Beyond Repetition: The Role of Gray Zone Alleles in the Upregulation of FMR1-Binding miR-323a-3p and the Modification of BMP/SMAD-Pathway Gene Expression in Human Granulosa Cells

The Fragile X mental retardation type 1 gene (<i>FMR1</i>) contains a CGG triplet cluster of varied length (30 repeats on average) located in its 5' UTR. In its premutated state (54-200 repeats), <i>FMR1</i> contributes to the pathogenesis of premature ovarian insufficiency (POI). Its gray zone alleles (41-54 repeats) are supposed to impair the ovarian function as well. In the case of a CGG repeat length > 200, Fragile X syndrome occurs. Post-transcriptional expression of <i>FMR1</i> is regulated by microRNAs. Although miR-323a-3p overexpression suppresses <i>FMR1</i> in various tissues, this relationship has not been evaluated in the human ovary. Additionally, this microRNA targets <i>SMAD</i>s, which are suggested regulators of ovarian cell proliferation, growth, and function. This study investigated how <i>FMR1</i> allele lengths with CGG repeat numbers <i>n</i> < 55 (normal and gray zone genotypes) relate to miR-323a-3p expression and how they may impact associated <i>SMAD</i> expression in human granulosa cells. COV434 cells and patient-derived GCs were used to evaluate <i>FMR1</i>, miR-323a-3p, and BMP/SMAD-pathway member expression levels. Briefly, miR-323a-3p was significantly upregulated in GCs of the gray zone group compared to the normal allele group (<i>p</i> < 0.0001), while the <i>FMR1</i> level did not vary. Furthermore, the gray zone group showed a significant upregulation of <i>BMPR2</i>, <i>SMAD1</i>, <i>SMAD4</i>, and <i>SMAD9</i>. In contrast, the miR-323a-3p transfection of COV434 cells significantly downregulated <i>SMAD3</i>, <i>SMAD4</i>, <i>SMAD5</i>, and <i>SMAD9</i>, while the <i>FMR1</i> and <i>SMAD1</i> levels remained stable. Our findings highlight a CGG repeat number-dependent upregulation of miR-323a-3p and an alteration of the BMP/SMAD pathway, suggesting that these changes happen and contribute to impaired ovarian function independently.