7.2. Variations and Add-Ons to Conventional PGT-A
A snapshot of today’s PGT-A landscape reveals WGA-based low-pass/shallow NGS as the workhorse of the industry. The technique has found a balance between three key factors: cost, ease of use, and quality of information generated. Nonetheless, numerous alterations to the standard PGT-A platform have recently emerged or are currently in development.
One is the aforementioned targeted amplification-sequencing method [104
], which increases sequencing depth in specific locations of the genome, but usually at the expense of breadth of genome coverage. Recent versions of this technique have taken advantage of repetitive sequences that are spread across the genome, permitting amplification using few primer pairs [188
]. Some families of repetitive elements contain appreciable sequence variation, meaning that using a single primer pair in the reaction (reagent savings, added simplicity) potentially provides widely distributed mapping of reads across the genome after bioinformatic analysis. The depth of sequencing is adequate to perform SNP analysis, in addition to quantitation. Allelic ratio analysis can improve confidence of aneuploidy calling, and can provide information regarding haploidy, triploidy, and UPD [189
There is substantial interest in developing methods to harness the sequence information gathered during NGS, aside from the usual copy number information. For example, Voet, Vermeesch and colleagues have developed haplarithmisis, a method that quantifies sequencing reads to perform PGT-A, but additionally uses the SNP information contained in the sequencing reads to determine haplotypes [190
]. This can be used to identify embryos with haplotypes carrying disease alleles, but it also reinforces confidence in calling of copy number changes, and reveals the parental and mechanistic origin of chromosomal abnormalities.
Several efforts are underway to combine PGT for chromosomal abnormalities and monogenic conditions (PGT-A/-M) in a single reaction. For example, Zimmerman and colleagues have described a qPCR method to perform simultaneous aneuploidy analysis and single gene testing [192
]. Farmer and colleagues have shown that combining a WGA reaction and specific primers targeting the 23 most common mutations in CFTR
do not compromise NGS analysis of chromosomal abnormalities, and can simultaneously provide accurate single nucleotide variant (SNV) and small indel information for the CFTR
]. Such a technique can be adapted to any mutation using appropriate primer spike-ins. Del Rey and colleagues have successfully combined NGS copy number analysis with large panels of common single gene conditions [194
]. Alcaraz and colleagues reported a technique that combines chromosomal copy number calling with a SNP-based analysis of a mutation specific to a patient’s genetic profile, showing its feasibility in over 150 different mutations [195
Handyside and colleagues have developed a technique called karyomapping [196
]. It relies on SNP arrays, which aside from providing copy number data, also harness the information contained in near 300,000 SNPs distributed in the genome, permitting linkage analysis for virtually any region of interest. Testing of the subject’s relatives reveals the segregation patterns of mutated loci and flanking informative SNPs, which subsequently provide the framework for linkage analysis in tested embryos. The universal applicability of the technology to virtually any single gene mutation is potentially very advantageous to classical analysis of monogenic conditions in embryos, where each mutation requires a specific assay. In addition to combining PGT-A and -M [197
], karyomapping allows detection of haploidy and triploidy, as well as UPD.
A recent array-based technology developed by Treff and colleagues combines copy number and SNP analysis, and can simultaneously perform PGT-A, -SR, -M, as well as testing for polygenic (-P) conditions [199
]. A polygenic risk score (PRS) is calculated for a panel of diseases, estimating the relative likelihood of an embryo to develop conditions such as diabetes or heart disease.
In an effort to improve ease-of-use and lower costs, Wei and colleagues have recently adapted nanopore technology in a MinION instrument (Oxford Nanopore) [201
] to PGT-A [202
]. While thorough validation is still pending, pilot experiments have shown it can detect aneuploidy and, to some degree, mosaicism in TE biopsies. Compared to standard NGS methods, this technology is estimated to increase sequencing speed by 15,000-fold, has a 99X lower capital equipment cost (~USD 1000), and an instrument footprint the size of a deck of cards.
The limited amount of genetic material in a TE biopsy renders ‘true’ whole genome sequencing a formidable task. However, there have been valiant efforts at obtaining a much broader genome coverage than with standard approaches, typically by coupling high-level sequencing with bioinformatic methods for linkage analysis with familial genomic data. Kumar and colleagues have described a method to infer the entire genome of an embryo from a TE biopsy without performing deep sequencing, instead using SNP linkage analysis with familial genome information [203
]. Two studies by Peters et al. [204
] and Murphy et al. [205
] have shown that detailed assessment of a TE biopsy by high-pass sequencing and linkage analysis can identify new, potentially disease-causing SNPs, in addition to producing copy number and single gene data (PGT-A/-M).
In summary, add-ons to PGT-A can provide valuable supplementary information and/or decrease costs, but should do so without compromising the very core question PGT-A seeks to answer: Are aneuploidies present in the sample? Many of the above tests still need to produce data that convincingly show their copy number analysis is as powerful as conventional WGA-based PGT-A, especially regarding mosaicism and segmental abnormalities.
7.3. The Prospect of Non-Invasive PGT-A
A method of PGT-A that does not require physical isolation of cells from the embryo would revolutionize the field. With the decreasing cost of sequencing, the most expensive component of PGT-A has become biopsy isolation, which requires hi-tech laser equipment and greatly skilled embryologists to perform the microsurgery. These hurdles likely prevent some clinics from adopting a PGT-A program. Even though studies have suggested negligible effect on blastocyst viability when TE biopsy isolation is performed correctly [90
], there is still risk of poorly executed, harmful, or botched procedures and other more nuanced detrimental effects, such as those associated with prolonged temperature and gas fluctuations during the procedure. Furthermore, some embryos do not get tested because poor morphology precludes TE biopsy collection, and a non-invasive method could increase the pool of assessed embryos.
The concepts of minimally invasive (mi-), or non-invasive (ni-)PGT-A became plausible after discovery of (presumably embryo-derived) DNA in blastocoel fluid (BF) [206
], and spent culture medium (SCM) [207
]. The ensuing question was whether those fluids contained sufficient good quality DNA to evaluate chromosomal copy number. The option involving BF was explored first.
BF is isolated by ‘blastocentesis’ [208
], in which a fine needle is inserted through the zona pellucida and between TE cell junctions into the blastocoel cavity, and fluid (< 1 µL) is aspirated. This collapses the blastocyst, which can subsequently be vitrified or left to re-expand. Gianaroli and colleagues have made an extensive exploration on the subject of concordance between BF and embryonic cells, publishing three studies based on WGA and aCGH technology [208
]. The latest, published in 2018, is their largest analysis (n
= 256 embryos), reporting 94% ploidy concordance between BF and a TE biopsy, and 66% full karyotype concordance [209
]. Nevertheless, BF amplification failed in 29% of cases, and of those that amplified a further 13% did not produce an informative chromosome copy number result. An interesting observation was that euploid embryos were significantly more likely to fail BF amplification, possibly pointing at a biological mechanism. The authors hypothesized that DNA in BF is likely to originate from cell apoptosis, which is more frequent in aneuploidy [209
]. Therefore, the quantity of DNA in BF might have some prognostic value regarding blastocyst competence.
Studies from other groups, one based on WGA and aCGH [211
] and two based on WGA and NGS [127
], have reported lower concordance of ploidy between BF and TE/ICM biopsy, between 62%–75%, as well as decreased full karyotype concordance, between 38%–48%. Crucially, failed amplification rates for BF showed a large range, between 13%–65%, possibly reflecting different approaches to blastocentesis, BF storage, and amplification chemistry [127
], but also differences in blastocyst population analyzed and true incidence of euploidy, mosaicism, and aneuploidy.
Several groups have recently explored the alternative method (which would be truly non-invasive): analysis of SCM. DNA contamination has been an obvious concern, since some media formulations contain foreign DNA, and there is potential for carryover maternal DNA in the culture drop. However, the observation that the total amount of DNA increases with developmental progression [213
] would indicate that the bulk DNA derives from the embryo. The first notable SCM study was performed by Shamonki and colleagues in 2016, reporting that their system (Qiagen’s Repli-G kit for amplification and aCGH) produced suboptimal data for 96% of the 56 samples analyzed [215
]. This confirmed that chemistries designed for chromosomal evaluation from cellular biopsy would unlikely be transferable to niPGT-A in their current formats. A series of studies followed, testing different methods of DNA amplification (MALBAC or SurePlex/PicoPlex, with various modifications), analysis (aCGH or NGS), and culturing protocols, expertly detailed by Leaver and Wells [216
The biggest caveat in many of the publications to date is that embryos were subjected to some form of manipulation before SCM was collected [217
]. Putting embryos through Day 3/4 assisted hatching, a freeze-thaw cycle, and/or a cellular biopsy prior to SCM collection provides a source of added DNA in the medium, confounding the subsequent analysis of amplification rate and concordance to the embryo. While it should not entirely invalidate such studies, these shortcomings must be carefully considered.
For the most part, studies with no embryo manipulation prior to SCM collection will be discussed here, as they can give a true sense of whether niPGT-A is applicable in the clinic. Liu and colleagues evaluated 88 SCM samples after continuous culture between Days 1–5, using MALBAC amplification (Yikon Genomics) and NGS on a HiSeq 2500 (Illumina) instrument, reporting a 91% amplification rate [222
]. Ploidy concordance with a TE biopsy was observed in 84% of cases (although mosaic and aneuploid results were conflated into one ‘abnormal’ group), and full karyotype concordance was 65%. Only later studies revealed that maternal DNA contamination from the oocyte retrieval procedure is a significant concern, and the uninterrupted culture from zygote to blastocyst stage likely maximizes that problem. The same testing method, namely MALBAC amplification (Yikon Genomics) and NGS on a HiSeq 2500 instrument (Illumina), was used in a study by Fang and colleagues, this time washing embryos at Day 3 of culture and moving to new media drops until Day 5 or 6 [223
]. The study reports a 97% amplification rate in the 170 samples analyzed. The authors did not collect a TE biopsy to perform concordance analysis, and instead embryos were transferred in the clinic, selecting them according to the result of the test (which they call ‘NICS’, for noninvasive chromosome screening). In total, 52 blastocysts that had been classified as normal were transferred to 50 patients, resulting in 30 implantations (58% rate), 3 miscarriages (10% rate), and 27 births (52% rate per embryo transfer). Those results could be regarded as favorable rates of positive clinical outcome, but unfortunately the study did not have a control arm, making the results difficult to evaluate. Are those outcomes better than if no niPGT-A had been performed? And would TE biopsy PGT-A have resulted in better outcomes? Two ongoing trials, in which NICS is compared to morphology assessment alone (ClinicalTrials.gov ID: NCT04339166) or to conventional PGT-A (ID: NCT03879265) will shed light on these questions.
A study by Vera-Rodriguez and colleagues [214
] explored three important issues concerning niPGT-A: (1) Amount of DNA, (2) Maternal DNA contamination, and (3) Embryonic mosaicism. To explore those concepts, the authors used a high-performance DNA quantitation method, SNP analysis to discern maternal from embryonic DNA, and FISH to evaluate each cell in a subset of blastocysts. The authors analyzed 56 SCM samples collected at Day 5 of culture (after having performed a media change at Day 3). Detailed DNA quantitation showed a median 6.7 pg DNA, which is approximately equivalent to the DNA content of one diploid cell- with half of the samples containing fewer amounts. In comparison, no-embryo control drops contained a median 1.4 pg. It must be stressed that this particular study did perform assisted hatching at Day 3, meaning that a truly non-invasive protocol might result in lower amounts of DNA per SCM. There was no difference in the amount of DNA between euploid and aneuploid embryos, in contrast to what was observed in BF studies [209
]. Strikingly, SNP analysis revealed maternal DNA presence in all samples tested, on average constituting a remarkable 92% of all DNA in SCM, even though a media change had been performed at Day 3. To explore the ploidy of SCM, the authors employed a double WGA amplification technique of SurePlex WGA (Illumina), followed by a complete IonReproseq (ThermoFisher) protocol and sequencing on an Ion PGM instrument (ThermoFisher). Ninety-one percent of samples yielded a result, compared to 100% using TE biopsies. FISH analysis of complete blastocyst cellular makeup revealed that SCM samples poorly reflected the status of embryos that were mosaics, and TE biopsy mosaicism was a far superior predictor of mosaicism in the remaining blastocyst. This was attributed to the possibility that in a mosaic context, cells with different ploidy release their DNA content into the SCM at different rates- rendering the SCM a poor representative of the embryo. Together, these findings clearly identified some of the hurdles that need to be addressed in developing niPGT-A for clinical use.
In 2019 the same group published a prospective blinded study, in which Rubio and colleagues analyzed 115 samples and showed a 95% amplification rate with 79% overall ploidy concordance to a TE biopsy and 64% full karyotype concordance (including segmental abnormalities) [224
]. To perform niPGT-A, the group used a modified version of the IonReproseq protocol (ThermoFisher), followed by sequencing on an Ion S5TM XL system (ThermoFisher). Embryo culture did not involve assisted hatching, and embryos were thoroughly washed at Day 4 through three media drops in hopes of removing carryover maternal DNA and lingering cumulus cells. Each embryo was subsequently placed in a new reduced media drop of 10 µL (to ensure the entire sample could be included in a WGA reaction) and cultured to Day 5, 6, or 7, at which time SCM was collected. Sub-analysis showed that extended culture to Day 6 or 7 (≥48 h) improved results (compared to just 24 h), increasing the overall ploidy concordance with a TE biopsy to 84% and full karyotype concordance to 72%, as well as rate of amplification to 100%. This considerable enhancement clearly showed that extended time in culture increases the amount of embryo-representative DNA in the SCM. This study went on to show in a small sample group (n
= 29) that embryos had an improved chance of implantation and lower miscarriage when TE biopsy and SCM concorded on euploidy status, compared to discordant cases for which TE biopsy indicated euploidy but SCM showed aneuploidy. If these results are replicated in a larger sample group, one could envision niPGT-A as an adjunct to conventional PGT-A to increase likelihood of favorable outcome. Two ongoing studies by this group (ClinicalTrials.gov IDs: NCT03520933 and NCT04000152) are further assessing the value of niPGT-A in the clinic.
A different approach altogether, combining BF and SCM, has so far been explored in a few studies that have all reported high rates of amplification 98%–100% [225
]. Perhaps the most compelling are the efforts by Kuznyetsov and colleagues [226
], since their protocol excluded freeze/thaw cycles prior to sample collection. In a publication from 2018, the Kuznyetsov group analyzed 19 fresh culture samples from 9 patients. Embryos were placed in new culture drops at Day 4 and left to grow until Day 5 or 6, at which point a TE biopsy was collected and the blastocyst was further collapsed with the use of lasers, allowing the BF to emanate into the SCM. The fact that a TE biopsy was collected prior to collection of fluids is a caveat, since the process likely releases DNA into the medium. An unspecified amount of the 25 µL combined BF and SCM sample was subsequently processed in one reaction by SurePlex amplification (Illumina) and VeriSeq NGS (Illumina) on a MiSeq instrument (Illumina) (the same protocol as the corresponding TE biopsies). The combined BF-SCM samples and corresponding TE biopsies had 100% ploidy concordance, and 100% full karyotype concordance for whole chromosome events (which decreased to 71% if segmental abnormalities and mosaicism were included).
Kuznyetsov and colleagues subsequently published a study in 2020 with a larger sample size (n
= 145) excluding the prior TE biopsy collection, reporting a 100% amplification rate and a 97.8% concordance (euploid/aneuploid) with TE samples [228
]. When mosaicism was considered (observed either in cell-free DNA or TE biopsy), rate of concordance declined. One parameter still required optimization: 88.2% of BF-SCM sample amplifications yielded informative NGS results, compared to 98% of TE biopsy amplifications. Overall, those are promising observations regarding the clinical application of this strategy. Since the protocol included assisted hatching at Day 4, an outstanding question is whether omitting this step (in an effort to further reduce the manipulations/invasiveness of the process) would influence the reaction. Other interesting findings from the study include that blastocyst morphology did not correlate with quantity of cell-free DNA, and the cell lysis step could be omitted from the WGA reaction (as it might contribute to maternal DNA contamination though carryover cumulus/corona cells).
There have also been efforts to quantify mtDNA levels in SCM samples. Stigliani and colleagues have reported that higher levels of cell-free mtDNA might be predictive of implantation in cleavage day embryo transfers [206
]. Compared to embryos that failed to implant, they observed a threefold increase in mtDNA quantity (on average) in embryos that implanted, but these findings are yet to be replicated with a larger sample size (n
= 51 not implanted, n
= 43 implanted embryos, p
= 0.0452). The same group also observed that higher quantities of cell-free mtDNA at Day 3 of culture have predictive power regarding the likelihood of reaching the blastocyst stage [229
]. Whether cell free mtDNA quantitation may serve as a biomarker of implantation for blastocyst stage transfers remains to be explored.
Valuable lessons have been learned from the studies so far: (1) Manipulation of embryos prior to sample collection (freeze-thaw cycle, biopsy collection, assisted hatching) might release DNA into media, compromising a study’s results regarding truly non-invasive PGT-A. Those manipulations should be avoided in future studies, (2) Fully euploid and aneuploid embryos might release similar amounts of DNA into the medium, but in a mosaic context, aneuploid cells might preferentially shed their content, adulterating the representativeness of the SCM in regards to the embryo, (3) The minute amounts of DNA require new adaptations of PGT-A chemistries and protocols that accommodate as much of the culture drop volume as possible, (4) Maternal DNA contamination is a big concern, (5) Longer cultures to Day 6 or 7 provide more cell-free (cf) DNA.
Still, several questions persist: (1) What is an acceptable rate of false or no results in niPGT-A (considering its many benefits)? (2) What is the biological mechanism that releases embryonic DNA into surrounding fluids? (3) Are SCM and/or BF better or worse representatives of the true ploidy status of the blastocyst, compared to a TE biopsy? (4) What is the capacity of niPGT to detect segmental abnormalities and mosaicism?
Together, the SCM and BF studies have revealed that the naturally small amount of DNA present in those fluids, possibly coupled with compromised integrity and maternal DNA contamination, present a real challenge in the development of niPGT-A. The reports have evidently pushed against the lower limits of detection of existing PGT-A platforms. There is however, cause for optimism; better cell-free DNA amplification chemistries and optimized bioinformatic analyses will bring us closer to an accurate niPGT-A screen suitable for routine clinical use.