Preimplantation genetic testing for aneuploidy (PGT-A) remains one of the most controversial topics in reproductive medicine. With more than 40% of in vitro fertilization cycles in the United States ...reportedly involving PGT, both those in favor of and those opposed to PGT-A have significant interest in the efficacy of PGT-A. Ongoing issues include what patient population, if any, benefits from PGT-A, the true frequency of chromosomal mosaicism, whether embryonic aneuploidies self-correct, and how practitioners manage embryos designated as “mosaic.” This review addresses several misconceptions and misinterpretations of data surrounding the genetic analysis and prediction of mosaicism in the preimplantation embryo.
Whole-chromosome aneuploidy screening has become a common practice to improve outcomes and decrease embryonic transfer order in patients undergoing treatment for infertility through in vitro ...fertilization. Despite implementation of this powerful technology, a significant percentage of euploid embryos fail to result in successful deliveries. As technology has evolved, detection of subchromosomal imbalances and embryonic mosaicism has become possible, and these serve as potential explanations for euploid embryo transfer failures. Cases involving a parent with a balanced translocation provide a unique opportunity to characterize the capabilities and limitations of detecting segmental imbalances with a variety chromosome screening platforms. Adaptation of these methods to de novo imbalances now represent an ongoing challenge in the field of preimplantation genetic screening as additional factors including mosaicism, clinical predictive value, and distinguishing true imbalances from technical artifacts must be more carefully considered.
Preimplantation genetic testing for aneuploidy (PGT‐A) reduces miscarriage risk, increases the success of IVF, shortens time to pregnancy, and reduces multiple gestation rates without compromising ...outcomes. The progression of PGT‐A has included common application of next‐generation sequencing (NGS) from single nucleotide polymorphism microarray, quantitative real‐time PCR, and array comparative hybridization platforms of analysis. Additional putative advances in PGT‐A capability include classifying embryos as mosaic and predicting the presence of segmental imbalance. A critical component in the process of technical validation of these advancements involves evaluation of concordance between reanalysis results and initial testing results. While many independent studies have investigated the concordance of results obtained from the remaining embryo with the original PGT‐A diagnosis, compilation and systematic analysis of published data has not been performed. Here, we review results from 26 primary research articles describing concordance in 1271 human blastocysts from 2260 pairwise comparisons. Results illustrate significantly higher discordance from PGT‐A methods which utilize NGS and include prediction of mosaicism or segmental imbalance. These results suggest caution when considering new iterations PGT‐A.
OBJECTIVE: To determine both the negative and positive predictive values of comprehensive chromosome screening (CCS) results for clinical outcome. DESIGN: Data obtained from two prospective, ...double-blinded, nonselection studies. SETTING: Academic center for reproductive medicine. PATIENT(S): One hundred forty-six couples with a mean maternal age of 34.0 ± 4.4 years and a mean paternal age of 37.3 ± 5.8 years. INTERVENTION(S): Embryo biopsy for DNA fingerprinting and aneuploidy assessment. MAIN OUTCOME MEASURE(S): Failure rate of embryos predicted aneuploid by CCS (negative predictive value) and success rate of embryos predicted euploid by CCS (positive predictive value). RESULT(S): A total of 255 IVF-derived human embryos were cultured and selected for transfer without influence from CCS analysis. Embryos were biopsied before transfer, including 113 blastomeres at the cleavage stage and 142 trophectoderm biopsies at the blastocyst stage. Comprehensive chromosome screening was highly predictive of clinical outcome, with 96% of aneuploid predicted embryos failing to sustain implantation and 41% sustained implantation from embryos predicted to be euploid. CONCLUSION(S): These nonselection data provide the first prospective, blinded, clinical study directly measuring the predictive value of aneuploidy screening for clinical outcome. The clinical error rate of an aneuploidy designation is very low (4%), whereas implantation and delivery rates of euploid embryos are increased relative to the entire cohort of transferred embryos.
Transfer of more than a single embryo in an IVF cycle comes with the finite possibility of a multiple gestation. Even a twin pregnancy confers significant risk to both mother and babies. The move to ...single-embryo transfer for all patients will be greatly facilitated by the ability to quantify embryo viability. Developments in time-lapse incubation systems have provided new insights into the developmental kinetics of the human preimplantation embryo. Advances in molecular methods of chromosomal analysis have created platforms for highly effective screening of biopsied embryos, while noninvasive analysis of embryo physiology reveals more about the embryo than can be determined by morphology alone.
Recent developments in time-lapse microscopy, molecular karyotyping and in proteomics and metabolomics have been assessed and presented here in a descriptive review.
New algorithms are being created for embryo selection based on their developmental kinetics in culture, and the impact of factors such as patient etiology and treatment are being clarified. Potential links between morphokinetic data and embryo karyotype are being elucidated. The introduction of new molecular methods of determining embryo chromosomal complement is proving to be accurate and reproducible, with the future trending toward CGH arrays or next generation sequencing as a rapid and reliable means of analysis, that should be suitable for each IVF clinic to adopt. A relationship between embryo metabolism and viability is established and is now being considered together with morphokinetic data to create more robust algorithms for embryo selection. Microfluidic devices have the capacity and potential to be used in human IVF clinics for the routine diagnosis of embryo biomarkers.
To determine the relationship between the age of the female partner and the prevalence and nature of human embryonic aneuploidy.
Retrospective.
Academic.
Trophectoderm biopsies.
Comprehensive ...chromosomal screening performed on patients with blastocysts available for biopsy.
Evaluation of the impact of maternal age on the prevalence of aneuploidy, the probability of having no euploid embryos within a cohort, the complexity of aneuploidy as gauged by the number of aneuploid chromosomes, and the trisomy/monosomy ratio.
Aneuploidy increased predictably after 26 years of age. A slightly increased prevalence was noted at younger ages, with >40% aneuploidy in women 23 years and under. The no euploid embryo rate was lowest (2% to 6%) in women aged 26 to 37, was 33% at age 42, and was 53% at age 44. Among the biopsies with aneuploidy, 64% involved a single chromosome, 20% two chromosomes, and 16% three chromosomes, with the proportion of more complex aneuploidy increasing with age. Finally, the trisomy/monosomy ratio approximated 1 and increased minimally with age.
The lowest risk for embryonic aneuploidy was between ages 26 and 30. Both younger and older age groups had higher rates of aneuploidy and an increased risk for more complex aneuploidies. The overall risk did not measurably change after age 43. Trisomies and monosomies are equally prevalent.
OBJECTIVE: To determine if cleavage- or blastocyst-stage embryo biopsy affects reproductive competence. DESIGN: Paired randomized clinical trial. SETTING: Academic-assisted reproduction program. ...PATIENT(S): Attempting conception through IVF. INTERVENTION(S): After selecting two embryos for transfer, one was randomized to biopsy and the other to control. Both were transferred within shortly thereafter. The biopsy was submitted for microarray analysis and single-nucleotide polymorphism (SNP) profiling. Buccal DNA obtained from the neonate after delivery had microarray analysis and SNP profile compared with that of the embryonic DNA. A match confirmed that the biopsied embryo implanted and developed to term, whereas a nonmatch indicated that the control embryo had led to the delivery. MAIN OUTCOME MEASURE(S): Paired analysis of the delivery rates of the transferred embryos. Either twin delivery or failure to deliver represents equivalent outcomes for the biopsied and control embryos. In contrast, singletons were determined to be from the biopsied or the control embryo. RESULT(S): Blastomere biopsy on day 3 of development resulted in a significant reduction in sustained implantation. Only 30% of biopsied embryos had sustained implantation and ultimately developed into live-born infants versus 50% of unbiopsied controls, a relative reduction of 39%. In contrast, sustained implantation rates were equivalent (51% vs. 54%) for biopsied and control blastocysts. CONCLUSION(S): Cleavage-stage biopsy markedly reduced embryonic reproductive potential. In contrast, trophectoderm biopsy had no measurable impact and may be used safely when embryo biopsy is indicated. CLINICAL TRIAL REGISTRATION NUMBER: NCT01219504.
To determine whether blastocyst biopsy and rapid quantitative real-time polymerase chain reaction (qPCR)-based comprehensive chromosome screening (CCS) improves in vitro fertilization (IVF) ...implantation and delivery rates.
Randomized controlled trial.
Academic reproductive medicine center.
Infertile couples in whom the female partner (or oocyte donor) is between the ages of 21 and 42 years who are attempting conception through IVF.
Embryonic aneuploidy screening.
Sustained implantation and delivery rates.
We transferred 134 blastocysts to 72 patients in the study (CCS) group and 163 blastocysts to 83 patients in the routine care (control) group. Sustained implantation rates (probability that an embryo will implant and progress to delivery) were statistically significantly higher in the CCS group (89 of 134; 66.4%) compared with those from the control group (78 of 163; 47.9%). Delivery rates per cycle were also statistically significantly higher in the CCS group. Sixty one of 72 treatment cycles using CCS led to delivery (84.7%), and 56 of 83 (67.5%) control cycles ultimately delivered. Outcomes were excellent in both groups, but use of CCS clearly improved patient outcomes.
Blastocyst biopsy with rapid qPCR-based comprehensive chromosomal screening results in statistically significantly improved IVF outcomes, as evidenced by meaningful increases in sustained implantation and delivery rates.
NCT01219283.
To determine whether performing comprehensive chromosome screening (CCS) and transferring a single euploid blastocyst can result in an ongoing pregnancy rate that is equivalent to transferring two ...untested blastocysts while reducing the risk of multiple gestation.
Randomized, noninferiority trial.
Academic center for reproductive medicine.
Infertile couples (n = 205) with a female partner less than 43 years old having a serum anti-Müllerian hormone level ≥1.2 ng/mL and day 3 FSH <12 IU/L.
Randomization occurred when at least two blastocysts were suitable for trophectoderm biopsy. The study group (n = 89) had all viable blastocysts biopsied for real-time, polymerase chain reaction–based CCS and single euploid blastocyst transfer. The control group (n = 86) had their two best-quality, untested blastocysts transferred.
The ongoing pregnancy rate to ≥24 weeks (primary outcome) and the multiple gestation rate.
The ongoing pregnancy rate per randomized patient after the first ET was similar between groups (60.7% after single euploid blastocyst transfer vs. 65.1% after untested two-blastocyst transfer; relative risk RR, 0.9; 95% confidence interval CI, 0.7–1.2). A difference of greater than 20% in favor of two-blastocyst transfer was excluded. The risk of multiple gestation was reduced after single euploid blastocyst transfer (53.4% to 0%), and patients were nearly twice as likely to have an ongoing singleton pregnancy (60.7% vs. 33.7%; RR, 1.8; 95% CI, 1.3–2.5).
In women ≤42 years old, transferring a single euploid blastocyst results in ongoing pregnancy rates that are the same as transferring two untested blastocysts while dramatically reducing the risk of twins.
NCT01408433.
OBJECTIVE: To develop and validate a quantitative real-time polymerase chain reaction (qPCR)–based method for blastocyst trophectoderm comprehensive chromosome screening (CCS) of aneuploidy. DESIGN: ...Prospective, randomized, and blinded. SETTING: Academic center for reproductive medicine. PATIENT(S): Nine cell lines were obtained from a commercial cell line repository, and 71 discarded human blastocysts were obtained from 24 IVF patients that underwent preimplantation genetic screening. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Consistency of qPCR diagnosis of aneuploidy compared with either conventional karyotyping of cell lines or microarray-based diagnoses of human blastocysts. RESULT(S): Samples from nine cell lines with well characterized karyotypes were diagnosed by qPCR with 97.6% (41/42) consistency. After applying a minimum threshold for concurrence, 100% consistency was achieved. Developmentally normal blastocysts designated as aneuploid or arrested blastocysts designated as euploid by single-nucleotide polymorphism microarray analyses were assigned identical 24 chromosome diagnoses by qPCR in 98.6% of cases (70/71). Overall euploidy (n = 37) and aneuploidy (n = 34) were assigned with 100% consistency. Data was obtained for both sample types in 4 hours. CONCLUSION(S): These data demonstrate the first qPCR technology capable of accurate aneuploidy screening of all 24 chromosomes in 4 hours. This methodology provides an opportunity to evaluate trophectoderm biopsies with subsequent fresh euploid blastocyst transfer. Randomized controlled trials to investigate the clinical efficacy of qPCR-based CCS are currently underway.
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