Extracellular Vesicles From Follicular Fluid in Infertile Women: Size, Morphology and miRNA Content Analysis

Abstract

The declining birth rates and fertility challenges in Europe have intensified global concerns over rising infertility, particularly among women. This study decisively investigates follicular fluid-related extracellular vesicles (FF-EVs) from infertile patients with polycystic ovary syndrome (PCOS) or diminished ovarian reserve (DOR) undergoing in vitro fertilization (IVF), comparing them to a healthy control group. We have identified significant variations in protein content and polydispersity in crude follicular fluid using UV-Vis absorption and dynamic light scattering (DLS) techniques. Furthermore, the morphology of the extracellular vesicles (EVs) and the patterns of non-coding RNA content, including miRNAs, reveal distinct differences in infertile patients. These findings offer critical insights into the molecular signatures associated with these conditions. This study plays a vital role in advancing reproductive healthcare by pinpointing potential targets that can enhance diagnosis and deepen our understanding of ovarian disorders.

Keywords: Cryo‐TEM; diminished ovarian reserve (DOR); extracellular vesicles (EVs); follicular fluid (FF); miRNA; morphology; polycystic ovarian syndrome (PCOS).

FIGURE 1

Isolation and purification of EVs from human follicular fluid (crude FF, total EVs, large EVs and small EVs).

FIGURE 2

Mean DLS count rate and absorbance at 280 nm according to the ovarian phenotype (healthy as a control, DOR or PCOS) in: (a,b) samples of individual patients (assay 1) and (c,d) pools of patients (assay 2). The square dot indicates the mean value and the horizontal bar inside the box indicates the median value.

FIGURE 3

Cryo‐TEM images illustrate the diversity of size and morphologies of EVs obtained from (a) healthy individuals, (b) patients with DOR and (c) patients with PCOS. All scale bars are 100 nm. (d) A morphological analysis of EVs was conducted based on a modified classification (Höög and Lötvall ; Neyroud et al. 2022) for healthy individuals and infertile patients with DOR or PCOS. This analysis was performed on 215 images containing a total of two 138EVs.

FIGURE 4

EV size distribution and Gauss fit in function of their morphology all ovarian phenotypes combined were obtained by Cryo‐TEM analysis.

FIGURE 5

EV's size distribution as obtained by analysis of the Cryo‐TEM images.

FIGURE 6

RNA nucleotide length profile: (a) Digital electrophoresis gels of small RNA (6–150 nucleotides [nt]) found in large EVs and small EVs samples. The corresponding gel cross‐section views are presented for patients aged 21–30 years in panels (b–d) large EVs and panels (e–g) small EVs.

FIGURE 7

Venn diagram of expression of miRNAs in large EVs (left) and small EVs (right). The expression values can be found in Table S4.

FIGURE 8

In silico pathway analysis of downregulated miRNAs in PCOS compared to DOR conducted using DIANA miRPath‐v4. Only significant results are plotted (p < 0.05).

FIGURE 9

Volcano plots of non‐coding RNA sequencing results comparing: (a) Large EVs to Small EVs (refer to the complete list in Table S8) and (b) DOR patients to healthy patients (see the complete list in Table S7). The y‐axis represents the negative logarithm of the p‐value (base 10), while the x‐axis shows the logarithm of the fold change (FC) (base 2). In the graph, red points indicate RNAs that are significantly overexpressed (log2FC > 1.0 and p‐value < 0.05), while green points represent RNAs that are significantly underexpressed (log2FC < −1.0 and p‐value < 0.05).