P-090 Genotypic sperm sorting: A less invasive Assisted Reproductive Technology (ART) to prevent genetic disorders in newborns

Abstract

Abstract Study question Is the prevention of genetic disorders in newborns possible by genotypically sorting sperm cells with specially characterized proteins on the sperm surface membrane? Summary answer For Cystic Fibrosis and X-linked Disorders prevention model, we utilized certain monoclonal antibodies to sort sperm cells by targeting proteins expressed on their surface membrane. What is known already Our study focused on Cystic Fibrosis (CF) and X-linked Diseases (XLD) which are genetic disorders inherited by offspring from parents who are healthy carriers of the autosomal recessive or allosomal genes. More than 10million Caucasians are healthy carriers of a mutant cystic-fibrosis gene (predominantly F508del) while 4% of all newborns are at risk of being born with an X-linked disease. The current clinically approved mitigation plan for preventing such genetic disorders in newborns – is to consider Preimplantation Genetic Testing for Monogenetic diseases (PGT-M). PGT-M involves an invasive microsurgical procedure that requires the removal of cells from 3-5day old embryos. Study design, size, duration For our study design, semen samples purchased from FairFax Cryobank from an anonymous CF-carrier and a non-carrier were utilized. The samples were assessed for the presence of H-Y male antigen and the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein, which are both selectively expressed on the sperm surface – to delineate genotypic sperm sort models for XLD and CF respectively. Participants/materials, setting, methods Methods utilized in the genotypic sperm sorting protocols included the use of phycoerythrin (PE) binding Tetrameric Antibody Complexes (TAC) coupled with magnetic beads. Immuno-magnetic sorting was conducted with a conventional sort method in tubes and 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). Main results and the role of chance Our study was able to elucidate a potential less invasive clinical “ART” application or method of genotypically sorting sperm cells with H-Y/CFTR monoclonal-antibody-functionalized microfluidic chips or conventional immuno-magnetic sorting protocols in sort tubes. Preliminary assessments showed that flow cytometry may be utilized to confirm sort purities of positively selected sperm populations (with HY or CFTR markers) from immuno-magnetic sort tubes – based on their morphological and cellular characteristics. For a genotypic sperm sort in the pathogenic CF-carrier sample, positively and negatively selected CF-sperm fractions (being CFTR-Wt and F508del-Mut sperm) from immuno-magnetic sort tubes could not be adequately distinguished by qPCR-Cq – because of their intrinsic and slightly insignificant genetic variabilities. However, the microfluidic sperm sorting chip fabricated for this study enabled a differential and positive immuno-magnetic selection of CFTR-Wt sperm with a comparably higher but statistically insignificant sort yield, relative to the population of the F508del-Mut sperm negatively selected – when assessed by qPCR-Cq (p value = 0.092; significant at < 0.05). Immunofluorescence and FiSH evaluations of genotypically sorted sperm cells in both XLD and CF models also showed pictorial comparisons with the observed genetic assessment outcomes. Limitations, reasons for caution One of the limitations of this study is the immuno-magnetic sort efficiency. The sorting efficiency may however be improved if freshly collected normospermic semen samples were utilized rather than a frozen-thawed sample which may have been subjected to the stress of cryopreservation and potential cellular damage with reduced motility. Wider implications of the findings This novel less invasive method of Assisted Reproductive Technology may also usher in a new “ART” called Intra-cytoplasmic Genotypically-selected Sperm Injection (IGSI). The IGSI method would potentially enable the selection of non-diseased sperm cells – for the sole purpose of helping to prevent genetic disorders in newborns. Trial registration number Not Applicable