Single-particle analysis of small extracellular vesicles from the follicular fluid of women undergoing fertility treatments reveals distinct PD-L1+populations

2024 · doi:10.1101/2024.12.17.628903
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The study investigated whether small extracellular vesicles (sEVs) bearing PD-L1 (PD-L1+ sEVs) exist as distinct subpopulations in human follicular fluid from women undergoing fertility treatments (n=10), using single-particle interferometric reflectance imaging sensing with antibody capture and immunofluorescence labeling for CD63, CD81, CD9, and PD-L1, plus atomic force microscopy for size distribution. The authors found that most tetraspanin-expressing EVs in follicular fluid were smaller than 50 nm, and that tetraspanins and PD-L1 showed distinct expression and colocalization profiles across cohort samples. They reported that substantial fractions of particles captured via anti-CD63, anti-CD81, or anti-CD9 antibodies were CD81-positive, and that PD-L1 was highest on CD9+ sEVs (about 5% average within the cohort). A major caveat explicitly noted is that further work is needed to determine the functional significance of PD-L1+ sEVs and whether they can serve as fertility biomarkers, and the study did not assess immune function directly. The paper does not explicitly discuss endometriosis or adenomyosis; it was included in the corpus via a keyword match in the upstream search index.

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Abstract

In some cell systems, small extracellular vesicles bearing PD-L1 (PD-L1 + sEVs) have been shown to be able to suppress T-cell immunity. We have herein investigated whether a distinct profile of PD-L1+ sEVs exists in human follicular fluid (FF). Single-particle interferometric reflectance imaging sensing combined with a single-particle antibody capture and immunofluorescence labelling were used to determine the expression and colocalization of CD63, CD81, CD9, and PD-L1 in sEVs derived from FF of women undergoing fertility treatments (n=10). In addition, the size distribution of sEVs was investigated via atomic force microscopy. Our data indicate that the bulk of tetraspanin-expressing EVs in human FF are less than 50 nm in size. Tetraspanins and PD-L1 exhibit distinct expression and colocalization profiles at sEV level across all cohort samples. A total of 42%, 46%, and 50% of all the particles captured by anti-CD63, anti-CD81, and anti-CD9 antibodies, respectively, were positive for CD81. PD-L1 was expressed at the highest level on CD9+ sEVs, with an average value of 5% within the cohort. The presence of distinct PD-L1+ sEV subpopulations suggests that they may play a role in regulating the immune response in the follicular microenvironment. Further research is needed to fully understand the functional significance of PD-L1+ sEVs in this context and their potential as biomarkers for predicting fertility outcomes. Abstract Figure
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Abstract In some cell systems, small extracellular vesicles bearing PD-L1 (PD-L1+ sEVs) have been shown to be able to suppress T-cell immunity. We have herein investigated whether a distinct profile of PD-L1+ sEVs exists in human follicular fluid (FF). Single-particle interferometric reflectance imaging sensing combined with a single-particle antibody capture and immunofluorescence labelling were used to determine the expression and colocalization of CD63, CD81, CD9, and PD-L1 in sEVs derived from FF of women undergoing fertility treatments (n=10). In addition, the size distribution of sEVs was investigated via atomic force microscopy. Our data indicate that the bulk of tetraspanin-expressing EVs in human FF are less than 50 nm in size. Tetraspanins and PD-L1 exhibit distinct expression and colocalization profiles at sEV level across all cohort samples. A total of 42%, 46%, and 50% of all the particles captured by anti-CD63, anti-CD81, and anti-CD9 antibodies, respectively, were positive for CD81. PD-L1 was expressed at the highest level on CD9+ sEVs, with an average value of 5% within the cohort. The presence of distinct PD-L1+ sEV subpopulations suggests that they may play a role in regulating the immune response in the follicular microenvironment. Further research is needed to fully understand the functional significance of PD-L1+ sEVs in this context and their potential as biomarkers for predicting fertility outcomes. Competing Interest Statement The authors have declared no competing interest. Footnotes Conflicts of interest: none barbara.bortot{at}burlo.trieste.it; roberta.diflorio{at}burlo.trieste.it; gabriella.zito{at}burlo.trieste.it; giuseppe.ricci{at}burlo.trieste.it francesco.valle{at}cnr.it; marco.brucale{at}cnr.it

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