Probing Proteoform Heterogeneity From Single Human Oocytes

Ovarian tissue cryopreservation is a fertility preservation strategy available to prepubertal patients undergoing gonadotoxic treatment who are at risk of developing premature ovarian insufficiency or for those whose treatment cannot be delayed. While cryopreserved tissue can be utilized for ovarian tissue transplantation to restore hormone function and fertility in some of these individuals, those with a high risk of malignant cell reintroduction currently have no options. In vitro maturation (IVM) of isolated immature oocytes from prepubertal patients is not yet successful enough for the clinic, necessitating study of the molecular factors influencing oocyte quality for IVM success. In this study, we characterized intact proteoforms from single human oocytes obtained from ovarian tissue cryopreservation and tissue donation in a donor cohort aged 2 to 33 years old to identify changes in the oocyte proteome across the pubertal transition. Utilizing single-cell proteoform imaging mass spectrometry (scPiMS), which employs a sampling probe to raster over single cells coupled to individual ion mass spectrometry detection, we identified 559 proteins and 769 unique proteoforms across 28 oocytes from eight donors, with an average of 78 unique proteoforms per oocyte. We used scPiMS to selectively sample oocytes and cumulus granulosa cells from single cumulus oocyte complexes to identify proteoforms specific to these cell types. Finally, we determined proteoform landscapes for members of the oocyte-specific subcortical maternal complex (SCMC), KHDC3, and OOEP, and found new proteoforms that differ with donor age. Together, these first-in-class observations provide a foundation for understanding protein-level changes in oocyte biology across puberty to ultimately improve the efficiency of IVM and make fertility restoration options accessible for more patients.