Pathogenic variants in subcortical maternal complex (SCMC) have been identified not only in mothers of Beckwith-Wiedemann syndrome (BWS) babies but also in women with reproductive disturbances such as failed pregnancy attempts and recurrent pregnancy loss. Based on the higher incidence of BWS in children born from Assisted Reproductive Technology (ART), this project aims to investigate incidence and molecular mechanism of pathogenic variants of SCMC in women with reproductive disorders. Study objectives will be (i) assess the incidence of these variants as a cause of differences in reproductive outcomes in the infertile female population and mothers of children with BWS; (ii) identify methylation changes in women with reproductive problems including those with offspring affected by BWS; (iii) determine the molecular causes underlying female infertility and imprinting disorder associated with damaging SCMC gene variants by employing a mouse model.
Cohort 1: healthy women with offspring affected by BWS and peculiar reproductive history from our population of clinically and molecularly diagnosed BWS families;
Cohort 2: women under 35 undergoing ART for infertility (defined as failure to achieve a pregnancy after 12 months or more of regular unprotected sexual intercourses) and unable to obtain a live birth after three completed cycles or after the transfer of at least 6 blastocysts;
Cohort 3: women under 35, with RPL (defined as the loss of two or more pregnancies before 24 weeks of gestation).
presence of conventional and molecular karyotype alterations
occurrence of known causes that can lead to decreased fertility or recurrent abortions: disorders of the ovaries, such as polycystic ovarian syndrome and other follicular disorders, disorders of the endocrine system causing imbalances of reproductive hormones levels, autoimmune conditions, male infertility, uterine or tubal dysfunctions and malformations, thrombophilic or noncorrected thyroid dysfunctions.
Parallel
WES analysis
GeneticWhole-exome sequencing will be performed as the first approach in all the recruited patients. First, we will analyze different subsets of genes, belonging to: 1. Maternal effect genes as SCMC components and other related genes; 2. Genes essential in the maturation of the oocyte and zygote progression through the early phases of the embryogenesis or highly expressed at different stages of oocyte maturation; 3. Genes with known and potential roles in the establishment and control of genomic imprinting and involved in DNA methylation reactions. Subsequently, variants with a high pathogenicity score will be analyzed, to identify any genes that may be associated with the phenomenon, but do not belong to the previously described categories of genes. Finally, we will conduct a whole genome sequencing (WGS) analysis on a selected subgroup of BWS mothers with peculiar clinical histories and negative WES analysis, to explore all the noncoding and regulatory regions not targeted by WES.
whole-genome methylation analysis
GeneticA whole-genome methylation analysis will be performed to identify methylation changes in women with reproductive problems including those with offspring affected by BWS
MEG Incidence
Incidence of MEGs, particularly pathogenetic variants of SCMC, in infertile female population and mothers of children affected by BWS
Compare DNA methylation
Whole-genome DNA methylation of blood leukocytes of the cohorts of women with reproductive problems including those with offspring affected by BWS and comparing it with that of a similar number of sex- and age-matched controls. Bioinformatics analysis will be used to infer differentially methylated regions from methylation data and to classify women status based on methylation profiles. Due to the complex distribution associated with whole-genome methylation profiles, dimensionality reduction techniques (e.g. PCA,MDS) and clustering methods (both hierarchical and centroidbased) will be used to assess the importance of specific regions (e.g. imprinted loci) to discriminate prioritized women from controls according to their methylation profiles. with reproductive problems including those with offspring affected by BWS
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Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico
Lead sponsor
Istituto Auxologico Italiano
Collaborator
University of Campania Luigi Vanvitelli
Collaborator
Federico II University
Collaborator
