Association of exosomal microRNAs in human ovarian follicular fluid
with oocyte quality
Di Zhang a, 1
, Jia Lv b, 1
, Rongxin Tang a
, Ying Feng c
, Yongfeng Zhao a
, Xiaoyang Fei c, d
,
Richeng Chian a, **
, Qigui Xie b, *
a
Center for Reproductive Medicine, Shanghai Tenth People’s Hospital of Tongji University, Shanghai, 200072, China
b
Department of Obstetrics and Gynecology, Shanghai Tenth People’s Hospital of Tongji University, Shanghai, 200072, China
c
Department of Reproductive Medicine, Hangzhou Women’s Hospital, Hangzhou, Zhejiang, 310008, China
d
Department of Reproductive Medicine, Jiangsu Province Hospital, Nanjing, Jiangsu, 210029, China
a r t i c l e i n f o
Article history:
Received 5 November 2020
Accepted 15 November 2020
Available online 28 November 2020
Keywords:
Exosome
microRNA
Ovarian follicular fluid
Oocyte quality
a b s t r a c t
The postponement of childbearing by women has led to an increase in infertility. The reproductive aging
process leads to a decrease in both the quantity and quality of oocytes. The aim of this study was to
investigate exosomal microRNAs in human ovarian follicular fluid and explore their potential association
with oocyte quality. We collected ovarian follicle fluid from 68 patients and assigned the patients to A
(superior oocyte quality) or B (poor oocyte quality) group according to their oocyte quality. Exosomal
miRNAs were extracted, library constructed and sequenced using the Illumina HiSeq platform. Subsequently,
we analyzed exosomal miRNA expression, predicted the miRNA target genes, and enriched Gene
Ontology terms using GOSeq. Kyoto Encyclopedia of Genes and Genomes pathway analysis was performed
using miRanda. A total of 47 miRNAs were found to be significantly differentially expressed
between group A and group B (p < 0.05). Among nine differentially expressed miRNAs that were previously
known, seven were upregulated in group B. In silico analysis indicated that several of these
exosomal miRNAs were involved in pathways implicated in oocyte quality. Analysis of the expression of
exosomal miRNAs in human ovarian follicular fluid showed that they were critical for maintaining oocyte
quality. Our findings provide the basis for further investigations of the functions of exosomal miRNAs in
the ovarian microenvironment and suggest that these exosomal miRNAs may be potential biomarkers for
evaluating oocyte quality. The findings are potentially important to maintain oocyte quality in clinical
settings.
© 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
In the past 40 years, females have tended to postpone their first
childbearing owing to social and economic changes [1], and this
trend continues. Studies have shown that oocyte quality is closely
related to female aging; women older than 35 years are more prone
to miscarriage, dysgenesis, and stillbirth than those younger than
35 [2,3]. However, some young women also experience the
degradation of oocyte quality, based on two frequently used indicators,
the anti-Müllerian hormone (AMH) and antral follicle
count (AFC) [4]. However, the onset of reproductive decline varies
among women and is difficult to determine.
Follicular development involves a complex network of interacting
cellular signals between somatic cells and oocytes [5]. In
particular, exosomes, which are nano-sized, membrane-bound
vesicles, are released by different cell types and serve as important
mediators of intercellular communication [6,7]. Exosomes contain
proteins, DNA, and RNA and can directly influence cellular processes,
causing intracellular changes [8,9]. Thus, exosomes are often
used as indicators of oocyte quality and competence [10].
MicroRNAs (miRNAs) are small noncoding RNAs that regulate
gene expression by binding to the 30-untranslated region of the
Abbreviations: microRNAs, miRNAs; AMH, anti-Müllerian hormone; AFC, antral
follicle count; TEM, Transmission electron microscopy; NTA, Nanoparticle tracking
analysis (NTA); GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and
Genomes.
* Corresponding author.
** Corresponding author.
E-mail addresses: ri-cheng.chian@mcgill.ca (R. Chian), Qigui_Xie@hotmail.com
(Q. Xie).
1
These authors contributed equally to this work.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
https://doi.org/10.1016/j.bbrc.2020.11.058
0006-291X/© 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Biochemical and Biophysical Research Communications 534 (2021) 468e473
target mRNA, resulting in mRNA degradation or posttranslational
silencing [11,12], thus playing a crucial role in the regulation of
development. Exosomal miRNAs are present in ovarian follicular
fluid and are involved in the exchange of genetic information between
cells [13]. The lipid membrane structure of exosomes acts as
a protective barrier against degradation of miRNAs by RNases,
making exosomes a reliable source of miRNAs for potential clinical
use. Exosomal miRNAs were also reported to affect embryonic
potential and oocyte fertilization [14,15]. However, research on
exosomal miRNAs in human ovarian follicular fluid and their association
with oocyte quality is limited, and the mechanisms underlying
their action remain unclear.
This study investigated the relationship between exosomal
miRNAs present in human ovarian follicular fluid and oocyte
quality. We analyzed exosomal miRNAs from ovarian follicular fluid
of women with different oocyte qualities and explored the potential
mechanisms underlying the action of miRNAs to provide support
for further functional studies of exosomal miRNAs.
2. Materials and methods
2.1. Ethical statement
Written informed consent was provided by all patients who
participated in this study. Ovarian follicular fluid was collected at
the First Affiliated Hospital of Nanjing Medical University. The
study was approved by the Ethics Committee of the First Affiliated
Hospital of Nanjing Medical University (Jiangsu Province Hospital;
No: 2016-SR-056) and conducted according to the Declaration of
Helsinki.
2.2. Study subjects
We recruited a total of 68 patients and allocated them to two
groups according to the quality of their retrieved oocytes. Group A
(n ¼ 33) comprised patients with a high oocyte quality, and group B
(n ¼ 35) comprised patients with a poor oocyte quality. The oocyte
quality was determined based on the AFC and AMH level. If the AFC
was >10 and the AMH level was >1.5 ng/mL, the oocyte quality was
considered high; if the AFC was 10 and the AMH level was
1.5 ng/mL, the oocyte quality was considered poor. This study was
conducted at the Reproductive Department of the First Affiliated
Hospital of Nanjing Medical University from May 2016 to July 2018.
2.3. Isolation of exosomes from ovarian follicular fluid
Ovarian follicular fluid was collected via transvaginal
ultrasound-guided puncture. Immediately following oocyte
retrieval, follicular fluid was collected and processed to remove the
blood. Follicular fluid was then centrifuged at 2,000 g for 10 min to
collect the supernatant. Thereafter, the supernatant was centrifuged
twice at 100,000 g for 70 min (Optima™ MAX-XP tabletop
ultracentrifuge; Beckman, Brea, CA, USA) to pellet the exosomes. All
centrifugations were conducted at 4 C. The pellets were resuspended
in 50 mL of phosphate-buffered saline (PBS; pH 7.4) at 4 C
and used for analysis.
2.4. Exosome characterization
2.4.1. Transmission electron microscopy (TEM)
The morphology of exosomes was determined using a transmission
electron microscope (HT7700; Hitachi, Fukuoka, Japan).
Briefly, the isolated exosome preparations were fixed in 1% aqueous
osmium tetroxide in cacodylate buffer overnight at 4 C. The pellets
were rinsed with sodium cacodylate (0.1 M, pH7.0) and dehydrated
with 75% ethanol. Exosomes were placed on copper grids, stained
with 2% uranyl acetate and lead citrate, and examined by TEM.
2.4.2. Nanoparticle tracking analysis (NTA)
Size distribution of the exosomes was determined by NTA
(NanoSight NS300; Malvern, UK). Briefly, the samples were diluted
with PBS and loaded into a NanoSight instrument, followed by
recording videos, which were analyzed to obtain the mean particle
size.
2.4.3. Western blotting
Exosomes were lysed with 1 Â RIPA buffer at 25 C. The protein
concentrations were determined using a bicinchoninic acid assay
(ThermoFisher Scientific, Waltham, MA, USA). Exosomal proteins
(50 mg) were separated by sodium dodecyl sulfate polyacrylamide
gel electrophoresis and then transferred onto polyvinylidene fluoride
membranes. The membranes were blocked and incubated with
a primary mouse monoclonal CD63 antibody (dilution 1:500;
#ab59479, Abcam, Cambridge, MA, USA) and a primary rabbit
polyclonal TSG101 antibody (dilution 1:1,000; #14497-1-AP, ProteinTech,
Rosemont, IL, USA) overnight at 4 C. The membranes
were subsequently washed with Tris buffer (pH 7.6) and incubated
with a secondary horseradish peroxidase-conjugated antibody
(Abcam) at 25 C for 1 h. The signals were detected using a
chemiluminescent substrate (ThermoFisher Scientific) and
analyzed using a ChemiDoc® gel documentation system (Bio-Rad
Laboratories, Hercules, CA, USA).
2.4.4. miRNA extraction
Total RNA was extracted using the miRNeasy mini kit (Qiagen,
Valencia, CA, USA) according to the manufacturer’s instructions.
RNA was dissolved in 30 mL of RNase-free water, and the RNA
concentrations were measured using a NanoDrop ND-1000 spectrophotometer
(NanoDrop Technologies, Wilmington, DE, USA).
The total RNA was electrophoresed in an agarose gel, and miRNAs
were retrieved according to their size (18e50 nucleotides).
2.5. Library construction and high-throughput sequencing
miRNA libraries were prepared using the TruSeq small RNA
sample prep kit (Illumina, San Diego, CA, USA) according to the
manufacturer’s instructions. Briefly, small RNAs were ligated with
30 and 50 adapters, followed by reverse transcription to produce
single-stranded cDNA. cDNA was amplified by PCR for 11 cycles. The
library was sequenced on a HiSeq 2500 platform using SE50 for 10
million clean reads per sample. Data from this study were deposited
to the National Center for Biotechnology Information Sequence
Read Archive under GEO accession number GSE154740.
2.6. miRNA expression analysis
miRNA expression profiles were analyzed using miRDeep2 [16].
Briefly, to obtain clean reads, sequences with a length between 18
and 26 nucleotides were selected and BLASTed against the miRNA
database RFam (https://rfam.xfam.org/) and Repbase database
(https://www.girinst.org/repbase/) to remove repeats, non-miRNA
families (tRNAs, rRNAs, etc.), and low-quality sequences.
Following quality control, the sequences were used for further
miRNA analysis. Differential expression analysis was conducted
using the DESeq/DESeq2 Bioconductor package (http://www.
bioconductor.org/packages/2.12/bioc/vignettes/DESeq/inst/doc/
DESeq.pdf). Following adjustment using the BenjaminieHochberg
approach to control the false discovery rate, the p-value was set
at < 0.05 to identify differentially expressed miRNAs.
D. Zhang, J. Lv, R. Tang et al. Biochemical and Biophysical Research Communications 534 (2021) 468e473
469
2.7. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and
Genomes (KEGG) pathway analysis
GOSeq (v1.34.1) was used to identify GO terms annotated to a
list of enriched genes with a significance of p < 0.05. Significant
differential gene expression was enriched in KEGG pathways, and
miRNAs targeting mRNAs were analyzed using miRanda (v3.3a)
(http://www.microrna.org).
3. Results
3.1. Exosome characterization
The average diameters of the exosomes isolated from follicular
fluid were in the range of 150e200 nm, as determined by NTA
(Fig. 1A), which was consistent with the characteristic sizes of
exosomes [17]. The morphology of the isolated exosomes was
confirmed by TEM (Fig. 1B). The isolated exosomes were also
characterized using two protein biomarkers, CD63 and TSG101
(Fig. 2).
3.2. Differential miRNA expression in exosomes from follicular fluid
A total of 47 miRNAs were found to be significantly differentially
expressed between the groups (p < 0.05). As shown in Fig. 3, there
were distinct miRNA expression patterns between the A and B
groups. Notably, nine miRNAs (Table 1) were previously known,
and seven of these were upregulated in the bad group. The
sequencing data was deposited in Gene Expression Omnibus portal
of National Center for Biotechnology Information with a GEO
accession number GEO154740.
3.3. GO analysis
Target genes of the significantly expressed miRNAs were annotated
using GO analysis, and their functions were categorized.
Among the most crucial biological processes involving target genes
of the miRNAs that were significantly differentially expressed between
the A and B groups were positive regulation of the transcription
from the RNA polymerase II promoter, signal
transduction, positive regulation of cell aging, and negative regulation
of calcium-dependent cellecell adhesion (Fig. 4).
3.4. KEGG pathway analysis
The KEGG pathway analysis showed that the most significant
pathways were the Notch signaling pathway, mitogen-activated
protein kinase (MAPK) signaling pathway, steroid hormone
biosynthesis, RNA degradation, gonadotropin-releasing hormone
(GnRH) signaling pathway, insulin secretion, calcium, tumor necrosis
factor (TNF), Toll-like receptor, PI3K/Akt, Ras, Wnt, ErbB,
nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB),
and Hippo signaling pathways, with p-values between
7.17EÀ23 and 4.33EÀ03 (Table 2).
Fig. 1. Characterization of exosomes isolated from the ovarian follicular fluid of patients. (A) Size distribution of exosomes determined by nanoparticle tracking analysis. (B)
Morphology of exosomes determined by transmission electron microscopy. Group A: high oocyte quality; Group B: poor oocyte quality.
Fig. 2. Western blot analysis of exosomes isolated from ovarian follicular fluid of patients.
STD: exosome standards; Group A: high oocyte quality; Group B: poor oocyte
quality.
D. Zhang, J. Lv, R. Tang et al. Biochemical and Biophysical Research Communications 534 (2021) 468e473
470
4. Discussion
Oocyte degradation is a complex process, and ovarian follicular
fluid provides a suitable microenvironment for maintaining oocyte
quality. Exosomes present in ovarian follicular fluid are required for
intercellular communication; they act as vectors carrying genetic
information. However, the mechanism by which exosomal miRNAs
affect oocyte quality is unclear. In this study, we investigated
miRNA expression in exosomes collected from the ovarian follicular
fluid of women with different oocyte qualities and found that there
were nine mature significantly differentially expressed miRNAs
between the A and B groups.
Four of the differentially expressed exosomal miRNAs that were
detected in this study are known to target several pathways
involved in the regulation of cell signaling, growth, secretion, and
biosynthesis, which suggests that exosomal miRNAs found in
ovarian follicular fluid are critical for transmitting signals that
regulate cell growth and differentiation. GO analysis showed that
signal transduction, cell aging, cell proliferation, metabolic processing,
and cellÀcell adhesion were among the most enriched
terms.
The Notch signaling pathway is a highly conserved signaling
mechanism; it is important for the processes of mammalian ovary
development, such as differentiation and proliferation, and was
shown to regulate steroidogenesis in mouse ovarian granulosa cells
[18]. The Hippo signaling pathway, although not confirmed to play
a role in mammalian ovary development, was reported to promote
the classical development-associated Notch signaling pathway and
play a critical role in regulating cell differentiation, proliferation,
and oocyte polarity in Drosophila [19]. It was also reported that
MAPK signaling pathway, which is regulated by GnRH signaling
pathway activity, is essential for normal fertility [20]. The calcium
signaling pathway potentially negatively affects the oocyte quality
by disrupting sequential biochemical changes that initiate egg
activation [21]. Kong et al. [22] found that TNF-a and a soluble Fas
ligand released by mouse cumulus cells accelerate oocyte aging
Fig. 3. Heatmap of significantly differentially expressed miRNAs in the A vs. B groups.
p < 0.05. Group A: high oocyte quality; Group B: poor oocyte quality.
Table 1
Known significantly differentially upregulated and downregulated miRNAs in the B
vs. A group (p < 0.05).
Gene_id logFC p-Value Regulation
hsa-miR-1246 14.0243 0.001 Up
hsa-miR-548ae-5p 13.0886 0.002 Up
hsa-miR-505-3p 10.7255 0.018 Up
hsa-miR-548t-3p 9.0489 0.021 Up
hsa-miR-513c-5p À10.5287 0.027 Down
hsa-miR-548au-5p 9.7746 0.037 Up
hsa-miR-320e 9.7407 0.039 Up
hsa-miR-548au-3p À9.6736 0.041 Down
hsa-miR-1303 9.2770 0.049 Up
FC: fold change. Group A: high oocyte quality; Group B: poor oocyte quality.
Fig. 4. GO analysis of target genes of significantly expressed miRNAs in follicular fluid exosomes from the A vs. B groups. Group A: high oocyte quality; Group B: poor oocyte quality.
D. Zhang, J. Lv, R. Tang et al. Biochemical and Biophysical Research Communications 534 (2021) 468e473
471
in vitro. According to Oad et al. [23], ErbB activation promotes
signaling pathways such as MAPK and PI3K, thus playing a role in
the stimulation of meiotic resumption by gonadotrophin. The NFkB
signaling pathway is also implicated in several processes, such as
cell adhesion, proliferation, differentiation, and apoptosis [24].
The miRNA hsa-miR-1246 has been implicated in several diseases,
especially cancer [25,26]. However, its functions in human
reproduction remain unclear. The role of miR-1246 in bovine embryos
was studied in vitro and in vivo by Ponsuksili et al. [27], who
found that a number of differentially expressed endometrial miRNAs
detected between days 3 and 7 of the bovine estrous cycle was
associated with the state of pregnancy. In our study, hsa-miR-1246
was found to be upregulated in exosomes from the bad group,
which suggests that it may be associated with the estrous cycle in
humans and affect fertility.
The involvement of hsa-miR-513c-5p in human reproduction
has not been investigated. However, miR-513-a-5p, miR-513b-5p,
and miR-513c-5p were reported to be the most upregulated miRNAs
in breast cancer, of which miR-513-a-5p acts as a risk factor for
breast cancer by inhibiting the expression of the progesterone receptor
[28]. The upregulation of hsa-miR-513c-5p in breast cancer
may also be related to hormonal dysregulation. In our case, its
downregulation in the bad group is expected to affect oocyte
quality.
The miRNA hsa-miR-320e belongs to the miR-320 family, which
includes miR-320aee. In human follicular fluid, miR-320 has been
reported to be associated with embryo quality in vivo [29]. Yin et al.
[30] demonstrated that miRNA-320 was one of the most downregulated
miRNAs in mouse ovarian granulosa cells following TGFb1
treatment; miR-320 overexpression was shown to inhibit
estradiol synthesis and the proliferation of mouse ovarian granulosa
cells by targeting E2F1 and SF-1, which affected the follicular
development in mice. In our study, hsa-miR-320e was upregulated
in the bad group, and we speculated that this upregulation might
affect the proliferation of ovarian granulosa cells and result in
oocyte degradation. However, the exact function of hsa-miR-320e
in this process requires further investigation.
Moreover, hsa-miR-1303 was upregulated in the bad group. It
was demonstrated that miR-1303 is involved in tumorigenesis and
the progression of several cancers, including prostate cancer [31]. In
our study, the hsa-miR-1303-targeted gene was enriched in the
Hippo signaling pathway. Hippo signaling is highly conserved and
is crucial for cell signaling in growth and development. It is also
responsible for the activation of primordial follicles in mice [32]. Li
et al. [33] demonstrated that the Hippo signaling pathway and
ovarian germline stem cells were associated with ovarian aging in
mice. Therefore, the upregulation of hsa-miR-1303 in the bad group
may explain, at least in part, the degradation of oocytes.
Furthermore, miR-505-3p represses the onset of puberty, and
female mice overexpressing miR-505-3p in the hypothalamus
present with delayed ovary maturation and lower fertility [34]. In
our study, the expression of hsa-miR-505-3p was higher in the B
group than in the A group, indicating that this miRNA possibly
causes low fertility.
The miRNA hsa-miR-548 belongs to a poorly conserved family,
which is widespread in all chromosomes, except chromosomes 19
and Y [35]. This miRNA gene family comprises 69 members, is
primate-specific, and is believed to have originated from transposable
elements. We detected the following miRNAs in this study:
hsa-miR-548ae-5p, hsa-miR-548t-3p, hsa-miR-548au-5p, and hsamiR-548au-3p.
Three of these were upregulated and one (hsa-miR-
548au-3p) was downregulated in the bad group, which indicates
the complexity of their possible roles in human ovarian follicular
fluid. Son et al. [36] reported that miRNA-548 might be involved in
the regulation of high-mobility group box 1 and the pathogenesis of
preterm birth. In the miR-548 family, nucleotide sequences are
highly divergent, and the occurrence of “seed-shifting” events is
not uncommon [35]. The various seed sequences play important
and different roles in multiple regulatory networks and signaling
pathways. Some miR-548 family members may interact with each
other under certain circumstances, and this phenomenon further
complicates studies of their exact functions. The exact roles of the
miR-548 genes identified in our study are unclear. However, these
genes were among the most significantly expressed miRNAs, which
implies that they may be crucial for the maintenance of oocyte
quality. It is thus necessary to explore their functions in future
studies.
In conclusion, we investigated the association of exosomal
miRNAs in human ovarian follicular fluid with oocyte quality.
Further functional studies of these exosomal miRNAs and their
mechanisms are warranted in the future. Our findings may pave a
way in maintaining oocyte quality in the ovarian microenvironment
and provide potential biomarkers for evaluating oocyte
quality in clinical settings.
Funding statement
This work was supported by the Ministry of Science and Technology
of China, National Key R&D Program of China (No.
2017YFC1001601) and Zhejiang Provincial Natural Science Foundation
of China (Grant No. LQ18H040009). The funder has no role in
the study design, data collection and analysis, manuscript writing
and decision to submit for publication.
Declaration of competing interests
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgements
N/A.
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