Preimplantation genetic diagnosis of hemophilia A.

Preimplantation genetic diagnosis (PGD) is a powerful tool to tackle the transmission of monogenic inherited disorders in families carrying the diseases from generation to generation. It currently remains a challenging task, despite PGD having been developed over 25 years ago. The major difficulty is it does not have an easy and general formula for all mutations. Different gene locus needs individualized, customized design to make the diagnosis accurate enough to be applied on PGD, in which the quantity of DNA is scanty, whereas timely laboratory diagnosis is mandatory if fresh embryo transfer is desired occasionally. Indicators for outcome assessment of a successful PGD program include the successful diagnosis rate on blastomeres (Day 3 cleavage-stage embryo biopsy) or trophectoderm cells (Day 5/6 blastocyst biopsy), the implantation rate per embryo transferred, and the livebirth rate per oocyte retrieval cycle. Hemophilia A (HA) is an X-linked recessive bleeding disorder caused by various types of pathological defects in the factor VIII gene (F8). The mutation spectrum of the F8 is complex, according to our previous report, including large segmental intra-gene inversions, large segmental deletions spanning a few exons, point mutations, and total deletion caused by chromosomal structural rearrangements. In this review, the molecular methodologies used to tackle different mutants of the F8 in the PGD of HA are to be explained, and the experiences of successful use of amplification refractory mutation system-quantitative polymerase chain reaction (ARMS-qPCR) and linkage analysis for PGD of HA in our laboratory are also provided.

A pilot proof-of-principle study to compare fresh and vitrified cycle preimplantation genetic screening by chromosome microarray and next generation sequencing.

BACKGROUND: Single embryo transfer (SET) has been utilized as a strategy to reduce the chance of multifetal gestations in in vitro fertilization (IVF) but lower pregnancy rate remains a concern. Recent studies showed that favorable outcome regarding SET can be achieved by selecting embryos with "more normal" genetic components. We explored the use of rapid array comparative genomic hybridization (aCGH) to select blastocysts for fresh SET and compared with the protocols adopting vitrified (ultrarapidly frozen) embryo transfer cycle. Validation of the rapid protocol of aCGH and comparison of the result with the regular protocol of aCGH and next generation sequencing (NGS) are also performed. RESULTS: First-time IVF patients with normal karyotype (n = 21) were enrolled for elective fresh SET cycle (n = 8; designated as fresh SET group) or vitrified embryo transfer cycle (n = 13; designated as vitrified ET group) coupling with comprehensive chromosomal screening by a 9-h rapid aCGH from Day 5 trophectoderm (TE) biopsy. In fresh SET group, 86 blastocysts (10.8 blastocysts/patient) were biopsied and analyzed. Aneuploidy was detected in 53.5 % (46/86) of the biopsied blastocysts. All patients had a single embryo transferred on the following day. The clinical pregnancy rate was 87.5 % (7/8) and the ongoing pregnancy rate was 62.5 % (5/8). In vitrified ET group, 58 blastocysts (4.5 blastocysts/patient) were biopsied and 56 blastocysts were analyzed. Aneuploidy was detected in 39.3 % (22/56) of biopsies. The patients accepted for SET or double embryos transfer (DET) in non-stimulated cycles. The clinical pregnancy rate and the ongoing pregnancy rate was 76.9 % (10/13) and 53.8 % (7/13) respectively. Spontaneous abortions occurred in both of the two patient groups. In the series of fresh SET group, no twin pregnancy was noted and at least one healthy baby had been born at gestational age (GA) 37(+6) weeks when submission. The results of PGS by rapid aCGH, regular aCGH and NGS were comparable in most occasions. CONCLUSION: This study evaluates the use of rapid aCGH to select blastocysts for fresh SET and demonstrates its feasibility in a real clinical IVF program. A successful livebirth is achieved and the favorable outcome is superior to the protocol adopting vitrified ET cycle in our own setting. Additional studies are needed to verify this pilot data and validate its application in large randomized trials.

Preimplantation genetic screening of blastocysts by multiplex qPCR followed by fresh embryo transfer: validation and verification.

BACKGROUND: Aneuploidy is an important etiology of implantation failure and quantitative real-time polymerase chain reaction (qPCR) seems a promising preimplantation genetic screening (PGS) technology to detect aneuploidies. This verification study aimed at verifying the impact on reproductive outcomes in in vitro fertilization (IVF) cycles using fresh embryo transfer (FET) in which the embryos were selected by blastocyst biopsy with qPCR-based PGS in our settings. RESULTS: A total of 13 infertile couples with more than once failed in vitro fertilization were enrolled during July to October of 2014. PGS was conducted by qPCR with selectively amplified markers to detect common aneuploidies (chromosomes 13, 18, 21, X, and Y). The design of the qPCR molecular markers adopted the locked nucleic acid (LNA) strategy. The blastocyst biopsy was performed on Day 5/6 and the PGS was done on the same day, which enabled FET. A total of 72 blastocysts were biopsied. Successful diagnoses were established in all embryos and the rate of successful diagnosis was 100 %. The aneuploidy rate was 38.9 % (28/72). 28 embryos were transferred. The clinical pregnancy rate was 61.5 % (8/13) per cycle. Early first trimester abortion was encountered in 1 and the ongoing pregnancy rate was 53.8 % (7/13) per cycle. CONCLUSION: This study verified the favorable outcome of adopting PGS with qPCR + FET in our own setting. Expanding the repertoire of aneuploidies being investigated (from a limited set to all 24 chromosomes) is underway and a randomized study by comparing qPCR and other PGS technologies is warranted.

Validating a rapid, real-time, PCR-based direct mutation detection assay for preimplantation genetic diagnosis.

Although co-amplification of polymorphic microsatellite markers is the current gold standard for preimplantation genetic diagnosis (PGD) of single-gene disorders (SGD), this approach can be hampered by the lack of availability of informative markers. We recently (2011) devised a novel in-house assay for PGD of aromatic L-amino acid decarboxylase deficiency, based on an amplification refractory mutation system and quantitative PCR (ARMS-qPCR). The objective of the present study was to verify ARMS-qPCR in a cohort of 20 PGD cycles with a diverse group of SGDs (15 couples at risk for 10 SGDs). Day-3 cleavage-stage embryos were subjected to biopsy and genotyping, followed by fresh embryo transfer (FET). The diagnostic rate was 82.9%; unaffected live births were achieved in 9 of 20 FET cycles (45%), with only one false negative (among 54 transferred embryos). Overall, the ARMS-qPCR had frequent allele-dropout (ADO), rendering it inappropriate as the sole diagnostic method (despite a favorable live-birth rate). Regardless, it has the potential to complement the current gold-standard methodology, especially when trophectoderm biopsy becomes a preferred option and genotyping needs to be timely enough to enable FET.