Francesca Spinella

Validation of a semiconductor next-generation sequencing-based protocol for preimplantation genetic diagnosis of reciprocal translocations.

We aim to validate a semiconductor next‐generation sequencing (NGS)‐based method to detect unbalanced chromosome translocation in preimplantation embryos.

The importance of determining the limit of detection of non‐invasive prenatal testing methods

Several non‐invasive prenatal testing (NIPT) methods, which analyze circulating fetal cell‐free DNA (cfDNA) in maternal plasma, suggest a fetal fraction (FF) ≥4% for a reportable result, with the assumption that fetal aneuploidies may not be detectable at lower FF. This study determined the actual limit of detection (LOD) of a massively parallel sequencing‐based NIPT method and evaluated its performance in testing samples with low FF.

The clinical utility of genome‐wide non invasive prenatal screening

In this study, we expanded conventional cell‐free fetal DNA (cfDNA)‐based non‐invasive prenatal testing (NIPT) to cover the entire genome. We aimed to compare the performance of the two tests in a large general population of pregnant women, in order to assess the clinical utility of the genome‐wide screening.

Extent of chromosomal mosaicism influences the clinical outcome of in vitro fertilization treatments

OBJECTIVE To assess whether the extent of chromosomal mosaicism can influence the success rate of IVF treatments. DESIGN Prospective study. SETTING Private genetic and assisted reproduction centers. PATIENT(S) The transfer of mosaic embryos was offered to 77 women for which IVF resulted in no euploid embryos available for transfer. INTERVENTION(S) All embryos were cultured to blastocyst stage; trophectoderm biopsy was performed on day 5/6 of development. Comprehensive chromosome screening was performed using either next-generation sequencing or array-comparative genomic hybridization methodologies. MAIN OUTCOME MEASURE(S) The clinical outcome obtained after transfer of mosaic embryos with low (<50%) and high (≥50%) aneuploidy percentage was compared with that resulting from a control group of 251 euploid blastocysts. RESULT(S) A significantly higher implantation rate (48.9% vs. 24.2%), clinical pregnancy rate/ET (40.9% vs. 15.2%), and live-birth rate (42.2% vs. 15.2%) were observed comparing embryos with mosaicism <50% and ≥50%. Mosaic embryos with high aneuploidy percentage (≥50%) showed a significantly lower clinical pregnancy rate/ET (15.2% vs. 46.4%), implantation rate (24.4% vs. 54.6%), and live-birth rate (15.2% vs. 46.6%) than euploid blastocysts. In contrast, embryos with lower aneuploidy percentage (<50%) have a clinical outcome similar to euploid embryos. CONCLUSION(S) The results of this study further confirm that mosaic embryos can develop into healthy euploid newborns. We demonstrated that the extent of mosaicism influences the IVF success rate. Mosaic embryos with low aneuploidy percentage have higher chances of resulting in the birth of healthy babies compared with embryos with higher mosaicism levels.

Genetic diseases and aneuploidies can be detected with a single blastocyst biopsy: a successful clinical approach

STUDY QUESTION Can simultaneous detection of aneuploidies and genetic diseases or chromosomal aberrations in blastocysts reduce the chance of transferring embryos with low implantation potential, guaranteeing good clinical outcomes? SUMMARY ANSWER The screening for chromosomal aneuploidies revealed that 50.6% of blastocysts diagnosed free of genetic disease or balanced, were aneuploid, therefore avoiding the transfer of blastocysts potentially resulting in implantation failures, miscarriages, or in some cases, in health affected live births. WHAT IS KNOWN ALREADY PGD is applied in patients at risk of transmitting genetically inheritable diseases to their offspring. It has been demonstrated that aneuploidies can involve chromosomes other than those investigated with PGD, affecting embryo implantation competence. Performing the biopsy at blastocyst level produces higher clinical outcomes allowing a more accurate diagnosis, compared to blastomere biopsy. STUDY DESIGN, SIZE, DURATION This consecutive case series study was performed from October 2011 to May 2016. Clinical and biological outcomes from 1122 blastocysts obtained in 304 PGD cycles for monogenic diseases (N = 163) or chromosomal rearrangements (N = 141) were analyzed. When the blastocyst resulted transferable after the PGD analysis or chromosomal rearrangement analysis, its ploidy status by mean of preimplantation genetic screening (PGS) was also detected using the same biopsy sample. Mean female age was 35.4 ± 4.2 years old. All biopsies were performed at blastocyst stage and analyzed by Whole Genome Amplification (WGA) followed by PCR for monogenic diseases, and by array-comparative genotype hybridization (array-CGH) for all cycles. PARTICIPANTS/MATERIALS, SETTING, METHOD All mature oocytes retrieved were injected and cultured individually until the blastocyst stage at 37°C, 6% CO2, 5% O2. When the blastocyst was formed, it was biopsied and vitrified, awaiting the genetic results. The frozen-thawed embryo transfer was performed in a subsequent cycle. In some cases, when the blastocyst was obtained within the morning of Day 5 of culture, it had been maintained in culture and transferred on Day 6, after receiving the genetic report. MAIN RESULTS AND THE ROLE OF CHANCE A total of 2809 (2718 fresh and 91 frozen-thawed) mature oocytes were injected with a fertilization rate of 75.5% (N = 2120), leading to the development of 2102 embryos. A further 24 frozen embryos, previously vitrified without any genetic testing, were successfully warmed for genetic screening. A total of 2126 embryos were cultured with a blastocyst formation rate of 52.8% (N = 1122); all of them were biopsied from Day 4 to Day 7 of culture. After the genetic analysis, 309 (27.5%) blastocysts resulted transferable, both for monogenic disease or translocation and for their ploidy status, 42 were diploid/aneuploid mosaic, 55 were no result and 716 were not transferable, due to genetic disease or chromosomal rearrangement and/or for their ploidy status. Of note, 316 (50.6% of transferable blastocysts after PGD and 28.2% of total number of biopsied blastocysts) of the blastocysts resulted healthy for the genetic disease or chromosomal rearrangement were aneuploid. Out of 304 PGD/PGS cycles performed, 28.6% (N = 87) resulted in no-transferable blastocysts after only PGD analysis; this percentage increased to 39.8% (N = 121) when also PGS was carried out (Mc Nemar test P < 0.001). A total of 202 embryo-transfers were performed, 53 fresh and 149 cryopreserved, in which 218 healthy or carrier euploid blastocysts were transferred. Clinical pregnancy, implantation and miscarriage rates were 49.0, 47.7 and 9.9%, respectively. To date, 66 deliveries occurred with 70 healthy babies born and 13 pregnancies are still ongoing. Finally, 91 euploid healthy blastocysts are still cryopreserved waiting to be transferred. LIMITATIONS, REASONS FOR CAUTION A higher than expected cycle cancellation rate could be found due to the double genetic analysis performed. For this reason, particular care should be taken in drafting and explaining informed consent, in order to avoid patient drop out. WIDER IMPLICATIONS OF THE FINDINGS When the biopsy has to be performed in order to prevent the transmission of an inheritable disease, it should be mandatory to analyze also the genetic status of the blastocyst, avoiding useless embryo-transfers in this particular category of patients. In our study, 316 aneuploid healthy blastocysts could have been transferred without performing PGS, leading to implantation failures, miscarriages, or in some cases, to live births affected by different syndromes. STUDY FUNDING/COMPETING INTEREST(S) No specific funding was obtained for this study. None of the authors have any competing interests to declare. TRIAL REGISTRATION NUMBER Not applicable.

Author's reply to Grati and Benn

We thank Dr. Grati and Dr. Benn for their interest in our article. We welcome the opportunity to provide clarification and further stimulate the discussion on this important topic. The authors raised concerns regarding the design of our study that, in their opinion, precludes accurate determination of analytic sensitivity, because karyotyping from apparently normal live-borns has not been performed. The reason for their criticism is related to the fact that several chromosomal abnormalities, including sex chromosome aneuploidies, some segmental imbalances and low level autosomal mosaic aneuploidies are not necessarily apparent at newborn physical examination. Wemay agree with Dr. Grati andDr. Benn that, to confidently exclude the presence of chromosomal abnormalities, the ideal clinical follow-up should involve karyotyping results from the newborns, and this limitation was clearly recognized in our paper. However, such a study design is very difficult to finalize, because of the related cost and the high proportion of patients that would be lost to follow-up. Even if we cannot rule out that chromosomal abnormalities, such as very small chromosomal rearrangements or low-level mosaicism occurrences, may have remained unnoticed, the possibility of genome-wide analysis false results going undetected remain low. Dr. Grati and Dr. Benn also raised concerns on the low number of abnormal pregnancies detected in our study cohort. In their opinion, these finding are suggestive of under-ascertainment of chromosomal abnormalities and, consequently, lower test sensitivity. Specifically, they remarked upon the lower number of rare autosomal trisomies (RATs) identified in our study, as compared with that expected from chorionic villus specimens or that reported in another study evaluating expanded NIPT. However, the authors start from the assumption that the outcome data from chorionic villus sampling (CVS) positive cases should be the same for the cases ascertained by genome-wide cfDNA screening, which is not necessarily correct. This was properly pointed out by Prof. Bianchi in the ‘open the kimono’ commentary, and may explain the difference in confined placental mosaicism (CPM) rate between our study and CVS data. There are multiple reasons supporting the latter hypothesis. First of all, there is likely more complete ascertainment by cfDNA sequencing. In fact, cfDNA likely derives from all placenta sites and has the potential to provide a more accurate representation of placenta heterogeneity. On the contrary, the biopsy material represents only a small fraction of the placenta and may only give a ‘snapshot’ of one portion of the tissue. Secondly, CVS is only performed on pregnant women who are already at high risk for fetal aneuploidy, while cfDNA screening is being performed on women with both high and general obstetrical risks for aneuploidy. In addition, the different number of RATs reported in our study could be explained by the method used to determine the fetal fraction (FF). At the time of testing, we performed two independent FF measurements: the first, based on sequencing data from different genomic regions, is independent of the aneuploidy status of the fetus; the second, applied only in cases where an aneuploidy has been detected, measures the fraction of aneuploid cfDNA calculating the sequence counts of that specific aneuploid region. The comparison of the two different FF estimates (whole genome vs aneuploid chromosome) represents a useful tool to predict the mosaic status of the placenta. In fact, in this case, the aneuploid fraction value will be markedly smaller than the whole genome estimate. In our study, the samples involving not concordant FF estimates were not scored as RAT and may represent the reason of the lower incidence of RATs as compared with other studies. Finally, the specific characteristics of the cohort of patients studied may also justify the above differences. Dr. Grati and Dr. Benn also concerned on the low numbers of abnormal cases with small segmental imbalances identified in our study, suggesting an under-ascertainment of pregnancies with such kind of chromosomal abnormalities. Although the genome-wide cfDNA screening test described in our paper has demonstrated able to detect small chromosome segments, it was designed to identify only copy-number variations (CNVs) larger than 7 Mb (the typical level of resolution reported for G-banded karyotyping), to assure high analytical sensitivity and minimize interpretation challenges. Smaller events were reported only when associated with a larger segmental deletion or duplication (e.g. Cases 2 and 3), or with a clinically relevant genomic region, limited to samples involving a high (>8%) FF (e.g. Case 6). In fact, testing for small clinically relevant CNV (~2–3 Mb) would require a much

Response to “The importance of determining the limit of detection of non-invasive prenatal testing methods”

We thank Dr Lüthgens and collaborators for their interest in our article. We welcome the opportunity to provide clarification and further stimulate the discussion on this important topic. The authors raised concerns regarding the accuracy of the method used in our study to determine the fetal fraction (FF). The reason for their criticism is related to the higher proportion of samples with an FF <4% reported in our article, compared with that observed in other studies. Different approaches are currently available for estimating the FF, either from the isolated DNA or the sequencing data. Among these methods, no two are alike. Thus, it is not surprising that these approaches show a relatively high variation in FF values, even at similar mean gestational age or when comparing similar populations. Recently, Gil et al., in a meta-analysis of published studies, reported that the incidence of test failures due to low FF (<4%) ranged from 0.5 to 6.1%. For instance, Pergament et al. described a 6.1% rate of the samples with FF <4% in a population of patients with a mean gestational age of 17.0weeks ± 4.1 days. This value is not so different from that reported in our study (8.5%), involving a significantly lower mean gestational age (12.8weeks ± 2.3 days). Lüthgens and collaborators found in their clinical practice only 0.6% of samples with FF <4%, a proportion that is also much lower than expected if compared with the 2 to 5% observed in the studies cited by the authors. This finding may be explained by the specific method used for FF determination or the characteristics of the population under evaluation (e.g. higher mean gestational age or lower mean maternal weight, as compared with our study). Alternatively, it could be assumed that FF is overestimated with their method, although no definitive conclusions can be drawn from the authors’ data set because such details have not been provided. The aforementioned methods for FF determination have not been compared with the same set of samples nor validated using standard reference DNAs; therefore, it is not currently possible to define which approach works best. In addition, each non-invasive prenatal testing (NIPT) approach uses a different bioinformatics method to compute aneuploidy risk, their own protocol for FF estimation, and different sequencing depths (i.e. the number of sequence tags counted for each chromosome tested). The combination of such parameters characterizes the limit of detection (LOD; i.e. the lowest FF with a detectable aneuploidy), which is different for each specific NIPT approach. Therefore, several NIPT protocols may need a minimum FF level of 4%. Other methods may be able to reliably detect aneuploidies at a lower FF, because they involve a higher sequencing depth or a more sensitive bioinformatics analysis. Lüthgens and collaborators believe that setting a lower threshold for FF below 4% will necessarily affect the sensitivity of the cell-free fetal DNA (cfDNA) testing. However, this assumption does not take into consideration the fact that the reliability of cfDNA testing results depends not only on the FF level but also on the sequencing depth employed in testing, both impacting the sensitivity or specificity of the cfDNA assay. If there is a low FF in the sample and a sufficiently high sequencing depth, then the assay can still provide accurate counting of the available chromosome fragments. The higher the number of sequence tags counted, the better the ability to distinguish euploid from aneuploid fetuses, thereby the better the test performance. Recently, Benn and Cuckle demonstrated that a low FF can partly be compensated by a higher sequencing depth, and the number of generated reads may thus overcome the statistical noise. In fact, when either depth or fetal fraction is high, expected cfDNA-based aneuploidy screening detection rates are high. However, when FF is low, deeper sequencing (at least ten millions of tags) is required to obtain high detection rates. The NIPT protocol used in our study relied on a high sequencing depth (an average of 16 ± 1.6 millions of unique tags). Such a level of resolution was sufficient to reliably detect aneuploidies in samples with low FF. This was demonstrated by the results of the LOD experiments and confirmed by the chromosomally abnormal pregnancies involving a 2%< FF< 4%, which were consistently identified, with no false negative results. It is unlikely that the two false positive cases that occurred in our study could be related with the low FF, as assumed by the authors. In fact, a low FF may potentially decrease the detection rate (or