Pioglitazone (PIO) enhances blastocyst vitrification outcomes via coordinated AQP3-mediated dehydration and antioxidant defense

Abstract

Blastocyst cryopreservation is a challenging process due to ice crystallization induced by blastocoel fluid. While aquaporin-3 (AQP3) overexpression improves cryotolerance, there is a lack of pharmacological regulators of AQP3 that can be easily applied in clinical settings. Notably the antidiabetic drug pioglitazone (PIO), despite its association with edema in patients, has been shown to potentially induce cellular dehydration through the modulation of AQP3. Here, we have demonstrated that PIO improves vitrification outcomes by orchestrating AQP3-mediated fluid expulsion and bolstering antioxidant defense mechanisms. Treatment of mouse blastocysts with 2 μM PIO in a hyperosmotic solution resulted in a notable contraction of blastocysts (reduced by 25.95 %, P < 0.01), correlating with 2.6-fold AQP3 up-regulation, compared to the control. This rapid dehydration yielded 95 ± 3.60 % post-thaw survival (vs. 82.5 ± 3.51 % Control, P < 0.001), while also reducing levels of Reactive oxygen species (ROS, P < 0.05) and Dihydroethidium (DHE) (P < 0.05) and increasing levels of glutathione (GSH, P < 0.05) and mitochondrial membrane potential (ΔΨm, P < 0.05) significantly. Mechanistically, PIO reduced DNA damage (γ-H2AX, P < 0.01) while increasing P53 expression (P < 0.01). Crucially, these effects were conserved in bovine Blastocysts, at the concentration of 4 μM, achieving 88.65 ± 8.31 % survival with PIO compared to 82.23 ± 7.48 % in the control group, highlighting the cross-species applicability of the findings Our findings position PIO as a dual-functional cryoadjuvant by coordinating rapid water efflux through AQP3 with antioxidant defense mechanisms. This repurposing approach, leveraging the anti-diabetic agent PIO as a cryo-adjuvant, offers an immediately implementable optimization pathway for the standardized enhancement of animal embryos vitrification protocols.