Mitochondria-related changes and metabolic dysfunction in low prognosis patients under the POSEIDON classification

What is the relationship between mitochondria of granulosa cells (GCs) and age and ovarian function in the patients under the POSEIDON classification?

Our results revealed obvious abnormal mitochondrial-related changes in low prognosis IVF population, where age and the function of ovarian reserve exerted a divergent effect on mitochondrial content and function.

Mitochondria have an important role in the cross-talk between GCs and oocytes. However, factors affecting mitochondria of GCs and related mechanisms are still poorly understood.

GCs samples were obtained from 119 infertile women undergoing IVF from September 2020 to February 2021. Six groups were investigated by the POSEIDON stratification: young with normal prognosis (C1), aging with normal prognosis (C2), young and low prognosis group with normal ovarian reserve (NOR) (G1), aging and low prognosis group with NOR (G2), young and low prognosis group with diminished ovarian reserve (DOR) (G3), and aging and low prognosis group with DOR (G4).

The morphology of GC mitochondria was observed by transmission electron microscopy. MtDNA copy number and mitochondrial replication-related genes were detected by real-time quantitative PCR (qPCR). Mitochondrial membrane potential (MMP) and cytosolic reactive oxygen species (ROS) were detected by confocal microscopy. Cellular glycolysis and aerobic respiratory capacity were analyzed by Seahorse XFe96 Analyzer, and related gene expression and protein levels were assessed by qPCR and Western blot.

Compared to the normal prognosis groups, mitochondrial morphology was impaired in the low prognosis groups, where the young groups (G1, G3) with low prognosis showed phenotypes undergoing oxidative stress (round, vacuolated, swollen with decreased matrix density) and the aging groups (G2, G4) revealed typical aging characteristics (an irregular shape with heterogeneous matrix density and cord-like cristae). Additionally, the degree of corresponding change and damage was more obvious in patients with DOR (G3, G4) regardless of age. For mitochondrial content, the mtDNA copy number in GCs was significantly negatively correlated with age in the low prognosis groups (β = -0.373, P = 0.005). Interestingly, the relationship between mtDNA copy number and anti-Mullerian hormone score differed between the two age groups with low prognosis, with a negative correlation in the young groups (β = -0.639, P = 0.049) and a positive correlation in the aging groups (β = 0.505, P = 0.039). In addition, significantly reduced mitochondrial activity (MMP, ROS) and cell metabolism (both glycolysis and OXPHOS) were observed in the low prognosis groups, with the most obvious decrease being observed in the DOR population. However, the metabolism of the GCs in normal prognosis aging women (C2) shifted from OXPHOS to anaerobic glycolysis.

Owing to the difficulties involved in primary GC collection and culture, the sample size was limited.

Mitochondrial abnormality is closely linked to the low prognostic outcome in IVF patients. Supplementing the functional mitochondrial content or improving mitochondrial function by autologous mitochondrial transfer or mitochondrial-related regulating drugs may help improve the clinical outcomes in patients with a low prognosis, especially for those with DOR.

This work was supported by the National Natural Science Foundation of China (No. 21737001), the Peking University Clinical Medicine + X Youth Project (PKU2020LCXQ011), the Research and Development Program of Peking University People's Hospital (No. RDH2017-03; No. RDX2019-06) and the Application of Clinical Features of Capital Special Subject (Z171100001017130). There were no competing interests.

This study was registered with the Chinese Clinical Trial Register (Clinical Trial Number: ChiCTR2100045531).