Human ovarian aging is characterized by oxidative damage and mitochondrial dysfunction

Abstract

Study question: Are human ovarian aging and the age-related female fertility decline caused by oxidative stress and mitochondrial dysfunction in oocytes?

Summary answer: We found oxidative damage in oocytes of advanced maternal age, even at the primordial follicle stage, and confirmed mitochondrial dysfunction in such oocytes, which likely resulted in the use of alternative energy sources.

What is known already: Signs of reactive oxygen species-induced damage and mitochondrial dysfunction have been observed in maturing follicles, and even in early stages of embryogenesis. However, although recent evidence indicates that also primordial follicles have metabolically active mitochondria, it is still often assumed that these follicles avoid oxidative phosphorylation to prevent oxidative damage in dictyate arrested oocytes. Data on the influence of ovarian aging on oocyte metabolism and mitochondrial function are still limited.

Study design, size, duration: A set of 39 formalin-fixed and paraffin-embedded ovarian tissue biopsies were divided into different age groups and used for immunofluorescence analysis of oxidative phosphorylation activity and oxidative damage to proteins, lipids, and DNA. Additionally, 150 immature oocytes (90 germinal vesicle oocytes and 60 metaphase I oocytes) and 15 cumulus cell samples were divided into different age groups and used for targeted metabolomics and lipidomics analysis.

Participants/materials, setting, methods: Ovarian tissues used for immunofluorescence microscopy were collected through PALGA, the nationwide network, and registry of histo- and cytopathology in The Netherlands. Comprehensive metabolomics and lipidomics were performed by liquid-liquid extraction and full-scan mass spectrometry, using oocytes and cumulus cells of women undergoing ICSI treatment based on male or tubal factor infertility, or fertility preservation for non-medical reasons.

Main results and the role of chance: Immunofluorescence imaging on human ovarian tissue indicated oxidative damage by protein and lipid (per)oxidation already at the primordial follicle stage. Metabolomics and lipidomics analysis of oocytes and cumulus cells in advanced maternal-age groups demonstrated a shift in the glutathione-to-oxiglutathione ratio and depletion of phospholipids. Age-related changes in polar metabolites suggested a decrease in mitochondrial function, as demonstrated by NAD+, purine, and pyrimidine depletion, while glycolysis substrates and glutamine accumulated, with age. Oocytes from women of advanced maternal age appeared to use alternative energy sources like glycolysis and the adenosine salvage pathway, and possibly ATP which showed increased production in cumulus cells.

Limitations, reasons for caution: The immature oocytes used in this study were all subjected to ovarian stimulation with high doses of follicle-stimulating hormones, which might have concealed some age-related differences.

Wider implications of the findings: Further studies on how to improve mitochondrial function, or lower oxidative damage, in oocytes from women of advanced maternal age, for instance by supplementation of NAD+ precursors to promote mitochondrial biogenesis, are warranted. In addition, supplementing the embryo medium of advanced maternal-age embryos with such compounds could be a treatment option worth exploring.

Study funding/competing interest(s): The study was funded by the Amsterdam UMC. The authors declare to have no competing interests.

Trial registration number: N/A.

Keywords: NAD+; mitochondrial dysfunction; oocyte quality; ovarian ageing; oxidative damage.

Figure 1.

Pyruvate dehydrogenase (PDH) activation status in different stages of folliculogenesis of human oocytes. Upper row: staining of different stages of folliculogenesis. Lower row: Immunofluorescence staining of PDHE (pyruvate dehydrogenase E1 component) in different stages of folliculogenesis. (A) Primordial follicle hematoxylin and eosin staining (scale bar 20 μm). (B) Primary follicle hematoxylin and eosin staining (scale bar 20 μm). (C) Secondary follicle hematoxylin and eosin staining (scale bar 50 μm). (D) Antral follicle hematoxylin and eosin staining (scale bar 200 μm). (E) Primordial follicle immunofluorescence staining (scale bar 10 μm). (F) Primary follicle immunofluorescence staining (scale bar 10 μm). (G) Secondary follicle immunofluorescence staining (scale bar 10 μm). (H) Antral follicle immunofluorescence staining (scale bar 100 μm). (I) Mean ratio of PDHE:PDHE1α pSer²⁹³ immunofluorescence staining as an indicator of oxidative phosphorylation activity per age group in oocytes. (J) Mean ratio of PDHE:PDHE1α pSer²⁹³ immunofluorescence staining as an indicator of oxidative phosphorylation activity per age group in cumulus cells. (K) Mean ratio of PDHE:PDHE1α pSer²⁹³ immunofluorescence staining as an indicator of oxidative phosphorylation activity per age group in oocytes from primordial follicles. (L) Mean ratio of PDHE:PDHE1α pSer²⁹³ immunofluorescence staining as an indicator of oxidative phosphorylation activity per age group in cumulus (granulosa) cells from primordial follicles. Gr: granulosa/cumulus cells; Th: theca cells. Channels: red: PDHE; yellow: phosphorylated PDHE (inactive PDH); blue: DAPI (DNA); green: wheat germ agglutinin (WGA; membrane). Noo: nucleus oocyte; Moo: mitochondria oocyte; Mgr: mitochondria granulosa cells.

Figure 2.

Reactive oxygen species (ROS) induced damage to proteins and lipids increases with age in primordial follicles. Immunofluorescence staining of different ROS damage markers in primordial follicles in human ovarian tissue and the simple linear regressions of the peak intensity of these markers with age in human oocytes. (A and B) ROS-induced protein oxidation staining (mouse anti 3-nitrotyrosine) significantly increased with increasing female age, R² = 0.4092, P = 0.002. (C and D) ROS-induced lipid peroxidation-staining (mouse anti 4-hydroxynonenal) significantly increased with increasing female age, R² = 0.2052, P = 0.0392. (E and F) ROS-induced DNA damage-staining (anti 8-Oxo-2'-deoxyguanosine) did not change with increasing female age, R² = 0.0073, P = 0.6983. Scale bars = 20 μm. Noo: nucleus oocyte; Gr: granulosa cells; Th: theca cells. Blue: DAPI, red: ROS-induced damage.

Figure 3.

Age-related metabolite changes in MI and GV oocytes. (A) PLS-DA (partial least square regression discriminant analysis) analysis of the metabolome of pooled human GV (germinal vesicle) oocytes distinguishes between different female age categories: 23–34, 35–37, and 38–42 years. (B) PLS-DA of the metabolome of pooled human MI (metaphase I) oocytes distinguishes between different female age categories: 23–34 and 35–42 years. (C) PLS-DA of the metabolome of single cumulus cell samples per female age category: 23–34, 35–37, and 38–42 years. There was no clear distinction between different age categories. (D) Volcano plot of relative changes in metabolites of MI oocytes between different age categories based on VIP scores >1, colored by pathway in which they play the most important role.

Figure 4.

Different signs of oxidative stress are seen in GV (germinal vesicle) and MI (metaphase I) oocytes. (A and B) Glutathione is depleted in aging oocytes during MI, while oxiglutathione accumulates, indicating an increase in oxidative stress. (C and D) Phosphatidylserine significantly decreased with age in GV oocytes and showed a decreasing trend in MI oocytes. Alkylphosphatidylethanolamine showed a decreasing trend with age in GV and MI oocytes. (E–I) Phosphatidylcholine, alkylphosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, and phosphatidylethanolamine show a decreasing trend with age, both in GV and MI oocytes. GV: GV oocytes; MI: MI oocytes.

Figure 5.

Metabolites associated with mitochondrial function are disturbed in aging oocytes, especially in MI oocytes. (A–D) Metabolites that fuel mitochondrial metabolism accumulate with age: glucose, pyruvate, lactate and glutamine. (E and F) Metabolites associated with energy status of the cell diminish in aging oocyte: NAD⁺ (nicotinamide adenine dinucleotide) and NMN (nicotinamide mononucleotide). (G–J) Monophosphate nucleotide intermediates that are dependent on mitochondrial metabolism show depletion with age: inosine monophosphate (IMP), guanosine monophosphate (GMP), adenosine monophosphate (AMP), and cytidine monophosphate (CMP) in MI oocytes. (K) Phosphocreatine, together with AMP, is associated with the adenosine salvage pathway, an alternative path to creating ATP, and both were reduced with age. (L) ATP increases in cumulus cells with age, possibly in an attempt to compensate for mitochondrial dysfunction in the aging oocyte. GV: germinal vesicle oocytes; MI: metaphase I oocytes; CC: cumulus cells.