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Effects of repeated controlled ovarian stimulation on the expression of aerobic glycolysis-related enzymes at various stages of early embryo development in mice

SHEN Lu-Lu^1,2, SHI Bei-Bei^1,2, XIONG Wen-Yan^1,2, SHI Rui-Gan^1,2, SONG Ya-Jing^1,2, DU Hui-Lan^1,2,3,*

¹Institute of Integrative Medicine, Hebei University of Chinese Medicine, Shijiazhuang 050091, China；²Hebei Key Laboratory of Integrative Medicine on Liver-Kidney Patterns, Shijiazhuang 050091, China；³Hebei Collaborative Innovation Center of Integrated Traditional and Western Medicine on Reproductive Disease, Shijiazhuang 050091, China

Abstract

This study aims to investigate whether the reduction in early embryonic development due to repeated controlled ovarian stimulation (COS) is associated with the expression levels of enzymes related to aerobic glycolysis. A repeated COS mouse model was established by injecting pregnant mare serum gonadotropin (PMSG) followed by human chorionic gonadotrophin (HCG) 48 h later in ICR mice. Repeated COS was performed by repeating this injection protocol. After pregnancy in these mice, 2- to 8-cell embryos and blastocysts were collected. The mRNA and protein expression levels of aerobic glycolysis-related enzymes, including hexokinase 2 (HK2), phosphofructokinase (PFK), pyruvate kinase M2 (PKM2), lactate dehydrogenase A (LDHA), as well as glucose transporter type 1 (GLUT1) and monocarboxylate transporter 4 (MCT4), were detected at each stage using qRT-PCR and immunofluorescence methods, along with the high-quality embryo rate. The results showed that, compared with the control group, there was no statistically significant difference in the high-quality embryo rate at each stage after a single stimulation (P > 0.05). As the number of stimulation cycles increased, embryo morphology deteriorated, and the high-quality embryo rate decreased. At the 2- to 8-cell stage, compared with the control group, a single stimulation had no significant effect on the mRNA and protein expression levels of HK2, PFK, PKM2, LDHA, GLUT1, and MCT4. However, two stimulations reduced the mRNA expression levels of PFK, PKM2, and GLUT1 at the 2-cell stage, the protein expression level of PFK, and also decreased the mRNA expression levels of LDHA and MCT4 and the protein expression levels of PKM2 and MCT4 at the 4-cell stage, as well as the mRNA and protein expression levels of PFK, LDHA, and MCT4 at the 8-cell stage. Three and four stimulations resulted in decreased mRNA and protein expression levels of HK2, PFK, PKM2, LDHA, GLUT1, and MCT4 at the 2-, 4-, and 8-cell stages (P < 0.05). At the blastocyst stage, a single stimulation had no significant effect on the mRNA and protein expression levels of HK2, PFK, PKM2, LDHA, GLUT1, and MCT4. Two stimulations reduced the protein expression level of LDHA, while three and four stimulations led to decreased mRNA and protein expression levels of HK2, PFK, PKM2, LDHA, GLUT1, and MCT4. These findings indicate that repeated COS reduces the expression levels of aerobic glycolysis-related enzymes at various stages of early embryonic development. Our results suggest that the reduction in early embryonic development due to repeated COS may be associated with the inhibition of aerobic glycolysis.

Key words: controlled ovarian stimulation; aerobic glycolysis; early embryo

Received: 　　Accepted:

Corresponding author: 杜惠兰　　E-mail:

DOI: 10.13294/j.aps.2026.0026

Citing This Article：

SHEN Lu-Lu, SHI Bei-Bei, XIONG Wen-Yan, SHI Rui-Gan, SONG Ya-Jing, DU Hui-Lan. Effects of repeated controlled ovarian stimulation on the expression of aerobic glycolysis-related enzymes at various stages of early embryo development in mice. Acta Physiol Sin 2026; 78 (3): 592-604 (in Chinese with English abstract).