P-107 MICROFLUIDIC SPERM SEXING: An XLD (X-linked Disorder) model for preventing genetic disorders in newborns

Abstract

Abstract Study question Can sperm sexing based on unique surface membrane markers – enable genotypic sperm sorting by allosomal content to prevent certain X-linked disorders in newborns? Summary answer In an X-Linked-Disorder prevention model, we utilized a microfluidic device functionalized with H-Y monoclonal antibodies attached to magnetic beads to enrich Y-chromosome bearing sperm sorts. What is known already About 4% of newborns are at risk of being born with an X-linked disease such as Fragile X Syndrome. The current clinically approved mitigation plan for preventing such genetic disorders is to consider Preimplantation Genetic Testing for Monogenetic diseases (PGT-M) which involves an invasive microsurgical procedure on fragile embryos. To minimize this invasiveness, we proposed a less invasive microsurgical exclusion approach to prevent such genetic disorders by genotypically sorting sperm cells that can be used for IUI/IVF/ICSI – with microfluidics – using their allosomal characteristics. Study design, size, duration Semen samples purchased from FairFax Cryobank from anonymous donors were used in immuno-magnetic microfluidic sorting protocols with the use of phycoerythrin (PE) binding Tetrameric Antibody Complexes (TAC) coupled with magnetic beads to tag H-Y monoclonal antibody (mab) binding activities on the sperm surface membrane. Participants/materials, setting, methods Immuno-magnetic sorting was conducted with specially fabricated microfluidic-sperm-sorting (MSS) chips. Sort yield and purities of positively selected sperm populations were assessed by flow-cytometry, immunofluorescence, fluorescent-in-situ-hybridization (FISH) and by quantitative-PCR (qPCR – Cq & Tm). Quantitative experiments were run in triplicates and the results were expressed as absolute means for comparison between paired variables. Statistical analyses were performed using Student’s t-test and a p-value of less than 0.05 was considered to be statistically significant. Main results and the role of chance For the assessment of H-Y sperm sorts, our preliminary data showed that flow cytometry was able to confirm the positive sperm selection (which were mostly Y-chromosome bearing sperm cells) in a sperm genotyping protocol – initially utilizing immuno-magnetic sort tubes prior to flowing through microfluidic chips. Both immuno-magnetic sort tubes and the microfluidic chips were able to optimally confirm from qPCR-Cq means – the negative selection – being the X-chromosome bearing sperm that may have been largely unselected by the H-Y-mab. Values for Cq means compared under H-Y-qPCR data sets were statistically significant with a p value of 0.000023 (p values is significant at < 0.05). Our study was able to elucidate a potential less invasive clinical application of genotypically sorting sperm cells with mab-functionalized microfluidic chips for an XLD-model. Molecular down stream applications like flow cytometry may therefore be utilized to experimentally confirm sort purities of positively selected sperm populations (with H-Y markers) – based on the utility of the SMCX and SMCY gene in the X and Y sperm chromosome bearing sperm cells respectively. Limitations, reasons for caution Physiological effect of magnetic fields of varying tesla or gauss strengths on sperm selected with immuno-magnetic beads may warrant further safety studies prior to actual clinical applications. Optimal selection results may be achieved with the use of freshly collected normospermic rather than frozen-thawed samples. Wider implications of the findings Although we had de-emphasized the clinical application of the microfluidic sperm sexing protocol for legitimate use in sex selection and gender balancing, this study design was solely focused on adapting a less invasive method of preventing X-linked genetic disorders with the potential exclusion of microsurgical procedures leading to PGT-M. Trial registration number NA