Anil Biricik

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.

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.

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.

Polymerase chain reaction-based detection of chromosomal imbalances on embryos: the evolution of preimplantation genetic diagnosis for chromosomal translocations

OBJECTIVE To develop and assess a polymerase chain reaction (PCR)-based preimplantation genetic diagnosis (PGD) approach for detection of chromosomal imbalances in embryos. DESIGN A prospective study of embryos derived from chromosome translocation carriers that have undergone PGD using a novel molecular-based approach. SETTING A reference molecular genetics laboratory specialized in the provision of transport PGD services and a private IVF clinic. PATIENT(S) Twenty-seven couples carrying 12 different reciprocal translocations and 2 Robertsonian translocations. INTERVENTION(S) Preimplantation genetic diagnosis from chromosome translocation carriers on blastomeres biopsied from cleavage stage embryos. MAIN OUTCOME MEASURE(S) Embryo diagnosis rate, pregnancy rate (PR), implantation rate, take-home-baby rate. RESULT(S) Overall, 241/251 (96.0%) embryos were successfully diagnosed for chromosome rearrangements. Preimplantation genetic screening was included in the protocol of 12 couples, involving analysis of 90 embryos, 84 (93.3%) of which were successfully diagnosed and 53 (63.1%) showed aneuploidies. Embryos suitable for transfer were identified in 24 cycles. Eighteen couples achieved a clinical pregnancy (75.0% PR/embryo transfer), with a total of 31 embryos implanted (59.6% implantation rate). Ten patients (1 triplet, 1 twin, and 8 singleton pregnancies) have delivered 13 healthy babies, and the other patients (3 twins and 5 singletons) have currently ongoing pregnancies. CONCLUSION(S) The PCR-based PGD protocol for translocations has the potential to overcome several inherent limitations of fluorescence in situ hybridization-based tests, providing potential improvements in terms of test performance, automation, turnaround time, sensitivity, and reliability.

Assessment of DNA fragmentation and aneuploidy on poor quality human embryos

In human assisted reproduction, low embryo quality due to retarded growth and abnormal cellular morphology results in fewer embryos suitable for transfer. This study aimed to assess the extent of DNA fragmentation and aneuploidy in spare slow growing or arrested human embryos. In 19 assisted reproduction cycles, a total of 57 embryos unsuitable for embryo transfer were used for simultaneous apoptosis and aneuploidy assessment. Among them, 31 (54.3%) showed DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) analysis. Among 26 embryos that were negative for TUNEL, interpretable fluorescence in-situ hybridization (FISH) results were obtained for 25 embryos (96.2%). Sixteen embryos were detected to be chromosomally abnormal (64.0%); three were found to be chaotic, six had complex aneuploidy, six had complete monosomy and one was polyploid. The results show that a high level of DNA fragmentation and aneuploidy are common in embryos with slow growth and/or low quality. More detailed studies are needed to assess the effect of factors such as ovarian stimulation regimens and in-vitro culture conditions. Moreover, application of simultaneous TUNEL and FISH techniques can be informative regarding DNA integrity and aneuploidy.

Results of preimplantation genetic diagnosis in patients with Klinefelter's syndrome.

With the application of preimplantation genetic diagnosis (PGD), a possible genetic contribution of spermatozoa obtained from 47,XXY non-mosaic Klinefelter patients on preimplantation embryos was analysed in eight couples. Interpretable fluorescence in-situ hybridization results were obtained for 28 out of 33 embryos biopsied (84.8%) and 23 blastomeres were analysed for chromosomes 13, 18, 21, X and Y. Nine out of 23 embryos were diagnosed as abnormal (39.1%). Five out of nine contained sex chromosome abnormalities (55.5%). Two were diagnosed as 47,XXY and three were found to have monosomy X. Besides sex chromosomal abnormalities, other abnormalities detected were haploidy, triploidy, monosomy 13, monosomy 18 and trisomy 13. Five blastomeres were analysed for sex chromosomes only and all of them were found to be normal. Overall, the rate of sex chromosome abnormality in biopsied embryos was found to be 17.8% (5/28). Moreover, among 33 embryos biopsied, five of the eight zygotes, which were classified as a poor prognosis group according to pronuclear morphology scoring, showed an impaired growth profile after biopsy and were found to be chromosomally abnormal. Elimination of abnormal embyos and transfer of normal ones resulted in four pregnancies in eight cycles (50%). Two pregnancies, one singleton and one twins resulted in healthy births. Two pregnancies, one singleton and one twins are continuing beyond the second trimester. These results show that there is in fact elevated chromosomal abnormality for both sex chromosomes and autosomes in embryos developed from Klinefelter males. Furthermore together with PGD, embryo scoring according to pronuclear morphology can give additional benefit for selecting chromosomally abnormal embryos. Therefore, PGD should be recommended in cases with Klinefelters syndrome and this information should be discussed with the couple when genetic counselling is given.

Embryo development characteristics in Robertsonian and reciprocal translocations: a comparison of results with non-translocation cases

The effect of translocations on embryo development was evaluated and results were compared in terms of embryo development with those of embryos obtained from standard intracytoplasmic sperm injection (ICSI) cycles. In 23 translocation carriers with 34 cycles, fertilization, pronuclear morphology scoring (PMS), developmental arrest, cleavage and blastocyst formation were evaluated and compared with embryos obtained from non-translocation cases undergoing ICSI (n = 98 cycles). In 28 cycles, preimplantation genetic diagnosis (PGD) was performed on prezygotes (first and second polar body biopsy for female carriers; n = 3) or on embryos having seven or more blastomeres (blastomere biopsy; n = 25). In six cycles for four couples, probes for translocated chromosomes were not available, so PGD could not be performed. Overall, in translocation cases, a lower fertilization rate, a higher rate of retarded embryo development, and a lower rate of blastocyst formation were observed compared with embryos of non-translocation cases. Fluorescence in-situ hybridization (FISH) analysis showed a 70.9% abnormality rate for reciprocal translocations and 55.0% for Robertsonian translocations respectively. In cases with Robertsonian and reciprocal translocation carriers, the probability of poor embryo development, which may be a result of high segregation abnormalities, may negatively affect the outcome of assisted reproductive techniques. This poor prognosis should also be considered when genetic counselling for translocation is given.