Abstract
Background
Clinically, hormone replacement therapy (HRT) is the main treatment for
primary ovarian insufficiency (POI). However, HRT may increase the risk
of both breast cancer and cardiovascular disease. Exosomes derived
from human umbilical cord mesenchymal stem cell (hUC-MSC) have been
gradually applied to the therapy of a variety of diseases through
inflammation inhibition, immune regulation, and tissue repair
functions. However, the application and study of hUC-MSC exosomes in
POI remain limited.
Methods
Here, we first constructed four rat animal models: the POI-C model (the
“cyclophosphamide-induced” POI model via intraperitoneal injection),
the POI-B model (the “busulfan-induced” POI model), the POI-U model
(the “cyclophosphamide-induced” POI model under ultrasonic guidance),
and MS model (the “maternal separation model”). Second, we compared the
body weight, ovarian index, status, Rat Grimace Scale, complications,
and mortality rate of different POI rat models. Finally, a
transabdominal ultrasound-guided injection of hUC-MSC exosomes was
performed, and its therapeuticy effects on the POI animal models were
evaluated, including changes in hormone levels, oestrous cycles,
ovarian apoptosis levels, and fertility. In addition, we performed
RNA-seq to explore the possible mechanism of hUC-MSC exosomes function.
Results
Compared with the POI-C, POI-B, and MS animal models, the POI-U model
showed less fluctuation in weight, a lower ovarian index, fewer
complications, a lower mortality rate, and a higher model success rate.
Second, we successfully identified hUC-MSCs and their exosomes, and
performed ultrasound-guided intraovarian hUC-MSCs exosomes injection.
Finally, we confirmed that the ultrasound-guided exosome injection
(termed POI-e) effectively improved ovarian hormone levels, the
oestrous cycle, ovarian function, and fertility. Mechanically, hUC-MSCs
may play a therapeutic role by regulating ovarian immune and metabolic
functions.
Conclusions
In our study, we innovatively constructed an ultrasound-guided ovarian
drug injection method to construct POI-U animal models and hUC-MSC
exosomes injection. And we confirmed the therapeutic efficacy of
hUC-MSC exosomes on the POI-U animal models. Our study will offer a
better choice for new animal models of POI in the future and provides
certain guidance for the hUC-MSCs exosome therapy in POI patients.
Graphical abstract
The schema of construction of different animal models, extraction and
identifying hUC-MSCs and exosomes, therapy of ultrasound-guided
hUC-MSCs exosome injection. Note: POI: premature ovarian insufficiency;
hUC-MSCs: Human umbilical cord mesenchymal stem cells; POI-C:
POI-cyclophosphamide; POI-B: POI-cyclophosphamide + Busulfan; POI-U:
POI-Ultrasonic guidance cyclophosphamide injection; MS: POI-Maternal
separation. POI-e: ultrasound-guided hUC-MSCs exosome injection; AMH:
Anti-müllerian hormone; LH: Luteinizing hormone; FSH:
Follicle-stimulating hormone; DA: dopamine; T: Testosterone; PRL:
prolactin; GnRH: Gonadotropin-releasing hormone.
[53]graphic file with name 13287_2024_3646_Figa_HTML.jpg
Supplementary Information
The online version contains supplementary material available at
10.1186/s13287-024-03646-y.
Keywords: Primary ovarian insufficiency, Ultrasound-guided injection,
POI animal models, hUC-MSCs, Exosomes
Introduction
Primary ovarian insufficiency (POI) affects 1–5% of women under
40 years of age [[54]1, [55]2]. POI, which mainly manifests as
irregular menstrual cycles, is often confirmed according to the
elevated serum levels of follicle-stimulating hormone (FSH) and
decreased levels of oestradiol and anti-Mullerian hormone (AMH)
[[56]3]. POI often leads to infertility, and its early onset can have a
negative impact on sexual health. Additionally, long-term oestrogen
deprivation has serious effects on women's health, particularly on bone
density, cardiovascular and nervous systems, and sexual health [[57]1].
Currently, clinical care for women with POI mainly includes hormone
replacement therapy (HRT) and psychological support. However, an
effective therapeutic solution for POI remains elusive.
POI is associated with chromosomal or genetic changes, infections,
metabolic disorders, autoimmune diseases, and iatrogenic factors
(ovarian surgery, radiotherapy, and chemotherapy, etc.) [[58]1, [59]4].
According to the aetiology of the disease, POI animal models can be
divided into the following four types: the “chemotherapy-induced”
model, “autoimmune” model, “mental stress” model, and “natural ageing”
model. Among these, the “chemotherapy-induced” model is the most used
animal model in research. However, there are big differences between
different models. Suitable and optimal animal models are essential
carriers for drug development and mechanistic research. In a previous
review, we compared the advantages and disadvantages of different POI
animal models [[60]5]. In this investigation, we constructed different
POI animal models and evaluated their strengths and weaknesses, which
included body weight and ovarian changes in rats, hormone level
fluctuations, model success rate, complications, and so on. Our study
aimed to build a suitable and ideal POI animal models for drug
development and mechanistic research.
In recent years, human umbilical cord mesenchymal stem cells (hUC-MSCs)
have attracted much attention as the potential cell therapy tools due
to their proliferation, multipotency, homing/migration abilities,
trophic effects, and immunosuppressive properties. These cells have
been demonstrated to possess therapeutic potential in various
autoimmune, inflammatory, and degenerative diseases [[61]6].
Accumulating studies have shown that intra-ovarian transplantation of
hUC-MSCs can restore fertility, recover serum hormone levels, and
facilitate follicle formation in a “chemotherapy-induced” POI rat model
[[62]4, [63]7]. However, poor engraftment efficiency and insufficient
viability of hUC-MSCs limits their application. Hence, various
strategies have been developed to improve the effectiveness of
hUC-MSCs. A 2022 study showed that the hUC-MSCs with autocross-linked
Hyaluronic acid gel can rescue ovarian reserve and fecundity in POI and
naturally aging mice [[64]8]. In addition to physical crosslinking
methods, hUC-MSCs derivatives have been extracted to restore the
function of ovary of POI.
Many studies reported that factors, including cytokines and exosomes,
can be secreted, which lead to the ovarian recovery of POI through
reducing apoptosis and inflammation, and inducing angiogenesis [[65]8,
[66]9]. A recent study from 2023 has shown that small membrane-coated
vesicles, namely MSCs cell exosomes, can protect granulosa cells from
chemotherapy-induced damage via venous injection [[67]10]. However, an
increasing number of studies suggest that the biological distribution
of hUC-MSCs in target organs via veins is rare [[68]6].
Ultrasound-guided drug injection is an efficient and safe method
[[69]11]. This study mainly focused on the therapeutic effect of
ultrasound-guided hUC-MSC exosomes on POI and explored the possible
mechanism by which hUC-MSC exosomes rescue the manifestations of POI.
In this work, we found a novel POI animal model, the
“cyclophosphamide-induced” POI model under ultrasonic guidance (POI-U
model), which has less complications and high model success rate.
Further, we successfully confirmed the therapeutic effects of hUC-MSC
exosomes administered via ultrasound-guided injection in the POI-U
model. Mechanistically, hUC-MSC exosomes may ameliorate ovarian
function by regulating the immune and metabolic systems.
Methods
Animals and experimental design
Ninety-four female Wistar rats (5 ~ 7 weeks old) were purchased from
Beijing Vital River Laboratory Animal Technology Co. Ltd., China. The
animals were raised in a specific pathogen-free (SPF) environment. The
rats were grouped into five groups: the control group
(comprising fifteen rats), the POI-C group (consisting of twenty-four
rats), the POI-B group (cyclophosphamide + busulfan, containing ten
rats), the POI-U group (including thirty rats), andthe MS group (also
including fifteen rats), according to different model methods. In
addition, there were fifteen rats in the ultrasound-guided exosome
injection group (POI-e and POI-2e). The specific experimental flow
chart is shown in Fig. [70]1.
Fig. 1.
[71]Fig. 1
[72]Open in a new tab
Schema of different premature ovarian insufficiency (POI) models. Note:
POI-C: POI-cyclophosphamide; POI-B: POI-cyclophosphamide + Busulfan;
POI-U: POI-Ultrasonic guidance cyclophosphamide injection; MS: Maternal
separation
The preparation methods of four animal disease models according to the
causes of POI were selected to evaluate the feasibility of the animal
models [[73]5, [74]12]. The grouping was as follows: (1) POI-C model:
Female Wistar adult rats aged 60 d were intraperitoneally injected with
CTX (50 mg/kg) on the first day, followed by 2 weeks of CTX (8 g/kg/d);
(2) POI-B model: Female Wistar adult rats approximately 60 days old
were given a single dose of CTX (120 mg/kg) via intraperitoneal
injection + busulfan (8 mg/kg) via subcutaneous injection; (3) POI-U
model: Female Wistar adult rats approximately 60 days old were injected
with CTX (50 μg/ovarin weight (g) ~50 μL) according to the weight of
ovary in both ovaries under the ultrasound guidance, and the same dose
of CTX was injected under ultrasound guidance 2 weeks later; (4) MS
model: On the 9 th day after birth, mother and infant rats were
separated for 1 d, which simulated early life stress stimulation
[[75]13]. Relevant indicators were monitored when the weight of the
young rats reached approximately 60 d. All animal studies were approved
by the ethics committee for laboratory animal welfare (IACUC) of Renmin
Hospital of Wuhan University [No. WDRM animal (f) No. 20210611A].
Ultrasound guided abdominal drug injection
The adult rats were fasted for 5–6 h. The rats were anesthetized with
isoflurane. Firstly, After the induction concentration was
appropriately adjusted (the induction concentration of isoflurane was
set to 3–4%), the induction box was filled with anesthetic for about
1 min. Then, the animal was put into the induction box, and the
induction box was closed, waiting for the animal to be completely
anesthetized. Secondly, after the maintenance concentration was
properly adjusted (2–2.5% isoflurane for rats), the animal was removed
from the induction box, and its head/nose was fixed in the anesthesia
mask. Finally, after the animal experiment was completed,
the evaporator was closed, and the rats were kept in pure oxygen for
about 5–10 min to facilitate the rapid recovery of the animal.
After anaesthesia, the hair on the abdomen was removed using a shaving
knife, a coupling agent was applied, and a low-frequency ultrasonic
probe (Ultrasonic probe 11L) was applied to search for the ovaries via
an ultrasound instrument (Voluson E10). The ovaries of normal rats
appeared as low-echo liquid dark areas of 0.5–1 cm, situated adjacent
to the kidneys, with abundant blood flow. Then, a 0.7 × 80 mm
needle/22 g (7/22 G) was used to penetrate into the ovarian substance
and both the left and right ovaries were treated with 50 μL
cyclophosphamide chemotherapy drugs (50 μg/ovarian weight (g)) with
fluorescent dyes and hUC-MSCs. The specific steps were as follows: (1)
The operator's puncture needle was placed directly under the probe in
the direction of the ovarian parenchyma to observe whether the blood
flow was near the puncture needle head; (2) the operator fixed the
needle position, and the assistant slowly injected 50 μL of
the fluorescent chemotherapeutic drug with a 1 mL syringe; (3) the
chemotherapy drug syringes was removed and the other needle with 50 μL
of saline irrigate the syringes ; (4) the ovary slightly enlarged
immediately after the injection, and a comparison of the ovarian
diameter before and after the injection was performed; and (5) one to
two animal samples were collected within 12 h post-injection, and the
fluorescence intensity of the ovary was observed by an in vivo imaging
apparatus. The ovarian diameter of the rats was measured under direct
ultrasound. The longest and shortest diameters measured by
the ultrasonic display were a cm and b cm, respectively. The area of
the ovary was calculated as
[MATH: Π∗a
2∗b/2 :MATH]
cm^2[.]
Culture and identification of hUC-MSCs
hUC-MSCs were provided from Hubei Levobank Biotechnology Co., Ltd., and
Seidet Biotechnology Development Co., Ltd. The cells were cultivated
using a specialized serum-free stem cell culture medium
supplemented with a primary cell culture additive (cat: NC0103, Youkang
Biotechnology (Beijing) Co., Ltd.). The morphology of hUC-MSCs was
photographed during the first 1–3 generations. Lipogenesis was induced
in hUC-MSCs as follows: (1) when hUC-MSCs reached 80 ~ 90% confluence,
digestion was performed. (2) hUC-MSCs were inoculated into six-well
plates at approximately 5 × 10^4 cells/well and 2 mL/well hUC-MSC
complete culture medium. (3) The fluid was changed every three days
(with complete culture medium of stem cell) until the cells reached
100% confluence. (4) Once the cells reached 100% confluence, the old
culture medium was removed, and lipogenic inducer A (2 mL/well) was
added for induction. (5) Three days later, lipogenic inducer B was
exchanged for maintenance. Twenty-four hours later, lipogenic inducer A
was exchanged for induction, and four-five cycles were carried out. (7)
Oil red O staining was performed to stain lipid droplets. Osteogenesis
was induced in hUC-MSCs. Specifically, (1) when hUC-MSCs reached
80 ~ 90% confluence, they were digested; (2) hUC-MSCs were inoculated
into six-well plates at approximately 4 × 10^3 cells/well, and
2 mL/well hUC-MSC complete culture medium was added, which was
incubated at 37 °C and 5% CO[2]. (3) After 24 h, the old culture medium
was removed, and pore osteogenic induction solution (2 mL) was added.
(4) The pore-osteogenic induction solution was changed every three days
for 2–3 weeks. (5) After 2–3 weeks, calcium nodules were formed, and
alizarin red staining was performed. hUC-MSC markers (FITC-CD90,
CD105-PerCP-Cy™5.5, CD73-APC, HLA-DR-PE) were detected by flow
cytometry (Beckman Coulter, CA, USA).
Extraction and characterization of hUC-MSC exosomes
After the supernatant from serum-free culture medium was collected, it
was concentrated using a 100 kDa ultrafiltration tube and then
centrifuged at 4 °C at 500 × g for 10 min, 2000 × g for 10 min and
20,000 × g for 20 min to remove cell debris and microvesicles. The
samples were then centrifuged at 100,000 × g for 120 min to remove the
supernatant. After DPBS resuspension and centrifugation at 100,000 × g
for 120 min, exosomes were obtained by transparent precipitation (Cat:
OptimaXE-100, Beckman Coulter) to extract the hUC-MSC exosomes. Then,
50 μL of rhodamine working liquid was added to every 100 μg of exosomes
(as calculated by the BCA method), and the exosomes were evenly blown
and mixed using a vortex oscillator for 1 min, incubated at room
temperature for 10 min, and resuspended in 1X PBS. The exosomes were
re-extracted by the overspeed centrifugal method to remove excess dye.
Rhodamine-labelled exosomes were used. Note that the above operation
should strictly avoid light, and rhodamine working liquid should be
available. An equal amount of PBS was slowly injected into the ovarian
tissue of the rats as a control group during the ultrasound-guided drug
injection.
The morphology of hUC-MSC exosomes was observed by transmission
electron microscopy (TEM, JEM-2100, JEOL, Japan). First, 20 μL of
exosome-suspended droplets was added to the electron microscope copper
mesh and left to stand for more than 1 min. Then, the sample was fixed
with 2% phosphotungstic acid solution for 1 ~ 10 min and air-dried at
room temperature. Finally, the sample was observed and photographed
under a biological transmission electron microscope. The zeta potential
and particle size of hUC-MSC exosomes were detected by a Zeta sizer
Nano ZS (Malvern, UK). The zeta potential analyser was preheated for
30 min in advance, and an appropriate amount of exosome suspension was
added into the quartz colorimetric plate to detect the zeta potential
to test the charge of the exosomes. Each sample was assayed at least 3
times.
Detection of body and ovarian weight of rats
The body weight of the rats was continuously monitored every day. The
rats were weighed gently to minimize stress. After the rats were killed
using sodium thiopental (50 mg/kg, i.p.), the volume of the drug
injected into the rat was about 0.5 mL. The skin and muscle layers were
cut from the abdomen to extract ovarian tissues. Their ovarian tissue
was separated, and the surrounding adipose tissue was carefully
stripped away for weighing. The ovary index was calculated as ovarian
weight (mg)/rat weight (g) × 100% [[76]12]. The ovarian tissue to be
fixed was carefully trimmed and fixed in 4% paraformaldehyde for tissue
sectioning. The tissues used for mRNA detection were directly frozen in
liquid nitrogen and then transferred to − 80 °C cryogenic refrigerator.
Examination of oestrous cycles
A daily vaginal smear was performed from 8:00 to 9:00 AM. Specifically,
we gently rinsed the rat's vagina with 7 μL of sterile normal saline
until the liquid became slightly cloudy and then uniformly spread onto
a slide. After the slides were air-dried, they were immersed in
absolute ethanol for 10 min and then dehydrated in gradient alcohol.
Haematoxylin was added at 37 °C for 10 min and rinsed off with running
tap water for 1 min. Subsequently, the slides were dyed with eosin
staining for 30 s ~ 1 min and repeatedly rinsed with running tap water.
The slides were observed under a light microscope after completely
drying (Olympus BX53; Olympus Corporation).
HE staining and counting of the ovarian follicles
Ovarian tissue was removed from the paraformaldehyde fixation solution
and then transferred to dissolved paraffin for fixation after
transparent dehydration. Subsequently, 4 μm thick tissue was cut from
the ovarian centre to make sections. Sections were stained with
haematoxylin for 5 min and then rinsed with running water several
times. Hydrochloric acid alcohol was used for differentiation for 1 s,
washed in warm water for a few seconds, and then cleaned with distilled
water at room temperature for 1 s. Finally, the slides were soaked in
eosin solution for 40 s and rinsed for 2 s, dehydrated with gradient
ethanol, cleared with xylene, and finally sealed with neutral rubber.
The number of follicles in each ovary in different groups was counted.
We counted ovarian follicles in the following categories: Follicles at
different developmental stages were divided into primordial follicles,
primary follicles, secondary follicles, and sinus follicles. Primordial
follicle: A single oocyte is surrounded by a layer of flattened
granulosa cells. Primary follicle: The oocytes are surrounded by a
single layer of cubic granulosa cells. Secondary follicle: The oocytes
are surrounded by more than two layers of granulosa cells, with no
follicular cavity. Sinus follicles: The follicles are further enlarged,
and follicular spaces are visible. Atretic follicle: The shape of the
follicle is irregular, and the oocyte has severe nuclear deviation,
shrinkage, zona pellucida collapse, loose granulosa cells and
follicular membrane cells, and cells that have atrophied and shed into
the follicular antrum.
Enzyme-linked immunosorbent assay
When the rats were anaesthetized, a disposable serum separation tube
was used to immediately take blood from the heart at the peak of the
heartbeat (3.5–5 mL) (Additional file [77]1: Fig. S1). The procedure
for taking blood from the heart is shown in Additional file [78]2:
video 1. After the blood collection vessel was inverted, it was left
for 1 h at room temperature. Then, the sample was centrifuged at
3000 rpm for 15 min, and the supernatant was collected and stored at
− 80 °C in separate containers to avoid repeated freeze‒thaw cycles.
Samples with haemolysis need to be discarded. Serum hormone levels (E2,
FSH, T, LH, AMH, GnRH, PRL, DA) were detected by enzyme-linked
immunosorbent assays. This procedure was repeated three times for each
sample according to the corresponding kit instructions (cat: ELK1208,
ELK1315, ELK1332, ELK2367, ELK4910, ELK5453, ELK7644, ELK7879, ELK
Biotechnology, Wuhan, China).
Fertility test
After modelling, female rats in different groups were mated with
3-month-old male rats in a 2:1 cage. The male rats were separated out
after 14 d of cages, and the expected delivery date was 21 d. The
production of rats was observed from the 18th d, and litter size was
counted for comparison between groups.
Real-time qPCR
Frozen ovarian tissue was removed and ground into powder in liquid
nitrogen. TRIzol (1 mL, Shanghai Yisheng Co., Ltd.) was added to
ovarian tissues. Then, RNA was extracted through a series of steps via
chloroform, isopropyl alcohol, and ethanol. Finally, the RNA was
dissolved in DEPC water. The concentration was determined with a
NanoDrop 2000, and reverse transcription was performed according to the
instructions of a reverse transcription kit. Quantitative PCR was
performed according to the quantitative PCR kit instructions (Shanghai
Yisheng Co., Ltd.). Table [79]1 lists the primers. The relative levels
of genes were calculated using the 2^−△△Ct method.
Table 1.
lists the primers that were utilized
Primers Sequence
r-gadph F ATGGCTACAGCAACAGGGT
R TTATGGGGTCTGGGATGG
r-bax F GAGGTCTTCTTCCGTGTGG
R GATCAGCTCGGGCACTTT
r-bcl2 F AGGAACTCTTCAGGGATGG
R GCGATGTTGTCCACCAG
[80]Open in a new tab
RNA-seq analysis
In each group of rats (POI and POI-e), ovarian tissue samples were
randomly selected from three rats. RNA-seq was performed by Guangzhou
Jidio Biotechnology Co., Ltd. Specially, Specially, total RNA was
extracted using the Trizol reagent (Invitrogen, Carlsbad, CA, USA).
Total RNA (1 μg per sample) was used to construct sequencing libraries.
Briefly, RNA quality was evaluated by an Agilent 2100 Bioanalyzer
(Agilent Technologies, Palo Alto, CA, USA) and checked using RNase free
agarose gel electrophoresis. After total RNA was extracted, the mRNA
was enriched by Oligo (dT) beads. Then the enriched mRNA was fragmented
into short fragments using a fragmentation buffer and reversely
transcribed into cDNA by using a NEB Next Ultra RNA Library Prep Kit
for Illumina (NEB #7530, New England Biolabs, Ipswich, MA, USA). The
purified double-stranded cDNA was end-repaired, A-tail was added, and
sequencing joints were connected. cDNA of about 200 bp was screened by
AMPure XP beads (1.0X) for PCR amplification, and PCR products were
purified by AMPure XP beads again. Finally, the resulting cDNA library
was sequenced using Illumina Novaseq 6000 by Gene Denovo Biotechnology
Co. (Guangzhou, China). Sequencing mode is double-ended sequencing
2 × 150 bp (PE 150), read length 150 bp.
The sequencing fragment process was obtained during sequencing, and
each fragment is called a read. The reads were further filtered by
fastp (V 0.18.0) to obtain quality clean reads. The steps for filtering
reads are as follows: (1) reads containing adapter were removed; (2)
reads containing more than 10% N were removed; (3) Remove reads that
are all A-base; (4) low-quality reads (base numbers with mass value
Q ≤ 20 account for more than 50% of the entire read) were removed.
Clean reads were then mapped to the rattus morvegicus reference genome
(Ensembl_release106) using HISAT (v2.2.4). Then, the mapped reads were
assembled by using StringTie (v1.3.1). The gene expression differences
between POI group and POI-e group were evaluated by the fragment per
kilobase of transcript per million mapped reads method (FPKM). The
calculation method of FPKM is
[MATH:
FPKM=106CNL<
/mi>/103 :MATH]
where C is the number of fragments (count) to be compared to the gene,
N is the total number of fragments to be compared to the reference
gene, and L is the effective length of the gene.
Differentially expressed genes (DEGs) were identified using DESeq2
software between two different groups. Fold change (FC) and difference
significance were used to screen the DEGs. DEGs with FC value greater
than 2 or lower than − 2, and a P value lower than 0.05 were considered
significant. Finally, GO function annotation and KEGG pathway
enrichment analysis were further carried out for DEGs via DAVID
database ([81]https://david.ncifcrf.gov/).
Statistical analysis
The experimental results were obtained by GraphPad Software (Version
7.0, United States). The measurement data were expressed as
mean ± standard deviation. The independent sample t test was used to
compare the normal distribution data between the two groups. The LSD
test in the one-way analysis of variance was used to compare the normal
distribution homogeneity of variance data between the three groups.
P < 0.05 indicates statistical difference.
Results
Comparison of body weight, ovarian index, and status of rats in four animal
models
The changes of body weight in the four models (POI-C, POI-B, POI-U, and
MS) were continuously monitored throughout the modelling period. We
found that the body weight of the rats exhibited a stable increase in
the initial stage. While, with the accumulation of drug concentrations
in the body, the body weight of the rats in the POI-C and POI-B groups
gradually decreased, especially in the POI-B group at approximately
12th day of administration (Fig. [82]2A–D). The phenomenon maybe that
systemic administration of chemotherapeutic drugs over a period can
lead to systemic malnutrition. The above findings indicate that
chemotherapy drugs had adverse reaction when they were administrated
throughout the body. It may be because that the systemic administration
of chemotherapy drugs will damage to stomach, digestive tract, liver,
and kidney, etc., resulting in loss of appetite, emaciation, and other
adverse reaction [[83]14]. After the rats were sacrificed, we compared
the ovarian weights of rats. The results displayed that the ovarian
weight of rats in the POI-C group was decreased significantly, while
the ovaries weight of rats in the POI-B, POI-U, and MS groups remained
unchanged. However, the ovaries weight of rats in the POI-B and POI-U
groups showed a declined trend (Fig. [84]2E). We further compared the
ovarian index, and the results indicated that the POI-U group showed a
highly decreased index, while other POI models had no significant
changes (Fig. [85]2F). The higher the value of the ovarian index, the
better the quality of the ootid and the better the ovarian function,
which is the best period of conception. On the contrary, the poor
quality of the ootid and the gradual decline of the ovarian function.
The results showed that POI-U animal has a huge damage to ovary
compared with other animal models, which maybe that local injections of
chemotherapy drugs have more damage to the ovaries than systemic
administration.
Fig. 2.
[86]Fig. 2
[87]Open in a new tab
Comparison of weight, ovarian index, and status of rats in four animal
models. A–D Body weight changes of POI-C, POI-B, POI-U and MS groups;
E–F Ovarian weight and index of POI-C, POI-B, POI-U and MS groups; G
Comparison of status, expression, hair, ovary, and complications of
rats in Con, POI-C, POI-B, POI-U, and MS groups
The status (RGS pain score, activity, expression, hair), complications,
and mortality of rats can indirectly reflect the effect of models. We
used the Rat Grimace Scale (RGS) scale to score the pain of the rats
[[88]15]. The higher the RGS score was, the worse the condition of the
rats were. The results showed that the RGS score of rats was 0 in the
POI-U and MS groups. However, the cumulative RGS scores of the POI-C
and POI-B groups were four and three, respectively (Table [89]2). In
addition, the rats were very active with shiny fur in the POI-U and MS
groups. However, the rats were depressed with dull fur and a
dishevelled appearance in the POI-C and POI-B groups (Fig. [90]2G). The
reason maybe that systemic administration increased pain and systemic
adverse reactions in rats.
Table 2.
The behavior and complications of different POI rat models
Group Social behavior Appearance RGS Hair Ovaries weight Complications
Death rate (%)
Orbital tightening Nose/cheek flattening Ear changes Whisker change
Control Active Normal 0 0 0 0 Smooth, lightness 0.083 ± 0.005 g / 0
POI-C model Dispirition Distress 2 1 1 0 Rough, dinginess,
0.068 ± 0.026 g Bone marrow suppression; Hepatic nodules; bleeding
(Nasal, orbital, Intestine) 37.5
POI-B model Dispirition Distress 1 1 1 0 Rough, dinginess 0.07 ± 0.28 g
Urinary retention, bleeding (Nasal, orbital, Intestine) 40
POI-U model Active Normal 0 0 0 0 Smooth, lightness 0.074 ± 0.016
Injured blood vessels 9
MS model Active Normal 0 0 0 0 Smooth, lightness 0.089 ± 0.008 g Mental
disorder (Schizophrenia or depression) 0
[91]Open in a new tab
NC3Rs: National Centre for the replacement Refinement & Reduction of
Animal in Research; POI-C (cyclophosphamide); POI-B (Busulfan); POI-U
(Ultrasound-guided cyclophosphamide injection); RGS: Rat Grimace Scale
For complications, there is a risk of bleeding at injection site in the
POI-U group. Rats in the MS group had obvious early life stress. In
addition, the rats in the POI-C and POI-B groups showed a tendency to
haemorrhage (such as nostril, orbital and intestinal haemorrhage). Some
rats in the POI-C group showed myelosuppression and hepatic nodules.
Two rats in the POI-B group had urinary retention complications.
Notably, the mortality rates of the POI-C, POI-B, POI-U and MS groups
were 0%, 37.5%, 40% and 9%, respectively. The ovarian weights of rats
in the Control, POI-C, POI-B, POI-U and MS groups were 0.080 ± 0.005 g,
0.068 ± 0.026 g, 0.07 ± 0.028 g, 0.074 ± 0.016 g and 0.089 ± 0.008 g,
respectively (Table [92]2), which may due to that Chemotherapy drugs do
more damage to the ovaries than early life stress. Above funding showed
that POI-U and MS animal had less complications and mortality, which is
a relatively safe way to construct animal models.
Comparison of ovarian follicles and hormone levels in the four animal models
Follicular development can be divided into four stages: primordial
follicles, primary follicles, secondary follicles, and mature
follicles. At primordial follicle, a single oocyte is surrounded by a
layer of flattened granulosa cells. At primary follicle, the oocyte is
surrounded by a single layer of cubic granulosa cells. At secondary
follicle, the oocyte is surrounded by large granulosa cells arrenged in
two layers, with no follicular cavity. At sinus follicles, the
follicles are further enlarged, and follicular spaces are visible.
Changes in follicles can also be evaluated as indicators of POI in
animal models. We further compared the follicular changes in the four
animal models. The results showed that the number of atretic follicles
increased significantly in the POI-C and POI-U groups, while there was
no significant change in the POI-B and MS groups (Fig. [93]3A, B). The
reason maybe that CTX is more injurious to the follicles than busulfan.
The results showed that cyclophosphamide have indeed huge damage to the
ovarian follicular.
Fig. 3.
[94]Fig. 3
[95]Open in a new tab
Comparison of ovarian follicles and hormones levels in four animal
models. A, B Comparison of rat ovarian follicles in Con, POI -C, POI-B,
POI-U and MS groups. C–K Comparison of serum AMH, FSH, LH, E2, FSH,
FSH/LH, T, GnRH, DA and PRL in Con, POI -C, POI-B, POI-U and MS groups
At present, the diagnosis of POI is mainly relies on alterations in
hormone levels. We further compared the serum levels of hormones in the
four animal models. Overall, compared with those of the control group,
serum AMH, E2, DA and PRL levels were significantly decreased in the
POI-C, POI-B and POI-U groups. Conversely, the serum levels of FSH and
LH were highly increased, and the change in the POI-U group was the
most obvious. There was no significant difference in FSH/LH, T or GnRH
levels in the four animal models (Fig. [96]3C–K). The reason may be
that the blood–brain barrier blocks the penetration of drugs into the
brain [[97]16]. GnRH secreted by the hypothalamus, FSH and LH produced
by pituitary gland remain unaffected in the POI-C, POI-B and POI-U
groups. However, compared with those of the control group, serum FSH
was decreased, while GnRH, T and DA were increased in the MS group
(Additional file [98]1: Fig. S2), which matched well with early life
stress model. MS serves as the model of early life stress, which can
lead to the schizophrenia-like phenotypes and persistent brain
abnormalities [[99]17]. In the study of MS model, there was a
significant increasing of DA level in MS rat [[100]13]. Our study of MS
model meets well with others investigations about early life stress. In
summary, the POI-C, POI-B, and POI-U animal models were successfully
constructed, while the MS model was unsuccessful.
Characterization of hUC-MSCs and exosomes
hUC-MSCs exhibit a fibroblast-like growth pattern, with cytoplasm
protruding outwards with protrusions of different lengths. After
passage, the cells demonstrate a homogeneous and swirl-like
morphology and have strong adherence. hUC-MSCs successfully induced
lipogenic and osteogenic differentiation in vitro (Fig. [101]4A). The
positive rates of CD105, CD73 and CD90 in hUC-MSCs were ≥ 90%, and
HLA-DR was negative (Fig. [102]4B). The morphology of hUC-MSC exosomes
was detected by TEM. The exosomes showed a homogeneous bilayer
structure. Zeta potential analysis showed that the exosomes were
negatively charged. The diameter of the exosomes was approximately
100 nm (Fig. [103]4C–E). The results confirm that the hUC-MSCs exosomes
have been successfully extracted and identified.
Fig. 4.
[104]Fig. 4
[105]Open in a new tab
Characterization of hUC-MSCs and exosomes. A Morphology of hUC-MSCs in
primary passage, Lipogenic and osteogenic morphology of hUC-MSCs; B
HLA-DR, CD90, CD105, and CD73 expression of hUC-MSCs via flow
cytometer; C The morphology of hUC-MSCs exosomes via TME; D Zeta
detects the charge of exosomes; E The particle size of exosomes was
detected by DLS
hUC-MSC-exosomes were injected under ultrasound guidance
Ultrasound-guided hUC-MSC exosome injection was performed by a skilled
sonographer. The ultrasound image confirmed that the needle was
successfully reached to the ovarian centre of the rat. At
post-injection, the ovarian diameter is directly increased compared
with that pre-injection state (Fig. [106]5A–D). At 2 h post-injection
of exosomes with fluorescent, the rats were sacrificed, and the ovarian
changes of rat were observed via IVIS Spectrum CT. Distinct red
fluorescence was found in the ovaries (Fig. [107]5E, F). By comparing
ovarian diameter and area, we found that the ovarian long diameter at
pre- and post-injection was 0.5433 ± 0.006667 cm and
0.6633 ± 0.01764 cm, respectively (P < 0.05). The short diameters at
pre- and post-injection were 0.2533 ± 0.008819 cm and
0.2833 ± 0.008819 cm, respectively (P > 0.05). The ovarian areas at pre
and post-injection were 0.1081 ± 0.004828 cm^2 and 0.1475 ± 0.00561
cm^2, respectively (P < 0.05) (Fig. [108]5G–K). The reason is that
drug-injection slowly diffuses in the ovary, which indicated the drug
was successfully injected into ovary of rat. The whole process of
ovarian puncture is shown in Additional file [109]3: video 2. The
ultrasound with hypoechoic shadow indicated that the drug had been
slowly injected into the ovaries, which is consistent with the criteria
of drug injection under ultrasound guidance [[110]18] The above results
indicated that our ultrasound-guided ovarian drug injection was
wonderful.
Fig. 5.
[111]Fig. 5
[112]Open in a new tab
The process of hUC-MSCs-exosomes were injected under ultrasound
guidance; A–C Imaging changes of ovaries before, during and after
ultrasound-guided injection; D Image of an ultrasound guided ovarian
injection; E, F Changes of ovarian fluorescence observed by in vivo
imager
The effect of ultrasound-guided hUC-MSC exosome injection on rats in POI-U
group
To evaluate the impact of hUC-MSC exosome injection on rats with POI,
our study also compared the variations of hormone levels in rats with
single-injection hUC-MSC exosomes (POI-e) and double-dose exosomes
(POI-2e). The results showed that compared with those of the POI-U
group, the levels of FSH, LH, T, DA, and PRL decreased, and AMH and E2
levels increased in the hUC-MSC injection groups (POI-e and POI-2e),
while there was no significant difference observed in FSH/LH or GnRH.
The reason maybe that hUC-MSC exosome can promote ovarian proliferation
and increased synthesis and secretion of steroid hormones [[113]19].
The fundings demonstrated that hUC-MSC exosome can effectively rescue
the hormone imbalances. Moreover, there was no substantial difference
in hormonal changes between the POI-e and POI-2e groups (Additional
file [114]1: Fig. S3). That may be that single intra-ovarian
administration is adequatet to achieve a therapeutic effect.
In proestrus, it mainly comprised nucleated epithelial cells
accompanied by a few keratinocytes and leukocytes. In oestrous, a
considerable number of defoliated keratinized epithelial cells and a
small proportion of nucleated epithelial cells were noted. In the
metestrus period, keratinized epithelial cells, nucleated epithelial
cells and white blood cells were observed. In the diestrus period, many
white blood cells and mucus and occasionally nuclear epithelial cells
were observed. The oestrus cycle statistics showed that the oestrus
cycle of the rats with POI mostly stayed in prooestrus and metaoestrus,
with less dioestrus. In contrast, the POI-e-treated rats spent less
time in diestrus. The number of litters was 11.9 ± 0.875, 8 ± 0.51, and
11.5 ± 1.28 in the Con, POI, and POI-U groups, respectively
(Fig. [115]6A–E). Eslami et al. [[116]20] also reported that
transplantation of cMSCs restored fertility in POI mouse models.
Altogether, these results suggested that POI-e treatment effectively
rescued ovarian function and fertility.
Fig. 6.
[117]Fig. 6
[118]Open in a new tab
The effect of ultrasound-guided hUC-MSCs exosome injection on POI-U
rats. A Representative photographs for proestrus, estrus, metestrus and
diestrus are shown (100 ×); B Duration of estrous cycle stage in Con,
POI, and POI-U groups. C HE staining of rat ovarian tissue in Con, POI,
and POI-U groups; D Changes of estrous cycle in Con, POI, and POI-U
groups. P: proestrus, E: estrous, M: metaestrus, D: Diestrus; E
Statistics of Litter number of rats; F The BCL2 and BAX mRNA expression
in Con, POI, and POI-U groups
To evaluate the impact of exosome injection on the level of ovarian
apoptosis of POI-U rats, we assessed the ovarian apoptosis levels, as
another indicator of POI in rats, in rats in the Con, POI-U and POI-e
groups. BCL2/BAX was decreased in the POI-U group compared with the Con
group. The higher the ratio of BCL2/BAX is, the stronger the
anti-apoptosis ability is. Above findings manifested that chemotherapy
drugs have a huge damage to the ovary and promote ovarian apoptosis.
These results confirmed that the POI model has been successfully
established. However, BCL2/BAX was significantly increased in the POI-e
group (Fig. [119]6F). The results showed that hUC-MSC exosomes
substantially mitigated ovarian damage in the POI-U group. Our result
is consistent with other studies that exosomes can promote ovarian
granulosa cell proliferation in ovaria-associated diseases [[120]21].
To sum up, our data demonstrated that hUC-MSC exosomes can effectively
restore the ovarian function and fertility of POI rat.
The potential mechanisms underlying hUC-MSC exosome treatment in rats with
POI
By comparing RNA-seq data from injured ovaries of the POI-U groupwith
those treated with hUC-MSC exosome, we generated volcano maps of genes
that were upregulated and downregulated post-treatment. There were 151
downregulated differentially expressed genes (DEGs) and 49 upregulated
DEGs (Fig. [121]7A). GO analysis revealed that the DEGs were enriched
in immune and metabolic pathways (Fig. [122]7B). Immune-related genes
such as Cd5, Ccr1, Cd247 and Tbx21 were highly expressed in the POI-e
group, and metabolism-related genes such as Gal1, Lcmt2, Aass, Car2,
and Aldh1a2 were upregulated in the POI-e group (Fig. [123]7C). Hence,
hUC-MSC exosomes may regulate immune and metabolic processes to improve
hormonal disorders, oestrous cycles, ovulation disorders and fertility
(Fig. [124]7D). Cao et al. [[125]22] demonstrated that Adipose
mesenchymal stem cell–derived exosome can enhance ovarian function and
reproduction of polycystic ovary syndrome via secreting cytokines in
metabolism and immunity. However, its specific mechanism needs to be
further elucidated.
Fig. 7.
[126]Fig. 7
[127]Open in a new tab
Gene analysis and mechanism of the POI-U model and hUC-MSCs exosome
treatment. A The volcano maps of upregulated and downregulated gene; B
GO pathway of DEGs; C Volcanic map of immune and metabolic pathways
from DEGs; D Schematic illustration of the mechanism underlying
hUC-MSCs exosome treatment. Note: DEGs: Differentially expressed genes
Discussion
Clinically, POI is closely related to genes, immune diseases, drugs,
surgery, and psychological factors [[128]23, [129]24]. Long-term HRT is
a common treatment strategy [[130]25]. However, clinical data show that
long-term HRT can increase the risk of breast cancer [[131]26] and
cardiovascular risk [[132]27]. In addition, HRT can only improve
patients' endometrial environment and menstrual cycle but cannot
improve AMH levels and fertility [[133]25]. Therefore, a reliable
therapy to replace HRT treatment is urgently needed.
Suitable and ideal animal models are essential carriers for drug
development and mechanism research. Our previous review summarized that
an ideal animal model would have the following characteristics: (1) the
pathogenic pathways and processes like those observed in humans; (2)
the pathological changes in the model can be reversed by drugs; (3) the
reproducibility of the results [[134]5]. To mimic the manifestations of
POI, we make full use of four animal models (POI-C, POI-B, POI-U, and
MS). We demonstrated that POI-C, POI-B, POI-U can effectively mimic the
manifestations of POI. Notably, the success rate of POI-U is increased
compared to POI-C and POI-B model. The possible reason is that local
chemotherapy injections are more toxic to the ovaries compared to
systemic injections of chemotherapy drugs. And complications were also
dramatically reduced in POI-U group. That's because local intra-ovarian
injections of chemotherapy drugs can greatly reduce their retention
levels in other organs. Moreover, POI-U requires only a single
administration and has fewer adverse reactions to other organs. The
strong tolerance of the body and high success rate of POI-U modelling
can effectively avoid the multiple organ damage, high death rate, long
cycle, complicated operation, and low efficiency caused by systemic
administration of chemotherapy drugs. This method is very suitable for
the study of chemotherapy drugs to construct a POI animal model. In the
future, ultrasound-guided drug injection technology will be more
conducive to model construction and drug therapeutic effect studies.
Therefore, we further explore the optimal drug therapy in POI-U animal
models.
MSCs, especially hUC-MSCs, have been shown to improve hormone levels
and fertility in animals with POI in numerous animal experiments since
2012 [[135]28]. Compared to other MSC resources, hUC-MSCs from
discarded umbilical cords showed enormous advantage owing to the fewer
ethical concerns, painless access, and lack of immunity [[136]29]. A
study from 2021, hUC-MSCs can restore the structure and function of
damaged ovarian tissue in chemotherapy-induced POI mice and improve
fertility. Moreover, the recovery effect of multiple hUC-MSCs
transplantation on ovarian function is better than that of single
hUC-MSCs transplantation [[137]30]. According to subsequent in-depth
research evidence, stem cells improve ovarian function due to their
paracrine action (including exosome, cytokines, and growth factors,
signalling lipids) rather than differentiation into specific cells.
Compared to hUC-MSCs, exosomes are small and easier to preserve,
penetrate the body's thick tissue barrier. Moreover, exosomes can
protect their contents from degradation [[138]29]. Exosomes are
membranous vesicles secreted by cells via the paracrine pathway and are
approximately 40–160 nm in diameter [[139]31]. Accumulating experiments
have proven that MSC exosomes play effectively therapeutic roles in
Alzheimer's disease [[140]32], bone defect repair [[141]33], polycystic
ovary syndrome [[142]34], etc. hUC-MSC exosomes have the functions of
inflammatory inhibition, immune regulation, and tissue repair. More
importantly, they have the advantages of higher safety, lower
immunogenicity and no tumorgenicity [[143]35]. Kang et al. demonstrated
that hUC-MSC exosomes can alleviate liver injury in non-alcohol related
steatohepatitis both in vivo and vitro [[144]35]. hUC-MSC exosomes can
regulate the cell–cell communication, cell signalling, and cell or
tissue metabolism [[145]29]. Feng et al. reported that hUC-MSC exosomes
can promote angiogenesis after cerebral ischaemia‒reperfusion injury
[[146]36]. However, it is unclear whether hUC-MSC exosomes have
beneficial therapeutic effects on ovarian injury and fertility in POI.
The ovary is the most important reproductive organ in women. The
ovarian physiological function and reproductive capacity are adversely
affected by follicle dysfunction or depletion in POI. In our
investigation, hUC-MSC exosomes were successfully extracted and
identified. Then, they were injected into POI animal under ultrasound
guidance. In vivo, we found that hUC-MSC exosomes can effectively
enhanced hormone levels, the oestrous cycle, ovarian function. The
therapeutic effect of hUC-MSC exosomes on POI rats was mainly due to
the improvement in local microenvironment within ovarian tissue,
including cellular vitality, inflammation, immune regulation, fibrosis,
and metabolism [[147]37]. Addressing fertility issues are the central
goal of POI treatment. Our results showed that the reproductive
function of POI rat from hUC-MSC exosomes therapy was significantly
improved, which collectively suggested that hUC-MSC exosomes showed
therapeutic effects on POI rats. However, the long-term effects of
hUC-MSC exosomes need to be evaluated, especially the progeny pregnancy
rate. At the same time, it is necessary to expand the number of animals
in further studies, and more complete results need to be confirmed in
preclinical or clinical trials. Elucidating the mechanism of hUC-MSC
exosomes-mediated therapy can ensure effective and targeted application
of exosomes. Hence, further sequencing revealed that hUC-MSC exosomes
might alleviate the symptoms of POI by regulating the ovarian immune
and metabolic environment. Of course, more preclinical studies are
needed to evaluate its efficacy and safety of hUC-MSC exosomes. Hence,
hUC-MSC exosomes may be a potential therapeutic agent for patients with
POI.
Importantly, the isolation and purification of hUC-MSC exosomes are
complex, and the amounts extracted are limited, posing it a major
challenge for clinical applications to achieve adequate amounts and
quality control of hUC-MSCs [[148]38]. Second, most hUC-MSC exosomes
are administered intravenously to reach the target organ via the homing
effect of hUC-MSC exosomes [[149]39, [150]40]. However, only a small
amount of hUC-MSC exosomes reach the treatment site owing to systemic
circulation and metabolic function, thus achieving limited therapeutic
effects. Therefore, selection of a reasonable administration mode is a
major challenge. Ultrasound has the advantages of being visual and
convenient [[151]41]. At present, ultrasound-guided in situ injection
is increasingly favoured in animal models and preclinical studies
[[152]42, [153]43]. In the ovary, local ovarian injection of
methotrexate ultrasound guided via transvaginal injection was applied
to the therapy of nontubal ectopic pregnancies [[154]44]. In our study,
we innovatively found that local ovarian injection of hUC-MSC exosomes
via abdominal ultrasound guidance can effectively treat animals with
POI and improve ovarian function.
Conclusion
In this study, we established POI-C, POI-B, POI-U and MS rat models,
among which POI-C, POI-B, POI-U models closely resembling the
manifestations of POI. By comprehensively comparing the body weight,
ovarian index, status, RGS, complications and model success rate of rat
model, we found that POI-U was the optimal animal model, which had high
success rate and low complications and mortality. Utilizing the POI-U
animal model, we successfully extracted and identified hUC-MSC exosomes
and confirmed that ultrasound-guided hUC-MSC exosomes injection can
effectively improve the oestrus cycle, hormone levels, pregnancy
outcome and ovarian apoptosis in rats with POI. Our study will become
fundamental for further clinical conduction of hUC-MSC
exosomes-medicated treatment for POI. In addition, ultrasound-guided
ovarian local injection of drugs plays a pivotal role in the
construction of animal models and hUC-MSC exosome injection. The
technique is simple to operate, can reduce the reaction of drugs to the
whole body, and achieve certain effects in a single time, which is
worthy of further promotion in animal studies. In conclusion, our data
propose a novel strategy based on hUC-MSC exosomes may be applied to
the treatment of POI disease in the future.
Supplementary Information
[155]13287_2024_3646_MOESM1_ESM.docx^ (33.5MB, docx)
Additional file 1. The procedure of cardiac extraction and serum
hormone detection. Fig 1. The procedure of taking blood from the heart.
Fig 2. Comparison of serum AMH, FSH, LH, E2, FSH, FSH/LH, T, GnRH, DA
and PRL in Con, POI -C, POI-B, POIU and POI-MS groups. Fig 3. The
comparison of serum AMH, FSH, LH, E2, FSH, FSH/LH, T, GnRH, DA and PRL
in POI -C, POI-e and POI -2e group compared with control group.
[156]Download video file^ (8.4MB, mp4)
Additional file 2. Heart extraction video.
[157]Download video file^ (23.1MB, mp4)
Additional file 3. The video of ultrasound guided in situ ovarian
puncture drug injection.
Acknowledgements