Chromosome testing strategies, such as preimplantation genetic testing for aneuploidy (PGT-A), improve initial IVF outcomes by avoiding unwitting transfer of aneuploid embryos in morphology-based ...selection practices. Newer technologies have revealed that some embryos may appear to have intermediate whole chromosome (or parts of a chromosome termed segmental) copy number results suggesting trophectoderm mosaicism. An embryo with a trophectoderm mosaic-range result may be the only option for transfer for some patients. Recent data suggest that such mosaic embryos can be transferred without added risk of abnormal birth outcomes but may be associated with increased implantation failure and miscarriage rates, with higher values of mosaicism appearing to be less favourable for producing good outcomes. In this Position Statement, we provide guidance to laboratories, clinics, clinicians and counsellors to assist in discussions on the utility and transfer of mosaic embryos.
Objective
To evaluate the clinical feasibility of noninvasive prenatal diagnosis (NIPD) for β‐thalassaemia using circulating single molecule amplification and re‐sequencing technology (cSMART).
...Design
Through carrier screening, 102 pregnant Chinese couples carrying pathogenic HBB gene variants were recruited to the study. Pregnancies were managed using traditional invasive prenatal diagnosis (IPD). Retrospectively, we evaluated the archived pregnancy plasma DNA by NIPD to evaluate the performance of our cSMART assay for fetal genotyping.
Setting
Chinese prenatal diagnostic centres specialising in thalassaemia testing.
Population
Chinese carrier couples at high genetic risk for β‐thalassaemia.
Methods
Fetal cell sampling was performed by amniocentesis and HBB genotypes were determined by reverse dot blot. NIPD was performed by a newly designed HBB cSMART assay and fetal genotypes were called by measuring the allelic ratios in the maternal cell‐free DNA.
Main outcome measures
Concordance of HBB fetal genotyping between IPD and NIPD and the sensitivity and specificity of NIPD.
Results
Invasive prenatal diagnosis identified 29 affected homozygotes or compound heterozygotes, 54 heterozygotes and 19 normal homozygotes. Compared with IPD results, 99 of 102 fetuses (97%) were correctly genotyped by our NIPD assay. Two of three discordant samples were false positives and the other sample involved an incorrect call of a heterozygote carrier as a homozygote normal. Overall, the sensitivity and specificity of our NIPD assay was 100% (95% CI 88.06–100.00%) and 97.26% (95% CI 90.45–99.67%), respectively.
Conclusions
This study demonstrates that our cSMART‐based NIPD assay for β‐thalassaemia has potential clinical utility as an alternative to IPD for pregnant HBB carrier couples.
Tweetable
A new noninvasive test for pregnancies at risk for β‐thalassaemia.
Tweetable
A new noninvasive test for pregnancies at risk for β‐thalassaemia.
Objective
To assess the diagnostic performance of a novel circulating single molecule amplification and re‐sequencing technology (cSMART) method for noninvasive prenatal testing (NIPT) of ...Phenylketonuria (PKU).
Design
Blinded NIPT analysis of pregnancies at high risk for PKU.
Setting
Shanghai Xinhua Hospital and Hunan Jiahui Genetics Hospital, China.
Population
Couples (n = 33) with a child diagnosed with PKU.
Methods
Trio testing for pathogenic PAH mutations was performed by Sanger sequencing. In second pregnancies, invasive prenatal diagnosis (IPD) was used to determine fetal genotypes. NIPT was performed using a PAH gene‐specific cSMART assay. Based on the plasma DNA mutation ratio relative to the fetal DNA fraction, fetal genotypes were assigned using a maximum‐likelihood algorithm.
Main outcome measures
Concordance of fetal genotyping results between IPD and NIPT, and the sensitivity and specificity of the NIPT assay.
Results
Compared with gold standard IPD results, 32 of 33 fetuses (96.97%) were accurately genotyped by NIPT. The sensitivity and specificity of the NIPT assay was 100.00% (95% CI 59.04–100.00%) and 96.15% (95% CI 80.36–99.90%), respectively.
Conclusions
The novel cSMART assay demonstrated high accuracy for correctly calling fetal genotypes. We propose that this test has useful clinical utility for the rapid screening of high‐risk and low‐risk pregnancies with a known history of PKU on one or both sides of the family.
Tweetable
NIPT of couples at high risk for PKU using a full‐coverage cSMART PAH gene test.
BACKGROUND
Trophectoderm biopsy at the blastocyst stage is an emerging approach in preimplantation genetic diagnosis (PGD). This study aimed to compare genotyping success and implantation rates in ...PGD cycles for β-thalassaemia following biopsy at the cleavage versus the blastocyst stage, with transfer of blastocysts.
METHODS
This pilot study included 20 cycles: Group A: 10 cycles, day 3 blastomere biopsy, day 5 transfer; Group B: 10 cycles, day 5 trophectoderm biopsy, day 6 transfer. Standard-assisted reproduction and laser biopsy procedures were used. Biopsied cells were genotyped using real-time PCR multiplexed with fluorescent microsatellite analysis.
RESULTS
In Group A, 131 fertilized eggs developed to 101 embryos suitable for single blastomere biopsy; 76/101 blastomeres were diagnosed (75.2%), 30 unaffected blastocysts were transferred resulting in six pregnancies (eight fetal hearts, 26.7% implantation rate). In Group B, 128 fertilized eggs developed to 53 blastocysts for trophectoderm biopsy (four to five cells), with 50/53 blastocysts diagnosed (94.3%), 21 unaffected blastocysts transferred and 6 pregnancies initiated (10 fetal hearts, 47.6% implantation rate). Overall, nine pregnancies reached >10 weeks gestation and were confirmed unaffected by prenatal diagnosis, with 12 healthy babies born.
CONCLUSIONS
This pilot study suggests that trophectoderm biopsy and blastocyst transfer may be more advantageous than cleavage stage biopsy with respect to outcome of PGD for monogenic diseases.
Men with Y chromosome (Yq) AZFc deletions lack all copies of the DAZ gene and have severe spermatogenic failure. A recently described gr/gr subdeletion of AZFc removes two of four copies of DAZ. To ...better understand the relative frequencies of AZFc and gr/gr deletions and their associated phenotypes, we analysed two large groups of infertile men. A total of 788 men from the Monash Male Infertility (MMI) database with a range of fertility disorders showed similar overall prevalences of AZFc (2.5%) and gr/gr deletions (3.4%). There was no association of gr/gr deletions with sperm density. In 234 control men of known or presumed fertility, only one gr/gr deletion was found. In a further 599 consecutive men presenting for assisted reproductive technologies, we detected 13 (2.2%) AZFc deletions and 28 (4.7%) gr/gr deletions. All AZFc deletions were seen with sperm densities <5 million/ml but again the gr/gr deletion occurred with similar frequency across all sperm density categories. These data show that gr/gr deletions are significantly associated with infertility in the Australian population (P = 0.0015) but not exclusively with reduced sperm density suggesting a complex interaction with other factors important for male fertility. Vertical transmission of gr/gr deletions from father to son by ICSI was demonstrated in four cases. Analysis of 130 ICSI-conceived sons revealed no de novo gr/gr deletions indicating that ICSI is not a risk factor. The data suggest that testing for gr/gr deletions should be considered in the routine genetic assessment of men with idiopathic infertility.
PGD is a well accepted reproductive choice for couples at genetic risk and involves the diagnosis and transfer of unaffected IVF embryos. PGD for monogenetic diseases is most commonly accomplished by ...the biopsy of one or two blastomeres from cleavage stage embryos, followed by PCR-based protocols. However, PCR-based DNA analysis of one or two cells is subject to several problems, including total PCR failure, or failure of one allele to amplify. Trophectoderm biopsy at the blastocyst stage enables the removal of more than two cells for diagnosis while being non-invasive to the inner cell mass which is destined for fetal development. The aim of this study was to develop a safe, reliable technique for the biopsy of trophectoderm cells from human blastocysts. This case report demonstrates that removal of trophectoderm cells prior to blastocyst transfer is compatible with implantation and development to term. Here we report successful PGD for β-thalassaemia following trophectoderm cell biopsy from blastocysts and the birth of a healthy infant.
Three-dimensional imaging of human stem cells using transmission soft X-ray tomography (SXT) is presented for the first time. Major organelle types—nuclei, nucleoli, mitochondria, lysosomes and ...vesicles—were discriminated at approximately 50 nm spatial resolution without the use of contrast agents, on the basis of measured linear X-ray absorption coefficients and comparison of the size and shape of structures to transmission electron microscopy (TEM) images. In addition, SXT was used to visualize the distribution of a cell surface protein using gold-labelled antibody staining. We present the strengths of SXT, which include excellent spatial resolution (intermediate between that of TEM and light microscopy), the lack of the requirement for fixative or contrast agent that might perturb cellular morphology or produce imaging artefacts, and the ability to produce three-dimensional images of cells without microtome sectioning. Possible applications to studying the differentiation of human stem cells are discussed.
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