Francesco Fiorentino

PGD for reciprocal and Robertsonian translocations using array comparative genomic hybridization

BACKGROUND Fluorescence in situ hybridization (FISH) is the most widely used method for detecting unbalanced chromosome rearrangements in preimplantation embryos but it is known to have several technical limitations. We describe the clinical application of a molecular-based assay, array comparative genomic hybridization (array-CGH), to simultaneously screen for unbalanced translocation derivatives and aneuploidy of all 24 chromosomes. METHODS Cell biopsy was carried out on cleavage-stage embryos (Day 3). Single cells were first lysed and DNA amplified by whole-genome amplification (WGA). WGA products were then processed by array-CGH using 24sure + arrays, BlueGnome. Balanced/normal euploid embryos were then selected for transfer on Day 5 of the same cycle. RESULTS Twenty-eight consecutive cycles of preimplantation genetic diagnosis were carried out for 24 couples carrying 18 different balanced translocations. Overall, 187/200 (93.5%) embryos were successfully diagnosed. Embryos suitable for transfer were identified in 17 cycles (60.7%), with transfer of 22 embryos (mean 1.3 ± 0.5). Twelve couples achieved a clinical pregnancy (70.6% per embryo transfer), with a total of 14 embryos implanted (63.6% per transferred embryo). Three patients delivered three healthy babies, during writing, the other pregnancies (two twins and seven singletons) are ongoing beyond 20 weeks of gestation. CONCLUSIONS The data obtained demonstrate that array-CGH can detect chromosome imbalances in embryos, also providing the added benefit of simultaneous aneuploidy screening of all 24 chromosomes. Array-CGH has the potential to overcome several inherent limitations of FISH-based tests, providing improvements in terms of test performance, automation, sensitivity and reliability.

What next for preimplantation genetic screening (PGS)? A position statement from the ESHRE PGD Consortium steering committee

Since 2004, there have been 11 randomized controlled trials (RCTs) mainly for advanced maternal age (AMA), which have shown no benefit of performing preimplantation genetic screening (PGS). Ten of the RCTs have been performed at the cleavage stage and one at the blastocyst stage. It is probable that the high levels of chromosomal mosaicism at cleavage stages, which may result in the tested cell not being representative of the embryo, and the inability to examine all of the chromosomes using fluorescence in situ hybridization, have contributed to the lack of positive outcome from the RCTs. We suggest that future RCTs should examine alternative biopsy timing (polar body and/or trophectoderm biopsy), and should apply technologies that allow more comprehensive testing to include all chromosomes (microarray-based testing) to determine if PGS shows an improvement in delivery rate. Currently there is no evidence that routine PGS is beneficial for patients with AMA and conclusive data (RCTs) on repeated miscarriage, implantation failure and severe male factor are missing. To evaluate benefits of PGS, an ESHRE trial has recently been started on patients with AMA using polar body biopsy and array-comparative genomic hybridization, which should bring more information on this patient group in the near future.

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.

Healthy Babies after Intrauterine Transfer of Mosaic Aneuploid Blastocysts

Implantation of 18 mosaic aneuploid blastocysts obtained through in vitro fertilization into 18 women resulted in six pregnancies of normal karyotype. These pregnancies proceeded to term, with the birth of six healthy babies.

Development and validation of a next-generation sequencing–based protocol for 24-chromosome aneuploidy screening of embryos

OBJECTIVE To validate a next-generation sequencing (NGS)-based method for 24-chromosome aneuploidy screening and to investigate its applicability to preimplantation genetic screening (PGS). DESIGN Retrospective blinded study. SETTING Reference laboratory. PATIENT(S) Karyotypically defined chromosomally abnormal single cells and whole-genome amplification (WGA) products, previously analyzed by array comparative genomic hybridization (array-CGH), selected from 68 clinical PGS cycles with embryos biopsied at cleavage stage. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Consistency of NGS-based diagnosis of aneuploidy compared with either conventional karyotyping of single cells or array-CGH diagnoses of single blastomeres. RESULT(S) Eighteen single cells and 190 WGA products from single blastomeres, were blindly evaluated with the NGS-based protocol. In total, 4,992 chromosomes were assessed, 402 of which carried a copy number imbalance. NGS specificity for aneuploidy call (consistency of chromosome copy number assignment) was 99.98% (95% confidence interval [CI] 99.88%-100%) with a sensitivity of 100% (95% CI 99.08%-100%). NGS specificity for aneuploid embryo call (24-chromosome diagnosis consistency) was 100% (95% CI 94.59%-100%) with a sensitivity of 100% (95% CI 97.39%-100%). CONCLUSION(S) This is the first study reporting extensive preclinical validation and accuracy assessment of NGS-based comprehensive aneuploidy screening on single cells. Given the high level of consistency with an established methodology, such as array-CGH, NGS has demonstrated a robust high-throughput methodology ready for clinical application in reproductive medicine, with potential advantages of reduced costs and enhanced precision.

Application of next-generation sequencing technology for comprehensive aneuploidy screening of blastocysts in clinical preimplantation genetic screening cycles

STUDY QUESTION Can next-generation sequencing (NGS) techniques be used reliably for comprehensive aneuploidy screening of human embryos from patients undergoing IVF treatments, with the purpose of identifying and selecting chromosomally normal embryos for transfer? SUMMARY ANSWER Extensive application of NGS in clinical preimplantation genetic screening (PGS) cycles demonstrates that this methodology is reliable, allowing identification and transfer of euploid embryos resulting in ongoing pregnancies. WHAT IS KNOWN ALREADY The effectiveness of PGS is dependent upon the biology of the early embryo and the limitations of the technology. Fluorescence in situ hybridization, used to test for a few chromosomes, has largely been superseded by microarray techniques that test all 24 chromosomes. Array comparative genomic hybridization (array-CGH) has been demonstrated to be an accurate PGS method and has become the de facto gold standard, but new techniques, such as NGS, continue to emerge. STUDY DESIGN, SIZE, DURATION The study consisted of a prospective trial involving a double blind parallel evaluation, with both NGS and array-CGH techniques, of 192 blastocysts obtained from 55 consecutive clinical PGS cycles undertaken during the period of September to October 2013. Consistency of NGS-based aneuploidy detection was assessed by matching the results obtained with array-CGH-based diagnoses. Primary outcome measure was accuracy of the chromosomal analysis; secondary outcome measures were clinical outcomes. PARTICIPANTS/MATERIALS, SETTINGS, METHODS Fifty-five patients (median age 39.3 years, range 32-46) undergoing PGS were enrolled in the study. All embryos were cultured to blastocyst stage; trophectoderm biopsy was performed on Day 5 of development or Day 6/7 for slower growing embryos. The method involved whole genome amplification followed by both NGS and array-CGH. The MiSeq control software, real-time analysis and reporter performed on-board primary and secondary bioinformatics analysis. Copy number variation analysis was accomplished with BlueFuse Multi software. MAIN RESULTS AND THE ROLE OF CHANCE A total of 192 blastocysts were blindly evaluated with the NGS-based protocol. Paired comparison between NGS and array-CGH from individual embryos showed concordant results in 191/192 (99.5%) of the blastocysts tested. In total 4608 chromosomes were assessed, 211 (4.6%) of which carried a copy number imbalance. NGS specificity for aneuploidy calling (consistency of chromosome copy number assignment) was 99.98% (4333/4334; 95% confidence interval [95% CI]: 99.87-100) with a sensitivity of 100% (211/211, 95% CI: 99.25-100). Despite one discordant result, NGS specificity and sensitivity for aneuploid embryo calling (24-chromosome diagnosis consistency) were both 100% since the discordant sample presented several other aneuploidies. Clinical application of the NGS-based approach revealed 74/192 (38.5%) euploid blastocysts. Following transfer of 50 embryos in 47 women, 34 women had positive hCG levels: 30 pregnancies continued, confirmed by at least one fetal sac and heart beat (63.8% clinical pregnancy rate/embryo transfer), 3 were biochemical and 1 miscarried. A total of 32 embryos implanted and led to the presence of a fetal sac (64.0% implantation rate). All pregnancies went to term resulting in the birth of 31 healthy babies. LIMITATION, REASON FOR CAUTION Although clinical results reported high pregnancy outcomes following transfer of screened embryos, further data and broad-based clinical application are required to better define the role of NGS in PGS. Before recommending widespread application, a randomized controlled trial confirming its clinical effectiveness is advisable. WIDER IMPLICATION OF THE FINDING This is the first study reporting extensive application of NGS-based comprehensive aneuploidy screening on embryos at blastocyst stage in a clinical setting versus array-CGH as test of reference. NGS has demonstrated a reliable methodology, with the potential to improve chromosomal diagnosis on embryos especially in terms of high-throughput, automation and ability to detect aneuploidy. NGS methodology may represent a valuable alternative to the other comprehensive aneuploidy screening techniques currently available. STUDY FUNDING/COMPETING INTERESTS No external funding was sought for this study. Drs F.K. and C.-E.M. are full-time employees of Illumina, Inc., which provided NGS library and sequencing reagents for the study. All other authors have no conflicts to declare. TRIAL REGISTRATION NUMBER Not applicable.

Sequential comprehensive chromosome analysis on polar bodies, blastomeres and trophoblast: insights into female meiotic errors and chromosomal segregation in the preimplantation window of embryo development

STUDY QUESTION What is the optimal stage from oocyte through preimplantation embryo development for biopsy and preimplantation genetic screening (PGS) to detect abnormal chromosome segregation patterns in eggs or embryos from advanced maternal age (AMA) patients? SUMMARY ANSWER Testing at the polar body (PB) stage was the least accurate mainly due to the high incidence of post-zygotic events. This suggests that postponing the time of biopsy to the blastocyst stage of preimplantation embryo development may provide the most reliable results for PGS. WHAT IS KNOWN ALREADY In the PGS field there is an ongoing debate about the optimal biopsy stage for PGS. This is a result of the lack of understanding of how aneuploidy arises in the human embryo. To date, most of the cytogenetic data obtained during PGS investigations have been derived through the analysis of cells at isolated points in the preimplantation window, thus potentially missing critical information on chromosomal segregation. Understanding the chromosome segregation patterns during preimplantation development holds the potential to significantly increase the success rates of IVF. In this study, a sequential comprehensive chromosome analysis of both the PBs and the corresponding embryos at both the cleavage and the blastocyst stages is presented. STUDY DESIGN, SIZE, DURATION This is a prospective longitudinal cohort study performed between October 2009 and August 2011 involving 9 infertile couples and 21 sets of complete comprehensive chromosomal screening data, including PB1, PB2, corresponding blastomeres and trophectoderm (TE) samples. PARTICIPANTS/MATERIALS, SETTING, METHODS Infertile couples undergoing IVF cycles with PGS where the female partner was older than 40 years and with a good response to controlled ovarian stimulation (>10 MII oocytes retrieved) were enrolled into the study. The exclusion criteria were (i) patients presenting with abnormal karyotype; (ii) specific ovarian pathologies including polycystic ovary syndrome, endometriosis grade III or higher and premature ovarian failure and (iii) severe male factor infertility (motile sperm count of <500 000/ml after preparation of a fresh ejaculate). The PBs, blastomere and TE samples were sequentially biopsied and analyzed by array comparative genomic hybridization (aCGH). The analysis of chromosome segregation patterns was performed to infer the origin of aneuploidy and to investigate the diagnostic accuracy of both PB and cleavage-stage PGS strategies. MAIN RESULTS AND THE ROLE OF CHANCE Twenty-one sets of complete data (PB1/PB2/blastomere/TE) including 84 aCGH experiments showed a pattern of multiple meiotic errors typically caused by sister chromatid separation errors and predominantly arising in the second meiotic division. Twenty-two of the 24 (91.7%) errors in the first meiotic division arose as a consequence of premature sister chromatid predivision. In half of these cases, the second meiotic division resulted in a balancing chromosome segregation event producing a normal female complement for that chromosome in the resulting embryo. Overall, only 62 out of 78 (79.5%) of the abnormal meiotic segregations had errors in the either one or both PBs consistent with the aneuploidies observed in their resulting embryos. Ten of the 21 (47.6%) embryos had aneuploidies other than female meiotic-derived ones, most of which detected on Day 3 and confirmed on Day 5 or 6 of embryo development (20/25) with chromosomal loss being three times more frequent than gains. Notably, as high as 20% of female-derived aneuploidies detected on PBs and confirmed on Day 3 were rescued at the blastocyst stage, mainly as a result of diploidization of trisomic chromosomes. On a per chromosome basis, the sensitivity in predicting blastocyst chromosomal complement was significantly lower for PB approach, 61.7%, compared with blastomeres analysis, 86.4% (P < 0.01). LIMITATIONS, REASONS FOR CAUTION The study was limited to the analysis of oocytes and embryos from AMA patients. Thus, these findings apply only to this patient group. Comparisons with other patient populations including patients with different indications for PGS should be made in future research. In addition, higher resolution and/or more accurate chromosomal screening tests could be used in future studies to corroborate the current findings. WIDER IMPLICATIONS OF THE FINDINGS These findings provide critical insights into the mechanisms causing errors during female meiosis and the preimplantation embryo development period to improve the design and treatment outcome of PGS.

Introducing array comparative genomic hybridization into routine prenatal diagnosis practice: a prospective study on over 1000 consecutive clinical cases

To assess the feasibility of offering array‐based comparative genomic hybridization testing for prenatal diagnosis as a first‐line test, a prospective study was performed, comparing the results achieved from array comparative genomic hybridization (aCGH) with those obtained from conventional karyotype.

What next for preimplantation genetic screening

Preimplantation genetic diagnosis for aneuploidy screening (preimplantation genetic screening-PGS) has been used to detect chromosomally normal embryos from subfertile patients. The main indications are advanced maternal age (AMA), repeated implantation failure, repeated miscarriages and severe male factor infertility. Many non-randomized PGS studies have been published and report an increase in implantation rate, and/or a decrease in miscarriage rate. Recently, two randomized controlled trials have been conducted on patients with AMA as the only indication. Neither study showed a benefit in performing PGS using live birth rate as the measure of success. The debate on the usefulness of PGS is ongoing; the only effective way to resolve the debate is to perform more well-designed and well-executed randomized clinical trials.

Short tandem repeats haplotyping of the HLA region in preimplantation HLA matching.

Recently, preimplantation genetic diagnosis (PGD) has been considered for several indications beyond its original purpose, not only to test embryos for genetic disease but also to select embryos for a nondisease trait, such as specific human leukocyte antigen (HLA) genotypes, related to immune compatibility with an existing affected child in need of a haematopoetic stem cell (HSC) transplant. We have optimized an indirect single-cell HLA typing protocol based on a multiplex fluorescent polymerase chain reaction (PCR) of short tandem repeat (STR) markers scattered throughout the HLA complex. The assay was clinically applied in 60 cycles from 45 couples. A conclusive HLA-matching diagnosis was achieved in 483/530 (91.1%) of the embryos tested. In total, 74 (15.3%) embryos revealed an HLA match with the affected siblings, 55 (11.4%) of which resulted unaffected and 46 (9.5%) have been transferred to the patients. Nine pregnancies were achieved, five healthy HLA-matched children have already been delivered and cord blood HSCs, were transplanted to three affected siblings, resulting in a successful haematopoietic reconstruction.