# Targeted hypoxia-inducible factor 1-alpha (HIF1A) stabilization during in vitro maturation of bovine cumulus-oocyte complexes increases blastocyst rates

## TL;DR

Stabilizing HIF1A during in vitro maturation of bovine oocytes improves blastocyst formation rates, suggesting a potential method to enhance embryo development.

## Contribution

The study demonstrates that low-dose Roxadustat treatment during in vitro maturation improves bovine blastocyst formation through HIF1A stabilization.

## Key findings

- Low-dose Roxadustat (25 µM) significantly enhances blastocyst formation (p < 0.01).
- HIF1A mRNA decreases in treated groups, but protein levels remain stable, indicating a feedback mechanism.
- PHD inhibition benefits are specific to the maturation phase, not fertilization or cleavage.

## Abstract

In vitro production (IVP) of bovine embryos frequently results in varying blastocyst yields, partly because culture conditions fail to support optimal oocyte and embryo development. In vivo, cumulus-oocyte complexes (COCs) mature under hypoxic conditions, and while in vitro protocols try to mimic the natural conditions, standard in vitro maturation (IVM) is typically performed under normoxia, guided largely by empirical outcomes rather than a biological understanding. We hypothesized that IVP efficiency would be improved by stabilization of hypoxia-inducible factor 1-alpha (HIF1A), via pharmacological inhibition of prolyl hydroxylase domain (PHD) activity, during IVM. To test this, we treated COCs with varying concentrations of Roxadustat, a PHD inhibitor. Low-dose treatment (25 µM) stabilized and significantly enhanced blastocyst formation (p < 0.01), while higher doses (100 µM) impaired maturation rates (p < 0.05). Interestingly, HIF1A mRNA expression decreased in treated groups, whereas protein levels remained stable, suggesting a feedback mechanism that may prevent excessive and potentially detrimental HIF1A activity. Additionally, we assessed the expression of markers related to cumulus expansion (HAS2, TNFAIP6), meiotic resumption (TMSB4), and cell proliferation (PCNA). However, their expression did not correlate with the increased blastocyst formation after Roxadustat treatment. Furthermore, PHD inhibition during in vitro culture (IVC) had no effect on cleavage rates (p > 0.05), but higher doses led to reduced blastocyst development (p < 0.01). These findings suggest that the benefits of HIF1A stabilization are specific to the maturation phase, likely by supporting cumulus-oocyte interactions critical for establishing developmental competence, rather than by directly influencing fertilization or early cleavage. These findings highlight the potential of fine-tuned HIF1A modulation to improve IVP efficiency. Further studies should elucidate the downstream molecular mechanisms and assess post-transfer development outcomes.

The online version contains supplementary material available at 10.1038/s41598-025-33894-8.