Search

Objective: Genomics Quality Assessment has provided external quality assessments (EQAs) for preimplantation genetic testing (PGT) for 12 years for eight monogenic diseases to identify sub-optimal PGT strategies, testing and reporting of results, which can be shared with the genomics community to aid optimised standards of PGT services for couples. Method: The EQAs were provided in two stages to mimic end-to-end protocols. Stage 1 involved DNA feasibility testing of a couple undergoing PGT and affected proband. Participants were required to report genotyping results and outline their embryo testing strategy. Lymphoblasts were distributed for mock embryo testing for stage 2. Submitted clinical reports and haplotyping results were assessed against peer-ratified criteria. Performance was monitored to identify poor performance. Results: The most common testing methodology was short tandem repeat linkage analysis (59%); however, the adoption of single nucleotide polymorphism-based platforms was observed and a move from blastomere to trophectoderm testing. There was a variation in testing strategies, assigning marker informativity and understanding test limitations, some clinically unsafe. Critical errors were reported for genotyping and interpretation. Conclusion: EQA provides an overview of the standard of preimplantation genetic testing-M clinical testing and identifies areas of improvement for accurate detection of high-risk embryos.

Chromosomal mosaicism detected during preimplantation genetic testing for aneuploidy (PGT-A) and its impact on embryo implantation have been widely discussed, and healthy live births from mosaic embryos were reported by many groups. On the other hand, only very few studies have focused on segmental chromosome aneuploidies and their clinical impact. Eighty-nine embryos with various PGT-A results (trophectoderm 1: TE1) were re-analysed using a second trophectoderm biopsy (TE2) and the rest of the embryo (RE) for testing. Of 19 euploid TE1 biopsies, 18 were concordant across TE2 and RE. Similarly, whole chromosomal aneuploidies were concordant in 59 of 62 TE1-TE2 and 58 TE1-RE. In contrast, from 31 segmental aneuploidies detected in TE1, only 15 were observed again in TE2 and 14 in RE. If a TE1 segmental abnormality appeared again in TE2, it was almost always present in RE (17/18) as well. Moreover, when a TE1 segmental abnormality was not detected in TE2, in 12 out of 13 cases RE was also unaffected. Similarly, only 1 of 26 TE1 whole chromosome mosaics were repeated in TE2 and 7 in RE. Our study confirms that euploid and whole chromosomal aneuploidy results are highly predictive of the embryo. In contrast, mosaicism has a very low concordance rate. Most importantly, re-biopsy of embryos with segmental aneuploidies demonstrated that they are mostly not uniform across the embryo. Finally, in the case of segmental aneuploidy, the second biopsy enables an accurate prediction of the real status of the embryo and could be offered to patients undergoing PGT-A.

What is the incidence and origin of meiotic whole and segmental aneuploidies detected by karyomapping at a blastocyst stage in human-derived IVF embryos? What is the distribution of various types of errors, including rare chromosomal abnormalities?The incidence of chromosomal aneuploidies was assessed in 967 trophectoderm biopsies from 180 couples who underwent 215 cycles of IVF with preimplantation genetic testing for monogenetic disease with a known causal mutation with a mean maternal age of 32.7 years. DNA from both parents and a reference sample was genotyped together with the analysed trophectoderm samples by karyomapping (single-nucleotide-polymorphism-based array).Chromosomal abnormalities were detected in 31% of the analysed samples. At least one whole chromosomal aneuploidy was detected in 27.1% of the trophectoderm biopsies, whereas a segmental aneuploidy was detected in 5.1% of the trophectoderm biopsies. Our results reveal that segmental aneuploidies predominantly affect paternally derived chromosomes (70.4%; P0.01) compared with whole chromosomal aneuploidies that more frequently affect maternally derived chromosomes (90.1%; P0.0001). Also, the frequency of meiosis I (MI) and meiosis II (MII) errors was established in meiotic trisomies; MI errors were observed to be more frequent (n = 102/147 [69.4%]) than MII errors (n = 45/147 [30.6%]).Karyomapping is a robust method that is suitable for preimplantation genetic testing for monogenetic disease and for detecting meiotic aneuploidies, including meiotic segmental aneuploidies, and provides complex information about their parental origin. Our results revealed that segmental aneuploidy more frequently affects paternal chromosomes compared with whole chromosomal aneuploidy in human IVF embryos at the blastocyst stage.

Introduction A potential impact of PGT-A on embryo implantation is often discussed in the reproduction of elderly women with a higher risk of oocyte aneuploidy while its impact is belied in women under 35 years at the same time. The authors studied the influence of PGT-A in egg donation program where the advanced reproductive age and potential oocyte incompetence is mostly excluded. Material and methods In total, 246 embryos were thawed and transferred in egg donation patients of the age category 35 plus in period January 2017 – December 2018. Group A consists of 188 patients with a comprehensive aneuploidy screening (PGT-A), group B consists of 58 patient with no PGT-A. There were performed elective single embryo transfers solely in both groups. Results In group A an implantation rate was 46% (86/188) while in group B 20%(12/58). Natural cycles with triggering ovulation were more successful than the HRT ones (IR 53% resp. 44%). Conclusions The results showed that PGT-A and transfer of euploid embryo improve an implantation rate in egg donation program in the age category 35 plus what is usually disregarded. The number of cycles to pregnancy was lower in case of a euploid embryo was transferred. The major factors involving the implantation rate besides the embryo quality and euploidy are an appropriate endometrial quality and striking the window of implantation (WOI). The implantation rate is slightly better in natural cycles compared to HRT (hormonal replacement treatment) cycles. Among other factors, we reached better results using a longer progesterone administration (minimum 5 days) and a time-lapse monitoring system used in synergy with an embryo selection for the biopsy.

Introduction Due to the rapid development in the PGT field, new and challenging issues including increased detection rates of embryos with segmental mosaicism have emerged. On the other hand, this rapid development brings us better technologies to address these issues. Even though karyomapping (SNP based array) is limited in aneuploidy testing and enables detection of high-level mosaic only, it provides us with interesting information whether mosaic aneuploidies affect mostly maternal or paternal chromosomes. We focused our attention oncomparing affected chromosomes in both mosaic segmentals and mosaic whole chromosomal aneuploidies with segmentals and whole chromosomal aneuploidies. Materials & methods The incidence and origin of chromosomal aneuploidies (including whole chromosomal aneuploidy, mosaic whole chromosomal aneuploidy, segmentals and mosaic segmentals) were assesed in 1206 trophectoderm biopsies. More than 97% (1172/1206) of samples were successfully analyzed by karyomapping (Illumina - HumanKaryomap 12 SNP bead chips, BlueFuse Multi software) in 249 PGT-M for various single gene conditions. All chromosomes of all embryos were evaluated on a B-llele frequency chart (BAF), a logR chart and haploblock charts. Mitotic aneuploidies including mosaics were called if BAF and logR chart were consistent, but aneuploidy was not indicated on a haploblock chart. Results At least one whole chromosomal aneuploidy was detected in 29.3% of the trophectoderm biopsies, while a segmental aneuploidy was detected in 5.7% of the trophectoderm biopsies. A uniparental disomy was detected 4 times (0.34%). Interestingly, our results reveal that segmental aneuploidies predominantly affect chromosomes of paternal origin (p Conclusions Our results show that both segmental aneuploidies and mosaic segmental aneuploidy affect mainly paternal chromosomes. The fact that mosaic segmentals are mostly detected in chromosomes of paternal origin as well as in full segmental aneuploidies strongly indicates that both originate in post-zygotic embryonal development and represent the same biological phenomenon rather than an artefact of whole genome amplification or SNP detection. Our findings support the hypothesis, that fragmentation during spermatogenesis might be the main reason why most of the segmental changes affect paternal chromosomes. Unrepaired fragments may be lost either during fertilization or during the first cell divisions in the early embryonic development, resulting in segmental loss and mosaic segmental loss. Further studies are needed for a better understanding of the biological significance of segmental mosaics and the clinical impact on the PGT outcome.

Bovine embryos are now routinely used in agricultural systems as a means of disseminating superior genetics worldwide, ultimately with the aim of feeding an ever-growing population. Further investigations, common for human IVF embryos, thus have priority to improve cattle IVF, as has screening for aneuploidy (abnormal chromosome number). Although the incidence and consequences of aneuploidy are well documented in human preimplantation embryos, they are less well known for the embryos of other animals. To address this, we assessed aneuploidy levels in thirty-one 2-cell bovine embryos derived from early- and late-cleaving zygotes. Contemporary approaches ( Whole Genome Amplification and next-generation sequencing) allowed aneuploidy assessment for all chromosomes in oocytes from donors aged 4-7 years. We also quantified mitochondrial DNA (mtDNA) levels in all blastomeres assessed, thereby testing the hypothesis that they are related to levels of aneuploidy. The overall incidence of aneuploidy in this cohort of bovine embryos was 41.1% and correlated significantly with the timing of cleavage (77.8% in late-cleaving vs. 31.8% in early-cleaving embryos). Moreover, based on mtDNA sequence read counts, we observed that the median mtDNA quantity is significantly lower in late-cleaving embryos. These findings further reinforce the use of the bovine system as a model for human IVF studies.

Introduction Karyomapping serves as the universal PGT-M platform based on Mendelian inheritance of SNP markers which assist in identifying haplotypes linked to a mutated gene. Moreover, karyomapping enables detection of aneuploidies by interrogating genome-wide SNP data in embryo samples and assists in a selection of euploid embryos for a transfer. We have found out that high quality SNP profiles, characterized by overall call rate ≥95%, might be reliably used for the identification of mitotic chromosome errors, aneuploidies in mosaic or segmental chromosome aberrations. Since karyomapping utilizes parental DNA samples, the origin of identified aneuploidies might be tracked in embryo samples. Our study describes the incidence and origin of aneuploidy, aiming at segmental chromosomes errors and mosaic aneuploidies which represents hot topic in respect to their clinical significance. Material & methods SNP data obtained by HumanKaryomap-12 BeadChips, Illumina SNP arrays were analysed in 2085 trophectoderm (TE) samples derived from 319 couples requesting PGT-M. Mean maternal age was 32.4 ± 4.5. Based on the karyomapping SNP data QC metrics, mean and median call rate was 90.4% and 95%, respectivelly. In total, 936 embryos fulfilled criteria of high quality SNP profiles (call rate ≥95%). A subset of twenty TE samples detected with mitotic trisomies, mosaic aneuploidies and segmental chromosome abnormalities by karyomapping were reanalysed using PG-Seq™ kit, which is the fully validated PGT-A platform in our laboratory. We have observed full concordance between both platforms. Results Analysing 936 high quality TE samples, we have observed that 617 (65.9%) of them were euploid. The remaining 319 TE samples were detected with the following aberrations: a) only aneuploid 234 (73.4%) samples; b) only mosaic 43 (13.5%) samples; c) aneuploid + mosaic 42 (13.2%) samples. In other words, overall mosaicism rate in 936 TE samples was 9.1% (85/936) in our study. This number tend to be underestimated, since we did not report low level mosaicism due to a lack of proper sensitivity validation of the karyomapping in last years. We have detected segmental aneuploidies in 12.6% (118/936) of TE samples, 56 (47.4%) of them were present in mosaic form. Regarding the origin of aneuploidies, 87% (220/253) of whole chromosome aneuploidies affected maternal chromosomes, the ratio difference was even more highlighted in whole chromosome trisomies (93%; 91 out of 98). In contrast, segmental chromosome abnormalities affected more frequently paternal chromosomes (61,3%; 38 out of 62). Notably, this trend towards paternally related segmental aneuploidies were also followed in segmental chromosome abnormalities in mosaic (60,7%; 34 out of 56). Conclusions 1. According to our validation, high quality SNP profiles (overall SNP call rate ≥95%) enables detection of mitotic trisomies, mosaicism at 25% sensitivity level and segmental chromosome abnormalities (in 5Mb resolution). 2. Meiotic whole chromosome trisomies prevail over mitotic whole chromosome trisomies (in full or mosaic state) in the ratio 4:1. 3. Segmental chromosome abnormalities (in full or mosaic state) affects more frequently paternal chromosomes (P value 4. Whole chromosome aneuploidies in mosaic affect equally maternal and paternal chromosomes.

Introduction Preimplantation genetic testing of aneuploidies (PGT-A) enables the selection of euploid embryos in order to increase implantation rates and reduce miscarriage rates after embryo transfer. Next Generation Sequencing (NGS) platforms provide higher dynamic range than other methods and are validated for the detection of copy number variations starting at 20% mosaicism. Sub-chromosomal segmental aneuploidies above 15 MBp are reported in routine PGT-A, and the resolution can be increased to 1-2 MBp when PGT targeted to a previously described structural rearrangement is carried out (PGT-SR). While calling whole chromosomal aneuploidy leads to the elimination of clearly non- viable embryos from transfer to the uterus, numerous healthy live-births have been reported after transferring mosaic embryos. Clinical impact of segmental aneuploidies detected in trophectoderm biopsies remains unclear. Material and methods All embryos were biopsied at day 5/6 and trophectoderm samples were analyzed by VeriSeq™ PGS Kit (Illumina). Discarded embryos dedicated to research with the consent of patients were de-vitrified and after blastocyst re-expansion a second trophectoderm sample of the same size was biopsied for analysis using the PG-Seq™ platform (RHS). In total, 95 embryos were re- analyzed and the results of both replicates were compared. After the first PGT-A, 73 embryos were classified aneuploid, 12 mosaic, and 10 euploid. Assessing the same set of samples by aberration type, 63 whole chromosomal aneuploidies were originally called using VeriSeq™, 28 whole chromosomal mosaics, 36 segmental aneuploidies and 9 segmental aneuploidy mosaics. Reproducibility of various aneuploidy types was evaluated after second biopsy analysis using PG-Seq™. Results In the group of 10 euploid embryos, the original status was confirmed in 9 embryos with one rebiopsied sample reporting a mosaic loss of chromosome 17. Aneuploidy was concordant in 63 out of 73 samples with 9/10 of the non-concordant results changing from segmental aneuploidy to euploid. Only 3 of 12 mosaic embryos remained mosaic with 6 rebiopsied mosaic embryos turning to euploid and 3 aneuploid. When evaluating each chromosomal change separately, reproducibility of 96.8% was observed for whole chromosomal aneuploidies (61/63), 50% for segmentals (18/36) and only 10.8% for mosaics (4/37). Particularly, none of the 9 segmental mosaics were detected in the second biopsy. In contrast to this, second biopsy testing revealed 2 whole chromosomal aneuploidies, 8 whole chromosomal mosaics and 5 segmental mosaics which were not detected in the previously analysed sample. Performance of both NGS platforms appeared to be comparable in terms of resolution and dynamic range for mosaic detection. Conclusions Our comparative study showed that concordance between two trophectoderm samples is about 97% when whole chromosomal aneuploidies are reported. This reflects the numbers described previously in studies comparing trophectoderm and inner cell mass of the embryo. In striking contrast, reproducibility of segmental aneuploidies and mosaic findings remains very poor even in the blastocyst stage and disables us to predict real developmental capacity of the embryo. Non-concordance is to be expected in mosaic embryos. Further clinical studies are needed to unravel whether segmental aneuploidies have similar potential to result in healthy live-births as already reported for mosaic embryos.

Introduction Preimplantation genetic testing of aneuploidies (PGT-A) using Next Generation Sequencing (NGS) platforms decreases significantly the risk of abortion from aneuploidy reason, nevertheless some fetuses are unfortunately aborted despite PGT-A from another sakes. NGS platforms provide high dynamic range and are able to detect copy number variations starting at 20% mosaicism. Material of trophectoderm biopsy includes several trophectoderm cells and it is quite often discussed whether such sample is representative enough to reflect the inner cell mass chromosomal status. The aim of this retrospective study is to reveal a representativeness and a significance of a trophectoderm biopsy sample evaluation with respect to the chromosomal constitution during the further embryonic development and assignments of concordance between trophectoderm biopsy day 5-6 and the finding in chorionic villi of aborted fetuses. Material and methods 59 chorionic villi samples were obtained from aborted fetuses during the surgical curettage for incomplete spontaneous or missed early abortions during a period January 2016 till March 2018. All samples were cleaned, washed and stored in a phosphate buffer and after DNA isolation the chromosome copy number was evaluated using 24sure array (Illumina). 42 samples were obtained from fetuses conceived after embryo transfers, 10 embryos were transferred without PGT-A and 32 were analyzed using PGT-A (VeriSeq™ PGS Kit by Illumina) before an embryo transfer. In all study cases a single embryo transfer was performed. All 32 PGT-A embryos were originally diagnosed as euploid. 17 samples were derived from spontaneously conceived embryos. The samples were divided into 3 groups. Group A consists of 10 non-PGT-A samples, group B of 17 samples from spontaneously conceived fetuses and group C of 32 samples from fetuses after PGT-A. Results In total, 23,7 % (14/59) chorionic villi samples were diagnosed as aneuploid. In the group A (non-PGT-A fetuses) 50% (5/10) were aneuploid, in the group B (spontaneous conceptions) 47% (8/17) samples were aneuploid while in the group C (PGT-A fetuses) only 3% (1/32) were aneuploid. Euploid status was concordant in 31 out of 32 PGT-A analyzed embryos (97%). Conclusions Our retrospective study showed that 50% of aborted fetuses after IVF with no PGT-A were aneuploid. This corresponds very well with the aneuploidy rate observed for products of spontaneous conception in infertile couples (47%). Chorionic villi samples from aborted fetuses derived from previously negatively tested blastocysts were aneuploid in 3% of cases (1/32). The concordance between an euploid status of trophectoderm samples and chorionic villi tissue from aborted fetuses is 97%. This confirms the figures referred previously in another studies comparing evaluation of trophectoderm and inner cell mass of the embryo and fully supports routine implementation of PGT-A into assisted reproductive techniques. The slight discordance is possible to be explained by unequal distribution of aneuploid/mosaic cell lines within the trophectoderm tissue at the blastocyst stage when the biopsy is performed.

The karyotype of the red river hog Potamochoerus porcus (2n = 34) differs from that of the domestic pig by the presence of 2 fusion chromosomes homologous to pig chromosomes 13/16 and 15/17. Moreover, chromosomes corresponding to pig chromosomes 13/16 and 1 are both acrocentric. Hybridization with region-specific painting probes confirmed tandem fusion of pig chromosomes 13 and 16, and a pericentric inversion of the pig chromosome 1p equivalent in P. porcus. The chromosome complement of the wart hog Phacochoerus africanus (2n = 34) differs from the pig karyotype in 2 centric fusions, 13/16 and 15/17. Karyotypic relationships among different Suidae species are discussed in the article. Besides fusions 13/16 and 15/17, which are common to several suids, another fusion of pig chromosomes 14 and 18 is suggested to exist in the karyotype of Sus cebifrons.

In pigs, in vitro production is difficult with a high occurrence of polyspermy and low blastocyst formation rates. To test the hypothesis that this may, at least in part, be due to chromosomal errors, we employed whole genome amplification and comparative genomic hybridization, performing comprehensive chromosome analysis to assess both cells of the two-cell stage in vitro porcine embryos. We thus described the incidence, nature and origin of chromosome abnormalities, i.e. whether they derived from incorrect meiotic division during gametogenesis or aberrant mitotic division in the zygote. We observed that 19 out of 51 (37%) of two-cell stage early pig IVP embryos had a chromosome abnormality, mostly originating from an abnormal division in the zygote. Moreover, we frequently encountered multiple aneuploidies and segmental chromosome aberrations. These results indicate that the pig may be particularly sensitive to in vitro production, which may, in turn, be due to incorrect chromosome segregations during meiosis and early cleavage divisions. We thus accept our hypothesis that chromosome abnormality could explain poor IVP outcomes in pigs.

Data on the frequency of aneuploidy in farm animals are lacking and there is the need for a reliable technique which is capable of detecting all chromosomes simultaneously in a single cell. With the employment of comparative genomic hybridization coupled with the whole genome amplification technique, this study brings new information regarding the aneuploidy of individual chromosomes in pigs. Focus is directed on in vivo porcine blastocysts and late morulas, 4.7% of which were found to carry chromosomal abnormality. Further, ploidy abnormalities were examined using FISH in a sample of porcine embryos. True polyploidy was relatively rare (1.6%), whilst mixoploidy was presented in 46.8% of embryos, however it was restricted to only a small number of cells per embryo. The combined data indicates that aneuploidy is not a prevalent cause of embryo mortality in pigs.

It is generally accepted that mammalian oocytes are frequently suffering from chromosome segregation errors during meiosis I, which have severe consequences, including pregnancy loss, developmental disorders and mental retardation. In a search for physiologically more relevant model than rodent oocytes to study this phenomenon, we have employed comparative genomic hybridization (CGH), combined with whole genome amplification (WGA), to study the frequency of aneuploidy in porcine oocytes, including rare cells obtained from aged animals. Using this method, we were able to analyze segregation pattern of each individual chromosome during meiosis I. In contrast to the previous reports where conventional methods, such as chromosome spreads or FISH, were used to estimate frequency of aneuploidy, our results presented here show, that the frequency of this phenomenon was overestimated in porcine oocytes. Surprisingly, despite the results from human and mouse showing an increase in the frequency of aneuploidy with advanced maternal age, our results obtained by the most accurate method currently available for scoring the aneuploidy in oocytes indicated no increase in the frequency of aneuploidy even in oocytes from animals, whose age was close to the life expectancy of the breed.

Although numerical chromosome errors are known to be prevalent in early human embryos and are likely to be a considerable factor influencing the mortality of early embryos and implantation failure, in domestic animals data about the frequency and nature of errors is limited. The objectives of this study were to investigate the whole chromosome set of in vivo obtained early pig embryos, applying methods of whole genome amplification and comparative genomic hybridization (CGH) and to contribute to the comprehensive understanding of the topic. The embryos were collected from gilts 72 h after insemination. Further, they were lysed and underwent whole genome amplification by multiple displacement amplification. In a subsequent CGH, amplified DNA was compared to control DNA using different fluorescent labeling and hybridization to male pig mitoses. 11 (14.3%) of the 77 pig embryos examined were observed to be aneuploid. We found chromosome errors comprising loss/gain of one or a few chromosomes (10.4%) but also extensive chromosome imbalances (3.9%). Chromosomes 8, 11, 12, 13, 17, and X were most frequently involved in aneuploidies, when compared to chromosomes 2, 9, and 18, which were rarely involved in chromosome errors.