Comparison of clinical information between the PCOS group and the control group.
To ensure the reliability of the results of this study, the selected samples of this study were strictly screened according to the Rotterdam criteria (2003). There were two groups (PCOS patients = 5, control patients = 5) of patients, and their follicular fluid exosome samples were analysed. All enrolled participants were diagnosed with primary infertility and received the same ovulation induction treatment programme; they were between 26 and 36 years of age with a duration of infertility between 1 and 5 years. The results showed that there was no significant difference between the PCOS patients and the non-PCOS patients in age, infertility, body mass index (BMI) and fasting blood glucose (FBG) levels. In addition, the number of follicles and anti-Mullerian hormone (AMH) levels were clearly upregulated in the PCOS patients, and there were patients with an LH/FSH > 1 in the PCOS group. The general clinical data of the two groups of patients are shown in Table 1.
Table 1
Information of PCOS patients and non-PCOS donors
Parameter
|
PCOS group (n = 5)
|
Non-PCOS roup (n = 5)
|
P value
|
Age
|
33.2 ± 2.4
|
30.4 ± 2.9
|
0.17
|
Infertility
|
2.4 ± 1.6
|
2.6 ± 1.6
|
0.84
|
BMI
|
23.402 ± 2.7
|
21.984 ± 1.7
|
0.39
|
FBG (mmol/L
|
4.85 ± 0.4
|
4.632 ± 0.3
|
0.41
|
E2(pg/ml)
|
49.8 ± 13.3
|
45.6 ± 12.6
|
0.66
|
Progesterone (ng/mL)
|
0.332 ± 0.13
|
0.46 ± 0.34
|
0.51
|
Testosterone (ng/mL)
|
0.502 ± 0.12
|
0.454 ± 0.14
|
0.63
|
FSH
|
5.704 ± 1.03
|
7.114 ± 0.8
|
0.07
|
PRL
|
10.788 ± 1.6
|
12.278 ± 3.1
|
0.41
|
LH
|
7.784 ± 3.8
|
4.992 ± 2.1
|
0.23
|
Number of follicles
|
25 ± 5.9
|
12.4 ± 5.2
|
0.01
|
AMH (ng/ml)
|
5.278 ± 2.1
|
1.94 ± 0.5
|
0.02
|
Abbreviations: BMI: body mass index; FBG: fasting blood glucose; LH: luteinizing hormone; AMH༚anti-Mullerian hormone; PRL: serum prolactin; E2: estradiol; FSH: follicle-stimulating hormone; |
Table 2
Information of validated miRNAs.
miRNA
|
miRbase ID
|
Primers (5’— 3’)
|
hsa-miR-200c-3p
|
MI0000650
|
TAATACTGCCGGGTAATGATGGA
|
hsa-miR-196a-3p
|
MI0000238
|
CGGCAACAAGAAACTGCCTGAG
|
hsa-miR-199a-5p
|
MI0000242
|
CCCAGTGTTCAGACTACCTGTTC
|
hsa-miR-143-5p
|
MI0000459
|
GGTGCAGTGCTGCATCTCTGGT
|
hsa-miR-483-3p
|
MI0002467
|
TCACTCCTCTCCTCCCGTCTT
|
hsa-miR-376a-3p
|
MI0000784
|
ATCATAGAGGAAAATCCACGT
|
hsa-miR-542-3p
|
MI0003686
|
TGTGACAGATTGATAACTGAAA
|
hsa-miR-21-5p
|
MI0000077
|
TAGCTTATCAGACTGATGTTGA
|
hsa-miR-4322
|
MI0015851
|
CTGTGGGCTCAGCGCGTGGGG
|
hsa-miR-132-3p
|
MI0000449
|
TAACAGTCTACAGCCATGGTCG
|
Differential expression of miRNA profiles in follicular fluid exosomes.
Exosomes were isolated from follicular fluid. Transmission electron microscopy (TEM) was used to detect exosomes approximately 50–200 nm in diameter from all samples (Fig. 1A). Western blot analysis was performed and revealed that two commonly used exosomal protein markers, namely, CD9 and TSG101, were highly enriched in the isolated exosomes relative to PBS (Fig. 1B). The results showed that exosomes from all follicular fluid samples were successfully purified. In total, 2457 miRNAs were identified in follicular fluid exosomes from both PCOS patients and controls in this study. Among them, 157 mature miRNAs in follicular fluid exosomes were significantly differentially expressed in the PCOS and control groups (P < 0.05, log2|FC|>1). The number of significantly upregulated miRNAs was 124, and the number of downregulated miRNAs was 33, as indicated by a volcano plot and a heatmap (Fig. 1C-D). To further validate the miRNA profiling results, ten miRNAs, hsa-miR-200c-3p, hsa-miR-196a-3p, hsa-miR-199a-5p, hsa-miR-143-5p, hsa-miR-483-3p, hsa-miR-376a-3p, hsa-miR-542-3p, hsa-miR-21-5p, hsa-miR-4322, and hsa-miR-132-3p, were randomly screened by RT-qPCR from PCOS patients and non-PCOS patients. According to the RT-qPCR results, the trends in the expression of the miRNAs determined by RT-qPCR were consistent with those obtained from RNA sequencing in PCOS and non-PCOS follicular fluid exosome samples (Fig. 1E-F).
Functional annotation and identification of the differentially expressed miRNA target genes.
KEGG pathway and GO analyses were performed to investigate the functions of 157 differentially expressed miRNA target genes. Furthermore, KEGG pathway enrichment revealed that the target genes were mainly involved in metabolic pathways, pathways in cancer, the PI3K-Akt signalling pathway, the MAPK signalling pathway, endocytosis, the Ras signalling pathway, the Hippo signalling pathway, and cellular senescence (Fig. 2A). GO enrichment analyses were also carried out to gain insight into the biological characteristics of the miRNAs. Metabolic processes were very prominent in both the significantly upregulated and downregulated miRNA target genes, including nucleic acid metabolic process, cellular macromolecule metabolic process, and heterocycle metabolic process (Fig. 2B-C). These results suggest that metabolic pathways possibly have great significance in the pathogenesis of PCOS.
Construction of the miRNA–lncRNA coexpression network
To further explore the epigenetic regulation of miRNAs, intersection analysis between miRNAs and lncRNAs was performed in follicular fluid exosomes from PCOS and non-PCOS patients (Fig. 3A). The differentially expressed lncRNAs in this study were strictly screened according to the previous research results of Liping Wang et al [19]. There were 29 differentially expressed miRNAs constructed with 2439 differentially expressed lncRNAs in the coexpression network via the data from starBase (Table 3). Subsequently, to further investigate the interconnections between the differentially expressed lncRNAs and miRNAs involved in the metabolic pathways (hsa01100) in PCOS, the network was represented, and the potential interaction was predicted (Fig. 3B). The results showed that the upregulated miRNAs, such as miR-369-3p, miR-139-5p, miR-371a-3p, miR-143-5p, miR-199a-5p, miR-196a-3p, and miR-26a-2-3p, reduced the expression of RDH10-AS1. In addition, NARF-IT1, AC090617.1, MZF1-AS1, AC009495.2, LINC01564, AQP4-AS1, L34079.3, OSBPL10-AS1, PIK3CD-AS2, LINC01181, LINC00907, and SP2-AS1 were regulated by the differentially expressed miRNAs. These findings suggest that these miRNAs and lncRNAs may play a role in the pathogenesis of PCOS.
Table 3
miRNAs in the miRNA–lncRNA coexpression network.
miRNA
|
logFC
|
PValue
|
DEG
|
hsa-miR-32-3p
|
6.254050282
|
5.30E-05
|
up_regulated
|
hsa-miR-200c-3p
|
-3.579123
|
0.000297
|
down_regulated
|
hsa-miR-196a-3p
|
4.452656099
|
0.000452
|
up_regulated
|
hsa-miR-199a-5p
|
3.455170178
|
0.000694
|
up_regulated
|
hsa-miR-143-5p
|
3.350954732
|
0.001784
|
up_regulated
|
hsa-miR-26a-2-3p
|
3.256636073
|
0.0036
|
up_regulated
|
hsa-miR-21-5p
|
2.052289441
|
0.008091
|
up_regulated
|
hsa-miR-106a-3p
|
5.883950978
|
0.010317
|
up_regulated
|
hsa-miR-132-3p
|
1.840853713
|
0.01079
|
up_regulated
|
hsa-miR-125b-1-3p
|
2.656313696
|
0.011578
|
up_regulated
|
hsa-miR-15a-3p
|
2.743713774
|
0.013771
|
up_regulated
|
hsa-miR-19a-3p
|
1.906020762
|
0.014684
|
up_regulated
|
hsa-miR-299-3p
|
2.111979855
|
0.017976
|
up_regulated
|
hsa-miR-24-1-5p
|
2.479764956
|
0.022368
|
up_regulated
|
hsa-miR-369-3p
|
2.247301418
|
0.023098
|
up_regulated
|
hsa-miR-30e-5p
|
1.467721548
|
0.023425
|
up_regulated
|
hsa-miR-139-5p
|
2.089759187
|
0.026395
|
up_regulated
|
hsa-miR-129-1-3p
|
3.7326463
|
0.028161
|
up_regulated
|
hsa-miR-34a-5p
|
1.607443155
|
0.028537
|
up_regulated
|
hsa-miR-369-5p
|
2.00127731
|
0.028573
|
up_regulated
|
hsa-miR-371a-3p
|
3.85268939
|
0.032162
|
up_regulated
|
hsa-miR-181b-2-3p
|
5.270233934
|
0.032403
|
up_regulated
|
hsa-miR-23a-5p
|
1.507630692
|
0.03562
|
up_regulated
|
hsa-miR-376c-3p
|
1.639249701
|
0.040714
|
up_regulated
|
hsa-miR-19b-3p
|
1.724336734
|
0.041056
|
up_regulated
|
hsa-miR-20a-5p
|
1.308580728
|
0.042476
|
up_regulated
|
hsa-miR-101-5p
|
1.6811069
|
0.045046
|
up_regulated
|
hsa-miR-376a-5p
|
2.293758046
|
0.046278
|
up_regulated
|
hsa-miR-199a-3p
|
1.52738784
|
0.047854
|
up_regulated
|