Endometrial Extracellular Vesicles From Women With Recurrent Implantation Failure Attenuate The Growth and Invasion of Embryos

ORIGINAL ARTICLES: REPRODUCTIVE SCIENCE
Endometrial extracellular vesicles

from women with recurrent
implantation failure attenuate the
growth and invasion of embryos
Chang Liu, M.D., Wen Yao, M.M., Junning Yao, M.D., Linshuang Li, M.M., Le Yang, M.D.,
Hanwang Zhang, Ph.D., and Cong Sui, M.D.
Reproductive Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology,
Wuhan, People’s Republic of China
Objective: To investigate whether endometrial extracellular vesicles (EVs) from patients with recurrent implantation failure (RIF)
attenuate the growth and invasion of embryos.
Design: In vitro experimental study.
Setting: University-affiliated hospital.
Patient(s): Ten RIF patients and seven fertile women.
Interventions(s): Endometrial cells isolated from endometrial tissues obtained from patients with RIF and fertile women were cultured
and modulated in vitro via hormones. Conditioned medium was collected for EV isolation.
Main Outcome Measure(s): EVs secreted by endometrial cells of patients with RIF (RIF-EVs) or fertile women (FER-EVs) were char-
acterized with the use of Western blotting, nanoparticle tracking analysis, and transmission electron microscopy. EVs from the two
groups were co-cultured with 2-cell murine embryos. Fluorescence-labeled EVs were used to visualize internalization by embryos.
Following co-culture, blastocyst and hatching rates were calculated. Blastocysts were stained with diamidino-2-phenylindole to
count the total cell number, and the hatched embryos were used to test invasion capacity.
Result(s): RIF-EVs and FER-EVs are bilayered vesicles 100 nm in size and enriched with TSG101, Alix, and CD9. EVs were
internalized within 12 hours. The blastocyst rates in the RIF-EV groups were significantly decreased compared with the FER-EV
groups at 5, 10, and 20 mg/mL. The hatching rates and total cell numbers of blastocysts also were decreased significantly in the
RIF-EV groups compared with the FER-EV groups at 10 and 20 mg/mL. Moreover, the invasion capacity of hatched embryos
decreased significantly in the RIF-EV group.
Conclusion(s): Endometrial EVs from patients with RIF attenuate the development and invasion of embryos. (Fertil SterilÒ 2020;114:
416–25. Ó2020 by American Society for Reproductive Medicine.)
El resumen está disponible en Español al final del artículo.
Key Words: Extracellular vesicles, recurrent implantation failure, embryonic development, implantation, maternal communication
Discuss: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/users/16110-fertility-
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E
mbryo implantation is an intri- trial luminal surface, initiating implan- two factors, the endometrium is consid-
cate physiological process. When tation. Synchronized communication ered to be more critical, because
an embryo reaches the blastocyst between a receptive endometrium and compromised endometrial factors are
stage, it migrates to the uterine cavity functional embryo is indispensable for responsible for two-thirds of implanta-
and adheres to and invades the endome- successful implantation. Among these tion failures in in vitro fertilization
(IVF) cycles (1). The endometrium not
Received January 29, 2020; revised March 27, 2020; accepted April 2, 2020; published online July 1, only provides an appropriate microenvi-
2020.
C.L. has nothing to disclose. W.Y. has nothing to disclose. J.Y. has nothing to disclose. L.L. has nothing ronment for the early development of an
to disclose. L.Y. has nothing to disclose. H.Z. has nothing to disclose. C.S. has nothing to disclose. embryo, but also acts as an active modu-
Supported by the National Natural Science Foundation of China (NSFC 81771582 and NSFC 81701450).
Reprint requests: Cong Sui, M.D., Reproductive Medicine Center, Tongji Hospital, Tongji Medicine Col- lator during implantation. It regulates
lege, Huazhong University of Science and Technology, Jiefang Avenue 1095#, Wuhan 430030, embryonic growth, attachment, adhe-
People’s Republic of China (E-mail: [email protected]).
sion, and trophoblast invasion (2).
Fertility and Sterility® Vol. 114, No. 2, August 2020 0015-0282/$36.00 Endometrial modulation of implanta-
Copyright ©2020 American Society for Reproductive Medicine, Published by Elsevier Inc. tion is conducted via multiple pathways,
https://doi.org/10.1016/j.fertnstert.2020.04.005
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such as soluble proteins, lipids, RNA moieties, and, notably, reserve, and premature ovarian failure), antiphospholipid
extracellular vesicles (EVs). antibody syndrome, or karyotype anomalies were excluded
Extracellular vesicles are bilayered membrane vehicles (3, from the RIF group. Because intrauterine abnormalities may
4). They package various molecules, such as proteins, lipids, contribute to implantation failure, RIF patients underwent
and RNAs, and transfer these cargos to recipient cells to hysteroscopy and endometrial biopsy to investigate the intra-
conduct intercellular communication (5, 6). EV-conducted uterine environment. For hysteroscopy examination, patients
communication between the maternal endometrium and the with vaginitis, Chlamydia trachomatis infection, or Myco-
conceptus has drawn much attention. Endometrially derived plasma genitalium infection were excluded. Furthermore,
EVs have been found in the uterine fluid during the window endometrial tissues were evaluated histologically for signs
of implantation (WOI) (7–9). These endometrial EVs enter of endometritis, for which they were also excluded from the
the embryonic cytoplasm and regulate its growth and study. The fertile group (n ¼ 7) included women who
implantation (7, 8, 10). conceived naturally and delivered a live birth via cesarean
Recurrent implantation failure (RIF) is defined as implan- section in the past 2 years without associated comorbidities.
tation failure after three or more consecutive transfers of These women expressed their desire to conceive with the
good-quality embryos (11–15). RIF is a major obstacle help of IVF-ET because they had previously had their fallo-
encountered with the use of artificial reproduction pian tubes ligated during cesarean section. Some clinicians
technologies (ART) (12, 16–18). Presently, the etiology of proposed that hysteroscopy and endometrial scratching
RIF remains unclear. Factors such as maternal age, should be performed before IVF-ET treatments as a means
immunologic disorders, inflammation, endometrial to improve the pregnancy outcomes (25, 26). Therefore,
receptivity, and embryo-related issues have been related to women who consented and intended to undergo hysteroscopy
RIF (16, 19, 20). The endometrial factor is a major cause of and endometrial sampling were recruited. Women in both
RIF after transfer of multiple good-quality embryos to reduce groups were under 40 years of age and had regular menstrual
embryonic issues (21–23). Many studies have investigated the cycles. Signed informed consents were obtained from all par-
discrepancies between the endometria of RIF and fertile ticipants. Endometrial tissues were obtained from biopsies
women, postulating that characterizing the normal conducted during hysteroscopy, which was performed on
endometrium as well as changes in RIF may enable day 9–11 of the menstrual cycle. Tissues were stored in ice-
associated pathologic mechanisms to be elucidated. Studies cold sterile phosphate-buffered saline (PBS) and transferred
comparing endometrial mRNA milieu between patients with to the laboratory immediately. All procedures were approved
RIF and control subjects have demonstrated that patients by the Institutional Review Board of Tongji Hospital
with RIF exhibit different endometrial gene profiles. These (TJ-IRB20190420).
altered genes influence various pathways, such as Wnt
signaling, cellular adhesion, cytoskeleton formation, cell Cell Culture
motility, and circadian rhythm (17, 21, 24). However,
Endometrial tissues were washed thrice in sterile PBS to remove
although such evidence confirms the aberrant nature of the
blood, minced into 1-mm pieces, and digested in 1 mg/mL IV
endometria of patients with RIF, the process by which such
collagenase (Servicebio) for 20 minutes at 37 C. The suspension
distortion of the endometrium and alteration of its function
was pipetted several times to disperse cells, and the reaction was
leads to implantation failure has not yet been clarified.
stopped by adding the same volume of culture medium. Both
Therefore, further studies may be required to establish a
endometrial stromal cells and epithelial cells are indispensable
direct association between the defective endometria of
for preparing the receptive endometrium for embryos to implant
patients with RIF and embryonic implantation.
(27). Therefore, both cell types were included in this study. Cell
The present study hypothesized that endometrial EVs of
suspension was filtered through a 70-mm-aperture sieve to re-
patients with RIF may differentially influence implantation
move debris, following which the endometrial cells were
compared with those of control subjects. We isolated endome-
cultured in DMEM/F12 (Boster) containing 10% fetal bovine
trial EVs from patients with RIF and fertile women and con-
serum (FBS; Sijiqing). In the WOI, endometrial cells underwent
structed a co-culture model using EVs and 2-cell murine
hormonal regulation to prepare the receptive status (1). To
embryos to investigate the regulatory role of endometrial
mimic the receptive phase, endometrial cells were treated with
EVs of women with RIF on embryonic development and
hormones as previously described (28). When cells reached
implantation.
80% confluence, medium was removed and confluent cells
were washed thrice with PBS to remove remaining FBS. The cells
MATERIALS AND METHODS were replenished with FBS-free DMEM/F12 primed with 108
Study Population and Sample Collection mol/L estrogen and 107 mol/L progesterone and treated for 2
days, and conditioned medium was collected every 24 hours
Seventeen women were recruited for the study. Patients with
for EV isolation.
RIF (n ¼ 10) were defined as women who experienced three or
more implantation failures after good-quality embryo trans-
fers. Patients presenting with uterine abnormalities, endocri- Isolation of Extracellular Vesicles
nologic disorders (including diabetes, impaired glucose EVs were isolated from the conditioned medium with the use
tolerance, insulin resistance, thyroid diseases, polycystic of a classic ultracentrifugation method (29). In brief, condi-
ovarian syndrome, hyperprolactinemia, diminished ovarian tioned medium was centrifuged at 2,000g for 20 minutes at
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ORIGINAL ARTICLE: REPRODUCTIVE SCIENCE
4 C to remove cells, followed by centrifugation at 10,000g for hours after hCG injection, pregnant mice were killed and their
30 minutes at 4 C to remove debris. The supernate was ultra- fallopian tubes collected to isolate 2-cell embryos.
centrifuged at 120,000g for 80 minutes at 4 C. The pellet was
resuspended in PBS and centrifuged at 120,000g for 80 mi- Embryo Culture
nutes at 4 C to collect EVs. The pellets were resuspended in
30 mL PBS and stored at 80 C. To investigate the effect of EVs on embryonic development,
embryos at the 2-cell stage were co-cultured with the EVs
Western Blotting from patients with RIF (RIF-EVs) or the EVs from fertile
women (FER-EVs). The concentrations of EVs were deter-
EVs or cells were rinsed in RIPA lysis buffer (Servicebio) con-
mined by means of bicinchoninic acid assay, and all EV sam-
taining a proteinase inhibitor cocktail (Servicebio) for 30 mi-
ples were adjusted to the same concentration with sterile PBS.
nutes at 4 C, and the resulting lysates were centrifuged at
EVs from the two groups were added to potassium-
12,000g for 30 minutes at 4 C. The protein concentration of
supplemented simplex-optimized medium (KSOM) to obtain
the supernate was measured with the use of a BCA Protein
final concentrations of 5, 10, and 20 mg/mL. KSOM treated
Assay Kit (Servicebio) according to the manufacturer’s proto-
with a similar volume of PBS (0 mg/mL) served as the negative
col. Protein in each sample (5 mg) was separated via sodium
control. EV-primed KSOM was divided into 50-mL droplets,
dodecyl sulfate–polyacrylamide gel electrophoresis and
plated on culture dishes, and covered with mineral oil. Each
transferred to a polyvinylidene fluoride membrane (Milli-
droplet contained five to six embryos from at least three
pore). After blocking with 5% skimmed milk, the membrane
different mice. The embryos were cultured in a 5% CO2/95%
was incubated with the antibodies CD9 (AF5139; Affinity;
air atmosphere at 37 C and 100% humidity for 75 hours.
1:1,000), Calnexin (Abcam; 1:1000), Alix (DF9027; Affinity;
Following co-culture, the numbers of initial 2-cell embryos,
1:1,000), and TSG101 (A2216; Abclonal; 1:1,000) at 4 C over-
blastocysts, and hatched embryos were recorded. Blastocyst
night, followed by incubation with secondary antibody (anti–
rate was calculated as the number of blastocysts (including
rabbit IgG horseradish peroxidase–linked antibody 7074S;
hatched embryos) divided by the total number of embryos,
CST; 1:1,000) for 1 hour at 37 C. Membranes were then
per treatment, and the hatching rate was defined as the num-
immersed in enhanced chemiluminescence substrate
ber of hatched embryos divided by the total number of em-
(abs920; Absin) and signals detected with the use of a Gene
bryos, per treatment.
Gnome XRQ chemiluminescence imaging system (Syngene).
Transmission Electron Microscopy Endocytosis of EVs by Murine Embryos

A 10-mL aliquot of fresh EVs was placed on a carbon-coated A 100-mL aliquot of EVs or PBS (negative control) was incu-
electron microscopy grid and extra fluid removed. EV samples bated with a membrane green dye, 3,30 -dioctadecyloxacarbo-
were then negatively stained with the use of 2% uranyl ace- cyanine perchlorate (DIO; Beyotime) according to the
tate. After dyeing, EV-attached grids were observed under a manufacturer’s instructions. After incubation, dyed EVs (or
Hitachi HT7700 transmission electron microscope (TEM). PBS) were resuspended in 26 mL PBS and ultracentrifuged
at 120,000g for 80 minutes to remove extra dye. Green
Nanoparticle Tracking Analysis fluorescence-labeled EVs (or PBS) were added to KSOM drop-
To characterize the diameter and distribution of EV samples, lets and co-cultured with the embryos for 12 hours under the
nanoparticle tracking analysis (NTA) was performed with same culture conditions as those used for embryo culture. The
the use of NanoSight (LM10) according to the manufacturer’s treated embryos were observed under a fluorescence micro-
instructions. EV samples were diluted with the use of distilled scope (Axio Observer A1; Carl Zeiss).
water at a ratio of 1:500 to acquire the concentration recom-
mended for the instrument. Each experiment was performed Development Evaluation and Invasion Assay of
in triplicate. Embryos
After co-culturing, unhatched blastocysts were aspirated
Animals and Treatment from the droplets, fixed in 4% paraformaldehyde, and dyed
All animal experiments were approved by the Institutional with diamidino-2-phenylindole (DAPI; Servicebio), following
Review Board of Tongji Hospital (TJ-A20190301). Fifty fe- which the total cell number of embryos were counted with the
male and six male Institute of Cancer Research mice, which use of a confocal microscope (Eclipse ci; Nikon). Hatched em-
were 7 weeks old, were purchased from the Animal Center bryos were placed on confluent Ishikawa cells for 24 hours to
of Tongji Hospital. The mice were maintained under 12- determine invasion capacity. Following co-culture, embryos
hour light-dark cycle conditions and had ad libitum access were observed with the use of a microscope and the invasion
to food and water. After a week of acclimation, female mice capacity was assessed according to a plate movement proto-
were injected with 10 IU pregnant mare’s serum gonadotropin col with modifications (30). Specifically, the plate was moved
(Sigma-Aldrich) intraperitoneally, followed by 10 IU hCG rapidly several times laterally and orthogonally, and tapped
(Sigma-Aldrich) 48 hours later. Superovulated mice were laterally. When moving or tapping the plate, embryos
mated with males at a ratio of 2:1, and the vaginal plug floating, rolling, or displaying loose contact with endometrial
was checked the next morning. The presence of the vaginal cells were considered to be uninvaded embryos. Whereas, em-
plug was considered to be a sign of pregnancy. Twenty-six bryos with no movements when the plate was moved or
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FIGURE 1
Isolation of EVs and determination of RIF-EVs and FER-EVs. (A) Western blotting shows that RIF-EVs and FER-EVs expressed classic EV protein
markers Alix, TSG101, and CD9. Representative shapes of (B) RIF-EVs and (C) FER-EVs detected with the use of transmission electron
microscopy. The size and distribution of (D) RIF-EVs and (E) FER-EVs examined with the use of nanoparticle tracking analysis. ECs ¼
endometrial cells; EVs ¼ extracellular vesicles; FER ¼ fertile women; RIF ¼ women with recurrent implantation failure.
Liu. Extracellular vesicles regulate embryos. Fertil Steril 2020.
tapped were considered to be invaded embryos. The invasion were evaluated via TEM and NTA, respectively. RIF-EVs and
rate was defined as the percentage of invaded embryos per to- FER-EVs were largely 100 nm in size (Figs. 1D and 1E) and ap-
tal number of embryos. peared as bilayers (Figs. 1B and 1C).
Statistical Analysis Internalization of EVs by Murine Embryos

Quantitative variables were expressed as mean SD, and the Internalization of EVs by murine embryos was visualized with
Student t test was performed to evaluate statistical signifi- the use of fluorescence-labeled EVs. Two-cell embryos were
cance. Chi-square tests followed by Bonferroni tests were co-cultured with labeled RIF-EVs, FER-EVs, or PBS (control)
used to determine significance of the blastocyst rate, hatching for 12 hours, and representative images of embryos from three
rate, and invasion rate. Statistical significance was set at different treatments arre shown in Figure 2A. Green fluores-
P< .05. cence signals in the RIF-EV– or FER-EV–treated embryos
were clear, whereas they were hardly visible in the control
RESULTS group. Undetectable fluorescence signals around the zona
pellucida excluded the possibility that EVs had adhered to
Characterization of Endometrial EVs
the membrane. These results confirmed that both RIF-EVs
The classic EV protein markers, Alix, TSG101, and CD9, were and FER-EVs were internalized by embryos.
detected in RIF-EVs and FER-EVs. Endometrial cell lysates
from RIF patients or fertile women served as control samples.
Images showed that these EV markers were enriched in RIF-EVs Attenuate the Development of Embryos
RIF-EVs and FER-EVs, whereas the endoplasmic reticulum– In RIF-EV–treated groups, the blastocyst rates in the 5 mg/mL
specific protein calnexin was almost undetectable in EV (35%), 10 mg/mL (38.1%), and 20 mg/mL (32.9%) groups were
samples (Fig. 1A). The morphology and distribution of EVs reduced compared with the 0 mg/mL group (51.6%). However,
VOL. 114 NO.

there was no significant difference between them. Although women with RIF are altered (17, 32–34). Therefore, it may
the blastocyst rates in the FER-EV–treated groups showed be hypothesized that differential molecular profiles are
an increasing trend with increasing concentration, no signif- responsible for inadequate endometrial receptivity in
icant difference was found. When RIF-EV and FER-EV groups patients with RIF. However, the possibility of dynamic
were compared, the blastocyst rates in FER-EV groups were communication between the endometrium and embryos has
significantly increased at concentrations of 5, 10, and 20 not been addressed. Therefore, to investigate whether
mg/mL (Fig. 2B). distorted endometria of patients with RIF interfered with
Hatching rates in the RIF-EV–treated groups decreased implantation or embryonic development, a co-culture model
with increasing concentration (Fig. 2C). It was significantly was constructed with the use of endometrial EVs and murine
decreased in the 20 mg/mL group (1.4%) compared with the embryos, which were previously confirmed to be an ideal
0 mg/mL group (22.6%). Hatching rates in the FER-EV–treated model for the functional investigation of endometrial EVs
groups increased with increasing concentration, although the of human origin (35). First, endometrial cells were isolated
differences were not significant. A comparison of data from from endometrial tissues and treated with hormones to mimic
both treatments indicated that the hatching rates in the the receptive phase during the WOI (28, 36). Via in vitro mod-
FER-EV groups were significantly increased at 10 mg/mL ulation, discrepancies between endocrinologic milieu among
(36.7%) and 20 mg/mL (43.3%). the population may be avoided. EVs were isolated from condi-
Total cell number in blastocysts is an indicator of embryo tioned media with the use of the use of classic ultracentrifu-
viability. It is used in the evaluation of embryonic develop- gation methods (29). EVs were marked with green
ment (31). Representative images of differently treated em- fluorescence to visualize internalization in embryos. Both
bryos are shown in Figure 3A. In RIF-EV groups, the total RIF-EVs and FER-EVs entered embryonic cytoplasm within
cell numbers in blastocysts were significantly decreased in 12 hours. These results confirmed that endometrial regulation
the 10 and 20 mg/mL groups compared with the 0 mg/mL plays a role in embryonic development via the EV pathway.
group (Fig. 3B). Total cell numbers were also significantly Moreover, our data indicated that internalization of EV took
decreased in the 10 and 20 mg/mL groups compared with less time than the 24–72 hours previously reported (7, 10).
the 5 mg/mL group. Total cell numbers in the FER-EV groups The regulatory role of EVs was then explored via a co-
increased in a dose-dependent manner and was increased culture experiment. The results showed that hatching rates
significantly in the 5, 10, and 20 mg/mL group compared and total cell numbers in a blastocyst were reduced in RIF-
with the 0 mg/mL group. When all of the data were compared, EV–treated embryos in a concentration-dependent manner.
the total cell numbers in the RIF-EV and FER-EV groups were The blastocyst rate was also decreased in the RIF-EV groups;
significantly different at concentrations of 10 and 20 mg/mL. however, a linear relationship was not observed. We assumed
that the reason for this phenomenon was that the formation of
RIF-EVs Decreased Embryonic Invasion Capacity blastocysts was a continuous process and generally charac-
terized by the formation of a cavity (37). The first small cavity
To evaluate the invasion capacity of embryos treated with could be observed at an early stage (at 20 5 cells), contin-
EVs, hatched embryos were aspirated from medium droplets uously increasing in size thereafter until reaching late blasto-
and placed on confluent Ishikawa cells. After incubating for cyst stage (104 cells) (37). Thus, the presence of a cavity
24 hours, embryos were observed under a microscope. indicates blastocyst formation, but it does not imply that blas-
Invasion-negative embryos either floated in the medium or tocysts are experiencing the same growth rate, which is
were weakly attached to the cell confluence. These could confirmed by the phenomenon that the total cell numbers
not be clearly visualized with the use of a phase-contrast mi- of blastocysts in the RIF-EV groups decreased with increasing
croscope because they were located at different planes relative concentration. Moreover, embryonic invasion capacity was
to cell confluence (Fig. 3C). Invading embryos were stably significantly down-regulated in embryos treated with RIF-
attached to the cell confluence and displayed outgrowth EVs. With the attenuated development and invasion in the
(Fig. 3D). Embryos hatched from 10 mg/mL RIF-EVs and 10 RIF-EV–treated embryos, we assumed that the contents in
mg/mL FER-EVs subgroups were compared for invasion ca- RIF-EVs might be responsible for the reduced growth poten-
pacity. The invasion rate was 30% in the RIF-EV group and tial of embryos in the RIF-EV groups. Moreover, the total
73.7% in the FER-EV group (Table 1). There was a significant cell numbers in the blastocysts of the RIF-EV groups was
difference between the two groups (P< .05). dose dependent, whereas no such trend was observed in the
FER-EV groups. This phenomenon also supported our
DISCUSSION assumption as stated above. Therefore, clarification of dis-
Recurrent implantation failure is a multifactorial anomaly in crepancies in the contents of RIF-EVs and FER-EVs was
ART, which affects many infertile women. The etiology of RIF considered to be important. Among all molecules in the
has been intensively studied. Although the detailed mecha- EVs, RNAs are considered to be potent mediators of the EV-
nism has not yet been clarified, many studies have reported directed communication, not only as a consequence of their
that endometrial factor is a major cause. Many researchers abundance in EVs (38), but also as a result of their reported
have attempted to identify the differences between the endo- functionality in recipient cells, even across species (5, 39).
metria of women with RIF and fertile women, and have EVs are classified into three types on the basis of biogen-
demonstrated that the protein, mRNA, microRNA (miRNA), esis and size. According to our data, endometrial EVs may be
and long noncoding RNA profiles in the endometria of exosomes, ranging from 30 to 150 nm in size (40, 41).
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FIGURE 2
RIF-EVs attenuated the development of embryos. (A) Images of DIO-labeled RIF-EVs, FER-EVs or controls internalized by embryos. The left panel
shows green fluorescence signals of labeled RIF-EVs, FER-EVs or control; the middle panel shows images of treated embryos at bright field; and
the right panel shows merged images. Embryos were treated with different concentration of RIF-EVs or FER-EVs respectively. Blastocyst rate (B)
and hatching rate (C) were calculated in each treatment. The number of initial 2-cell embryos, blastocysts and hatched embryos in each
treatment were indicated in the figures. Data were analyzed by Chi-square tests and Bonferroni test. #Data were significantly different between
the linked groups (P<.05); N.S.no significance. DIO ¼ 3,30 -dioctadecyloxacarbocyanine perchlorate; FER-EVs ¼ extracellular vesicles from fertile
women; RIF-EVs ¼ extracellular vesicles from women with recurrent implantation failure.
Although the packaging mechanism of exosomes has not nected alterations in RIF patients’ endometria reported in the
been illustrated, it has been reported that the RNA profiles literature with dynamic early maternal communication via
of EVs are similar to those of the cells from which these EVs the EV pathway.
originated (5, 42). Besides, Kim et al. proposed that exosomes However, the above assumptions are limited by being
may function as tools in maintaining the homeostasis of based on the precondition that endometrial EVs carry con-
cellular RNA expression (43). In this regard, differently ex- tents that are similar to those of the cells from which they
pressed RNA molecules in the endometria of RIF patients originate. Some exceptions to this have been reported. For
may be delivered to embryonic cells via EVs. Revel et al. example, Ng et al. reported that exosomes/microvesicles con-
reported that miR-145 was up-regulated in the secretory tained specific miRNAs (45). A similar observation was re-
endometria of RIF patients (32). In addition, overexpressed ported for exosomal mRNAs. Ratajczak et al. demonstrated
miR-145 in trophoblasts impeded their migrating and inva- that mRNAs of several pluripotent transcription factors
sion capacity (44). This indicates that EV-based transfer of were enriched in embryonic stem cell microvesicles compared
miR-145, which is highly enriched in the endometria of RIF with those in the embryonic stem cells themselves (46). In
patients, may exert a negative effect on trophoblasts. Accord- addition, Skog et al. reported that 4,700 different mRNAs
ing to this hypothesis, molecules up-regulated in the endome- were exclusive to microvesicles (47). Furthermore, instead
tria of RIF patients and negatively regulated trophoblasts may of random secretion, a selective packaging mechanism ap-
serve as candidate elements in RIF-EVs. Thus, our study con- pears to underlie the secretion of EV contents. Accordingly,
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FIGURE 3
RIF-EVs inhibit the growth of blastocyst and invasion of embryos. Blastocysts treated with different doses of RIF-EVs and FER-EVs were fixed and
dyed with DAPI and the total cell numbers of blastocysts were counted. (A) Representative confocal microscopy images of dyed blastocysts are
shown. (B) The total cell numbers of blastocysts were compared in different treatments. Representative phase-contrast microscopy images of
(C) an invasion-negative embryo and (D) an invaded embryo. Black arrows indicate the embryos. *Significantly different (P<.05) from the
respective control group (0 mg/mL); asignificantly different from respective dose in FER-EVs group (P<.05); &data were significantly different
between the two linked groups (P<.05). DAPI ¼ diamidino-2-phenylindole; FER-EVs ¼ extracellular vesicles from fertile women; RIF-EVs ¼
extracellular vesicles from women with recurrent implantation failure.
the contents in RIF-EVs are difficult to predict, and therefore, pathogenesis of implantation failure. Further studies will be
the mechanisms underlying the negative regulation of embry- needed to determine the differences between the contents of
onic development may require further exploration. Moreover, RIF-EVs and FER-EVs, and to illustrate detailed mechanisms
because our EVs were isolated from a conditioned medium underlying such differences.
in vitro, these EVs may exhibit subtle differences compared
with EVs secreted in vivo. In summary, this study indicates
that EVs isolated from the endometria of women with RIF TABLE 1
attenuate the development of embryos, thereby providing
The effects of extracellular vesicles on the invasion of embryos.
new insight into the pathophysiology of RIF.
Variable RIF-EVs FER-EVs
No. of invaded embryos 3 14
CONCLUSION Total no. of embryos 10 19
Invasion rate, % 30 73.7*
Considering all of the factors evaluated in the present study, it Note: Invasion rate was defined as the number of invaded embryos divided by the total
is proposed that RIF-EVs attenuate embryonic development number of embryos per treatment. Data were analyzed by means of the chi-square test
and statistical significance set at P< .05. FER-EVs ¼ extracellular vesicles from fertile women;
by inhibiting blastocyst formation, decreasing the total cell RIF-EVs ¼ extracellular vesicles from women with recurrent implantation failure.
* P< .05.
number of embryos as well as embryonic invasion capacity.
Such dysregulation in embryos may be associated with the
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tant in Animal Center of Tongji Hospital, for the immense tential marker pathways in the endometrium that may cause recurrent im-
plantation failure. Reprod Sci 2019;26:879–90.
help provided during the animal experiments.
22. Dhaenens L, Lierman S, De Clerck L, Govaert E, Deforce D, Tilleman K, et al.
Endometrial stromal cell proteome mapping in repeated implantation failure
and recurrent pregnancy loss cases and fertile women. Reprod Biomed On-
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424
by Elsevier
Las vesículas extracelulares endometriales de mujeres con fallo recurrente de implantaci uan el crecimiento y la invasi
on aten on de los
embriones.
Objetivo: investigar si las vesículas extracelulares (EVs) endometriales de pacientes con fallos recurrentes de implantaci
on (RIF)
aten
uan el crecimiento y la invasi
on de los embriones.
~o: Estudio experimental in vitro.
Disen
Lugar: Hospital afiliado a la universidad.
Paciente (s): Diez pacientes con RIF y siete mujeres fertiles.
Intervenciones: las celulas endometriales aisladas de tejidos endometriales obtenidos de pacientes con RIF y mujeres fertiles fueron
cultivadas y moduladas in vitro mediante hormonas. El medio acondicionado se recogi o para el aislamiento VE.
Principales medidas de resultado: las VE secretadas por las celulas endometriales de pacientes con RIF (RIF-VE) o mujeres fertiles
(FER-VE) fueron caracterizadas con el uso de Western blot, analisis de seguimiento de nanopartículas y microscopía electr onica de
transmision. Las VE de los dos grupos se cultivaron conjuntamente con embriones murinos de 2 celulas. Las VE marcadas con fluores-
cencia se usaron para visualizar la internalizacion de los embriones. Despues del co-cultivo, se calcularon las tasas de blastocisto y
eclosi
on. Los blastocistos se ti~
neron con diamidino-2-fenilindol para contar el n
umero total de celulas, y los embriones eclosionados
se usaron para evaluar la capacidad de invasi on.
Resultado (s): RIF-VE y FER-VE son vesículas bicapa de 100 nm de tama~ no y enriquecidas con TSG101, Alix y CD9. Las VE se in-
ternalizaron dentro de las 12 horas. Las tasas de blastocisto en los grupos RIF-VE disminuyeron significativamente en comparaci on con
los grupos FER-VE a 5, 10 y 20 mg / ml. Las tasas de eclosi on y el numero total de celulas de los blastocistos tambien disminuyeron
significativamente en los grupos RIF-VE en comparacion con los grupos FER-VE a 10 y 20 mg / ml. Ademas, la capacidad de invasi on
de los embriones eclosionados disminuy o significativamente en el grupo RIF-VE.
Conclusion (es): las VE endometriales de pacientes con RIF aten
uan el desarrollo y la invasi
on de embriones.
VOL. 114 NO.


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ORIGINAL ARTICLES: REPRODUCTIVE SCIENCE

Endometrial extracellular vesicles

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Transmission Electron Microscopy Endocytosis of EVs by Murine Embryos

Statistical Analysis Internalization of EVs by Murine Embryos

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