Faculty Bibliography

PURPOSE: To determine if Aneuploidy Risk Classification Models are predictive of euploidy/aneuploidy amongst IVF facilities. METHODS: We retrospectively applied key time lapse imaging events of embryos (Campbell et al.[5, 6]) to stratify embryos into 3 groups: low, medium and high risk of aneuploidy. The actual ploidy results (from array comparative genomic hybridization) were compared with expectations [5, 6]. Sources of variability in morphokinetic parameters were determined using Analysis of Variance (ANOVA). RESULTS: The model failed to segregate euploid embryos from aneuploid embryos cultured at our facility. Further analysis indicated that the variability of embryos among patients was too great to allow selection of euploid embryos based on simple morphokinetic thresholds. Clinical selection of embryos based on morphokinetics alone is unlikely to identify euploid embryos accurately for transfer or yield higher rates of live delivery. CONCLUSIONS: The use of non-invasive morphokinetics is unlikely to discriminate aneuploid from euploid embryos. Further, it does not approach the accuracy of preimplantation genetic screening with array comparative genomic hybridization.

BACKGROUND:The majority of human embryos created using in vitro fertilisation (IVF) techniques are aneuploid. Comprehensive chromosome screening methods, applicable to single cells biopsied from preimplantation embryos, allow reliable identification and transfer of euploid embryos. Recently, randomised trials using such methods have indicated that aneuploidy screening improves IVF success rates. However, the high cost of testing has restricted the availability of this potentially beneficial strategy. This study aimed to harness next-generation sequencing (NGS) technology, with the intention of lowering the costs of preimplantation aneuploidy screening. METHODS:Embryo biopsy, whole genome amplification and semiconductor sequencing. RESULTS:A rapid (<15 h) NGS protocol was developed, with consumable cost only two-thirds that of the most widely used method for embryo aneuploidy detection. Validation involved blinded analysis of 54 cells from cell lines or biopsies from human embryos. Sensitivity and specificity were 100%. The method was applied clinically, assisting in the selection of euploid embryos in two IVF cycles, producing healthy children in both cases. The NGS approach was also able to reveal specified mutations in the nuclear or mitochondrial genomes in parallel with chromosome assessment. Interestingly, elevated mitochondrial DNA content was associated with aneuploidy (p<0.05), a finding suggestive of a link between mitochondria and chromosomal malsegregation. CONCLUSIONS:This study demonstrates that NGS provides highly accurate, low-cost diagnosis of aneuploidy in cells from human preimplantation embryos and is rapid enough to allow testing without embryo cryopreservation. The method described also has the potential to shed light on other aspects of embryo genetics of relevance to health and viability.

PURPOSE: This multicentered retrospective study analyzed whether the quantity of euploid blastocysts in a given cohort after comprehensive chromosomal screening can be used to identify candidates for single embryo transfer. METHODS: Blastocysts from 437 patients underwent trophectoderm biopsy followed by array comparative genomic hybridization. Embryos were then selected for single or double embryo transfer. The number of euploid blastocysts produced and transferred for each patient was recorded, as was clinical pregnancy rate and multiple gestation rate. RESULTS: In patients with /= 4 euploid blastocysts, clinical pregnancy rate was not reduced with single embryo transfer was performed, whereas the multiple gestation rate was greatly reduced. CONCLUSIONS: Size of the euploid embryo cohort is a marker for success in single embryo transfer cycles. Patients who produce at least four euploid blastocysts are outstanding candidates for single embryo transer.

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.

The chief cause for failure of in vitro fertilization (IVF)-assisted reproductive treatments may be the high frequency of aneuploid preimplantation embryos, especially among women of advanced reproductive age. It has been hypothesized that pregnancy loss with advancing maternal age can be prevented by selective transfer of euploid embryos. Preimplantation genetic diagnosis (PGD) for aneuploidy was first attempted more than 20 years ago. It was hoped that screening embryos for aneuploidy and transferring only those found to be euploid would increase implantation and pregnancy rates and reduce pregnancy loss rates. Initial attempts to detect aneuploidy used fluorescence in situ hybridization analysis (first-generation PGD). However, several randomized controlled trials found no benefit for first-generation PGD or even a negative impact on implantation, pregnancy, or loss rates. A more accurate version of PGD was needed. Array comparative genomic hybridization (aCGH) is a second-generation PGD. Use of this technique in randomized controlled trials improved pregnancy rates. The major drawback of aCGH is the need for at least 3 full days for an analysis to be completed. With the introduction of vitrification (freezing), safe cryopreservation of embryos that underwent biopsy became possible, allowing delay of embryo biopsy from 3 days (gastrula stage) to 5 to 6 days (blastocyst stage), which is less detrimental to embryo development. This multicenter retrospective study assessed the relationship between maternal age, chromosome abnormality, implantation, and pregnancy loss. The aim of the study was to determine whether aCGH followed by selective transfer of euploid embryos would mitigate the age-related decline in implantation rates observed in IVF cycles. Preimplantation chromosome screening was performed in women undergoing IVF at a number of fertility clinics in the United States. Implantation rates across different maternal ages were examined with embryo biopsy on day 3 or day 5/6 followed by aCGH. The primary outcome measures were aneuploidy, implantation, pregnancy, and loss rates. Aneuploidy rates increased with advancing maternal age from 53% to 93% for day 3 biopsies and from 32% to 85% for day 5/6 biopsies. Implantation rates for euploid embryos for ages 35 to 42 years were maintained after PGD; rates ranged from 44% to 32% for day 3 biopsies and 51% to 40% for day 5 biopsies. Ongoing pregnancy rates per transfer remained stable for maternal ages younger than 42 years, ranging from 48.5% to 38.1% for day 3 biopsies and 64.4% to 54.5% for day 5 biopsies. For patients 42 years or older, implantation rates were 23.3% with day 3 biopsies and 27.7% with day 5/6, and the ongoing pregnancy rate was 9.3% for day 3 biopsies and 10.3% for day 5. These data show no significant difference in implantation and pregnancy rates after selective transfer of euploid embryos between reproductively younger and older patients up to 42 years old. The enhanced embryo selection afforded by methods such as aCGH cannot improve pregnancy rates in patients who fail to produce at least 1 euploid embryo, a situation increasingly common with advancing maternal age. These findings and mounting data from other studies suggest that aneuploidy is the primary reason for the marked decline in IVF treatment success rates in women of advanced reproductive age when PGD is not used.