Assessment of aneuploidy concordance between clinical trophectoderm biopsy and blastocyst.

Victor AR ，Griffin DK ，Brake AJ ，Tyndall JC ，Murphy AE ，Lepkowsky LT ，Lal A ，Zouves CG ，Barnes FL ，McCoy RC ，Viotti M ... -  《-》

The use of copy number loads to designate mosaicism in blastocyst stage PGT-A cycles: fewer is better.

How well can whole chromosome copy number analysis from a single trophectoderm (TE) biopsy predict true mosaicism configurations in human blastocysts? When a single TE biopsy is tested, wide mosaicism thresholds (i.e. 20-80% of aneuploid cells) increase false positive calls compared to more stringent ones (i.e. 30-70% of aneuploid cells) without improving true detection rate, while binary classification (aneuploid/euploid) provides the highest diagnostic accuracy. Next-generation sequencing-based technologies for preimplantation genetic testing for aneuploidies (PGT-A) allow the identification of intermediate chromosome copy number alterations potentially associated with chromosomal mosaicism in TE biopsies. Most validation studies are based on models mimicking mosaicism, e.g. mixtures of cell lines, and cannot be applied to the clinical interpretation of TE biopsy specimens. The accuracy of different mosaicism diagnostic thresholds was assessed by comparing chromosome copy numbers in multiple samples from each blastocyst. Enrolled embryos were donated for research between June 2019 and September 2020. The Institutional Review Board at the Near East University approved the study (project: YDU/2019/70-849). Embryos showing euploid/aneuploid mosaicism (n = 53), uniform chromosomal alterations (single or multiple) (n = 25), or uniform euploidy (n = 39) in their clinical TE biopsy were disaggregated into five portions: the inner cell mass (ICM) and four TE segments. Collectively, 585 samples from 117 embryos were analysed. Donated blastocysts were warmed, allowed to re-expand, and disaggregated in TE portions and ICM. PGT-A analysis was performed using Ion ReproSeq PGS kit and Ion S5 sequencer (ThermoFisher). Sequencing data were blindly analysed with Ion Reporter software to estimate raw chromosome copy numbers. Intra-blastocyst comparison of copy number data was performed employing different thresholds commonly used for mosaicism detection. From copy number data, different case scenarios were created using more stringent (30-70%) or less stringent criteria (20-80%). Categorical variables were compared using the two-sample z test for proportions. When all the five biopsies from the same embryo were analysed with 30-70% thresholds, only 8.4% (n = 14/166) of patterns abnormal in the original analysis revealed a true mosaic configuration, displaying evidence of reciprocal events (3.6%, n = 6/166) or confirmation in additional biopsies (4.8%, n = 8/166), while most mosaic results (87.3% of total predicted mosaic patterns) remained confined to a single TE specimen. Conversely, uniform whole chromosome aneuploidies (28.3% of total patterns, n = 47/166) were confirmed in all subsequent biopsies in 97.9% of cases (n = 46/47). When 20-80% thresholds were employed (instead of 30-70%), the overall mosaicism rate per biopsy increased from 20.2% (n = 114/565) to 40.2% (n = 227/565). However, the use of a wider threshold range did not contribute to the detection of additional true mosaic patterns, while significantly increasing false positive mosaic patterns from 57.8% to 79.5% (n = 96/166; 95% CI = 49.9-65.4 vs n = 271/341; 95% CI = 74.8-83.6, respectively) (P < 0.00001). Moreover, the shift of the aneuploid cut-off from 70% to 80% of aneuploid cells resulted in mosaicism overcalling in the high range (50-80% of aneuploid cells), impacting the accuracy of uniform aneuploid classification. Parametric analysis of thresholds, based on multifocal analysis, revealed that a binary classification scheme with a single cut-off at a 50% level provided the highest sensitivity and specificity rates. Further analysis on technical noise distribution at the chromosome level revealed a greater impact on smaller chromosomes. While enrolment of a population enriched in embryos showing intermediate chromosome copy numbers enhanced the evaluation of the mosaicism category compared with random sampling such study population selection is likely to lead to an overall underestimation of PGT-A accuracy compared to a general assessment of unselected clinical samples. This approach involved the analysis of aneuploidy chromosome copy number thresholds at the embryo level; future studies will need to evaluate these criteria in relation to clinical predictive values following embryo transfers for different PGT-A assays. Moreover, the study lacked genotyping-based confirmation analysis. Finally, aneuploid embryos with known meiotic partial deletion/duplication were not included. Current technologies can detect low-intermediate chromosome copy numbers in preimplantation embryos but their identification is poorly correlated with consistent propagation of the anomaly throughout the embryo or with negative clinical consequences when transferred. Therefore, when a single TE biopsy is analysed, diagnosis of chromosomal mosaicism should be evaluated carefully. Indeed, the use of wider mosaicism thresholds (i.e. 20-80%) should be avoided as it reduces the overall PGT-A diagnostic accuracy by increasing the risk of false positive mosaic classification and false negative aneuploid classification. From a clinical perspective, this approach has negative consequences for patients as it leads to the potential deselection of normal embryos for transfer. Moreover, a proportion of uniform aneuploid embryos may be inaccurately categorized as high-level mosaic, with a consequent negative outcome (i.e. miscarriage) when inadvertently selected for transfer. Clinical outcomes following PGT-A are maximized when a 50% threshold is employed as it offers the most accurate diagnostic approach. The study was supported by Igenomix. The authors not employed by Igenomix have no conflicts of interest to declare. N/A.

Girardi L ，Figliuzzi M ，Poli M ，Serdarogullari M ，Patassini C ，Caroselli S ，Pergher I ，Cogo F ，Coban O ，Boynukalin FK ，Bahceci M ，Navarro R ，Rubio C ，Findikli N ，Simón C ，Capalbo A ... -  《-》

Non-invasive preimplantation genetic testing for putative mosaic blastocysts: a pilot study.

What is the potential of applying non-invasive preimplantation genetic testing (niPGT) for chromosome abnormalities in blastocysts reported with a mosaic trophectoderm (TE) biopsy? niPGT of cell-free DNA in blastocyst culture medium exhibited a good diagnostic performance in putative mosaic blastocysts. Advances in niPGT have demonstrated the potential reliability of cell-free DNA as a resource for genetic assessment, but information on mosaic embryos is scarce because the mosaicism may interfere with niPGT. In addition, the high incidence of mosaicism reported in the context of PGT and the viability of mosaic blastocysts raise questions about whether mosaicism really exists. The study was performed between May 2020 and July 2020. First, clinical data collected by a single-center over a 6-year period on PGT for chromosome aneuploidies (PGT-A) or chromosomal structural rearrangements (PGT-SR) were analyzed. After confirming the reliability of niPGT, 41 blastocysts classified as mosaics by trophectoderm (TE) biopsy were re-cultured. The chromosomal copy number of the blastocyst embryo (BE, the gold standard), TE re-biopsy, and corresponding cell-free DNA in the culture medium was assessed. Data on patients enrolled for PGT at a single center from 2014 to 2019 were collected and the cycles with available putative mosaic blastocysts were evaluated. To verify the diagnostic validity of niPGT, eight aneuploid blastocysts were thawed and re-cultured for 14-18 h. The concordance of the niPGT diagnosis results and the whole blastocyst testing results was analyzed. Forty-one blastocysts reported as mosaics from 22 patients were included and re-cultured for 14-18 h. The genetic material of the BE, TE re-biopsy, and corresponding cell-free DNA in the culture medium was amplified using multiple annealing and looping-based amplification cycles. The karyotype data from niPGT and TE re-biopsy were compared with that from the whole blastocyst, and the efficiency of niPGT was assessed. Data on 3738 blastocysts from 785 PGT-A or PGT-SR cycles of 677 patients were collected. According to the TE biopsy report, of the 3662 (98%) successfully amplified samples, 24 (0.6%) yielded no results, 849 (23.2%) were euploid, 2245 (61.3%) were aneuploid, and 544 (14.9%) were mosaic. Sixty patients without euploid blastocysts opted for a single mosaic blastocyst transfer, and 30 (50%) of them obtained a clinical pregnancy. With the BE chromosome copy number as the gold standard, niPGT and TE re-biopsy showed reliable detection ability and diagnostic efficiency in eight putative aneuploid blastocysts. Of the 41 putative mosaic blastocysts re-cultured and re-tested, 35 (85.4%) showed euploid BE results. All but two of the blastocysts previously diagnosed with segmental chromosomal mosaic were actually euploid. In addition, all blastocysts previously classified as low degree (20-50%) mosaics were identified as euploid by BE PGT, whereas four of the six putative high degree (50-80%) mosaic blastocysts showed chromosomal abnormalities. The raw concordance rates of spent culture medium (SCM) and TE re-biopsies compared with BE were 74.4% and 82%, respectively, in terms of overall ploidy and 96.2% and 97.6%, respectively, per single chromosome when considering all degree mosaic results as true positives. However, when we set a mosaicism identification threshold of 50%, the concordance rates of SCM and TE re-biopsies compared with BE were 87.2% and 85% at the overall ploidy level and 98.8% and 98.3% at the chromosomal level, respectively. At the full ploidy level, the sensitivity and false negative rates for niPGT were 100% and 0, respectively. After adjustment of the threshold for mosaicism, the specificity of niPGT increased from 69.7% to 84.8% in terms of overall ploidy and from 96.1% to 98.9% at the chromosomal level. The primary limitation of this study is the small sample size, which decreases the strength of our conclusions. If possible, identifying the clinical outcome of niPGT on reassessed mosaic blastocysts would be further progress in this field. This study is the first to explore the practicability of niPGT in diagnostic reassessment of putative mosaicism. The present study provides a novel opportunity for patients with only mosaic blastocysts and no euploid blastocysts, regardless of the technical or biological basis of mosaicism. Employing niPGT after 14-18 h of re-culturing might be a superior option for the best use of blastocysts because of its minimally invasive nature. This work was supported by grants from National Key Technology Research and Development Program of China (No. 2017YFC1002004), the Central Guiding the Science and Technology Development of the Local (2018080802D0081) and College Natural Science Project of Anhui Province (KJ2019A0287). There are no competing interests to declare. N/A.

Li X ，Hao Y ，Chen D ，Ji D ，Zhu W ，Zhu X ，Wei Z ，Cao Y ，Zhang Z ，Zhou P ... -  《-》

Extended in vitro culture of human embryos demonstrates the complex nature of diagnosing chromosomal mosaicism from a single trophectoderm biopsy.

What is the accuracy of preimplantation genetic testing for aneuploidies (PGT-A) when considering human peri-implantation outcomes in vitro? The probability of accurately diagnosing an embryo as abnormal was 100%, while the proportion of euploid embryos classified as clinically suitable was 61.9%, yet if structural and mosaic abnormalities were not considered accuracy increased to 100%, with a 0% false positive and false negative rate. Embryo aneuploidy is associated with implantation failure and early pregnancy loss. However, a proportion of blastocysts are mosaic, containing chromosomally distinct cell populations. Diagnosing chromosomal mosaicism remains a significant challenge for PGT-A. Although mosaic embryos may lead to healthy live births, they are also associated with poorer clinical outcomes. Moreover, the direct effects of mosaicism on early pregnancy remain unknown. Recently, developed in vitro systems allow extended embryo culture for up to 14 days providing a unique opportunity for modelling chromosomal instability during human peri-implantation development. A total of 80 embryos were cultured to either 8 (n = 7) or 12 days post-fertilisation (dpf; n = 73). Of these, 54 were PGT-A blastocysts, donated to research following an abnormal (n = 37) or mosaic (n = 17) diagnosis. The remaining 26 were supernumerary blastocysts, obtained from standard assisted reproductive technology (ART) cycles. These embryos underwent trophectoderm (TE) biopsy prior to extended culture. We applied established culture protocols to generate embryo outgrowths. Outgrowth viability was assessed based on careful morphological evaluation. Nine outgrowths were further separated into two or more portions corresponding to inner cell mass (ICM) and TE-derived lineages. A total of 45 embryos were selected for next generation sequencing (NGS) at 8 or 12 dpf. We correlated TE biopsy profiles to both culture outcomes and the chromosomal status of the embryos during later development. Of the 73 embryos cultured to 12 dpf, 51% remained viable, while 49% detached between 8 and 12 dpf. Viable, Day 12 outgrowths were predominately generated from euploid blastocysts and those diagnosed with trisomies, duplications or mosaic aberrations. Conversely, monosomies, deletions and more complex chromosomal constitutions significantly impaired in vitro development to 12 dpf (10% vs. 77%, P < 0.0001). When compared to the original biopsy, we determined 100% concordance for uniform numerical aneuploidies, both in whole outgrowths and in the ICM and TE-derived outgrowth portions. However, uniform structural variants were not always confirmed later in development. Moreover, a high proportion of embryos originally diagnosed as mosaic remained viable at 12 dpf (58%). Of these, 71% were euploid, with normal profiles observed in both ICM and TE-derived lineages. Based on our validation data, we determine a 0% false negative and 18.5% false positive error rate when diagnosing mosaicism. Overall, our findings demonstrate a diagnostic accuracy of 80% in the context of PGT-A. Nevertheless, if structural and mosaic abnormalities are not considered, accuracy increases to 100%, with a 0% false positive and false negative rate. The inherent limitations of extended in vitro culture, particularly when modelling critical developmental milestones, warrant careful interpretation. Our findings echo current prenatal testing data and support the high clinical predictive value of PGT-A for diagnosing uniform numerical aneuploidies, as well as euploid chromosomal constitutions. However, distinguishing technical bias from biological variability will remain a challenge, inherently limiting the accuracy of a single TE biopsy for diagnosing mosaicism. This research is funded by the Ghent University Special Research Fund (BOF01D08114) awarded to M.P., the Research Foundation-Flanders (FWO.KAN.0005.01) research grant awarded to B.H. and De Snoo-van't Hoogerhuijs Stichting awarded to S.M.C.d.S.L. We thank Ferring Pharmaceuticals (Aalst, Belgium) for their unrestricted educational grant. The authors declare no competing interests. N/A.

Popovic M ，Dhaenens L ，Taelman J ，Dheedene A ，Bialecka M ，De Sutter P ，Chuva de Sousa Lopes SM ，Menten B ，Heindryckx B ... -  《-》

The clinicians´ dilemma with mosaicism-an insight from inner cell mass biopsies.

How reliable are cleavage stage and trophectoderm (TE) biopsies compared to inner cell mass (ICM) biopsies? The reliability of TE biopsy compared to ICM biopsy is almost perfect, but only substantial between cleavage stage biopsy and ICM biopsy. One of the prevailing reasons for implantation failure is presumed to be chromosomal aneuploidy in human preimplantation embryos. Preimplantation genetic testing for aneuploidies (PGT-A) has been introduced into assisted reproduction in an effort to increase pregnancy rates. Increasing evidence indicates that genetic results obtained following blastomere or TEbiopsy may not accurately reflect the true genetic status of the embryo due to the presence of embryonic mosaicism, and therefore the reliability of PGT is highly controversial. This was an observational descriptive study, performed in a private infertility centre from August 2016 to January 2017. The mean female age was 33.9 years, ranging from 24 to 46 years, and the mean number of biopsied embryos per couple was 2.2 (range 1-7 embryos). Blastomere biopsies had been performed at cleavage stage on Day 3 (D3) due to the turnover time of genetic testing and the inability to cryopreserve embryos in accordance with the local law governing ART. To confirm the genetic results in embryos not chosen for transfer, additional biopsies of the TE at blastocyst stage (BLASTO-TE) as well as of the ICM (BLASTO-ICM) were performed on D5. Only surplus blastocysts, which had not been selected for transfer and were not cryopreserved in accordance with the law governing ART, had been included. Comparison of all biopsies (D3/BLASTO-ICM/BLASTO-TE) per embryo demonstrated that 50 (59.5%) out of 84 embryos showed concordance in all three results (= full concordance). Thirty-four (40.4%) embryos had at least two discordant results between the three biopsies, regardless of whether the embryo diagnosis (aneuploid/euploid) was discordant or not, or in aneuploid embryos, whether the chromosomal patterns were inconsistent. Nine (= 10.7%) embryos had complete discordance between all three biopsies. False positive results between D3/BLASTO-TE, D3/BLASTO-ICM and BLASTO-TE/BLASTO-ICM were 26.4%/30.2% and 7.5%, respectively, while the Kappa agreement between the different approaches was 0.647, 0.553 and 0.857, respectively. Therefore the reliability of D3/BLASTO-TE, D3/BLASTO-ICM and BLASTO-TE/BLASTO-ICM can be interpreted as substantial, as moderate and as almost perfect. The limitation of this study is the possible bias in the concordance/discordance rate because embryos that had been selected for transfer did not undergo biopsy on D5. The obvious discordance between the three different approaches for PGT-A underlines the limitations of genetic testing and highlights the importance of ongoing research in order to improve the accuracy of PGT-A results. Until then reproductive specialists will continue to make challenging decisions on whether to transfer or discard an embryo in light of current evidence questioning the reliability of genetic results. This study was supported by Igenomix. The funder provided support in the form of salary for R.C. The co-author R.C. is an employee of Igenomix. She participated in the blinded analysis of the samples; however the final data collection and statistical analysis of the results, as well as the decision to publish, was taken by B.L, I.E. and H.F. The authors B.L., I.E., A.L., A.B., A.A., N.D. and H.F. have no competing interests. The funder did not have any additional role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. The commercial affiliation of R.C. did not play any role in the study. This study was approved by the Ethics Committee of IVIRMA Middle East Fertility Clinic, Abu Dhabi, UAE (Research Ethics Committee IVI-MEREFA009a/2017).

Lawrenz B ，El Khatib I ，Liñán A ，Bayram A ，Arnanz A ，Chopra R ，De Munck N ，Fatemi HM ... -  《-》