P-791 The 4-step ASCOT technique modifies the plasma proteome of patients with premature ovarian insufficiency to resemble that of normoresponder women
Authors: Buigues A , Pellicer N , Martínez J , Ramírez-Martín N , Rodríguez-Hernández C , Blázquez P , Pellicer A , Herraiz S 
Published: July 2024

Abstract

Study question
Does the 4-step ASCOT technique induce any modification in the plasma proteomic profile of women with premature ovarian insufficiency (POI)?

Summary answer
The 4-step ASCOT technique elicits substantial changes in plasma proteome of POI patients, leading to a more similar composition to that observed in normoresponder women.

What is known already
Nowadays, treatment of POI women is still challenging and different approaches have been proposed to improve their reproductive potential, including stem cells (SC) and platelet-rich plasma (PRP). Previous studies showed that plasma treatments containing a combination of both SC-secreted and platelet-enclosed factors are the most promising regenerative treatment in mouse and human ovarian tissues. Therefore, we designed the 4-step ASCOT technique, consisting in: 1) SC mobilization from bone marrow to peripheral blood to enrich plasma with SC secreted factors, 2) Platelet concentration, 3) platelet activation to deliver additional growth factors and 4) direct ovarian injection.

Study design, size, duration
Experimental study with plasma samples of 10 POI women undergoing the 4-step ASCOT reactivation technique (NCT04475744) and 5 normoresponder fertile women. Peripheral plasma samples from POI women were collected at recruitment (Pre) and three months after SC-PRP ovarian injection (Post). Proteomic profiles of Pre and Post plasma samples were compared between them and with the normoresponder samples.

Participants/materials, setting, methods
Proteins were isolated from all plasmas and quantified by the data-independent acquisition parallel accumulation serial fragmentation (diaPASEF) approach. Principal component analysis was performed to compare the plasma proteomic profile of pre- and post-treatment samples, applying the vip function to determine which proteins explained the differences between both time-points (vip&gt;1.5). Then, ElasticNet and Limma methods were employed to determine the statistically significant changes and functional analysis performed to establish the enriched KEGG pathways (false discovery rate&lt;0.05).

Main results and the role of chance
Plasma proteome of POI patients at recruitment exhibited a distinctly different proteomic pattern from that of normoresponder women. However, principal component analysis revealed a clear distinction between plasma samples obtained from POI patients at recruitment and three months after the 4-step ASCOT technique (principal component 1 (PC1) accounting for 20% of explained variance, and PC2 for a 15.4%). Differences between both time-points were mainly determined by 56 proteins involved in processes with a key role in ovarian function, such as complement and coagulation cascades, platelet activation, Hippo pathway, cell cycle, oocyte meiosis and PI3K-Akt signaling pathway. Of these, fifteen proteins exhibited significantly differential expression between both time points, including the age-related APOF protein and the GC vitamin D binding protein, which was the most prominently changed protein after the treatment.
Interestingly, the 4-step ASCOT technique allowed the plasma proteome of patients with POI to reach a similar profile to that observed in normoresponder women. In fact, a detailed analysis uncovered 72 differentially expressed proteins between pre-treatment and normoresponder samples, with only 28 in post-treatment vs. normoresponders. Moreover, the magnitude of expression changes, when compared to normoresponder women, was more significant in pre-treatment than in post-treatment samples.

Limitations, reasons for caution
Further research will be necessary to determine the extent of the regenerative effects of the identified differentially expressed proteins within the ovaries, and their direct implications in ovarian aging and function.

Wider implications of the findings
Our findings highlight that the 4-step ASCOT technique induces changes in plasma proteome of POI patients, reaching similar values to normoresponders. These changes, that remain for few months, might contribute to ovarian reactivation, being the stepping stone to design more effective and personalized treatments for patients with impaired ovarian reserves.

Trial registration number
NCT04475744