Morphological, Ultrastructural, and Molecular Aspects of In Vitro
Mouse Embryo Implantation on Human Endometrial Mesenchymal
Stromal Cells in The Presence of Steroid Hormones as An
This experimental study aimed to evaluate the effects of 17β-estradiol (E2) and progesterone (P4) on the interaction
between mouse embryo and human endometrial mesenchymal stromal cells, and gene expressions related to implantation
Materials and Methods
In this experimental study, the endometrial stromal cells were isolated enzymatically and
mechanically, and cultured to the fourth passage. Next, their immunophenotype was confirmed by flow cytometric
analysis as mesenchymal stromal cells. The cells were cultured as either the experimental group in the presence of E2
(0.3 nmol) and P4 (63.5 nmol) or control group without any hormone treatment. Mouse blastocysts were co-cultured
with endometrial mesenchymal stromal cells in both groups for 48 hours. Their interaction was assessed under an
inverted microscope and scanning electron microscopy (SEM). Expressions of
Similar observations were seen in both groups by light microscopy and SEM. We observed the presence of
pinopode-like structures and cell secretions on the apical surfaces of endometrial mesenchymal stromal cells in both
groups. The trophoblastic cells expanded and interacted with the mesenchymal monolayer cells. At the molecular
level, expression of
This study has shown that co-culture of endometrial mesenchymal stromal cells with mouse embryo in
media that contained E2 (0.3 nmol) and P4 (63.5 nmol) could effectively increase the expression of
Implantation is a complex process that involves fine coordination and dialogue between the embryo and endometrium (1). Embryonic development to the blastocyst stage and uterine differentiation to the receptive phase are both essential for initiation and progression of a successful implantation (2). The process of implantation consists of apposition, adhesion, and the invasion of the blastocyst to the uterine wall (3).
In addition to the physical interaction between the
embryo and uterine cells, this process is influenced by
maternal steroidal hormones, growth factors, and cytokines
in a paracrine manner that play a vital role in embryonic
signaling (4). Uterine differentiation to support embryo
implantation is coordinated by progesterone (P4) and
17ß-estradiol (E2) (5, 6). In mice and rats both maternal
P4 and E2 are critical to implantation. However, in most
species such as hamsters, rabbits, and pigs, implantation
can occur in the presence of P4 alone (7). The implantation
process involves different factors and proteins such as
leukemia inhibitory factor (
The highest level of
Integrins are a family of transmembrane glycoproteins
with two subunits, a and ß. They act as receptors for
extracellular matrix components and other cells (16).
Integrin expressions increase in the phase of receptivity
of the endometrium and are considered markers of the
implantation window (9). The cycle-specific expression
patterns of endometrial integrins indicate their hormonal
regulation (17). These proteins are expressed on the
endometrium and the blastocyst. The human blastocyst
Ethical restrictions and experimental limitations prevent
direct evaluation of interactions between the embryo and
endometrium at the morphological and molecular levels.
So, the application of
Our previous studies demonstrated that passage-4 endometrial mesenchymal stromal cells expressed typical markers of mesenchymal stromal stem cells. They could differentiate into different cell lines (21, 22). According to our knowledge, there is scant information about the establishment of implantation models using endometrial stromal cells. Recently, Fayazi et al. (23) showed that the CD146+ endometrial mesenchymal cells could differentiate to endometrial epithelial-like cells. However, in this study, the researchers did not evaluate the interaction of these epithelial-like cells with embryos.
Ovarian hormones have critical roles during embryo
implantation. These hormones regulate the specific
gene products that may play important roles in embryo
implantation (24). The profile of genes expression in rodents
and human endometrium using
In our recent pilot study, we examined the effects
of different dosages of E2 (0.3, 0.7, and 1 nmol) in
combination with P4 (63.5 nmol) on the proliferation
and survival rate of human endometrial stromal cells.
Our data showed that 0.3 nmol of E2 with 63.5 nmol of
P4 had a significantly higher proliferation rate than the
other examined dosages of E2. By using 0.3 nmol of E2
with 63.5 nmol of P4 in another part of this experiment,
our molecular observation demonstrated that despite any
significant difference in expression of
According to the role of implantation models to facilitate
evaluation of the implantation process, the present study
aimed to determine the effects of E2 (0.3 nmol) and P4
(63.5 nmol) on the interaction between mouse embryo
and human endometrial mesenchymal cells, and the gene
expressions related to implantation (
Materials and Methods
Reagents and materials of this research were obtained from Sigma Aldrich (Munich, Germany), unless mentioned otherwise.
Human endometrial samples
The Ethics Committee of the Medical Faculty of Tarbiat Modares University (no. 1394.137) approved this experimental study. Written informed consent was taken from all patients. The endometrial samples were obtained from healthy fertile women aged 25-35 years (n=10) during the proliferative phase who underwent hysteroscopy for non-pathological conditions. The patients did not have any exogenous hormone treatment for 3 months before the surgery. The normal morphology and normal menstrual cycle of the endometrial tissue was proven by histological examination and confirmed by an experienced histopathologist.
Cell isolation and culture
The tissues were washed in phosphate-buffered saline (PBS), cut into small 1 mm pieces in Dulbecco’s modified Eagle’s medium/Hams F-12 (DMEM/F-12, Invitrogen, UK) that contained 100 mg/ml penicillin G sodium, 100 mg/ml streptomycin sulfate B, and 10% fetal bovine serum (FBS, Invitrogen, UK). The tissues were then subjected to mild enzymatic digestion according to a method by Chan et al. (28). Collagenase type 1 (300 µg/ ml) and deoxyribonuclease type I (40 µg/ml) were used to digest the tissue fragments into single cells along with the mechanical methods. In order to remove glandular and epithelial components, the resulting suspension were passed through 100 and 40 sieve meshes (Becton Dickinson, USA). Finally, endometrial stromal cells were cultured to the fourth passage using DMEM/F-12 that contained antibiotics and 10% FBS, and incubated at 37°C in 5% CO2.
Flow cytometric analysis of endometrial cells
After the fourth passage, we confirmed the immunophenotype of the endometrial cells using flow cytometric analysis to evaluate mesenchymal (CD90, CD73, and CD44) and hematopoietic markers (CD45 and CD34). A total of 1×105 endometrial cells were suspended in 50 µl of PBS and incubated with direct fluorescein isothiocyanate (FITC)-conjugated antibodies (anti-human CD90, CD44, and CD45, 1:50 dilutions) and direct phycoerythrin (PE)-conjugated antibodies (antihuman CD73 and CD34; 1:50 dilutions) at 4°C for 45 minutes. Finally, 200 µl of PBS was added and the cells were examined with a FACSCalibur apparatus (Becton Dickinson, USA).
Preparation of the media and cell culture
After the fourth passage, the mesenchymal stromal cells were collected and divided into two groups, experimental and control. The cells were cultured in the presence of 0.3 nmol E2 and 63.5 nmol P4 (27) (Aburaihan, Iran) in the experimental group. The cells were cultured in the absence of any hormone treatment in the control group.
In order to prepare an initial concentration, E2 and P4 were dissolved in 100% ethanol and then suspended in media that contained 10% FBS to achieve a final working concentration (29, 30). The media that contained the hormones was allowed to incubate overnight in order to evaporate the ethanol. In each group, endometrial mesenchymal stromal cells were cultured in 48-well (15×103 cells per well) plates using DMEM/F-12 that contained antibiotics and 10% FBS for 5 days. On the fifth day of culture, these cells were co-cultured with mouse embryos at the blastocyst stage.
Superovulation and blastocyst collection
Adult female (8-10 weeks old, n=25) and male (8-12 weeks old, n=10) National Medical Research Institute (NMRI) mice were used in this study. The mice were housed under 12 hour light/12 hour dark conditions at 20-25°C with enough humidity, water and food in the laboratory animals house at Tarbiat Modares University (Iran).
The adult female mice were superovulated with an intraperitoneal injection of 7.5 IU pregnant mare serum gonadotropin (PMSG, Folligon, Intervet, Australia) followed by an intraperitoneal injection of 10 IU human chorionic gonadotropin hormone (hCG, Choragon, Germany) 48 hours later. Then, the mice were individually mated with fertile males. Normal morphology blastocyst embryos were collected from the uterine horns and transferred on the cultured endometrial mesenchymalstromal cells in both groups (3 embryos per well and 3 wells per group) for a period of 48 hours.
During culture period and after embryo transfer, the endometrial mesenchymal stromal cell proliferation and implantation process was followed by inverted microscope assessments every 12 hours in both groups.
Scanning electron microscopy
The samples in the experimental and control groups were examined by scanning electron microscopy (SEM) for ultrastructural assessment of embryo implantation. The specimens (3 embryos per well and 3 wells per group) were fixed in two steps of 2.5% glutaraldehyde in PBS and 1% osmium tetroxide in the same buffer for 2 hours, respectively. After dehydration with ethanol, the specimens were dried, mounted, and coated with gold particles (Bal-Tec, Switzerland), and examined by SEM (Philips XL30, Netherland).
RNA isolation and reverse transcription reaction
RNA was isolated from endometrial mesenchymal stromal cells after co-culture with embryos in each group of 3 embryos per well and 3 wells per group using the RNeasy Mini Kit (Qiagen, Germany). The RNA samples were treated with DNase to eliminate any genomic DNA contamination just prior to cDNA synthesis. The RNA concentration was determined by spectrophotometry. Then, the cDNA was synthesized in a total volume of 20 µl using a cDNA kit (Fermentas, EU) and stored at -80°C until use. All experiments were repeated three times.
Quantitative real-time reverse transcription- polymerase chain reaction assays
The primers for real time reverse transcription-
polymerase chain reaction (RT-PCR) were newly
designed using GenBank (
Statistical analysis was performed with SPSS version 22.0 software. Quantitative variables were expressedas mean ± SD. The results of real-time RT-PCR were compared by the independent samples t test. P=0.05 were considered statistically significant.
Flow cytometric analysis
Immunophenotype of cultured endometrial cells after the fourth passage showed the following: 1.5% ± 97.7 (CD73), 87.3 ± 2.1% (CD90), 69.1 ± 2% (CD44), 1.99 ± 0.1% (CD34), and 1.03 ± 0.06% (CD45, Fig .1,).
|Target gene||Primer pair sequences (5´-3´)||Accession number||Fragment size (bp)||T (˚C)|
The morphology of the co-cultured mouse embryos on the top of endometrial mesenchymal stromal cells as seen under an inverted microscope. The morphology in the two studied groups was similar and demonstrated in the Figure 2,. The endometrial cells showed a flattened monolayer. As these micrographs indicated, the embryonic cells were spread on the endometrial mesenchymal stromal cell layer and attached tightly to these cells. The trophoblastic cells were outgrowth around the embryo.
Scanning electron microscopy
The scanning electron micrographs of cultured endometrialmesenchymal stromal cells and mouse embryos were seenin the Figure 3A-C. The ultrastructural observations did notshow the prominent difference between the two groups. Themesenchymal stromal cells had a spindle shape and flattenedcells which attached to the floor of plate. In both groups, weobserved the presence of pinopodes-like structures (yellowarrowhead in Fig.3C) and cell secretions on the apical surfaces of endometrial mesenchymal stromal cells (yellow arrow in Fig.3A,).
Real-time reverse transcription-polymerase chain reaction
At the molecular level, we noted the following ratio
In this study, we sought to improve an implantation model by using steroidal hormone-treated human stromal endometrial cells that followed our previous study. We have evaluated the interaction between mouse embryo and endometrial mesenchymal stromal cells under the influences of E2 and P4 at the morphological, ultrastructural, and molecular levels. For embryo implantation, alterations in the structure and function of endometrial cells are critical.
Our observations have shown some signs of receptive endometrial characteristics on the apical surfaces of the endometrial mesenchymal stromal cells such as cell secretions and the presence of the pinopode-like structures. It has been determined that the steroidal hormones play an important role in embryo implantation (24). However, our observations did not show any obvious morphological and ultrastructural differences between the steroid hormone treated group to the non-treated group. These observations might be related to the insufficient dosage of hormones used in this study. It has been shown that the effects of steroid hormones are mainly dose-dependent which agrees with this suggestion (4). More studies would be necessary to confirm this suggestion. On the other hand, the secreted factors by embryo impact the differentiation and preparation of endometrial mesenchymal stromal cells for attachment to the embryo. However, more studies need to prove this suggestion.
In the current study, we performed quantitative analysis to detect ultrastructural changes. In order to better evaluate the effects of these hormones, additional experiments would be required. Evidences exist that expression of pinopodes and other ultrastructural changes in the endometrial cells are hormone dose-dependent (4). Probably the dosages of E2 and P4 used in this study were not adequate to show remarkable ultrastructural changes. Stavreus-Evers et al. (32) reported the importance of increased P4 serum levels of P4 in pinopode development. An association existed between formation of pinopodes to the concentrations of P4 in the human endometrium. Ma et al. observed that estrogen at different physiological concentrations could initiate implantation of an embryo but the implantation window remained open for an extended period at lower estrogen levels and rapidly closed at higher E2 levels (33).
In the current study, for the first time, we evaluated the
expression of some genes related to implantation in the
presence of steroid hormones. Our molecular analysis
showed that despite an increase in
The aim of the present study was to examine the effect
of an embryo co-culture with these hormone-treated cells.
Thus it could be concluded that these different expression
pattern of genes related to implantation might be due to
the presence of the embryos. The trophectoderm of an
embryo is the main source of P4 and a number of other
hormones that could be secreted thus it could change
the level and balance of hormones within the media. In
agreement with this suggestion, some reports indicated
that E2 and P4 differently modulate the expression of
genes related to the implantation in a dose-dependent
Horcajadas et al. (36), in an in vivo
study, assessed expressions of four genes in the human
endometrium under the influence of E2. They observed
that during the implantation window only three genes
Dassen et al. (37), with an in vitro culture of a human endometrial explant in the presence of E2 and P4, reported that the expression of some genes associated with embryo implantation such as IL1RL1 and CRABP2 depended on the duration of E2 exposure.
Defects in the expression of genes related to implantation result in implantation failure during the receptive phase by changing the dosage of hormones or lack of steroidal hormone signaling (33, 38).
According to the best of our knowledge, limited studies have evaluated the expression of genes related to implantation in the in vitro model. The results are influenced by the use of different assay methods, the use of different protocols for sample preparation, differences between species, and the manner of steroid usage.
The authors would like to express their appreciation to Mr. Pour Beiranvand for his technical assistance. This research was financially supported by Tarbiat Modares University as part of a Ph.D. thesis and the Iranian Stem Cell Network. The authors have no conflicts of interest relevant to this article.
M.R.; Has done the expriments, analyzed the data and contributed to writing the manuscript. M.S.; Has supervised the study and contributed to writing the manuscript. M.J.; Has involved to preparation the samples. All authors read and approved the final manuscript.