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Single-Cell Sequencing Study Suggests Antioxidant Pathway Role in Female Fertility Declines

NEW YORK – Investigators from the US and China have identified potential biomarkers for ovarian aging in humans and other primates, along with other molecular insights aimed at understanding and eventually treating female infertility.

"This study provides a comprehensive understanding of the specific mechanisms of primate ovarian aging at single-cell resolution," corresponding author Guang-Hui Liu, a membrane biology, biomacromolecule, and brain researcher affiliated with the Chinese Academy of Sciences and the Xuanwu Hospital Capital Medical University, said in a statement, noting that the findings "will hopefully lead to the development of new tools to aid in the rejuvenation of aged ovarian cells."

Liu and colleagues used a modified single-cell tagged reverse transcription (STRT) sequencing method to assess transcriptome profiles in more than 2,500 individual ovarian cells from four young and four old cynomolgus monkeys, uncovering seven gene expression-based cell types. In the aging non-human primates, they saw expression shifts in the egg-producing oocytes at distinct developmental stages, including a dip in the activity of genes from antioxidant and stress response pathways in early-stage oocytes and the granulosa cells that support their development.

The team subsequently saw similar patterns when it did reactive oxygen species profiling, small interfering RNA-based gene knockdown experiments, or RNA sequencing on ovarian granulosa cells from healthy human women between the ages of 21 and 46 years old. Those and other findings from the study appeared in Cell on Thursday.

"We found that oxidative stress, the cellular stress that damages cells, is a key player in ovarian aging," co-corresponding author Juan Carlos Izpisua Belmonte, chair at the Salk Institute for Biological Studies Gene Expression Laboratory, said in a statement. "This discovery provides valuable insight into the mechanisms by which ovaries age and eventually become infertile."

Past work suggests human female fertility declines in the years leading up to menopause, the team explained. Even so, the ovarian expression changes that accompany fertility declines in females have not been fully documented, prompting the current single-cell analyses.

"Our goal was to analyze each ovarian cell type along with patterns in gene expression in order to better understand exactly how ovaries age," explained corresponding author Jing Qu, a stem cell and reproductive biology researcher affiliated with the Chinese Academy of Sciences and the University of Chinese Academy of Science, in a statement. "This systematic approach provides a better understanding of the mechanisms of healthy ovarian aging."

Using multiplexed STRT-seq, the researchers initially did single-cell RNA analyses on 2,600 oocyte and somatic ovarian cells isolated from four juvenile monkeys (four or five years old) and four aged monkeys between the ages of 18 and 20 years old.

"As species of the genus Macaca undergo menopause at approximately 25 years of age … the aged monkeys analyzed here were in the period of pre-menopause or peri-menopause," the authors explained, "and thus suitable for studying ovarian aging."

In the non-human primates, the team saw dozens of genes with rising or diminishing expression in the older animals. In particular, it focused in on age-related expression changes affecting antioxidant genes such as IDH1 and NDUFB10 in oocytes and granulosa cells, suggesting the antioxidant pathway activity and adequate stress response could contribute to age-related changes in the ovary's ability to produce and support functional egg cells.

In human granulosa cells, meanwhile, the researchers found that dialing down the levels of IDH1 and NDUFB10 led to muted cell proliferation. Their preliminary results pointed to an uptick in reactive oxygen species and apoptosis in the cells obtained from older women, and expression profiles for the cells overlapped to some extent with those identified in the non-human primates.

The data indicate that the functional decay of cell-type-specific redox regulatory networks has "profound effects on ovarian aging," and reveal targets for novel therapeutic interventions to treat aging-associated ovarian disorders and female infertility, the authors concluded.