In this study, we used scRNA-seq to investigate the follicle microenvironment of normal individuals, older individuals, and POI patients. The follicle microenvironment contains T cells, B cells, neutrophils, basophils, MPs, and GCs. Our analysis revealed that when compared with samples from normal individuals, the proportions of granulosa cells 6 and monocytes were increased, while the proportion of GDTCells was decreased in both POI patients and older individuals, suggesting these cell subclusters may contribute to POI pathogenesis.
In physiological conditions, adequate inflammatory stress is necessary for normal follicular development and ovulation [27]. Before ovulation, GCs possess an inflammatory and immune-like phenotype that produces prostaglandins, inflammatory cytokines, and chemokines, which promote ovulation and fertilization [28]. However, ovarian biopsies of POI patients reveal elevated lymphocytic infiltration and immune responses, and the excess inflammation might influence ovarian aging and POI [29]. The proportion of granulosa cells 6, which are characterized by the high expression levels of MALAT1, NEAT1, XIST, KCNQ1OT1, and AC016831.5, was higher in samples from older individuals and POI patients. Most of these genes, including MALAT1 [30], KCNQ1OT1 [31], NEAT1 [32], are implicated in inflammatory responses. The expression of KCNQ1OT1, which is also involved in DNA methylation, is also reported to be significantly reduced in vitrified oocytes [33], suggesting KCNQ1OT1 may be involved in the regulation of ovarian function. Using scRNA-seq data from preimplantation embryos, Cidral et al. [34] demonstrated that XIST is upregulated at the morula stage and that its expression is higher in female embryos than in male embryos. Moreover, we find that the DEGs identified in granulosa cells 6 from old individuals and POI patients when compared with the normal group, were all enriched in the phagosome and antigen processing and presentation pathways. Consistently, Wang et al. [35] compared protein expression in the ovarian follicular fluid and cumulus cells from patients with diminished ovarian response (DOR) vs healthy controls, and found that the differentially expressed proteins were mostly enriched in the phagosome process. Although antigen processing and presentation is an immune-related pathway, it is also implicated in the regulation of ovarian follicle development [36]. Our preliminary findings suggest that excess inflammation plays a crucial role in both ovarian aging and POI. This possibility was further supported by observations that T cell subtypes as dysfunctional signature scores of NK, CD8Teff, and CD8Tem subgroups, and immunosuppressive signature scores of NK cells were significantly lower in samples from older individuals and POI patients.
Additionally, we found that the proportion of GDTCells, which express high levels of TRGC1 and TRDC, was decreased in samples from older individuals and POI patients. The genes that were found to be upregulated in samples from older individuals and POI patients when compared with the normal group targeting the GDTCells were enriched for ribosomal factors and protein localization to ER. Consistently, we found that the genes that were upregulated in monocytes from older individuals and POI patients, versus the normal group, were also enriched in ribosomal factors and ER-related pathways, such as nonsense-mediated decay protein targeting to ER, establishment of protein localization to ER, and protein localization to ER. The ribosome is a multi-unit complex that translates mRNA into protein. Ribosomal biogenesis plays an essential role in cell proliferation, differentiation, apoptosis, development, and transformation [37]. Mounting evidence indicates that interference with ribosomal biogenesis causes cancer, aging, and age-related degenerative diseases [37, 38]. A recent study revealed that the ribosomes of aging cells move more slowly and periodically, thereby “stalling” translation and causing ribosome collisions and accumulation of new peptides, which worsens ribosomal dysfunction and aging [39]. Based on single-cell quantification of ribosome occupancy and proteomics data, Ozadam et al. [40] recently reported that ribosome occupancy in germinal vesicle-stage oocytes is the predominant determinant of protein abundance in the zygote. The ER plays a crucial role in triggering the deterioration of oocyte quality during oocyte aging [41–43]. Interestingly, ribosome-rich ER membranes expanded, which might enhance TNF biosynthesis [26]. Thus, we hypothesize that ribosome enrichment may cause ER expansion, thereby triggering inflammation, POI, and natural ovarian aging.
Moreover, we observed a loss of VEGFA/FLT1 interaction between monocytes and monocytes in samples from old individuals and POI patients when compared with the normal group. Fms-like tyrosine kinase-1 (FLT1), also known as VEGFR1, which is abundantly expressed on the membranes of macrophage cell lineages, such as monocytes, transduces signals for migration and cytokine/chemokine production by these cells. The VEGF/FLT1-macrophage signaling axis stimulates seemingly non-inflammatory and inflammatory responses in various tissues and promotes a variety of diseases, including cancer (via proangiogenesis and lymphangiogenesis), arthritis, and atherosclerosis [44]. Thus, we hypothesize that the loss of VEGFA/FLT1 interaction in monocytes, along with the enhanced ER and ribosome pathways, may drive excess inflammation, which accelerates GC senility and the state of infertility. A limitation of this study is that we did not use in vivo and/or in vitro experiments to validate this hypothesis.
In this study, we have described the follicular microenvironment of follicular fluid samples from normal individuals, older individuals, and POI patients, including T cells, B cells, neutrophils, basophils, MPs, and GCs. Further analysis revealed higher proportions of granulosa cells 6 and monocytes, and lower proportions of GDTCells in samples from POI patients and older individuals when compared with normal individuals. We hypothesize that the loss of VEGFA/FLT1 interaction in monocytes, along with enhanced ER and ribosome pathways, may contribute to excess inflammation, which accelerates GC senility and the state of infertility. This study provides novel insights into the pathogenesis of POI and aging, and highlights targeting VEGFA/FLT1 interaction as a potential strategy for inhibiting inflammation and treating POI.