Chromosomal mosaicism in human blastocysts: a cytogenetic comparison of trophectoderm and inner cell mass after next-generation sequencing.

RESEARCH QUESTION: What is the incidence of chromosomal mosaicism in human blastocysts and can a single trophectoderm (TE) biopsy accurately predict the chromosomal constitution of the inner cell mass (ICM)? DESIGN: Observational study in 46 surplus cryopreserved preimplantation embryos of unknown chromosomal constitution. For each embryo, a TE biopsy was performed and the ICM was collected separately. Both samples underwent next-generation sequencing (NGS) for cytogenetic analysis and were classified as chromosomally normal, abnormal or mosaic. Mosaic samples were classified as low or high mosaic, based on the majority dominance of either normal or abnormal cells in the biopsied sample. Findings within each embryo were compared. RESULTS: Chromosomal mosaicism was detected in 59% (n = 27/46) of the embryos, with a cytogenetic concordance rate between TE and corresponding ICM of 48% (n = 22/46). Concordance was higher from a clinical perspective: in 86% of embryos with a high-mosaic or abnormal TE, the ICM was also high-mosaic or abnormal. In 88% of the blastocysts with a normal or low-mosaic TE biopsy, a normal or low-mosaic ICM was observed. CONCLUSION: Despite the low cytogenetic concordance rate due to chromosomal mosaicism present in blastocysts, it was found that a single TE biopsy could correctly predict whether the ICM consists of mostly normal or abnormal cells in the majority of cases.

Clinical impact of additional findings detected by genome-wide non-invasive prenatal testing: Follow-up results of the TRIDENT-2 study.

In the TRIDENT-2 study, all pregnant women in the Netherlands are offered genome-wide non-invasive prenatal testing (GW-NIPT) with a choice of receiving either full screening or screening solely for common trisomies. Previous data showed that GW-NIPT can reliably detect common trisomies in the general obstetric population and that this test can also detect other chromosomal abnormalities (additional findings). However, evidence regarding the clinical impact of screening for additional findings is lacking. Therefore, we present follow-up results of the TRIDENT-2 study to determine this clinical impact based on the laboratory and perinatal outcomes of cases with additional findings. Between April 2017 and April 2019, additional findings were detected in 402/110,739 pregnancies (0.36%). For 358 cases, the origin was proven to be either fetal (n = 79; 22.1%), (assumed) confined placental mosaicism (CPM) (n = 189; 52.8%), or maternal (n = 90; 25.1%). For the remaining 44 (10.9%), the origin of the aberration could not be determined. Most fetal chromosomal aberrations were pathogenic and associated with severe clinical phenotypes (61/79; 77.2%). For CPM cases, occurrence of pre-eclampsia (8.5% [16/189] vs 0.5% [754/159,924]; RR 18.5), and birth weight <2.3rd percentile (13.6% [24/177] vs 2.5% [3,892/155,491]; RR 5.5) were significantly increased compared to the general obstetric population. Of the 90 maternal findings, 12 (13.3%) were malignancies and 32 (35.6%) (mosaic) pathogenic copy number variants, mostly associated with mild or no clinical phenotypes. Data from this large cohort study provide crucial information for deciding if and how to implement GW-NIPT in screening programs. Additionally, these data can inform the challenging interpretation, counseling, and follow-up of additional findings.

TRIDENT-2: National Implementation of Genome-wide Non-invasive Prenatal Testing as a First-Tier Screening Test in the Netherlands.

The Netherlands launched a nationwide implementation study on non-invasive prenatal testing (NIPT) as a first-tier test offered to all pregnant women. This started on April 1, 2017 as the TRIDENT-2 study, licensed by the Dutch Ministry of Health. In the first year, NIPT was performed in 73,239 pregnancies (42% of all pregnancies), 7,239 (4%) chose first-trimester combined testing, and 54% did not participate. The number of trisomies 21 (239, 0.33%), 18 (49, 0.07%), and 13 (55, 0.08%) found in this study is comparable to earlier studies, but the Positive Predictive Values (PPV)-96% for trisomy 21, 98% for trisomy 18, and 53% for trisomy 13-were higher than expected. Findings other than trisomy 21, 18, or 13 were reported on request of the pregnant women; 78% of women chose to have these reported. The number of additional findings was 207 (0.36%); these included other trisomies (101, 0.18%, PPV 6%, many of the remaining 94% of cases are likely confined placental mosaics and possibly clinically significant), structural chromosomal aberrations (95, 0.16%, PPV 32%,) and complex abnormal profiles indicative of maternal malignancies (11, 0.02%, PPV 64%). The implementation of genome-wide NIPT is under debate because the benefits of detecting other fetal chromosomal aberrations must be balanced against the risks of discordant positives, parental anxiety, and a potential increase in (invasive) diagnostic procedures. Our first-year data, including clinical data and laboratory follow-up data, will fuel this debate. Furthermore, we describe how NIPT can successfully be embedded into a national screening program with a single chain for prenatal care including counseling, testing, and follow-up.

Cytogenetic confirmation of a positive NIPT result: evidence-based choice between chorionic villus sampling and amniocentesis depending on chromosome aberration.

It has been shown that in non-invasive prenatal testing (NIPT) there is a small chance of a false-positive or false-negative result. This is partly due to the fact that the fetal cell-free DNA present in maternal plasma is derived from the cytotrophoblast of chorionic villi (CV), which is not always representative for the fetal karyotype due to chromosomal mosaicism. Therefore, a positive NIPT result should always be confirmed with invasive testing, preferably amniocentesis, in order to investigate the fetal karyotype. However, since this invasive test can only be safely performed after 15.5 weeks of gestation while NIPT can be done from the 10(th) week of gestation, this potentially means an unacceptable long waiting time for the prospective parents to receive a definitive result. Based on our experience with cytogenetic investigations in CV and the literature, we determined whether CV sampling may be appropriate for confirmation of an abnormal NIPT result.

Application of SNP array for rapid prenatal diagnosis: implementation, genetic counselling and diagnostic flow.

We report on the validation and implementation of the HumanCytoSNP-12 array (Illumina) (HCS) in prenatal diagnosis. In total, 64 samples were used to validate the Illumina platform (20 with a known (sub) microscopic chromosome abnormality, 5 with known maternal cell contamination (MCC) and 39 normal control samples). There were no false-positive or false-negative results. In addition to the diagnostic possibilities of arrayCGH, the HCS allows detection of regions of homozygosity (ROH), triploidy and helps recognising MCC. Moreover, in two cases of MCC, a deletion was correctly detected. Furthermore we found out that only about 50 ng of DNA is required, which allows a reporting time of only 3 days. We also present a prospective pilot study of 61 fetuses with ultrasound abnormalities and a normal karyotype tested with HCS. In 4 out of 61 (6.5%) fetuses, a clinically relevant abnormality was detected. We designed and present pre-test genetic counselling information on categories of possible test outcomes. On the basis of this information, about 90% of the parents chose to be informed about adverse health outcomes of their future child at infancy and childhood, and 55% also about outcomes at an adult stage. The latter issue regarding the right of the future child itself to decide whether or not to know this information needs to be addressed.

The fate of the mosaic embryo: chromosomal constitution and development of Day 4, 5 and 8 human embryos.

BACKGROUND: Post-zygotic chromosome segregation errors are very common in human embryos after in vitro fertilization, resulting in mosaic embryos. However, the significance of mosaicism for the developmental potential of early embryos is unknown. We assessed chromosomal constitution and development of embryos from compaction to the peri-implantation stage. METHODS: From 112 cryopreserved Day 4 human embryos donated for research, 21 were immediately fixed and all cells were analysed by fluorescent in situ hybridization (FISH) for chromosomes 1, 7, 13, 15, 16, 18, 21, 22, X and Y. The remaining 91 embryos were thawed, with 54 embryos undergoing biopsy of one or two cells which were fixed and analysed by FISH. Biopsied embryos were kept in standard culture conditions for 24 h. Embryos arrested before cavitation (n = 24) were fixed whereas developing Day 5 blastocysts (n = 24) were co-cultured for a further 72 h on an endometrial monolayer followed by fixation. Cell numbers were counted and all nuclei were analysed by FISH. Data from a previous FISH analysis on cryopreserved good-quality Day 5 blastocysts (n = 36) were also included in the present study. RESULTS: FISH analysis was successful for 18 Day 4 fixed embryos and, according to our definition, 83% were mosaic and 11% showed a chaotic chromosomal constitution. FISH analysis of two blastomeres from Day 4 developing embryos showed that 54% were mosaic, 40% were normal and 6% were abnormal. Analysis of Day 4, 5 and 8 whole embryos showed a decrease in incidence of mosaicism over time, from 83% on Day 4 to 42% on Day 8. A significant positive correlation was observed between the total cell number and the percentage of normal cells in developing Day 5 and Day 8 embryos but not in developing Day 4 or embryos arrested before cavitation. CONCLUSIONS: These data suggest that both the developmental arrest of a significant proportion of mosaic embryos on Day 4, and the cell death or reduced proliferation of aneuploid cells within an embryo may be responsible for the observed decrease of aneuploid blastomeres from compaction to the peri-implantation stage.

Milder ovarian stimulation for in-vitro fertilization reduces aneuploidy in the human preimplantation embryo: a randomized controlled trial.

BACKGROUND: To test whether ovarian stimulation for in-vitro fertilization (IVF) affects oocyte quality and thus chromosome segregation behaviour during meiosis and early embryo development, preimplantation genetic screening of embryos was employed in a prospective, randomized controlled trial, comparing two ovarian stimulation regimens. METHODS: Infertile patients under 38 years of age were randomly assigned to undergo a mild stimulation regimen using gonadotrophin-releasing hormone (GnRH) antagonist co-treatment (67 patients), which does not disrupt secondary follicle recruitment, or a conventional high-dose exogenous gonadotrophin regimen and GnRH agonist co-treatment (44 patients). Following IVF, embryos were biopsied at the eight-cell stage and the copy number of 10 chromosomes was analysed in 1 or 2 blastomeres. RESULTS: The study was terminated prematurely, after an unplanned interim analysis (which included 61% of the planned number of patients) found a lower embryo aneuploidy rate following mild stimulation. Compared with conventional stimulation, significantly fewer oocytes and embryos were obtained following mild stimulation (P < 0.01 and < 0.05, respectively). Consequently, both regimens generated on average a similar number (1.8) of chromosomally normal embryos. Differences in rates of mosaic embryos suggest an effect of ovarian stimulation on mitotic segregation errors. CONCLUSIONS: Future ovarian stimulation strategies should avoid maximizing oocyte yield, but aim at generating a sufficient number of chromosomally normal embryos by reduced interference with ovarian physiology.

A familial inverted duplication 2q33-q34 identified and delineated by multiple cytogenetic techniques.

We describe a unique family with two children having a delay in psychomotor development. In both children we identified an interstitial duplication dup(2)(q34q33) using multiple, complementary molecular cytogenetic techniques. Comparative genomic hybridisation (CGH) and array-CGH were used to determine the size and the location of the duplicated region, the orientation of the duplicated region was identified with fluorescence in situ hybridisation (FISH). Both parents demonstrated a normal karyotype and normal CGH and array-CGH-profiles. However, FISH on peripheral blood cells from the mother showed the inv dup(2) in 9% of metaphases and 19% of interphase nuclei. To our knowledge this is the first report of a mosaic carrier of duplication in the long arm of chromosome 2. The finding of chromosomal mosaicism of at least 19% in the mother increases the recurrence risk. The exact characterisation of the inv dup(2) with FISH probes enabled us to offer a reliable prenatal FISH test. Comparison of the clinical features of the two children with those of previously described cases supports the hypothesis that the characteristic facial phenotype is linked to the distal part of the 2q33-q37 region. This report illustrates that in case of two sibs with an identical structural chromosomal abnormality the possibility of parental chromosomal mosaicism must be thoroughly investigated.

(Potential) false-negative diagnoses in chorionic villi and a review of the literature.

OBJECTIVES: To assess the incidence of (potential) false-negative findings of cytogenetic diagnosis in STC-villi and/or LTC-villi and to determine the best strategy for karyotyping chorionic villi in order to avoid false-negative results. METHODS: 2476 chorionic villus samples were received for prenatal cytogenetic investigations. Karyotyping was routinely performed on STC- and LTC-villi preparations by G-banding. Fluorescence in situ hybridization (FISH) analyses were performed in addition to standard chromosome analysis when necessary. Sometimes follow-up investigations like amniocentesis were performed before a definite prenatal cytogenetic result could be reported. RESULTS: In 2389/2476 (96.5%) of the cases, both STC- and LTC-villi were investigated. Normal STC- with abnormal LTC-villi results and finally an abnormal fetal karyotype were detected in ten cases (10/2389; 0.42%); in 9/10 of the cases the indication was fetal ultrasound abnormalities. Normal STC- and LTC-villi and finally an abnormal fetal karyotype were detected in two cases (2/2389; 0.08%). CONCLUSION: The most reliable technique for prenatal diagnosis after chorionic villus sampling (CVS) is the combination of the analysis of both STC- and LTC-villi to reduce the incidence of false-negative findings to a minimum. In the case of fetal ultrasound abnormalities with a small amount of villi available, the investigation of LTC-villi is recommended over that of STC-villi.

The development of cytogenetically normal, abnormal and mosaic embryos: a theoretical model.

Assisted reproduction and preimplantation genetic diagnosis (PGD) involve various complicated techniques, each of them with its own problems. However, the greatest problem with PGD for chromosome abnormalities is not of a technical nature but is a biological phenomenon: chromosomal mosaicism in the cleavage stage embryo. Here, we present a hypothetical, quantitative model for the development of chromosomally normal, abnormal and mosaic embryos. The arising of mosaicism in 2-8-cell embryos was described by a binomial probability model on the occurrence of mitotic events inducing chromosomal changes in the blastomeres. This model converted the 'mean' rate of mosaicism found in cross-sectional studies (60%) into an equal rate of mosaic embryos at arrival at the 8-cell stage (59.8%). The disappearance of > 90% of the mosaic embryos or the mosaicism itself from surviving embryos during the morula stage was explained by mitotic arrest of most of the mitotically changed cells under increasing cell cycle control. In our model, 25.9 and 14.3% of the embryos at the 8-cell stage are normal and abnormal respectively. The remaining 59.8% of the embryo shows mosaicism: 34.6% of abnormal/normal cells and 25.2% of abnormal/abnormal cells. The high proportion of abnormal and mosaic embryos together explains the high rate of abnormal laboratory findings in PGD for chromosomal abnormalities and aneuploidy screening. The poor representation of a 1- or 2-cell biopsy for the 7- or 6-cell post-biopsy embryo in the case of mosaicism explains the high rate of false-negative and false-positive results.