Clinical and molecular characteristics of hemophilia A affected individuals and carriers: A 24 years experience from three centers.

Hemophilia A is a rare bleeding disorder with variable expressivity and allelic heterogeneity. Despite the advancement of prenatal diagnostics and molecular studies, the number of studies reviewing the reproductive choices of hemophilia A carriers and affected individuals remains limited. Through this retrospective review, we hope to gain a deeper understanding of hemophilia A-affected individuals' clinical and molecular characteristics, as well as the reproductive choices of the at-risk couples. A total of 122 individuals harboring likely causative F8 gene alterations from 64 apparently unrelated families attending three centers between 3/2000 and 3/2023 were included in this study. Their clinical and molecular findings as well as reproductive choices were gathered in a clinical setting and verified through the electronic medical record database of the public health system. Forty-seven affected males and 75 female heterozygous carriers were included in the analysis. Among 64 apparently unrelated families, 36 distinct pathogenic/likely pathogenic variants were identified, of which 30.6% (11/36) of variants were novel. While the majority of clinical findings and genotype-phenotype correlations appear to be in accordance with existing literature, female carriers who had no fertility intention were significantly more likely to have affected sons than those who had fertility intention (5/19 vs. 4/5; p = 0.047). Through this retrospective review, we summarized the clinical and molecular characteristics of 122 individuals harboring pathogenic/likely pathogenic F8 variants, as well as their fertility intentions and reproductive outcomes. Further studies are required to look into the considerations involved in reproductive decision-making.

Optimizing non-invasive preimplantation genetic testing: investigating culture conditions, sample collection, and IVF treatment for improved non-invasive PGT-A results.

PURPOSE: This study aimed to optimize the non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) in the laboratory by comparing two collection timing of the spent culture medium (SCM), two embryo rinsing protocols, and the use of conventional insemination instead of intracytoplasmic sperm injection (ICSI). METHODS: Results of two embryo rinsing methods (one-step vs sequential) and SCM collected on day 5 vs day 6 after retrieval were compared against trophectoderm (TE) biopsies as reference. Results from day 6 SCM in cycles fertilized by conventional insemination were compared with PGT-A using ICSI. RESULTS: The rate of concordance was higher in day 6 samples than in day 5 samples when the sequential method was used, in terms of total concordance (TC; day 6 vs day 5: 85.0% vs 60.0%, p = 0.0228), total concordance with same sex (TCS, 82.5% vs 28,0%, p < 0.0001), and full concordance with same sex (FCS, 62.5% vs 24.0%, p = 0.0025). The sequential method significantly out-performed the one-step method when SCM were collected on day 6 (sequential vs one-step, TC: 85.0% vs 64.5%, p = 0.0449; TCS: 82.5% vs 54.8%, p = 0.0113; FCS: 62.5% vs 25.8%, p = 0.0021). There was no significant difference in niPGT-A results between cycles fertilized by the conventional insemination and ICSI. CONCLUSION: We have shown a higher concordance rate when SCM was collected on day 6 and the embryos were rinsed in a sequential manner. Comparable results of niPGT-A when oocytes were fertilized by conventional insemination or ICSI. These optimization steps are important prior to commencement of a randomized trial in niPGT-A.

Knowledge, attitudes and intention on fertility preservation among breast cancer patients.

Breast cancer is the most common cancer in reproductive age women. The aim of this study is to assess the knowledge, attitude and intention on fertility preservation among women diagnosed to have breast cancer. This is a multi-centre cross-sectional questionnaire study. Reproductive age women diagnosed with breast cancer attending Oncology, Breast Surgery and Gynaecology Clinics and support groups were invited to participate. Women filled in paper or electronic form of the questionnaire. 461 women were recruited and 421 women returned the questionnaire. Overall, 181/410 (44.1%) women had heard of fertility preservation. Younger age and higher education level were significantly associated with increased awareness of fertility preservation. Awareness and acceptance of the different fertility preservation methods in reproductive age women with breast cancer was suboptimal. However, 46.1% women felt that their fertility concerns affected their decision for cancer treatment in some way.

Distinguishing between carrier and noncarrier embryos with the use of long-read sequencing in preimplantation genetic testing for reciprocal translocations.

Balanced reciprocal translocation carriers are usually phenotypically normal but are at an increased risk of infertility, recurrent miscarriage or having affected children. Preimplantation genetic testing on chromosomal structural rearrangement (PGT-SR) offers a way to screen against unbalanced embryos. Here, we demonstrated a new method to distinguish carrier from noncarrier embryos. Translocation breakpoints were first delineated by nanopore sequencing followed by polymerase chain reaction (PCR) across breakpoints. High-resolution breakpoint mapping was successful in all (9/9) balanced reciprocal translocation carriers. Retrospective analysis of their embryo biopsies with breakpoint PCR showed 100% concordant results with PGT-SR on trophectoderm biopsies (40/40) and 53% concordance on blastomere biopsies (8/15). The low concordant rate in blastomeres was due to failure in the amplification of derivative chromosomes involving large deletions. Breakpoint PCR also showed 100% concordant results with prenatal/postnatal outcomes on 5 pregnancies, indicating that our new method can accurately distinguish carrier from noncarrier embryos.

High-resolution mapping of reciprocal translocation breakpoints using long-read sequencing.

Long-read nanopore sequencing enables direct high-resolution breakpoint mapping on balanced carriers of reciprocal translocation. The mean sequencing depth on the translocated chromosomes to achieve accurate mapping of breakpoints ranged from 2.5-fold to 6.2-fold. To speed up determination of the breakpoints from long-read sequencing data, alignment reads on the translocated chromosomes were extracted before piped into NanoSV. Checking the position of breakpoints on Interactive Genomics Viewer (IGV) was crucial to successful design of breakpoint PCR primers, especially when large deletion was involved at the breakpoints. •Long-read sequencing enables accurate breakpoint mapping with base-pair resolution•Splitting bam files by translocated chromosomes drastically speeded up the breakpoint determination•IGV helps to identify the breakpoint positions and facilitate the design of breakpoint PCR primers.