocytes were observed, with some having high rates and others low. Oocytes with respiration rates of between 0.48 and 0.55 nl O(2)/h were viable, with lower rates consistent with lack of continued in-vitro maturation or atresia. This technology may have a future in the clinical laboratory as a predictor of oocyte health and ability to develop into an embryo with greater potential of delivery.

Analysis of HLA-G in maternal plasma, follicular fluid, and preimplantation embryos reveal an asymmetric pattern of expression.

Soluble HLA-G (sHLA-G) secretion by human preimplantation embryos in culture has been associated with successful embryo development, and therefore has potential to serve as a noninvasive marker of embryo viability. We have examined the spatial and temporal expression of HLA-G in embryos of varying developmental competence and the role of maternal factors in human embryonic HLA-G expression. Embryos that reached blastocyst stage on day 5 showed a higher frequency of sHLA-G secretion than those at morula or arrested stages (p < 0.05). There was no significant difference in sHLA-G secretion between normal embryos and those diagnosed as chromosomally abnormal by preimplantation genetic diagnosis. HLA-G detected in maternal plasma and follicular fluid did not appear to correlate with HLA-G expressed in the embryo or embryo supernatants. Confocal microscopy analysis indicated that HLA-G protein expression in embryos was not homogeneous; mostly, it was confined to blastocysts localized on trophectoderm and trophectoderm projections. Single-particle fluorescent imaging analysis of HLA-G on the cell surface of JEG-3 cells showed that HLA-G particles were mostly monomeric, but dimeric and higher order oligomers were also observed. These results suggest that HLA-G play an important role in preimplantation embryo development. However, the observed expression of HLA-G in arrested and chromosomally abnormal embryos indicates that HLA-G testing should be used with caution and in conjunction with conventional methods of embryo screening and selection.

The analysis of one or two blastomeres for PGD using fluorescence in-situ hybridization.

BACKGROUND: The analysis of one or two blastomeres for PGD using fluorescence in-situ hybridization (FISH) is debated. The proportion of analysable embryos, false negatives, false positives, sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV) and efficiency were evaluated when one or two blastomeres were analysed. METHODS: Embryos of patients having PGD for aneuploidy screening were assigned non-randomly to two groups: group I (n = 413), more slow cleaving embryos with one nucleus for analysis, and group II (n = 1366), regularly cleaving embryos with two nuclei for analysis. A two-round FISH procedure was performed investigating seven chromosomes; 486 embryos were reanalysed. RESULTS: The proportion of analysable embryos was significantly higher in group II (98.2 versus 95.9%) (P = 0.04). Despite the apparently increased false-positive rate (group I: 25.6% and group II: 13.6%) and the decreased PPV (group I: 91.9% and group II: 96.7%), specificity (group I: 74.4% and group II: 86.4%) and efficiency (group I: 93.5% and group II: 97.3%) in group I, no significance was reached (P = 0.11, P = 0.053, P = 0.11 and P = 0.06, respectively). CONCLUSIONS: Although the analysis of one blastomere generates statistically significantly fewer embryos with a diagnosis than does the analysis of two blastomeres, the 2% difference may not be clinically relevant. The diagnostic accuracy is not significantly different between the two groups, hence not favouring the analysis of one or two blastomeres.

[Uterine Doppler study for pre-implantation prognosis of the embryo]. / Doppler utérin dans le pronostic pré-implantatoire de l'embryon.

To assess if a simple US criteria was present allowing pre- and post-implantation evaluation of the quality of the embryo. Measurement of the pulsatility index (PI) of the uterine arteries in association with evaluation of the type of curves was useful for follow-up. PI correlates with the number of follicules, the number of cells at Day 2, and the likelihood of pregnancy whereas the type of curve directly correlates with the appearance of the oocytes and embryo before the transfer. These results should be confirmed by larger studies but they could lead to early detection and a treatment of these abnormalities of vascularization.

Practical considerations of embryo manipulation: preimplantation genetic typing.

In the past years, research in embryo technologies is moving to the establishment of preimplantation genetic typing or also denominated preimplantation genetic diagnosis (PGD). The objectives of these tests are the prevention of genetic diseases transmission and the prediction of phenotypic characteristics, as well as sex determination, genetic disorders and productive and reproductive profiles, prior to the embryo transfer or freezing, during early stages of development. This paper points out the state-of-the-art of PGD, mainly in cattle and discuss the perspectives of multiloci genetic analysis of embryos.