OBJECTIVE: To evaluate whether a second biopsy, following a first diagnostic failure on blastocysts tested for preimplantation genetic testing for monogenic diseases (PGT-M), allows to obtain genetic diagnosis and to what extent this procedure can influence clinical pregnancy and live birth rates compared to the PGT-M process with a successful genetic diagnosis from the first biopsy.
METHODS: Embryos from women who underwent PGT-M in an infertility centre and who had been transferred after two biopsies for genetic analysis (n = 27) were matched in a 1:1 ratio accordingly to women\'s age (± 1 year) and fertility status (fertile vs infertile), as well as with the study period, with embryos who were transferred after receiving a conclusive PGT result straight after the first biopsy (n = 27). The main evaluated outcome was clinical pregnancy rate following embryo transfers in which healthy embryos were transferred after only one biopsy and those in which an embryo was transferred after being re-biopsied. Live birth rate was the secondary outcome.
RESULTS: Clinical pregnancy rate was 52% (95% CI: 34-69) following the transfer of a single-biopsy blastocyst and 30% (95% CI: 16-48) following the transfer of a re-biopsied blastocyst. The likelihood to have a healthy baby was 33% (95% CI: 19-52) following the transfer of a blastocyst biopsied once and 22% (95% CI: 11-41) following the transfer of a re-biopsied blastocyst.
CONCLUSIONS: The re-biopsy intervention seems to considerably reduce the pregnancy potential of a blastocyst. However, a greater sample size is necessary to clarify this issue definitively.

OBJECTIVE: This study aimed to evaluate the value of long-read sequencing for preimplantation haplotype linkage analysis.
METHODS: The genetic material of the three β-thalassemia mutation carrier couples was sequenced using single-molecule real-time sequencing in the 7.7-kb region of the HBB gene and a 7.4-kb region that partially overlapped with it to detect the presence of 17 common HBB gene mutations in the Chinese population and the haplotypes formed by the continuous array of single-nucleotide polymorphisms linked to these mutations. By using the same method to analyze multiple displacement amplification products of embryos from three families and comparing the results with those of the parents, it could be revealed whether the embryos carry disease-causing mutations without the need for a proband.
RESULTS: The HBB gene mutations of the three couples were accurately detected, and the haplotype linked to the pathogenic site was successfully obtained without the need for a proband. A total of 68.75% (22/32) of embryos from the three families successfully underwent haplotype linkage analysis, and the results were consistent with the results of NGS-based mutation site detection.
CONCLUSIONS: This study supports long-read sequencing as a potential tool for preimplantation haplotype linkage analysis.

Background: Congenital disorder of glycosylation (CDG) is a severe morphogenic and metabolic disorder that affects all of the systems of organs and is caused by a mutation of the gene PMM2, having a mortality rate of 20% during the first months of life. Results: Here we report the outcome of an in vitro fertilisation (IVF) cycle associated with preimplantation genetic testing for monogenic diseases (PGT-M) in a Romanian carrier couple for CDG type Ia with distinct mutations of the PMM2 gene. The embryonic biopsy was performed on day five of the blastocyst stage for six embryos. The amplification of the whole genome had been realized by using the PicoPLEX WGA kit. Using the Array Comparative Genomic Hybridisation technique, we detected both euploid and aneuploid embryos. The identification of the PMM2 mutation on exon 5 and exon 6 was performed for the euploid embryos through Sanger Sequencing with specific primers on ABI 3500. Of the six embryos tested, only three were euploid. One had compound heterozygosity and the remaining two were simple heterozygotes. Conclusion: PGT-M should be strongly considered for optimising embryo selection in partners with single-gene mutations in order to prevent transmission to the offspring.

β-thalassemia is a common single-gene disorder in India, with hematopoietic stem cell transplantation (HSCT) being the only cure. HSCT with matched unrelated donor is less successful, whereas finding a human leukocyte antigen (HLA)-matched related donor is difficult. Preimplantation genetic testing for monogenic diseases (PGT-M) with HLA matching is a novel option to have a matched sibling for HSCT for couples having an affected child. We present the first such case report in India. A couple, both carriers of β-thalassemia and having an affected son, underwent PGT-M with HLA matching combined with preimplantation genetic testing for aneuploidies of embryos to have a β - thalassemia-free child. This resulted in birth of a 10/10 HLA-matched sibling.