Human umbilical cord mesenchymal stem cells improve the reserve function of perimenopausal ovary via a paracrine mechanism
Li et al. Stem Cell Research & Therapy
Human umbilical cord mesenchymal stem cells improve the reserve function of perimenopausal ovary via a paracrine mechanism
Jia Li 0
QiuXian Mao 0
Zhi Zhang 1
ChunYan Yin 0
0 Department of Obstetrics and Gynecology, Guangdong No.2 Provincial People's Hospital , NO.466 Xingangdong Road, Guangzhou 510317 , China
1 Department of Laboratory Medicine, Guangdong No.2 Provincial People's Hospital , NO.466 Xingangdong Road, Guangzhou 510317 , China
Background: Human umbilical cord mesenchymal stem cells (hUCMSCs) are a type of pluripotent stem cell which are isolated from the umbilical cord of newborns. hUCMSCs have great therapeutic potential. We designed this experimental study in order to investigate whether the transplantation of hUCMSCs can improve the ovarian reserve function of perimenopausal rats and delay ovarian senescence. Method: We selected naturally aging rats confirmed by vaginal smears as models of perimenopausal rats, divided into the control group and the treatment group, and selected young fertile female rats as normal controls. hUCMSCs were transplanted into rats of the treatment group through tail veins. Enzyme-linked immunosorbent assay (ELISA) detected serum levels of sex hormones, H&E staining showed ovarian tissue structure and allowed follicle counting, immunohistochemistry and western blot analysis revealed ovarian expression of hepatocyte growth factor (HGF), vascular endothelial cell growth factor (VEGF), and insulin-like growth factor-1 (IGF-1), polymerase chain reaction (PCR) and western blot analysis revealed hUCMSCs expression of HGF, VEGF, and IGF-1. Results: At time points of 14, 21, and 28 days after hUCMSCs transplantation, estradiol (E2) and anti-Müllerian hormone (AMH) increased while follicle-stimulating hormone (FSH) decreased; ovarian structure improved and follicle number increased; ovarian expression of HGF, VEGF, and IGF-1 protein elevated significantly. Meanwhile, PCR and western blot analysis indicated hUCMSCs have the capacity of secreting HGF, VEGF, and IGF-1 cytokines. Conclusions: Our results suggest that hUCMSCs can promote ovarian expression of HGF, VEGF, and IGF-1 through secreting those cytokines, resulting in improving ovarian reserve function and withstanding ovarian senescence.
hUCMSCs; Perimenopausal; Ovarian reserve function; HGF; VEGF; IGF-1; Paracrine
Menopause is the permanent termination of
menstruation because of loss of ovarian follicular activity.
Perimenopause means the coming of menopause, which
manifests itself as menstrual irregularity and vasomotor
symptoms, it ends in the 12 months after the final
menstrual period . Most women experience
perimenopause classically between the ages of 45 and 55 years .
Perimenopause can last for several years or even
decades, and brings many perimenopausal symptoms such
as hot flushes, vaginal atrophy, osteoporosis, depression,
etc. [3, 4]. Currently, hormone replacement therapy
(HRT), symptomatic supporting treatment, and
treatment with phytoestrogens or herbal remedies are the
three main treatments of perimenopausal symptoms.
However, HRT may have a long-term effect on
increasing the risk of breast cancer, endometrial cancer, and
ovarian cancer; symptomatic supporting treatment cures
the symptoms, not the disease; and treatment with
phytoestrogens or herbal remedies lacks data on the
mechanism and long-term safety [1, 4]. The delay of
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
childbearing as an important social change has led to an
increasing number of women desiring late menopause,
and the improvement of the quality of life means women
want to avoid the trouble of perimenopausal symptoms
and to slow down the rapidity of ovarian aging. In
addition, follicles have limited numbers. Several million
non-growing follicles (NGFs) are established by the
ovary at around 5 months of gestational age, then
decline to approximately 1000 when the menopause starts,
and are finally exhausted through atresia and apoptosis
after 12–14 years of menopause [5, 6]. Therefore, how to
make the best use of NGFs, delay ovarian senescence,
and cure perimenopausal syndrome fundamentally are a
serious problem in today’s society.
Human mesenchymal stem cells (MSCs) have
attracted great interest recently, due to their huge
therapeutic potential. Human umbilical cord mesenchymal
stem cells (hUCMSCs) are obtained directly from the
Wharton’s jelly of a human umbilical cord, and are also
called human Wharton’s jelly mesenchymal stem cells
(WJ-MSCs). Fewer ethical issues, being obtained
painlessly from abandoned umbilical cord, and being
hypoimmunogenic are the prominent advantages of
hUCMSCs compared to other sources of MSCs . The
ability to modulate immune responses makes hUCMSCs
an important stem cell source for allogeneic
transplantation therapy without immunological rejection .
Troyer and Weiss  concluded that there was no
evidence for direct immunological rejection of
undifferentiated hUCMSCs in vivo and they would be accepted well
in allogeneic transplantation. Besides, Gong et al. 
discovered that there were no signs of immunologic
response and no evidence in the dosage escalation and
frequencies of hUCMSCs used in patients. Furthermore,
hUCMSCs have multipotent stem cell characteristics,
which can differentiate into multiple lineages under
different differentiation conditions . Some studies also
showed [12, 13] that hUCMSCs differentiated into
oocyte-like structures and expressed both mRNA and
protein of germ cell-specific markers. hUCMSCs
increased the proliferation of damaged human endometrial
stromal cells (ESCs) and decreased the apoptosis
percentage significantly when cultured with them. Yang et
al.  suggested that hUCMSCs may restore
endometrial damage through secreting vascular endothelial
growth factor (VEGF) and anti-apoptosis. Zhu et al. 
confirmed that hUCMSCs transplantation could restore
ovaries damaged by chemotherapy in rats. This
experiment was lately verified by Song et al. . They all
proposed that the improved ovarian function in
premature ovarian failure (POF) rat model was more
likely due to the cytokines produced by hUCMSCs
via a paracrine mechanism rather than directly
differentiating to germ cells.
Early follicle-stimulating hormone (FSH) was a main
endocrine feature of perimenopause, reported by
Sherman and Korenman in 1975 for the first time ,
and has been used since the 1990s as a biomarker of
reproductive potential . Mean FSH levels between the
earliest menopausal phase of every definition showed
statistically significant differences, and serum estradiol
(E2) levels decrease and FSH levels increase with
growing age in midlife women . Therefore, Gracia et al.
 suggested that delicate changes in blood may be
helpful in recognizing the earliest hormonal changes
during the transition to menopause. However, the
increase of FSH levels only happens around 10 years
before the menopause in which infertility perhaps starts.
Thus, a markedly raised FSH is considered a relatively
late predictor for menopausal transition .
AntiMüllerian hormone (AMH), as a member of the
transforming growth factor beta (TGF-β) family, can affect
the transition from NGFs to growing follicles. Hence it
is viewed currently as the best available predictor of
ovarian reserve . AMH levels remain relatively stable
over the menstrual cycle; therefore, measurement does
not need to be conducted on a specific cycle day. In
such a way, AMH has the advantage over FSH .
Individual AMH serum level reflects the size of the antral
follicles pool, representing the quantity of NGFs
accurately . Since AMH production decrease is in accord
with the age-related decline in the number of antral
follicles, AMH levels can be used as a label for ovarian
Based on the precedents discussed above, we designed
this experiment to investigate the therapeutic potential
of hUCMSCs using perimenopausal rats. We established
the perimenopausal sample by selecting naturally aging
rats, confirmed by vaginal smears and the level of serum
hormone. The sample is in accordance with the status of
perimenopausal women. In addition, we chose serum
level of E2, AMH, and FSH as the evaluation index of
ovarian reserve function. Our study aimed to identify
the potential of hUCMSCs in perimenopausal treatment
and the mechanism involved.
The naturally aging female Sprague-Dawley rats (SPF
class, weight 410–450 g, 12–14 months old) and young
female Sprague-Dawley rats with fertility (SPF class, weight
280–320 g, 3–5 months old) were provided by Guangdong
Medical Laboratory Animal Center (Foshan City, China).
They were bred at a temperature of 30 ± 2 °C with a
12hour light/dark cycle. Vaginal smears of rats were taken to
determine estrous cycle at 11:00 am daily. Only aging rats
with disorganized estrous cycles and young rats with
normal estrous cycles were chosen [27, 28]. Before the
experiment, 1 ml of blood was gathered from each rat’s
orbital during dioestrus. We centrifuged blood samples
for 10 minutes at 2000 rpm after 45 minutes’ standing and
reserved the upper serum at -80 °C. Initial levels of rat E2,
FSH, and AMH were accessed for each serum sample.
Aging rats were distributed into the control group and the
treatment group (n = 15 per group) randomly. Young rats
were set as the normal control group (n = 15).
Identification of hUCMSCs phenotype
The P1 generation cell lines of hUCMSCs were obtained
from ChongQing HuaYa Stemcell Technology
Corporation (ChongQing, China). hUCMSCs were isolated from
human umbilical cords of newborns. Maternal blood
passed the etiological examination, proving that they were
not infected by treponema pallidum, hepatitis B virus, and
human immunodeficiency virus. Flow cytometry was used
to identify the phenotype of hUCMSCs.
CD73(eBioscience, Inc., San Diego, CA, USA, 12-0739-41), CD90
(eBioscience, 11-0909-41), CD105 (eBioscience,
12-105741), CD14 (eBioscience, 11-0149-41), CD34 (eBioscience,
12-0349-41), CD45 (eBioscience, 11-0459-41), CD79a
(eBioscience, 12-0792-41) and HLA-DR (eBioscience,
119952-41) monoclonal antibodies were used for detection.
Mouse IgG monoclonal antibody was used as negative
control. Cells at a concentration of 2 × 106 cells/ml were
incubated with 5 ul antibodies (for each) at 4 °C for 30
minutes and were analyzed by flow cytometer (Beckman
Coulter, Brea, CA, USA, MoFlo Astrios EQ).
hUCMSCs were cultured in complete medium (DEME/
F12 with 10% FBS and 1% penicillin-streptomycin) until
they fulfilled the quantity. And then the P3 generation of
hUCMSCs was suspended in phosphate-buffered saline
(PBS) at a concentration of 1 × 106 cells/ml. The
hUCMSCs suspension was injected into the treatment
group via the tail vein (1 ml per rat). After 48 hours, the
treatment group was again injected with 1 ml hUCMSCs
suspension. To avoid cell clusters causing rats
thrombosis, cell suspension should be adequately scattered
before injection. The control group was injected with 1 ml
PBS per rat via the tail vein at the same time. No
treatment was administered to the normal control group. At
14, 21, and 28 days after the final cell transplantation,
five rats from each group were selected randomly and
euthanized. Blood was taken from orbits and ovaries
were removed for analysis.
Measurement of rats’ E2, FSH, and AMH levels
Blood samples were standing for 45 minutes and
centrifuged for 10 minutes at 2000 rpm. The supernatant
serum was collected and reserved at -80 °C. Levels of
sera E2, FSH, and AMH were measured by
enzymelinked immunosorbent assay (ELISA) kit E2 (Cusabio
Biotech Co., Ltd, Wuhan, China, CSB-E05110r), FSH
(CUSABIO, CSB-E06869r), and AMH (CUSABIO,
H&E staining showed ovarian tissue structure and allowed
Left ovaries were removed for fixation in 10% formalin
to prepare the paraffin sections. Each section was 3 um
thick. Sections were stained with hematoxylin and eosin
(H&E) and observed under a light microscope. The
number of follicles at different stages was counted
according to definition. Primordial follicles were defined as
an oocyte surrounded by a single fusiform granule cell.
Primary follicles were an oocyte surrounded by a single
layer of cuboidal granulosa cells. Secondary follicles were
surrounded by six to eight layers of cuboidal granulosa
cells, with no visible antrum. Antral possessed a clearly
defined antral space .
PCR analysis hUCMSCs HGF, VEGF, and IGF-1 mRNA
The P3 generation of hUCMSCs was cultured in culture
dishes until they grew to 90% confluence. After that,
culture media were abandoned and cells were washed in
PBS three times. The total RNA of the cells was
extracted using E.Z.N.A.Total RNA Kit I (Omega Bio-tech,
Inc., Norcross, GA, USA, R6834-01). Before RNA was
transcribed, DNase I (Beyotime, Shanghai, China,
D7076) had been added to remove the genome DNA.
Ultraviolet spectrophotometry was used to determinate
the concentration of total RNA. RNA was transcribed
reservedly to cDNA using PrimeScript™ 1st Strand
cDNA Synthesis Kit (Takara Bio Inc., Kyoto, Japan,
6110A), and cDNA was amplified by Premix Taq™
version 2.0 plus dye kit (Takara Bio USA Inc., Mountain
View, CA, USA, RR901Q). Distilled water was used to
replace cDNA as the blank control of quality when
amplified. Human hepatocyte growth factor (HGF), VEGF
and insulin-like growth factor-1 (IGF-1) primer (both
from Sangon Biotech, Shanghai, China) was designed
according to GeneBank, primer sequences are as below.
HGF: forward (5′-GCAATTAAAACATGCGCTGA-3′),
reverse (5′-TGGAATTTGGGAGCAGTAGC-3′), overall
length is 269 bp; VEGF: forward (5′-GGGGAGGAG
GAAGAAGAGAA-3′), reverse (5′-GTGGAGGTAGAG
CAGCAAGG-3′), overall length is 315 bp; IGF-1:
forward (5′-AGGGTATGGCTCCAGCAGTC-3′), reverse
(5′-GAGGGGTGCGCAATACATCT-3′), overall length
is 106 bp. 5 ul purpose gene amplification product was
taken for electrophoresis on 2% agarose gel. Observation
under the gel imaging analysis system was conducted,
with photographs taken.
Western blot analysis of hUCMSCs expression of HGF,
VEGF, and IGF-1
The P3 generation of hUCMSCs was cultured in
culture dishes until they grew to 90% confluence. After
that, the culture media was abandoned and cells
were washed in PBS three times. The total protein
was extracted from the cells using a total protein
extraction kit (Nanjing Keygen Biotech Co. Ltd,
Nanjing, China, KGP250) and the protein content
was measured by BCA protein quantitation assay kit
(Keygen Biotech, KGP904). Protein was taken
according to the quantitation for electrophoresis on 12%
SDS-PAGE gel. We used 5% skimmed milk with
TBST and closed PVDF membrane after
transmembrane. The primary antibodies used HGF (1:1500
dilution; ab83760; Abcam, Cambridge, MA, USA),
VEGF (1:1200 dilution; ab46154; Abcam), IGF-1
(1:600 dilution; ab176523; Abcam) for the reaction at
4 °C overnight. The second antibodies used
HRPconjugated Affinipure Goat Anti-Mouse IgG (H + L)
or HRP-conjugated Affinipure Goat Anti-rabbit IgG
(H + L) (1:8000 dilution; SA00001-1 or SA00001-2;
Proteintech, Wuhan, China) for the reaction at room
temperature for 1 hour. Chemiluminescence used the
Tanon 5200 analysis system (Tanon Science &
Technology Co Ltd., Shanghai, China).
Immunohistochemistry analysis of ovarian expression of
HGF, VEGF, and IGF-1
The ovary sections were immunohistochemically stained
for HGF, VEGF, and IGF-1 antibodies in order to explore
the cytokine expression in ovaries after hUCMSCs
transplantation. Antigen was retrieved by microwave antigen
retrieval in sodium citrate buffer (pH6.0).
Immunohistochemistry (IHC) assay used the S-P method (ZSGB-Bio,
Beijing, China, SP-9000-D). The primary antibodies
HGF (1:300 dilution; ab83760; Abcam), VEGF (1:300
dilution; ab46154; Abcam) and IGF-1 (1:50 dilution;
ab176523; Abcam) were used for the reaction at 4 °C
overnight. The primary antibody was replaced by PBS as
the negative control. DAB kit (ZSGB-Bio, ZLI-9017) was
used for staining. All sections were observed and
semiquantitatively analyzed under a light microscope by two
pathologists independently. The immunoreactive score
(IRS) is according to their staining intensity and positive
range at high magnification (×400). Staining intensity
was divided into four levels (negative, weak,
intermediate, strong), and given 0–3 scores respectively. The
positive range was divided into five levels and given 0–4
scores respectively according to the positive cells’
percentage of the microscopic view (0–10%, 11–25%, 26–
50%, 51–75%, or 76–100%). Ten view-zones were chosen
for each section, which avoided overlaps on observation.
Fig. 1 Flow cytometry analysis of phenotype characterization of hUCMSCs. Phenotype of CD73, CD90, CD105, CD14, CD34, CD45, CD79a and
HLA-DR of hUCMSCs was detected by flow cytometry. Intensity ≥ 95% represented strong expression while ≤2% represented low or
The score of staining intensity multiplying the score of
the positive range decides the score of a view zone and
the average score of ten view zones determines the score
of a staining section.
Western blot analysis of ovarian expression of HGF, VEGF,
Right ovaries were washed in saline and stored at -80 °C.
Each ovary’s total protein was extracted for the
Fig. 2 Serum hormone level analysis. Levels of E2 (a), AMH (b), and
FSH (c) were measured by ELISA at 0 (when the perimenopausal
model was established), 14, 21, and 28 days after hUCMSCs
transplantation. *P < 0.05 vs control group. AMH anti-Müllerian
hormone, E2 estradiol, FSH follicle-stimulating hormone
Table 1 The number of primordial, primary, secondary, and
antral follicles at 14 days after transplantation
28.40 ± 4.15* 13.80 ± 3.56* 12.60 ± 3.91
Normal control 34.60 ± 6.07* 16.00 ± 5.05* 14.40 ± 4.22* 8.20 ± 1.79*
Data are shown as mean ± SD. (n = 5)
*P < 0.05 vs. control group
expression of HGF, VEGF, and IGF-1 assay by western
blotting. The experiment method was the same as the
cells’ western blot assay method. The Tanon 5200
Chemiluminescence Imaging System was used to analyze
image intensity. The standardization ratio of target
protein intensity to GAPDH (1:8000 dilution; 3777R-100;
BioVision, Inc., Milpitas, CA, USA) intensity was viewed
as the reference index of expression quantity.
All data were analyzed using IBM Statistical Program
for Social Sciences 20.0 (IBM Corp., Armonk, NY,
USA). The results were shown as the mean ±
standard deviation. Data from the same group at different
time points and data from multiple groups at the
same time point were analyzed by one-way ANOVA
test. The contrast between two groups used Students’
t test. P values were considered significant when less
Phenotype characterization of hUCMSCs
Flow cytometry test results showed that hUCMSCs
express the specific markers of mesenchymal cell such as
CD73, CD90, CD105 (≥95%) while they do not express
CD14, CD34, CD45, CD79a and HLA-DR (≤2%) (Fig. 1).
Table 2 The number of primordial, primary, secondary, and
antral follicles at 21 days after transplantation
26.80 ± 2.77* 12.00 ± 1.22* 10.60 ± 2.70* 4.60 ± 1.14*
Normal control 33.00 ± 7.75* 16.40 ± 4.62* 14.60 ± 4.04* 8.40 ± 1.67*
Data are shown as mean ± SD. (n = 5)
*P < 0.05 vs. control group
Data are shown as mean ± SD. (n = 5)
*P < 0.05 vs. control group
13.80 ± 2.77* 10.60 ± 1.82* 5.00 ± 1.00*
hUCMSCs transplantation does not cause rats’ graft
There was no difference between the three groups of rats
in their mental status, diet, hair color, and activity after
transplantation. Moreover, there were no deaths, bleeding,
hemiplegia, convulsions, and other graft rejection
reactions in the treatment group. This implies that hUCMSCs
transplantation is safe for Sprague-Dawley rats.
hUCMSCs transplantation improves rats’ ovarian reserve
Before hUCMSCs transplantation, rats’ serum E2 and
AMH level of the control group and the treatment
group significantly decreased, while FSH increased
compared to the normal control group. On the other
Table 3 The number of primordial, primary, secondary, and
antral follicles at 28 days after transplantation
Normal control 35.00 ± 5.57* 17.00 ± 3.74* 15.20 ± 3.77* 8.20 ± 1.92*
Fig. 3 H&E staining analysis of ovarian structures. a Control group. b Treatment group at 14 days after hUCMSCs transplantation. c Treatment
group at 21 days after hUCMSCs transplantation. d Treatment group at 28 days after hUCMSCs transplantation. e Normal control group. Scale
bar = 500 um
Fig. 4 Analysis of hUCMSCs mRNA expression and protein
expression. a Gel imaging analysis of hUCMSCs mRNA expression.
Lanes 1–4 were respectively for HGF, VEGF, IGF-1 mRNA, and blank
control of quality. b Western blot images of HGF, VEGF, and IGF-1
protein expression of hUCMSCs. HGF hepatocyte growth factor, IGF-1
insulin-like growth factor-1, VEGF vascular endothelial cell
hand, there was no significant difference between the
two perimenopausal groups. After hUCMSCs
transplantation, however, rats’ serum E2 and AMH of the
treatment group increased and FSH decreased. This
tendency has clear differences compared with the
control group (Fig. 2; P < 0.05), yet has no significant
difference between each time period of 14, 21, and 28
days after hUCMSCs treatment. Rats’ sera hormone
level of the normal control group has no change at
each time point.
hUCMSCs transplantation improves rats’ ovary structure
and follicle counting
Primordial, primary, secondary, and antral follicles were
classified and counted according to the previous
description and definition (Tables 1, 2, and 3). In the normal
control group, histomorphology of normal ovaries were
observed (Fig. 3e). Conversely, in the control group, we
found that ovarian stromal showed densification, the
number of follicles at different periods were decreased
(Fig. 3a). Expectedly, in the treatment group,
developmental condition of follicles tended to improve, the
number of ovarian follicles was increased (Fig.3b, c, d).
The forms of rats’ ovarian tissue at different time points
after transplantation were similar in the treatment
hUCMSCs can secrete HGF,VEGF, and IGF-1
Polymerase chain reaction (PCR) results suggested that
hUCMSCs expressed HGF, VEGF, and IGF-1 mRNA
(Fig. 4a). Meanwhile, western blot results suggested that
Fig. 5 Immunohistochemistry (IHC) analysis for cytokines HGF, VEGF, and IGF-1 in ovaries after hUCMSCs transplantation. Images for control group
treatment group and normal control group at three time points are listed. A brown-yellow coloring of the cytoplasm of the cells was deemed to
be positive staining. It can be seen that treatment with hUCMSCs enhanced HGF, VEGF, and IGF-1 expression in ovaries. Scale bar = 200 um. HGF
hepatocyte growth factor, IGF-1 insulin-like growth factor-1, VEGF vascular endothelial cell growth factor
hUCMSCs expressed HGF, VEGF, and IGF-1 protein
(Fig. 4b). Western blot results were in accordance with
PCR results, proving that hUCMSCs can secrete HGF,
VEGF, and IGF-1 cytokines.
HGF, VEGF, and IGF-1 expression in rats’ ovaries were
improved after hUCMSCs transplantation
We found that HGF, VEGF, and IGF-1 were massively
detected in rats’ ovarian granulosa cells, theca cells, and
stromal cells through IHC. In addition, the expression of
HGF, VEGF, and IGF-1 in the normal control group was
frequently observed, while the control group showed a
lower trend. After hUCMSCs transplantation, however,
HGF, VEGF, and IGF-1 protein expression apparently
rose compared to the control group (Fig. 5). The IRS of
the three groups was statistically different (Tables 4, 5
and 6). This was later confirmed by western blot assay
(Fig. 6). The standardization ratio of protein expression
of HGF, VEGF, and IGF-1 in the treatment group and
the normal control group significantly showed that
ovarian expression of HGF, VEGF, and IGF-1 proteins of
perimenopausal rats tended to be close to young rats
after hUCMSCs treatment, compared to the control
While many researchers focus attention on the
treatment of POF rats using adipose-derived mesenchymal
stem cells (A-MSCs) , bone marrow mesenchymal
stem cells (BMSCs) , and hUCMSCs [15, 16], our
study focuses on the treatment of perimenopausal rats
using hUCMSCs. In our study, the serum level of E2 and
AMH decreased while FSH increased when the
perimenopausal group of rats was selected. After hUCMSCs
transplantation, we detected the serum index of E2,
AMH, and FSH again and observed ovary tissue
structures. The results suggested that hUCMSCs
transplantation improved ovarian reserve function of
perimenopausal rats. Although hUCMSCs have the
ability to differentiate into germ cells, modern theories tend
to deem [15, 16] that hUCMSCs do not change to
oocyte-like structures or cells in vivo. Additionally,
Table 5 Comparison of the IHC scores for VEGF
2.90 ± 0.89* 2.78 ± 0.45* 3.02 ± 0.38* 0.190
Normal control 3.06 ± 0.62* 3.12 ± 0.54* 2.98 ± 0.66* 0.067
hUCMSCs labelled with fluorochrome mainly
concentrated on the ovarian stroma, not on the ovarian follicle.
Follicle development needs the support of the vascular
network in the ovary, hence inadequate ovarian stroma
vessels may lead to a decline of oocyte quality with aging
. The development of vascular networks in the theca
cell layer of the follicle is induced by angiogenic
cytokines. In the ovary, the angiogenic factors produced by
granulosa cells help to maintain the vasculature and
health of the dominant follicles [33, 34]. A research
study reported  that increasing age along with the
reduction of ovarian stromal blood flow is a relatively
late phenomenon, occurring only in women aged ≥41
years. As for angiogenic cytokines, VEGF is an effective
mitogen for vascular endothelium  and it also
stimulates vascular permeability . Improving VEGF
expression during the follicular stage may be helpful in
increasing ovarian angiogenesis and the number of
predominant follicles doomed for ovulation [38–41].
Beyond that, VEGF is a powerful survival factor for ovarian
granulosa cell apoptosis and ovarian follicular atresia
[42, 43]. Apart from VEGF, HGF is an important
element of the internal follicular environment that accelerates
the viability of growing follicles and enhances the
proliferation of ovarian surface epithelium in order to
replenish the area damaged due to expulsion of the ovum
during ovulation [44, 45]. HGF, expressed both in thecal
cells and granulosa cells of rat ovaries, may play its
function as a modulator of the mesenchymal-epithelial cell
reciprocities between theca cells and granulosa by
facilitating cell proliferation and steroid hormone production
. A complete HGF system also supports granulosa
2.92 ± 0.53* 3.30 ± 0.42* 3.10 ± 0.22* 1.080
Table 4 Comparison of the IHC scores for HGF
Table 6 Comparison of the IHC scores for IGF-1
2.20 ± 0.45* 2.12 ± 0.33* 2.10 ± 0.20* 0.118
Normal control 3.04 ± 0.25* 3.12 ± 0.16* 3.12 ± 0.16* 0.274
Normal control 2.64 ± 0.30* 2.66 ± 0.28* 2.52 ± 0.24* 0.377
Fig. 6 Images (a) and analysis of ovarian expression of HGF (b), VEGF (c), and IGF-1 (d) protein by western blot at different time points after hUCMSCs
transplantation. GAPDH expression was used as internal reference. The protein expression in the control group was normalized as 1. Relative ratio of
treatment group or normal control group to control group was viewed as the index of protein expression quantity. Data were shown in mean ± SD.
*P < 0.05 vs. control group. HGF hepatocyte growth factor, IGF-1 insulin-like growth factor-1, VEGF vascular endothelial cell growth factor
cells growing via an anti-apoptotic effect . Besides,
as for other important cytokines in the ovary, IGF-1 is
expressed in growing granulosa cells and healthy
follicles, which cannot be detected in atretic follicles 
and is necessary for the proliferation of granulosa cells
at the early stage of folliculogenesis . According to
studies [50, 51], ovarian IGF-1 expression stimulates
progesterone and estradiol production, and enhances
granulosa cell FSH reactivity by improving FSH receptor
expression. A study reported  that serum and
follicular fluid levels of IGF-1 are decreased in reproductive
aging women aged 40–45 years compared to young
women aged 20–25 years. Thus, IGF-1 plays a crucial
role in follicular development. In addition, a study
suggested  that the amount of hUCMSCs in the rat
ovarian tissue was basically constant without obvious
proliferation for at least 8 weeks. This conforms to the
results of our study; both serum hormone concentration
and ovarian expression of HGF, VEGF, and IGF-1
protein remaining stable to 28 days after hUCMSCs
hUCMSCs can secrete cytokines such as VEGF, HGF,
and IGF-1. In addition, the ovarian expression of VEGF,
HGF, and IGF-1 distinctly increased after hUCMSCs
transplantation into perimenopausal rats via the tail vein.
The results implied that hUCMSCs transplantation
improved ovarian reserve function of perimenopausal
rats through a paracrine mechanism.
Through this study we suggest that hUCMSCs may be
localized to the ovarian stroma and secreted cytokines
after transplantation, affecting ovaries via a paracrine
mechanism persisting at least 28 days. We can consider
that hUCMSCs have therapeutic effects for
perimenopausal rats to improve ovarian reserve function via a
paracrine mechanism. However, based on these results,
the time should be extended to more than 28 days to
understand the timeliness of the therapeutic effects of
hUCMSCs transplantation. Furthermore, whether the
fertility of perimenopausal rats could be recovered after
hUCMSCs transplantation needs further observation
and testing. We hope this study can provide a theoretical
foundation for perimenopausal treatment.
AMH: Anti-Müllerian hormone; A-MSCs: Adipose-derived mesenchymal stem
cells; BMSCs: Bone marrow mesenchymal stem cells; E2: Estradiol;
ELISA: Enzyme-linked immunosorbent assay; ESCs: Endometrial stromal cells;
FSH: Follicle-stimulating hormone; H&E: hematoxylin and eosin;
HGF: Hepatocyte growth factor; HRT: Hormone replacement therapy;
hUCMSCs: Human umbilical cord mesenchymal stem cells; IGF-1: Insulin-like
growth factor-1; IHC: Immunohistochemistry; IRS: Immunoreactive score;
MSCs: human mesenchymal stem cells; NGF: Non-growing follicles;
PBS: Phosphate-buffered saline; PCR: Polymerase chain reaction;
POF: Premature ovarian failure; TGF-β: Transforming growth factor beta;
VEGF: Vascular endothelial cell growth factor; WJ-MSCs: Human Wharton’s
jelly mesenchymal stem cells
JL contributed to the experimental operation, manuscript writing, and
literature review. QXM was responsible for animal modeling. JJH and HQS
were responsible for data gathering and analysis. ZZ and CYY contributed to
funding support and final editing of the manuscript content. All authors read
and approved the final manuscript.
Ethics approval and consent to participate
Our investigation using experimental animals was conducted on the basis of
the GuangDong Medical Laboratory Animal Center’s specific guidelines and
This study was approved by the ethics committee of Guangdong No.2
Provincial People’s Hospital (GD2H-QR-KJ-018).
1. Rees M. Management of the menopause: integrated health-care pathway for the menopausal woman . Menopause Int . 2011 ; 17 : 50 - 4 .
2. Brotherston J. Contraception meets HRT: seeking optimal management of the perimenopause . Brit J Gen Pract . 2015 ; 65 : e630 - 2 .
3. Weber MT , Maki PM , McDermott MP . Cognition and mood in perimenopause: A systematic review and meta-analysis . J Steroid Biochem Mol Biol . 2014 ; 142 : 90 - 8 .
4. Ortmann O , Dören M , Windler E. Hormone therapy in perimenopause and postmenopause (HT) . Arch Gynecol Obstet . 2011 ; 284 : 343 - 55 .
5. Richardson SJ , Senikas V , Nelson JF . Follicular depletion during the menopausal transition: evidence for accelerated loss and ultimate exhaustion . J Clin Endocrinol Metab . 1987 ; 65 : 1231 - 7 .
6. Faddy MJ , Gosden RG , Gougeon A , Richardson SJ , Nelson JF . Accelerated disappearance of ovarian follicles in mid-life: implications for forecasting menopause . Hum Reprod . 1992 ; 7 : 1342 - 6 .
7. Bongso A , Fong C. The therapeutic potential, challenges and future clinical directions of stem cells from the Wharton's jelly of the human umbilical cord . Stem Cell Rev . 2013 ; 9 : 226 - 40 .
8. Fong C , Chak L , Biswas A , Tan J , Gauthaman K , Chan W , Bongso A. Human Wharton's jelly stem cells have unique transcriptome profiles compared to human embryonic stem cells and other mesenchymal stem cells . Stem Cell Rev . 2011 ; 7 : 1 - 16 .
9. Troyer DL , Weiss ML . Concise review: Wharton's jelly-derived cells are a primitive stromal cell population . Stem Cells . 2008 ; 26 : 591 - 9 .
10. Gong W , Han Z , Zhao H , Wang Y , Wang J , Zhong J , Wang B , Wang S , Wang Y , Sun L , Han Z. Banking human umbilical cord-derived mesenchymal stromal cells for clinical use . Cell Transplant . 2012 ; 21 : 207 - 16 .
11. Karahuseyinoglu S , Cinar O , Kilic E , Kara F , Akay GG , Demiralp DÖ , Tukun A , Uckan D , Can A. Biology of stem cells in human umbilical cord stroma: in situ and in vitro surveys . Stem Cells . 2007 ; 25 : 319 - 31 .
12. Qiu P , Bai Y , Pan S , Li W , Liu W , Hua J. Gender depended potentiality of differentiation of human umbilical cord mesenchymal stem cells into oocyte-Like cells in vitro . Cell Biochem Funct . 2013 ; 31 : 365 - 73 .
13. Huang P , Lin LM , Wu XY , Tang QL , Feng XY , Lin GY , Lin X , Wang HW , Huang TH , Ma L. Differentiation of human umbilical cord Wharton's jelly-derived mesenchymal stem cells into germ-like cells in vitro . J Cell Biochem . 2010 ; 109 : 747 - 54 .
14. Yang X , Zhang M , Zhang Y , Li W , Yang B. Mesenchymal stem cells derived from Wharton jelly of the human umbilical cord ameliorate damage to human endometrial stromal cells . Fertil Steril . 2011 ; 96 : 1029 - 36 .
15. Zhu SF , Hu HB , Xu HY , Fu XF , Peng DX , Su WY , He YL . Human umbilical cord mesenchymal stem cell transplantation restores damaged ovaries . J Cell Mol Med . 2015 ; 19 : 2108 - 17 .
16. Song D , Zhong Y , Qian C , Zou Q , Ou J , Shi Y , et al. Human umbilical cord mesenchymal stem cells therapy in cyclophosphamide-induced premature ovarian failure rat model . Biomed Res Int . 2016 ; 2016 : 1 - 13 .
17. Sherman BM , Korenman SG . Hormonal characteristics of the human menstrual cycle throughout reproductive life . J Clin Invest . 1975 ; 55 : 699 - 706 .
18. Steiner A. Biomarkers of ovarian reserve as predictors of reproductive potential . Semin Reprod Med . 2013 ; 31 : 437 - 42 .
19. Randolph JF , Sowers M , Bondarenko IV , Harlow SD , Luborsky JL , Little RJ . Change in estradiol and follicle-stimulating hormone across the early menopausal transition: effects of ethnicity and age . J Clin Endocrinol Metab . 2004 ; 89 : 1555 - 61 .
20. Gracia CR , Sammel MD , Freeman EW , Lin H , Langan E , Kapoor S , Nelson DB. Defining menopause status: creation of a new definition to identify the early changes of the menopausal transition . Menopause . 2015 ; 12 : 128 - 35 .
21. van Montfrans JM , Hoek A , van Hooff MH , de Koning CH , Tonch N , Lambalk CB . Predictive value of basal follicle-stimulating hormone concentrations in a general subfertility population . Fertil Steril . 2000 ; 74 : 97 - 103 .
22. Sowers MR , Eyvazzadeh AD , McConnell D , Yosef M , Jannausch ML , Zhang D , Harlow S , Randolph JJ. Anti-mullerian hormone and inhibin B in the definition of ovarian aging and the menopause transition . J Clin Endocrinol Metab . 2008 ; 93 : 3478 - 83 .
23. van Rooij IA , Broekmans FJ , Te Velde ER , Fauser BC , Bancsi LF , de Jong FH , Themmen AP . Serum anti-Müllerian hormone levels: a novel measure of ovarian reserve . Hum Reprod . 2002 ; 17 : 3065 - 71 .
24. Broer SL , Broekmans FJM , Laven JSE , Fauser BCJM . Anti-Mullerian hormone: ovarian reserve testing and its potential clinical implications . Hum Reprod Update . 2014 ; 20 : 688 - 701 .
25. van Rooij IA , Tonkelaar I , Broekmans FJ , Looman CW , Scheffer GJ , de Jong FH , Themmen AP , Te VE . Anti-mullerian hormone is a promising predictor for the occurrence of the menopausal transition . Menopause . 2004 ; 11 : 601 - 6 .
26. van Rooij IAJ , Broekmans FJM , Scheffer GJ , Looman CWN , Habbema JDF , de Jong FH , Fauser BJCM , Themmen APN , Te Velde ER . Serum antimüllerian hormone levels best reflect the reproductive decline with age in normal women with proven fertility: A longitudinal study . Fertil Steril . 2005 ; 83 : 979 - 87 .
27. Ishii M , Yamauchi T , Matsumoto K , Watanabe G , Taya K , Chatani F. Maternal age and reproductive function in female Sprague-Dawley rats . J Toxicol Sci . 2012 ; 37 : 631 - 8 .
28. Marcondes F , Bianchi F , Tanno A. Determination of the estrous cycle phases of rats: some helpful consideration . Braz J Biol . 2002 ; 62 : 609 - 14 .
29. Myers M , Britt KL , Wreford NGM , Ebling FJP , Kerr JB . Methods for quantifying follicular numbers within the mouse ovary . Reproduction . 2004 ; 127 : 569 - 80 .
30. Takehara Y , Yabuuchi A , Ezoe K , Kuroda T , Yamadera R. The restorative effects of adipose-derived mesenchymal stem cells on damaged ovarian function . Lab Invest . 2012 ; 93 : 181 - 93 .
31. Fu X , He Y , Xie C , Liu W. Bone marrow mesenchymal stem cell transplantation improves ovarian function and structure in rats with chemotherapy-induced ovarian damage . Cytotherapy . 2008 ; 10 : 353 - 63 .
32. Tatone C , Amicarelli F , Carbone MC , Monteleone P , Caserta D , Marci R , Artini PG , Piomboni P , Focarelli R. Cellular and molecular aspects of ovarian follicle ageing . Hum Reprod Update . 2008 ; 14 : 131 - 42 .
33. Shimizu T , Jiang JY , Sasada H , Sato E. Changes of Messenger RNA Expression of angiogenic factors and related receptors during follicular development in gilts . Biol Reprod . 2002 ; 67 : 1846 - 52 .
34. Lam PM , Haines C. Vascular endothelial growth factor plays more than an angiogenic role in the female reproductive system . Fertil Steril . 2005 ; 84 : 1775 - 8 .
35. Ng EH , Chan CC , Yeung WS , Ho PC . Effect of age on ovarian stromal flow measured by three-dimensional ultrasound with power Doppler in Chinese women with proven fertility . Hum Reprod . 2004 ; 19 : 2132 - 7 .
36. Leung DW , Cachianes G , Kuang WJ , Goeddel DV , Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen . Science . 1989 ; 246 : 1306 - 9 .
37. Senger DR , Galli SJ , Dvorak AM , Perruzzi CA , Harvey VS , Dvorak HF . Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid . Science . 1983 ; 219 : 983 - 5 .
38. Danforth DR , Arbogast LK , Ghosh S , Dickerman A , Rofagha R , Friedman CI . Vascular endothelial growth factor stimulates preantral follicle growth in the rat ovary . Biol Reprod . 2002 ; 68 : 1736 - 41 .
39. Shimizu T , Jiang JY , Iijima K , Miyabayashi K , Ogawa Y , Sasada H , Sato E. Induction of follicular development by direct single injection of vascular endothelial growth factor gene fragments into the ovary of miniature gilts . Biol Reprod . 2003 ; 69 : 1388 - 93 .
40. Wulff C , Wilson H , Wiegand SJ , Rudge JS , Fraser HM . Prevention of thecal angiogenesis, antral follicular growth, and ovulation in the primate by treatment with vascular endothelial growth factor trap R1R2 . Endocrinology . 2002 ; 143 : 2797 - 807 .
41. Iijima K , Jiang JY , Shimizu T , Sasada H , Sato E. Acceleration of follicular development by administration of vascular endothelial growth factor in cycling Female Rats . J Reprod Dev . 2005 ; 51 : 161 - 8 .
42. Kosaka N , Sudo N , Miyamoto A , Shimizu T. Vascular endothelial growth factor (VEGF) suppresses ovarian granulosa cell apoptosis in vitro . Biochem Biophys Res Commun . 2007 ; 363 : 733 - 7 .
43. Quintana R , Kopcow L , Marconi G , Sueldo C , Speranza G , Barañao RI . Relationship of ovarian stimulation response with vascular endothelial growth factor and degree of granulosa cell apoptosis . Hum Reprod . 2001 ; 16 : 1814 - 8 .
44. Gutman G , Barak V , Maslovitz S , Amit A , Lessing JB , Geva E. Regulation of vascular endothelial growth factor-A and its soluble receptor sFlt-1 by luteinizing hormone in vivo: implication for ovarian follicle angiogenesis . Fertil Steril . 2008 ; 89 : 922 - 6 .
45. Zachow RJ , Weitsman SR , Magoffin DA . Hepatocyte growth factor regulates ovarian theca-interstitial cell differentiation and androgen production . Endocrinology . 1997 ; 138 : 691 - 7 .
46. Ito M , Harada T , Tanikawa M , Fujii A , Shiota G , Terakawa N. Hepatocyte growth factor and stem cell factor involvement in paracrine interplays of theca and granulosa cells in the human ovary . Fertil Steril . 2001 ; 75 : 973 - 9 .
47. Uzumcu M , Pan Z , Chu Y , Kuhn PE , Zachow R. Immunolocalization of the hepatocyte growth factor (HGF) system in the rat ovary and the antiapoptotic effect of HGF in rat ovarian granulosa cells in vitro . Reproduction . 2006 ; 132 : 291 - 9 .
48. Oliver JE , Aitman TJ , Powell JF , Wilson CA , Clayton RN . Insulin-like growth factor I gene expression in the rat ovary is confined to the granulosa cells of developing follicles . Endocrinology . 1989 ; 124 : 2671 - 9 .
49. Kadakia R , Arraztoa JA , Bondy C , Zhou J. Granulosa cell proliferation is impaired in the Igf1 null ovary . Growth Horm IGF Res . 2001 ; 11 : 220 - 4 .
50. Taketani T , Yamagata Y , Takasaki A , Matsuoka A , Tamura H , Sugino N. Effects of growth hormone and insulin-like growth factor 1 on progesterone production in human luteinized granulosa cells . Fertil Steril . 2008 ; 90 : 744 - 8 .
51. Zhou J , Kumar TR , Matzuk MM , Bondy C. Insulin-like growth factor I regulates gonadotropin responsiveness in the murine ovary . Mol Endocrinol . 1997 ; 11 : 1924 - 33 .
52. Klein NA , Battaglia DE , Miller PB , Branigan EF , Giudice LC , Soules MR . Ovarian follicular development and the follicular fluid hormones and growth factors in normal women of advanced reproductive age . J Clin Endocrinol Metab . 1996 ; 81 : 1946 - 51 .