In briefSeverely deficient levels of vitamin D (VD) affect ovarian cellular function reducing production of androstenedione and oestradiol and insulin receptor expression; although mildly deficient levels have no effect.AbstractNumerous studies have investigated the link between VD deficiency and reproductive outcomes with contradictory results. VD regulates steroidogenic enzymes crucial for human granulosa and cumulus cell function. This study investigated whether deficient levels of 1,25-(OH)2-D3 altered ovarian cell function, and if the ovary could obtain bioactive 1,25-(OH)2-D3 via local enzymatic expression of CYP27B1 to counteract systemic deficiency. A variety of cells and tissues were used for the in vitro experiments. We have shown for the first time an increase in VDR expression in theca of large compared to small follicles, which along with the ability of 1,25-(OH)2-D3 to decrease anti-Müllerian hormone expression, supports a role for 1,25-(OH)2-D3 in theca and granulosa cell function. Conversely, very low levels of 1,25-(OH)2-D3 equivalent to hypovitaminosis inhibited thecal production of androstenedione and cAMP-driven oestradiol production. Human thecal and un-luteinised GC are incredibly hard to obtain for research purposes, highlighting the uniqueness of our dataset. We also demonstrated that deficient levels of 1,25-(OH)2-D3 downregulated insulin receptor expression, potentially reducing insulin sensitivity. We have shown that the ovary expresses CYP27B1, potentially allowing it to make local bioactive 1,25-(OH)2-D3, which along with the upregulation in VDR expression in ovarian cellular compartments could be protective locally in counteracting systemic VD deficiency. To conclude, a severely deficient VD environment (<2 nM or <1 ng/mL) could contribute to impaired ovarian cell function, and hence, potentially affect folliculogenesis/ovulation, but levels associated with mild deficiency may have less impact, apart from in the presence of hyperinsulinaemia and insulin resistance.