Blastocyst-stage comprehensive chromosome screening (CCS) to select euploid embryos is the key to successful elective single embryo transfer (SET).
Illustration by Alex Baker, DNA Illustrations, Inc.Dr. Forman is a Reproductive Endocrinologist, Reproductive Medicine Associates of New Jersey, Morristown and Basking Ridge, and Assistant Professor, Department of Obstetrics, Gynecology & Reproductive Sciences, Rutgers Robert Wood Johnson Medical School, New Brunswick, New Jersey.
Dr. Scott is Laboratory and Scientific Director at Reproductive Medicine Associates of New Jersey, Basking Ridge, Professor, Department of Obstetrics, Gynecology & Reproductive Sciences and Division Director and Fellowship Director, Division of Reproductive Endocrinology & Infertility, Rutgers Robert Wood Johnson Medical School, New Brunswick, New Jersey. He is also Board President of the Foundation for the Assessment and Enhancement of Embryonic Competence, which utilizes the method for relative quantitation of chromosomal DNA that he developed.
Neither author has a conflict of interest to report with respect to the content of this article.
Kathy is a 39-year-old G1P0010 with 2 years of unexplained infertility after a miscarriage. After 3 cycles of injectable gonadotropins with timed intrauterine insemination (IUI) she progressed to treatment with in vitro fertilization (IVF). In her first cycle, she produced 4 blastocysts and the 2 of the highest quality were transferred. She was discharged to her obstetrician at 9 weeks’ gestation with a twin intrauterine pregnancy. At 20 weeks’ gestation, she had preterm contractions and precipitously delivered nonviable twins. She subsequently had a frozen single-embryo transfer (SET) that resulted in a negative pregnancy test and a second frozen SET, which resulted in a clinical miscarriage. The fetus was found to have trisomy 16 on cytogenetic analysis. Kathy would like to undergo another IVF cycle. How should she be managed?
Like most couples, Kathy and her husband did not plan to have twins when they decided to start a family. It was only after many months of trying to conceive and the disappointment of one negative pregnancy test after another that the concept of twins became appealing. The inefficiency of treatment options has led to the approximately 20-fold increased risk of multiple pregnancy becoming desirable to many patients requiring IVF for infertility.1
Twin pregnancies are at an increased risk of virtually every obstetrical and neonatal complication. Preterm multiple delivery is the principal complication of modern assisted reproductive technologies (ART) and it results in an estimated $1 billion annual burden on the healthcare system.2 Despite these unassailable facts, some have continued to argue that this nonphysiological state of pregnancy is a desirable option for couples seeking pregnancy through ART.3 Because singleton pregnancies are safer, more natural, and have a lower risk of complications, they should be the desired goal of infertility treatment as with spontaneous conception. Therefore, the goal of IVF should be to identify the embryo with the highest reproductive potential from among a cohort of embryos in order to maximize the chance of singleton delivery after a single embryo is transferred to the uterus.
The first IVF baby born in 1978 was the product of SET.4 Since then, due to the inefficiency of human reproduction and the near-impossibility of using visual inspection to predict which embryo is most likely to implant and develop into a normal baby, superovulation with transfer of multiple embryos has become standard practice. With each transfer, the physician and patient hope that at least one embryo (and not more than two) will implant. Unfortunately, the minority of transfers replicate the physiological normal outcome of a singleton pregnancy. But the landscape of IVF is rapidly changing.
The Blastocyst Euploid Selective Transfer (BEST) Trial was the first randomized trial to demonstrate that SET could be performed without compromising delivery rates.5 This trial made use of blastocyst-stage comprehensive chromosome screening (CCS) to assess whether the embryo selected for SET was chromosomally normal. This was the culmination of several years of validation studies to demonstrate that DNA could safely be obtained from embryos, analyzed rapidly, and used to select a single chromosomally normal (euploid) blastocyst for transfer.
Aneuploidy is the primary reason that a viable-appearing embryo does not always result in a normal baby. It is logical that if a euploid embryo could reliably be selected for transfer, it would have a better chance of resulting in a live birth than an untested embryo that may or may not have the correct complement of chromosomes. Being able to safely and accurately identify the chromosomal status of an embryo has long been a challenge for the ART field. After years of preclinical validation, clinical trials, and embryology advancements, accurate aneuploidy testing is finally a reality. Improvements in the clinical embryology laboratory have allowed for extended culture to the blastocyst stage with improved selection of viable embryos. Safer and more accurate methods of biopsying embryos and obtaining DNA for aneuploidy testing have been developed and validated. Ultra-rapid cryopreservation using the vitrification technique has made it possible to preserve individual high-quality embryos for future SET.
Until now, even the youngest, best-prognosis patients have been reluctant to embrace elective single embryo transfer (eSET), as evidenced by its low utilization in the United States (Figure 1a). The risk of triplet pregnancies after IVF has declined significantly, but the risk of twins remains high across age groups (Figure 1b). It stands to reason that when transferring 2 embryos, each has a chance to implant and will result in a higher delivery rate than eSET. This fact has been borne out by several randomized controlled trials (RCTs) of eSET versus double embryo transfer (DET). Each trial included in a recent meta-analysis showed lower delivery rates after eSET when compared to DET (Figure 2).6
Some have advocated that patients should undergo consecutive eSETs if their first transfer is unsuccessful.7 While cumulative pregnancy rates in a randomized trial were similar using this approach,8 it requires patients to undergo more failed cycles. In clinical practice, patients and physicians are likely to transfer more embryos after a failed eSET.9 There is a critical need to maximize patients’ chances of conceiving after the first eSET. To that end, we sought to determine whether transferring a single euploid embryo could rival the high delivery rates seen when 2 embryos are transferred.
Before embarking on such a clinical trial, it first had to be demonstrated that the chromosomal status of embryos could be accurately determined. In order to do this, a method of sampling DNA from embryos had to be developed and proven safe. The analysis of the chromosomal status had to be accurate and correlate with the true state of the embryo. Finally, the result had to be clinically meaningful, such that embryos with the potential to result in live births would not be discarded because of abnormal test results.
Unlike amniocentesis or chorionic villus sampling, in which thousands of cells are available, in the case of embryos, the number of copies of each chromosome must be determined using only picogram quantities of DNA from single cells. Culturing for karyotype is not technically feasible. Fluorescence in situ hybridization (FISH) works well when there is an abundance of cells such as amniocytes but its accuracy was never adequately validated on embryo samples. Thus, after several years and many RCTs, FISH-based aneuploidy screening was largely abandoned in IVF.10 The concept of selectively transferring euploid embryos, however, remained theoretically valid.
In addition to the questionable diagnostic accuracy of FISH, the technique relied on an invasive biopsy of a single cell from a cleavage-stage embryo that typically has only 6 to 8 cells on the third day of in vitro development. Even a highly accurate method of aneuploidy screening may not be able to overcome a detrimental developmental impact of an invasive biopsy. When an invasive biopsy is necessary to detect a congenital disorder such as cystic fibrosis, the benefit of prevention may outweigh the risk of biopsy. But the explicit goal of aneuploidy screening is to improve IVF efficiency. Thus, the impact of the biopsy and accuracy of the screening result must be considered together.
In parallel with increased utilization of extended culture, a technique of safely biopsying blastocysts was developed.11 This biopsy technique, termed trophectoderm biopsy, involves laser dissection of approximately 5 cells that are destined to develop into placental tissue. Due to the relative increase in DNA available from this type of biopsy, it was shown to be more accurate for single-gene preimplantation genetic diagnosis (PGD) than day-3 biopsy.12 Furthermore, a paired randomized trial using DNA fingerprinting found that day-3 biopsy was actually harmful, decreasing the chance an embryo would implant and develop into a baby by 39%, whereas trophectoderm biopsy did not meaningfully affect the ability to implant.13
The next problem to tackle was improving the diagnostic accuracy of the chromosomal screening technology. On the one hand, FISH overdiagnosed aneuploidy due to signal artifact; on the other, it only assessed a handful of chromosomes even though it is well known that all chromosomes can undergo malsegregation during meiosis. What was needed was a method that accurately and rapidly assessed whether each chromosome was present in the correct copy number of 2.
The molecular biology division at Reproductive Medicine Associates of New Jersey (RMA-NJ) first validated a screening technology using an array platform that employed whole genome amplification (WGA) and hybridization to a chip that analyzes 260,000 single nucleotide polymorphisms (SNPs) across the 22 autosomes and the X chromosome.14 This technique was proven to be accurate in predicting the chromosomal status of the embryo, and the prediction correlated with reproductive potential. A “nonselection” study was performed before introducing this method into clinical practice. In that study, embryos were selected as per routine and the aneuploidy screening result was revealed only after the embryos were transferred. Embryos predicted to be aneuploid had a negligible chance of resulting in live births.15
Although trophectoderm biopsy at the blastocyst stage was shown to be the safest and most accurate way to obtain DNA from embryos, that approach did not allow sufficient time to process a SNP array and perform an embryo transfer in the same cycle. For these reasons, a more rapid but equally accurate option was necessary. These goals were achieved with real-time, quantitative polymerase chain reaction (qPCR). Using qPCR, a strategy of 24-chromosome aneuploidy screening was devised whereby specific loci would be interrogated on each of the 22 autosomes and the 2 sex chromosomes (Figure 3).16 The accuracy of this approach was proven and CCS was born. It became possible to rapidly screen embryos in a safe and highly reliable manner. This option was no longer restricted to the poorest-prognosis patients and could be applied to optimizing the efficiency and safety of IVF in general. First it was demonstrated that using this technology to select 2 blastocysts for transfer resulted in improved implantation and delivery rates;17 however, there was an unacceptably high twin risk with that approach.
Once qPCR-based CCS was introduced into clinical practice, patients increasingly opted for SET or had only one euploid embryo available for transfer. A retrospective analysis revealed that patients fared significantly better when transferring a single euploid blastocyst compared with a nonbiopsied blastocyst (55% vs 42%).18 This improvement was even more significant considering that at that time, most patients who chose to use CCS were of advanced reproductive age or had prior IVF failures. Other molecular methods of CCS, such as array comparative genomic hybridization (aCGH), have been introduced to clinical practice and are being widely used; however, none has been as extensively validated as qPCR-based CCS.
Once the safety and efficacy of blastocyst-stage CCS was demonstrated and the improvement in single blastocyst transfers appeared likely, an RCT was necessary. Even among high-quality blastocysts, SET is much less advantageous than DET. For example, if each blastocyst independently has a 50% chance of implanting, then DET would be expected to result in an approximately 25% higher delivery rate than SET (75% vs 50%) at the cost of a twin rate of approximately 33%. There is some evidence to suggest that multiple embryos have a synergistic effect,19 enhancing one another’s ability to implant, providing an additional advantage to DET. But if it were known with near-certainty that the blastocyst selected for SET was euploid, this improvement in embryo selection could level the playing field. Every SET would then include a euploid embryo, whereas DET without biopsy and CCS sometimes would include 2 aneuploid embryos (with the proportion of those transfers increasing with maternal age).
The BEST Trial sought to compare euploid eSET against untested double-embryo transfer (DET). The goal was to achieve similar delivery rates with the selection advantage afforded by CCS, overcoming the enormous disadvantage of transferring one fewer high-quality embryo. The BEST Trial effectively showed that euploid eSET was noninferior to untested DET with a delivery rate of 61% versus 65% after the first embryo transfer to 175 randomized patients.5 The difference in the risk of multiple gestation was dramatic (0% vs 48%), which translated into a significantly reduced risk of preterm delivery (13% vs 29%), low-birth-weight newborns (11% vs 33%), and NICU admission (11% vs 26%).20 More patients randomized to euploid eSET were able to replicate the physiologic norm of human reproduction, with 60% having a term, singleton delivery compared with 31% after untested DET
Similar delivery rates imply that even the highest-quality blastocysts have a risk of being aneuploid. Among 521 blastocysts tested in the BEST trial, 31% were abnormal, with the risk ranging from 21% for patients younger than 35 years to 56% for those older than 40 years. A prospective study demonstrated that even the best-quality embryo in a cohort (ie, the one that a senior embryologist would have chosen for day-5 eSET) has a significant risk of aneuploidy, with more than a 40% risk in women older than 35 years.21 Therefore, a validated form of CCS has the potential to spare couples from undergoing an eSET of an aneuploid embryo that is destined for failure, possibly causing them to drop out of infertility care due to stress and disappointment. Aneuploid embryo transfer may also cause miscarriages that require surgical intervention. After a failed cycle, patients often request transfer of 2 embryos at the next cycle, despite the persistently high twin risk in that setting.
The concept of transferring a single embryo in each IVF cycle is not novel. Although the argument that patients want twins is often presented, for the vast majority of couples, twins were not in their plans when they initially attempted to conceive. Even for couples who would like to eventually have 2 or more children, a twin pregnancy is not the safest pathway. To use an example from another area of medicine, the goal of growth hormone (GH) treatment in children with GH deficiency is to use the lowest necessary dose to achieve a height close to what would be otherwise expected; giving excessive amounts to achieve an above-average height would be viewed as reckless even though there are clear advantages to increased height. Similarly, striving for twins when safer alternative exists can no longer be justified.
Surveys suggest that most couples would embrace eSET if their chances for delivery were not compromised.22 In practice, with the success of euploid eSET, this approach has become increasingly used across age groups. In 2013, 55% of transfers at RMA-NJ were single-blastocyst transfers, many performed after CCS. Patients with diminished ovarian reserve who wish to have more than one child are able to cryopreserve additional euploid blastocysts, helping them resist the temptation to try to have both children from one embryo transfer. To date, the delivery outcomes, as evidenced by birth weight, pregnancy complications, and risk of birth defects, have been reassuring and similar to deliveries from embryos that were not biopsied.
One limitation to universal acceptance of this new treatment paradigm has been lack of insurance coverage for embryo biopsy and genetic analysis, even for patients for whom the IVF cycle is covered. Because the increased maternal and pediatric charges arising from multiple gestation typically are covered, there is the potential for global health care savings if the biopsy and CCS were covered with the caveat that eSET was to be performed.
Research is under way in our laboratory to apply next-generation sequencing (NGS) technology to CCS. The ability to process multiple samples on a single sequencing chip holds the potential to reduce the per-embryo cost of CCS and perhaps make it more accessible. A drawback is that the laboratory processing for NGS takes significantly longer than qPCR and would require embryo cryopreservation, because test results could not be obtained in time for a fresh embryo transfer within the window of uterine receptivity. This approach may also provide the opportunity to concurrently screen for clinically relevant microdeletions and duplications, which have been found to occur in approximately 1 in 60 karyotypically normal ongoing pregnancies.23
The landscape of IVF treatment is rapidly changing. The field is experiencing a seismic shift that could virtually eliminate multiple gestation, the biggest complication of ART. Because of the safer outcomes and the benefit of embryo cryopreservation, IVF with euploid eSET is poised to replace controlled ovarian hyperstimulation with IUI as first-line therapy for couples with unexplained infertility. This paradigm change has become possible through the combination of improvements in the ability to reliably culture human embryos to the blastocyst stage, to safely perform micromanipulation techniques (ie, intracytoplasmic sperm injection laser-assisted hatching of the zona pellucida and trophectoderm biopsy), and to apply genetic technologies allowing for a minimally invasive, highly accurate assessment of the chromosomal composition of human embryos before implantation. With these techniques now well-established, the long-held goal of transferring one embryo and having one healthy baby after IVF is finally becoming a reality.
Kathy underwent a second IVF stimulation and produced 3 blastocysts that were biopsied on day 5. Two were predicted to be euploid and one aneuploid (trisomy 22). A single euploid blastocyst was transferred and the other euploid blastocyst was vitrified for a potential future transfer. Kathy conceived and had an uncomplicated vaginal delivery of a healthy, 3300-g baby girl at 39 weeks.
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