A heatmap for expected cumulative live birth rate in preimplantation genetic testing for monogenic disorders and chromosomal structural rearrangements.

PURPOSE: Our objective is to predict the cumulative live birth rate (CLBR) and identify the specific subset within the population undergoing preimplantation genetic testing for monogenic disorders (PGT-M) and chromosomal structural rearrangements (PGT-SR) which is likely to exhibit a diminished expected CLBR based on various patient demographics. METHODS: We performed a single-centre retrospective cohort study including 1522 women undergoing 3130 PGT cycles at a referral centre for PGT. A logistic regression analysis was performed to predict the CLBR per ovarian stimulation in women undergoing PGT-M by polymerase chain reaction (PCR) or single-nucleotide polymorphism (SNP) array, and in women undergoing PGT-SR by SNP array, array comparative genomic hybridization (CGH) or next-generation sequencing (NGS). RESULTS: The mean age of women was 32.6 years, with a mean AMH of 2.75 µg/L. Female age and AMH significantly affected the expected CLBR irrespective of the inheritance mode or PGT technology. An expected CLBR < 10% was reached above the age of 42 years and AMH ≤ 1.25 µg/L. We found no significant difference in outcome per ovarian stimulation between the different PGT technologies, i.e. PCR, SNP array, array CGH and NGS. Whereas per embryo transfer, we noticed a significantly higher probability of live birth when SNP array, array CGH and NGS were used as compared to PCR. CONCLUSION: In a PGT-setting, couples with an unfavourable female age and AMH should be informed of the prognosis to allow other reproductive choices. The heatmap produced in this study can be used as a visual tool for PGT couples.

Cleavage-stage or blastocyst-stage embryo biopsy has no impact on growth and health in children up to 2 years of age.

BACKGROUND: Studies show conflicting results on neonatal outcomes following embryo biopsy for PGT, primarily due to small sample sizes and/or heterogeneity in the timing of embryo biopsy (day 3; EBD3 or day 5/6; EBD5) and type of embryo transfer. Even fewer data exist on the impact on children's health beyond the neonatal period. This study aimed to explore outcomes in children born after EBD3 or EBD5 followed by fresh (FRESH) or frozen-thawed embryo transfer (FET). METHODS: This single-centre cohort study compared birth data of 630 children after EBD3, of 222 EBD5 and of 1532 after non-biopsied embryo transfers performed between 2014 and 2018. Follow-up data on growth were available for 426, 131 and 662 children, respectively. RESULTS: Embryo biopsy, either at EBD3 or EBD5 in FET and FRESH cycles did not negatively affect anthropometry at birth, infancy or childhood compared to outcomes in non-biopsied FET and FRESH cycles. While there was no adverse effect of the timing of embryo biopsy (EBD3 versus EBD5), children born after EBD3 followed by FET had larger sizes at birth, but not thereafter, than children born after EBD3 followed by FRESH. Reassuringly, weight and height gain, proportions of major congenital malformations, developmental problems, hospital admissions and surgical interventions were similar between comparison groups. CONCLUSION: Our study indicated that neither EBD3 nor EBD5 followed by FRESH or FET had a negative impact on anthropometry and on health outcomes up to 2 years of age.

Analysis of parental contribution for aneuploidy detection (APCAD): a novel method to detect aneuploidy and mosaicism in preimplantation embryos.

RESEARCH QUESTION: Can (mosaic) aneuploidy be reliably detected in preimplantation embryos after multiple displacement amplification and single nucleotide polymorphism detection, independent of haplotyping and copy number detection, with a new method 'analysis of parental contribution for aneuploidy detection' or 'APCAD'? DESIGN: This method is based on the maternal contribution, a parameter that reflects the proportion of DNA that is of maternal origin for a given chromosome or chromosome segment. A maternal contribution deviating from 50% for autosomes is strongly indicative of a (mosaic) chromosomal anomaly. The method was optimized using cell mixtures with varying ratios of euploid and aneuploid (47,XY,+21) lymphocytes. Next, the maternal contribution was retrospectively measured for all chromosomes from 349 Karyomapping samples. RESULTS: Retrospective analysis showed a skewed maternal contribution (<36.4 or >63.6%) in 57 out of 59 autosome meiotic trisomies and all autosome monosomies (n = 57), with values close to theoretical expectation. Thirty-two out of 7436 chromosomes, for which no anomalies had been observed with Karyomapping, showed a similarly skewed maternal contribution. CONCLUSIONS: APCAD was used to measure the maternal contribution, which is an intuitive parameter independent of copy number detection. This method is useful for detecting copy number neutral anomalies and can confirm diagnosis of (mosaic) aneuploidy detected based on copy number. Mosaic and complete aneuploidy can be distinguished and the parent of origin for (mosaic) chromosome anomalies can be determined. Because of these benefits, the APCAD method has the potential to improve aneuploidy detection carried out by comprehensive preimplantation genetic testing methods.

Multiple vitrification-warming and biopsy procedures on human embryos: clinical outcome and neonatal follow-up of children.

STUDY QUESTION: Does double vitrification and warming of human blastocysts having undergone biopsy once or twice have an impact on the clinical outcome? SUMMARY ANSWER: The clinical pregnancy rate obtained with double vitrification single biopsy blastocysts was comparable to that obtained with single vitrification single biopsy blastocysts in our center in the same time period (46%; 2016-2018), whereas that obtained with double-vitrified double-biopsied blastocysts seemed lower and will need further study. WHAT IS KNOWN ALREADY: Genetic testing on cryopreserved unbiopsied embryos involves two cryopreservation procedures. Retesting of failed/inconclusive-diagnosed blastocysts inevitably involves a second round of biopsy and a second round of vitrification as well. To what extent this practice impacts on the developmental potential of blastocysts has been studied to a limited extent so far and holds controversy. Additionally, the obstetrical/perinatal outcome after the transfer of double-vitrified/single or double-biopsied blastocysts is poorly documented. STUDY DESIGN, SIZE, DURATION: This retrospective observational study included 97 cycles of trophectoderm biopsy and preimplantation genetic testing (PGT) on vitrified-warmed embryos followed by a second round of vitrification between March 2015 and December 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS: In 36 warming cycles, no biopsy was performed on the embryos before the first vitrification (single biopsy group). In 61 warming cycles, the embryos had been biopsied on Day 3 (n = 4) or on Day 5/6 (n = 57) before the first vitrification (double biopsy group). A second biopsy was mostly indicated in cycles of failed or inconclusive diagnosis at the first biopsy. Two cycles involved a more specific mutation test for X-linked diseases on male embryos and one cycle involved testing for a second monogenic indication supplementary to a previously tested reciprocal translocation. Post-warming suitability for biopsy, availability of genetically transferable embryos and clinical outcome of subsequent frozen-thawed embryo transfer (FET) cycles were reported. Neonatal follow-up of the children was included. MAIN RESULTS AND THE ROLE OF CHANCE: In total, 91 cleavage-stage embryos and 154 blastocysts were warmed, of which 34 (37.4%) and 126 (81.8%), respectively, were of sufficient quality to undergo trophectoderm biopsy and were subsequently vitrified for a second time. Out of these, 92 underwent biopsy for the first time (single biopsy), whereas 68 underwent a second biopsy (double biopsy). After diagnosis, 77 blastocysts (48.1%) were revealed to be genetically transferable (44 in the single biopsy group and 33 in the double biopsy group). In 46 warming cycles, 51 blastocysts were warmed and 49 survived this second warming procedure (96.0%). Subsequently, there were 45 FET cycles resulting in 27 biochemical pregnancies and 18 clinical pregnancies with fetal heartbeat (40.0% per FET cycle: 44.0% in the single biopsy group and 35.0% in the double biopsy group, P = 0.54). Thirteen singletons were born (eight in the single biopsy group and five in the double biopsy group), while three pregnancies were ongoing. A total of 26 embryos (13 in each group) remain vitrified and have the potential to increase the final clinical pregnancy rate. The neonatal follow-up of the children born so far is reassuring. LIMITATIONS, REASONS FOR CAUTION: This is a small retrospective cohort, thus, the implantation potential of double vitrification double biopsy blastocysts, as compared to double vitrification single biopsy blastocysts and standard PGT (single vitrification, single biopsy), certainly needs further investigation. Although one could speculate on birthweight being affected by the number of biopsies performed, the numbers in this study are too small to compare birthweight standard deviation scores in singletons born after single or double biopsy. WIDER IMPLICATIONS OF THE FINDINGS: PGT on vitrified-warmed embryos, including a second vitrification-warming step, results in healthy live birth deliveries, for both single- and double-biopsied embryos. The neonatal follow-up of the 13 children born so far did not indicate any adverse effect. The present study is important in order to provide proper counseling to couples on their chance of a live birth per initial warming cycle planned and concerning the safety issue of rebiopsy and double vitrification. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: N/A.

PREIMPLANTATION GENETIC TESTING: Clinical experience of preimplantation genetic testing.

Thirty years of rapid technological advances in the field of genetic testing and assisted reproduction have reshaped the procedure of preimplantation genetic testing (PGT). The development of whole genome amplification and genome-wide testing tools together with the implementation of optimal hormonal stimulation protocols and more efficient cryopreservation methods have led to more accurate diagnoses and improved clinical outcomes. In addition, the shift towards embryo biopsy at day 5/6 has changed the timeline of a typical PGT clinical procedure. In this paper, we present an up-to-date overview of the different steps in PGT from patient referral to baby follow-up.

Concurrent whole-genome haplotyping and copy-number profiling of single cells.

Methods for haplotyping and DNA copy-number typing of single cells are paramount for studying genomic heterogeneity and enabling genetic diagnosis. Before analyzing the DNA of a single cell by microarray or next-generation sequencing, a whole-genome amplification (WGA) process is required, but it substantially distorts the frequency and composition of the cell's alleles. As a consequence, haplotyping methods suffer from error-prone discrete SNP genotypes (AA, AB, BB) and DNA copy-number profiling remains difficult because true DNA copy-number aberrations have to be discriminated from WGA artifacts. Here, we developed a single-cell genome analysis method that reconstructs genome-wide haplotype architectures as well as the copy-number and segregational origin of those haplotypes by employing phased parental genotypes and deciphering WGA-distorted SNP B-allele fractions via a process we coin haplarithmisis. We demonstrate that the method can be applied as a generic method for preimplantation genetic diagnosis on single cells biopsied from human embryos, enabling diagnosis of disease alleles genome wide as well as numerical and structural chromosomal anomalies. Moreover, meiotic segregation errors can be distinguished from mitotic ones.

Auxotrophic Mutations Reduce Tolerance of Saccharomyces cerevisiae to Very High Levels of Ethanol Stress.

Very high ethanol tolerance is a distinctive trait of the yeast Saccharomyces cerevisiae with notable ecological and industrial importance. Although many genes have been shown to be required for moderate ethanol tolerance (i.e., 6 to 12%) in laboratory strains, little is known of the much higher ethanol tolerance (i.e., 16 to 20%) in natural and industrial strains. We have analyzed the genetic basis of very high ethanol tolerance in a Brazilian bioethanol production strain by genetic mapping with laboratory strains containing artificially inserted oligonucleotide markers. The first locus contained the ura3Δ0 mutation of the laboratory strain as the causative mutation. Analysis of other auxotrophies also revealed significant linkage for LYS2, LEU2, HIS3, and MET15. Tolerance to only very high ethanol concentrations was reduced by auxotrophies, while the effect was reversed at lower concentrations. Evaluation of other stress conditions showed that the link with auxotrophy is dependent on the type of stress and the type of auxotrophy. When the concentration of the auxotrophic nutrient is close to that limiting growth, more stress factors can inhibit growth of an auxotrophic strain. We show that very high ethanol concentrations inhibit the uptake of leucine more than that of uracil, but the 500-fold-lower uracil uptake activity may explain the strong linkage between uracil auxotrophy and ethanol sensitivity compared to leucine auxotrophy. Since very high concentrations of ethanol inhibit the uptake of auxotrophic nutrients, the active uptake of scarce nutrients may be a major limiting factor for growth under conditions of ethanol stress.

Chromosome fragility at FRAXA in human cleavage stage embryos at risk for fragile X syndrome.

Fragile X syndrome (FXS), the most common inherited intellectual disability syndrome, is caused by expansion and hypermethylation of the CGG repeat in the 5' UTR of the FMR1 gene. This expanded repeat, also known as the rare fragile site FRAXA, causes X chromosome fragility in cultured cells from patients but only when induced by perturbing pyrimidine synthesis. We performed preimplantation genetic diagnosis (PGD) on 595 blastomeres biopsied from 442 cleavage stage embryos at risk for FXS using short tandem repeat (STR) markers. In six blastomeres, from five embryos an incomplete haplotype was observed with loss of all alleles telomeric to the CGG repeat. In all five embryos, the incomplete haplotype corresponded to the haplotype carrying the CGG repeat expansion. Subsequent analysis of additional blastomeres from three embryos by array comparative genomic hybridization (aCGH) confirmed the presence of a terminal deletion with a breakpoint close to the CGG repeat in two blastomeres from one embryo. A blastomere from another embryo showed the complementary duplication. We conclude that a CGG repeat expansion at FRAXA causes X chromosome fragility in early human IVF embryos at risk for FXS.

Hereditary breast and ovarian cancer and reproduction: an observational study on the suitability of preimplantation genetic diagnosis for both asymptomatic carriers and breast cancer survivors.

Preimplantation genetic diagnosis (PGD) is a reproductive option for BRCA1/2 mutation carriers wishing to avoid transmission of the predisposition for hereditary breast and ovarian cancer (HBOC) to their offspring. Embryos obtained by in vitro fertilisation (IVF/ICSI) are tested for the presence of the mutation. Only BRCA-negative embryos are transferred into the uterus. The suitability and outcome of PGD for HBOC are evaluated in an observational cohort study on treatments carried out in two of Western-Europe's largest PGD centres from 2006 until 2012. Male carriers, asymptomatic female carriers and breast cancer survivors were eligible. If available, PGD on embryos cryopreserved before chemotherapy was possible. Generic PGD-PCR tests were developed based on haplotyping, if necessary combined with mutation detection. 70 Couples underwent PGD for BRCA1/2. 42/71 carriers (59.2 %) were female, six (14.3 %) of whom have had breast cancer prior to PGD. In total, 145 PGD cycles were performed. 720 embryos were tested, identifying 294 (40.8 %) as BRCA-negative. Of fresh IVF/PGD cycles, 23.9 % resulted in a clinical pregnancy. Three cycles involved PGD on embryos cryopreserved before chemotherapy; two of these women delivered a healthy child. Overall, 38 children were liveborn. Two BRCA1 carriers were diagnosed with breast cancer shortly after PGD treatment, despite negative screening prior to PGD. PGD for HBOC proved to be suitable, yielding good pregnancy rates for asymptomatic carriers as well as breast cancer survivors. Because of two cases of breast cancer shortly after treatment, maternal safety of IVF(PGD) in female carriers needs further evaluation.

Genetic defects in the development of the skull vault in humans and mice.

Using a genetic approach, an increasing number of genes has been shown to be important for proper skull vault development. In this review, we discuss the genes involved in disorders of the dermal skull vault in humans, including craniosynostosis and skull ossification defects, and supplement this with data from transgenic and knockout mouse models. These studies have shown the importance of signaling mediated by fibroblast growth factors (FGFs), bone morphogenic proteins (BMPs), and transforming growth factor (TGF)-beta. In addition, some insights into the disease mechanisms leading to skull vault disorders are beginning to be discovered.

Clinical and molecular analysis of nine families with Adams-Oliver syndrome.

Adams-Oliver syndrome (AOS) is defined by the combination of limb abnormalities and scalp defects, often accompanied by skull ossification defects. We studied nine families affected with AOS, eight of which have not been clinically described before. In our patients, scalp abnormalities were most often found, followed by limb and skull defects. The most common limb abnormalities appeared to be brachydactyly, syndactyly of toes 2 and 3 and hypoplastic toenails. Additional features observed were cutis marmorata telangiectatica congenita, cryptorchidism and cardiac abnormalities. In an attempt to identify the disease-causing mutations in our families, we selected two genes, ALX4 and MSX2, which were considered serious candidates based on their known function in skull and limb development. Mutation analysis of both genes, performed by direct sequencing, identified several polymorphisms, but no disease-causing mutations. Therefore, we can conclude that the AOS in our set of patients is not caused by mutations in ALX4 or MSX2.