Research On Pathogenic Genes And Molecular Mechanisms Of Ovulation Disorders

Screening and identification of pathogenic genes for premature ovarian insufficiency based on consanguineous familiesObjective: To identify the pathogenic gene and mutation of three consanguineous family with premature ovarian insufficiency(POI)or ovarian dysfunction,and provide new clues and evidence for understanding genetic causes for POI and help patients by genetic counseling.Materials and methods: Whole-exome sequencing technology was used in the POI proband,bioinformatics analysis was carried out to identify the potential genetic cause in this pedigree.Sanger sequencing analyses were performed to validate the segregation of the variant within the pedigree.In silico analysis was also used to predict the effect and pathogenicity of the variant.Results:(1)Whole-exome sequencing analysis identified novel and rare homozygous mutation associated with POI,namely mutation in FIGLA(c.2 T > C,start codon shift).The mutation in the start codon of the FIGLA gene alters the open reading frame,leading to a FIGLA knock-out like phenotype.(2)HELQ(c.781C>T;p.R261G)was found as a pathogenic mutation in a non-syndromic POI consanguineous pedigree.Bioinformatics analysis suggested mutations may affect the high-level structure of proteins and fail to normally bind to DNA,finally result in weakened DNA damage repair capabilities;(3)Whole-exome sequencing analysis identified the first homozygous POLG mutation(c.2890C>T;p.R964C)associated with ovarian dysfunction.These homozygous mutations were also harbored by the proband or affected siblings and were segregated within the pedigree.And bioinformatics analysis suggested that the mutations were rare and pathogenic.Conclusions: Biallelic mutations and recessive inheritance pattern may be associated with the occurrence of POI.This study has improved our understanding genetic mutations and the mode of inheritance of certain sequence variants in POI or ovarian dysfunction.This information will assist genetic counseling and precision medicine in the future.The study of activin A on the upregulation of PAI-1 in human granulosa lutein cellsObjective: In the mammalian ovary,the proteolysis of the extracellular matrix is dynamically regulated by plasminogen activator and plasminogen activator inhibitor(PAI),and it is a critical event that influences various physiological and pathological processes.Activin A is a member of the transforming growth factor-? superfamily and is expressed at a high level in human luteal cells.At present,it is not known whether activin A can regulate the expression and production of PAI in human granulosa lutein(h GL)cells.The aim of present study was to examine the effects of activin A on the expression and production of intraovarian PAI-1 and the underlying molecular mechanisms,as well as to understand whether this regulation exists under female physiological and pathological conditions.Materials and methods: The selection of a suitable material for functional in vitro studies is critical to comprehensively understand the cellular activities and the underlying molecular mechanisms of ovarian biology.In our study,we choose the immortalized h GL cells as cell mode.These h GL cells provide an attractive model for studying the cellular activities related to luteal function during luteal development.Collect follicular fluid from normal healthy women of reproductive age and patients with polycystic ovary syndrome,and detect the levels of activin A and PAI-1.Results:(1)Using primary and immortalized h GL cells as the cell model,we demonstrated that activin A upregulated the expression of PAI-1 and increased the production of PAI-1 in an autocrine/paracrine manner.Additionally,using a dual inhibition approach(molecular inhibitors and si RNA-mediated knockdown),we showed that this biological function is mediated by the ALK4-mediated SMAD3-SMAD4-dependent signaling pathway.Our findings suggest that activin A may be involved in the regulation of luteal function via the induction of PAI-1 expression and an increase in PAI-1 production.(2)There is no significant correlation between activin A and PAI-1 level in the follicular fluid from normal healthy women.Compared to the control,the level of activin A in the follicular fluid of patients with PCOS is significantly lower,while the level of PAI-1 is higher but not statistically different between both groups.Conclusions: Our findings demonstrated that activin A may play an important role in the regulation of ovulation and luteal function via the induction of PAI-1 production through ALK4-SMAD3/SMAD4 pathway in the human ovary.However,the upregulation of PAI-1 induced by activin A was not observed among normal healthy women or patients with PCOS,which may be caused by the inconsistence to the actual intra-ovarian environment.It is speculated that there may be spatiotemporal regulation of multiple molecular or multiple pathways in ovarian endocrinal environment in vivo,while the regulation of PAI-1 by activin A may not be the dominant role.