Dagan Wells

Clinical application of comprehensive chromosomal screening at the blastocyst stage

OBJECTIVE To evaluate a new strategy for comprehensive chromosome screening at the blastocyst stage. DESIGN Clinical research study. SETTING An IVF clinic and a specialist preimplantation genetic diagnosis laboratory. PATIENT(S) Forty-five infertile couples participated in the study. The mean maternal age was 37.7 years, and most couples had at least one previous unsuccessful IVF treatment cycle (mean 2.4). INTERVENTION(S) This study used a novel chromosome screening approach, combining biopsy of several trophectoderm cells on day 5 after fertilization and detailed analysis of all 24 types of chromosome using comparative genomic hybridization. MAIN OUTCOME MEASURE(S) Proportion of embryos yielding a diagnostic result, aneuploidy rate, implantation rate, and pregnancy rate. RESULT(S) A diagnosis was obtained from 93.7% of embryos tested. The aneuploidy rate was 51.3%. The probability of an individual transferred embryo forming a pregnancy reaching the third trimester/birth was 68.9%, an implantation rate 50% higher than contemporary cycles from the same clinic. The pregnancy rate was 82.2%. CONCLUSION(S) The comprehensive chromosome screening method described overcomes many of the problems that limited earlier aneuploidy screening techniques and may finally allow preimplantation genetic screening to achieve the benefits predicted by theory. The high embryo implantation rate achieved is particularly encouraging and, if confirmed in subsequent studies, will be of great significance for IVF clinics attempting to reduce the number of embryos transferred or to implement single embryo transfer.

Validation of microarray comparative genomic hybridization for comprehensive chromosome analysis of embryos

OBJECTIVE To validate and determine the best array-comparative genomic hybridization (aCGH; array-CGH) protocols for preimplantation genetic screening (PGS). DESIGN Embryos had one cell removed as a biopsy specimen and analyzed by one of two array-CGH protocols. Abnormal embryos were reanalyzed by fluorescence in situ hybridization (FISH). SETTING Reference laboratory. PATIENT(S) Patients donating embryos or undergoing PGS. INTERVENTION(S) Embryo biopsy, array-CGH, FISH reanalysis. MAIN OUTCOME MEASURE(S) Diagnosis, no result rate and error rate. RESULT(S) Method one produced 11.2% of embryos with no results and a 9.1% error rate compared with 3% and 1.9% for method two, respectively. Thereafter, only method two was used clinically. The aneuploidy rate for cleavage-stage embryos was 63.2%, significantly increasing with maternal age. The chromosomes most involved in aneuploidy were 16, 22, 21, and 15. We report the first live births after array-CGH combined with single blastomere biopsy. CONCLUSION(S) Array-CGH is proved to be highly robust (2.9% no results) and specific (1.9% error rate) when applied to rapid (24-hour) analysis of single cells biopsied from cleavage-stage embryos. This comprehensive chromosome analysis technique is the first to be validated by reanalyzing the same embryos with another technique (e.g., FISH). Unlike some alternative techniques for comprehensive chromosome screening, array-CGH does not require prior testing of parental DNA and thus advance planning and careful scheduling are unnecessary.

First clinical application of comparative genomic hybridization and polar body testing for preimplantation genetic diagnosis of aneuploidy.

Abstract Objective: To develop a preimplantation genetic diagnosis (PGD) protocol that allows any form of chromosome imbalance to be detected. Design: Case report employing a method based on whole-genome amplification and comparative genomic hybridization (CGH). Setting: Clinical IVF laboratory. Patient(s): A 40-year-old IVF patient. Intervention(s): Polar body and blastomere biopsy. Main Outcome Measure(s): Detection of aneuploidy. Result(s): Chromosome imbalance was detected in 9 of 10 polar bodies. A variety of chromosomes were aneuploid, but chromosomal size was found to be an important predisposing factor. In three cases, the resulting embryos could be tested using fluorescence in situ hybridization, and in each case the CGH diagnosis was confirmed. A single embryo could be recommended for transfer on the basis of the CGH data, but no pregnancy ensued. Conclusion(s): Evidence suggests that preferential transfer of chromosomally normal embryos can improve IVF outcomes. However, current PGD protocols do not allow analysis of every chromosome, and therefore a proportion of abnormal embryos remains undetected. We describe a method that allows every chromosome to be assessed in polar bodies and oocytes. The technique was accurate and allowed identification of aneuploid embryos that would have been diagnosed as normal by standard PGD techniques. As well as comprehensive cytogenetic analysis, this protocol permits simultaneous testing for multiple single-gene disorders.

The relationship between blastocyst morphology, chromosomal abnormality, and embryo gender.

OBJECTIVE To assess correlation between blastocyst morphology and chromosomal status. DESIGN Observational research study. SETTING An IVF clinic and a specialist preimplanation genetic diagnosis (PGD) laboratory. PATIENT(S) Ninety-three couples undergoing IVF treatment in combination with chromosome screening of embryos. INTERVENTION(S) Five hundred blastocysts underwent trophectoderm biopsy and comprehensive chromosome screening using comparative genomic hybridization (CGH). The morphology of the embryos was evaluated using standard methods. MAIN OUTCOME MEASURE(S) Association of aneuploidy and morphologic score. RESULT(S) A total of 56.7% of blastocysts were aneuploid. One-half of the grade 5/6 blastocysts were euploid, compared with only 37.5% of embryos graded 1/2, suggesting an effect of aneuploidy on blastocyst development. Aneuploidy also had a negative effect on inner cell mass and trophectoderm grades. Morphologically poor blastocysts had a higher incidence of monosomy and abnormalities affecting several chromosomes. The gender ratio was significantly skewed in relation to morphology. A total of 72% of blastocysts attaining the highest morphologic scores (5AA and 6AA) were found to be male, compared with only 40% of grade 3 embryos. CONCLUSION(S) Morphology and aneuploidy are linked at the blastocyst stage. However, the association is weak, and consequently, morphologic analysis cannot be relied on to ensure transfer of chromosomally normal embryos. A significant proportion of aneuploid embryos are capable of achieving the highest morphologic scores, and some euploid embryos are of poor morphology. Gender was associated with blastocyst grading, male embryos developing at a significantly faster rate than females.

Cytogenetic analysis of human blastocysts with the use of FISH, CGH and aCGH: scientific data and technical evaluation

BACKGROUND Recent studies have suggested that biopsy of several trophectoderm (TE) cells from blastocysts followed by comparative genomic hybridization (CGH) analysis might represent an optimal strategy for aneuploidy detection, but few data on accuracy are available. The main question concerns the rate of mosaicism at the blastocyst stage, and to what extent this might cause misdiagnoses. We assessed blastocyst aneuploidy and mosaicism rates and evaluated the accuracy and efficiency of CGH and microarray-CGH (aCGH) for TE analysis. METHODS A total of 52 blastocysts, from 20 couples, were biopsied and their chromosomes examined by CGH. The remaining cells were spread and tested by fluorescent in situ hybridization (FISH). Of the 52 blastocysts, 20 underwent a second TE biopsy and were tested using aCGH. RESULTS CGH and aCGH produced results for 98% of TE samples. 42.3% of blastocysts were uniformly euploid, 30% were uniformly aneuploid and 32.4% were mosaic. Of the mosaic embryos, 15.4% were found to be composed of a mixture of different aneuploid cell lines, while 17% contained both normal and aneuploid cells. Mosaic diploid-aneuploid blastocysts with >30% normal cells accounted for <6% of analysed embryos. CONCLUSIONS Comprehensive chromosome screening and follow-up assessment of large numbers of cells provided a unique insight into the cytogenetics of human blastocysts. Meiotic and post-zygotic errors leading to mosaicism were common. However, most mosaic blastocysts contained no normal cells. Hence, CGH or aCGH TE analysis is an accurate aneuploidy detection tool and may assist in identifying viable euploid embryos with higher implantation potential.

Comprehensive molecular cytogenetic analysis of the human blastocyst stage

BACKGROUND The high frequency of chromosomal abnormalities observed in human gametes and embryos is unlike that seen in other mammalian species and is of great clinical significance, leading to high rates of pregnancy loss, and live-born children with aneuploid syndromes. Although much is known concerning the aneuploidy rates of oocytes, cleavage stage embryos and fetuses during pregnancy, the chromosomal status of blastocysts has been relatively little investigated. METHODS A total of 158 good quality blastocysts were examined using micromanipulation, whole genome amplification and comparative genomic hybridization. RESULTS From the obtained data, it was evident that the aneuploidy rate (38.8%) is significantly lower for blastocysts than for embryos at earlier stages (51%). However, in many cases, chromosome errors, including monosomy, imbalance affecting the larger chromosomes and complex aneuploidy persisted to this final stage of preimplantation development. CONCLUSIONS This study represents the first attempt to gain a detailed insight into the extent and type of chromosome errors seen at the blastocyst stage, using a comprehensive molecular cytogenetic method. Our data suggest that the blastocyst stage does not represent an absolute selective barrier, and that the majority of aneuploid embryos are lost at the time of implantation or shortly thereafter.

Diminished effect of maternal age on implantation after preimplantation genetic diagnosis with array comparative genomic hybridization.

OBJECTIVE To assess the relationship between maternal age, chromosome abnormality, implantation, and pregnancy loss. DESIGN Multicenter retrospective study. SETTING IVF centers in the United States. PATIENT(S) IVF patients undergoing chromosome screening. INTERVENTION(S) Embryo biopsy on day 3 or day 5/6 with preimplantation genetic diagnosis (PGD) by array comparative genomic hybridization. MAIN OUTCOME MEASURE(S) Aneuploidy, implantation, pregnancy, and loss rates. RESULT(S) Aneuploidy rates increased with maternal age from 53% to 93% for day 3 biopsies and from 32% to 85% for blastocyst biopsies. Implantation rates for euploid embryos for ages <35-42 years did not decrease after PGD: ranges 44%-32% for day 3 and 51%-40% for blastocyst. Ongoing pregnancy rates per transfer did not decrease for maternal ages <42 years after PGD with day 3 biopsy (48.5%-38.1%) or blastocyst biopsy (64.4%-54.5%). Patients >42 years old had implantation rates of 23.3% (day 3), 27.7% (day 5/6), and the pregnancy rate with day 3 biopsy was 9.3% and with day 5 biopsy 10.3%. CONCLUSION(S) Selective transfer of euploid embryos showed that implantation and pregnancy rates were not significantly different between reproductively younger and older patients up to age 42 years. Some patients who start an IVF cycle planning to have chromosome screening do not have euploid embryos available for transfer, a situation that increases with advancing maternal age. Mounting data suggests that the dramatic decline in IVF treatment success rates with female age is primarily caused by aneuploidy.

Comprehensive chromosome screening of polar bodies and blastocysts from couples experiencing repeated implantation failure.

OBJECTIVE To identify and transfer cytogenetically normal embryos after screening all chromosomes of first and second polar bodies (PBs) or trophectoderm samples with the use of comparative genomic hybridization. DESIGN Clinical research study. SETTING In vitro fertilization clinic referring samples to a specialist preimplantation genetic diagnosis laboratory. PATIENT(S) Thirty-two couples with repeated implantation failure. INTERVENTION(S) Zygotes from patients with repeated implantation failure and poor response to ovarian stimulation underwent PB biopsy. Patients with repeated implantation failure who were candidates for blastocyst transfer received trophectoderm biopsy. Zygotes or blastocysts were vitrified while chromosome analysis took place. Euploid embryos were transferred during a subsequent cycle. MAIN OUTCOME MEASURE(S) Cytogenetic status and implantation and pregnancy rates. RESULT(S) The oocyte and blastocyst aneuploidy rates were 65.5% and 45.2%, respectively. Abnormalities affecting all chromosomes were detected. Implantation and pregnancy rates for the patients with PB biopsy were 11.5% and 21.4%, respectively, whereas for patients receiving blastocyst analysis they were 58.3% and 69.2%. CONCLUSION(S) Initial results for patients of advanced maternal age (39.8 years) with repeated implantation failure and poor ovarian response were encouraging. However, further study is required to confirm whether or not screening is beneficial. Blastocyst analysis was associated with high pregnancy rates, suggesting that comprehensive chromosome screening may assist patients with repeated implantation failure capable of producing blastocysts in achieving pregnancies.

Use of comprehensive chromosomal screening for embryo assessment: microarrays and CGH

One of the most important factors influencing embryo viability is chromosome imbalance (aneuploidy). Embryos derived from aneuploid gametes have little potential for forming a viable pregnancy, but cannot be distinguished from normal embryos using standard morphological evaluation. For more than a decade, preimplantation genetic screening (PGS) has been used to assist in the identification of aneuploid embryos. However, current strategies, based upon cell biopsy followed by fluorescent in situhybridization, allow less than half of the chromosomes to be screened. In this review, we discuss methods that overcome the limitations of earlier PGS strategies and provide screening of the entire chromosome complement in oocytes and embryos. In recent months, there has been a rapid growth in the number of PGS cycles utilizing one such method, comparative genomic hybridization (CGH). Data from IVF cycles utilizing CGH must be considered preliminary, but appear to indicate a dramatic increase in embryo implantation following comprehensive chromosomal screening. It is expected that methods based upon microarrays will yield similar clinical results and may be sufficiently rapid to permit comprehensive screening without the need for embryo cryopreservation. Some microarray platforms also offer the advantage of embryo fingerprinting and the potential for combined aneuploidy and single gene disorder diagnosis. However, more data concerning accuracy and further reductions in the price of tests will be necessary before microarrays can be widely applied.

Maternal age-related differential global expression profiles observed in human oocytes

The age-related decline in female fertility has been attributed to a variety of causes including progressive oocyte depletion, meiotic irregularities and mitochondrial dysfunction. However, additional factors could potentially be involved. To explore this possibility, comprehensive analysis of gene expression in human oocytes, discarded following IVF procedures and segregated by age, was undertaken using microarray methods. These findings indicate that the expression of oocyte genes, in a variety of major functional categories including cell cycle regulation, cytoskeletal structure, energy pathways, transcription control, and stress responses, are influenced by maternal age. These results are corroborated by a complementary extensive study using mouse oocytes.