1 Department of Gynecology and Obstetrics, Faculty of Medicine, Kyoto University and 2 Department of Obstetrics and Gynecology, Japan Baptist hospital, Sakyo-ku, Kyoto, Japan 3 To whom correspondence should be addressed at: Department of Gynecology and Obstetrics, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto, 606-8507, Japan. e-mail: fuji{at}kuhp.kyoto-u.ac.jp
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: attachment/BeWo/endometrium/implantation failure/PBMC
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To examine this interaction, several in-vitro models have been proposed for human implantation (Bischof and Campana, 1996). For example, co-cultures of human blastocysts and human EECs were used for assessment of uterine receptivity (Simon et al., 1997
). Since the use of human blastocysts for experimentation is generally limited, murine blastocysts have also been used in co-cultures with EECs for attachment assays. Based on results obtained using this model, crosstalk between the human embryo and EECs through the interleukin (IL)-1 system was proposed (Simon et al., 1998
). In addition, it was demonstrated that co-cultures between human choriocarcinoma cell lines such as BeWo, JAR and JEG-3 and endometrial carcinoma cell lines provided useful models for examining the mechanisms of attachment between these cells (John et al., 1993
; Thie et al., 1996
, 1997
, 1998
; Hohn et al., 2000
). In these assays, the authors obtained spheroids which mimic blastocysts derived from choriocarcinoma cell lines by gentle continuous rolling culture. They also developed a sophisticated three-dimensional culture model using human endometrium in the secretory phase (Grummer et al., 1994
).
Although these experimental models are useful for basic investigations, there are few available in-vitro models available to assess clinically the endometrial receptivity of individual patients suffering from implantation failure. In this study, the use of BeWo cell-derived spheroids in place of human blastocysts was investigated in assays to analyse the interaction with EEC monolayer cultures, the purity of which was confirmed by the two specific cell markers, CD9 for EECs (Park et al., 2000a,b) and CD13 for endometrial stromal cells (Imai et al., 1992a
,c). It has been reported previously that spleen cells derived from pregnant mice or thymocytes derived from non-pregnant mice promote murine embryo implantation by regulating endometrial receptivity (Takabatake et al., 1997
; Fujita et al., 1998
). It was also shown that human peripheral blood mononuclear cells (PBMCs) enhance murine embryo spreading and invasion in vitro (Nakayama et al., 2002
), suggesting the promoting effects of immune cells on early events of embryo implantation. Therefore, the effects of PBMCs on human endometrial receptivity were also investigated in an assay using EEC monolayer cultures and BeWo-cell spheroids. Furthermore, to analyse the regulatory mechanism for endometrial receptivity, the effects of PBMCs on the differentiation of EECs were examined by analysing one of the useful differentiation markers for EECs, dipeptidyl peptidase IV (DPPIV), using Northern blot analysis and an enzyme assay (Imai et al., 1992b
; Fujiwara et al., 1994
).
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antibodies
The mouse anti-human CD9 mAb (ALB-6, IgG1 class), anti-human CD13 mAb (MCS-2, IgG1 class) and anti-CD45 (leukocyte common antigen) mAb (T29/33, IgG1 class) were purchased from Cosmo Bio Co. Ltd. (Tokyo, Japan), Nichirei Co. (Tokyo, Japan) and Dako Japan Co. (Kyoto, Japan) respectively. Anti-trinitrophenyl (TNP) mouse mAb (unrelated mAb, IgG1 class) was used as a negative control (Tsujimura et al., 1990). Fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse immunoglobulin (Dako Japan Co., Kyoto, Japan) was used as the second antibody in immunofluorescence staining.
Monolayer cultures of EECs
Isolation of EECs was performed essentially as described previously (Vigano et al., 1993), with several modifications. Endometrial tissues were minced into small pieces of <1 mm3 and incubated in RPMI supplemented with 10% fetal calf serum (FCS; Dainippon Pharmaceutical Co., Osaka, Japan), 0.5% collagenase I (Wako Pure Chemicals, Osaka, Japan) and 0.005% deoxyribonuclease I (DNAase I; Sigma, St. Louis, MO, USA) at 37°C for 1 h. After this enzymatic digestion, most endometrial stromal cells (including immune cells) were present as single cells or small aggregates, whereas most of the epithelial cells remained in larger clumps. The single-cell fraction containing endometrial stromal cells was removed by three rounds of differential sedimentation at unit gravity (i.e. keeping tubes static) (Kariya et al., 1991
), and the remaining fraction of the large clumps containing epithelial cells was collected. These cells were re-subjected to digestion using collagenase and DNAase. After this digestion, the fraction containing the large clumps was further incubated on a 10-cm dish (Corning, New York, USA) for 1 h at 37°C to remove adherent stromal cells. The non-adherent cell clumps were collected again and treated with 0.05% trypsin, 0.02% EDTA and 0.005% DNAase I for 5 min to obtain single cells. After washing, the cells were inoculated in the wells of a collagen type IV-coated 24-well plate (Becton Dickinson, Bedford, MA, USA) at a concentration of 1x106 cells/ml in RPMI 1640 medium supplemented with 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin. After the cells had been cultured for 24 h, the medium was changed and the unattached cells were discarded. The remaining cells were further cultured in the presence or absence of PBMCs for 72 h and then subjected to attachment assays.
Preparation of autologous PBMCs
On the day after the operation, blood samples were obtained from individual patients and autologous PBMCs were isolated using Ficoll-Hypaque centrifugation as described previously (Hashii et al., 1998). After centrifugation, PBMCs were collected from the interphase layer and washed four times with RPMI 1640. PBMCs (5x105 in 500 µl) suspended in RPMI 1640 supplemented with 10% FCS were inoculated in a basket-type culture well unit (Intercell, 0.45 µm diameter pores; Kurabo, Tokyo, Japan) that was placed in each well of the culture plates containing 1.5 ml of culture medium. The micropore membrane located in the bottom of the Intercell prevented the direct interaction of EECs with PBMCs, but allowed the transfer of soluble factors such as cytokines. In the control group, EECs were cultured with 2 ml of medium in the absence of PBMCs. After co-culturing for 72 h, the Intercells containing PBMCs were removed and the remaining EECs were subjected to attachment assays.
Spheroid formation by BeWo cells
BeWo cells, a continuous cell line established from a human choriocarcinoma (Patillo and Gey, 1968), were obtained from the Japanese Cancer Research Resources Bank and cultured in RPMI 1640 medium supplemented with 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin. The cells were maintained as monolayers in 25 cm2 flasks at 37°C in a humidified atmosphere of 5% CO2 in air.
Spheroid mass formation by BeWo cells was induced as described previously, but with slight modifications (John et al., 1993). BeWo cells were harvested by incubating them in a solution of 0.05% trypsin and 0.02% EDTA for 15 min at 37°C in 5% CO2 in air. The suspensions of BeWo cells (8x105 cells/8 ml) were placed in aliquots into 25 ml flasks and incubated on a shaker (Thermo-shaker model E-7; Thermonics, Tokyo, Japan) in 5% CO2 in air for 68 h. By using this procedure, multicellular spheroids of
100 µm diameter were obtained.
Attachment assay
Following the 72-h culture, each well containing cultured EECs was thoroughly washed with phosphate-buffered saline (PBS) twice and supplemented with fresh culture medium. The BeWo-cell spheroids were then gently placed in the centre of each well (20 spheroids per well) in triplicate and incubated at 37°C for 1 h. After incubation, the culture plate was shaken at 180 r.p.m. for 1 min using a shaker (multi shaker MMS, Tokyo Rikakikai Co., LTD., Tokyo, Japan). The medium containing unattached spheroids was collected, and fresh medium was added to each well. The above procedure was repeated, after which the number of attached BeWo-cell spheroids remaining in each well was counted using a phase-contrast microscope (Olympus Optical Co., Ltd., Tokyo, Japan). The number of BeWo-cell spheroids in the collected medium (unattached spheroids) was also counted. All attachment assays were performed in triplicate.
Flow cytometric analysis of human EECs
Freshly isolated EECs and cultured EECs were analysed by flow cytometry as described previously (Fujiwara et al., 1994). EECs which were cultured with or without PBMCs were dispersed with 0.05% trypsin and 0.02% EDTA, and sedimented by centrifugation. The sedimented EECs were incubated with CD9 mAb (100 µg/ml, 10 µl), CD13 mAb (100 µg/ml, 10 µl), CD45 mAb (100 µg/ml, 10 µl) or anti-TNP mAb (100 µg/ml, 10 µl) for 30 min at 4°C. After washing with Hanks balanced salt solution (HBSS), the cell pellet was incubated with FITC-conjugated rabbit anti-mouse immunoglobulin (diluted 1:40, 20 µl), for 30 min at 4°C in the dark. After washing with HBSS, the cells were resuspended in the same solution, and viable cells were gated and analysed by flow cytometry (FACScan; Becton Dickinson Immunocytometry Systems Japan, Tokyo, Japan).
Assay for DPPIV activity in cultured EECs
DPPIV activity was measured using glycyl-prolyl-p-nitroanilide hydrochloride (Sigma) as substrate, according to a slight modification of a previously described method (Fujiwara et al., 1994). After 72 h of culturing in the presence or absence of PBMCs, each well containing EECs obtained from the late-proliferative phase (n = 6) or early secretory phase (n = 7) was washed three times with PBS to remove unattached cells. Glycyl-prolyl-p-nitroanilide hydrochloride (0.4 mmol/l) in 1 ml of PBS containing Ca2+ and Mg2+ was added to each well and incubated for 60 min at 37°C in humidified air containing 5% CO2. The reaction was stopped by adding 1 ml of cold sodium acetate-acetic acid buffer (pH 4.2). The reaction product, p-nitroaniline, was measured by determining optical absorbance at 385 nm, and the concentration was extrapolated from a standard curve run in each assay. After the reaction, the cells were dispersed and the number of cells was determined. Enzymatic activity was expressed as p-nitroaniline produced per 105 cells in 60 min.
RNA extraction and RT-PCR analysis of DPPIV mRNA expression in cultured EECs
After culturing with or without PBMCs, the EECs were washed three times with PBS, and total RNA was extracted using a commercial kit (TRIzol; Gibco BRL, Gaithersburg, MD, USA). Total RNA (5 µg) from the EECs were reverse-transcribed with random primers using a commercial kit (First Strand cDNA Synthesis Kit; Pharmacia, Inc., Piscataway, NJ, USA). The resulting cDNA mixtures were subjected to 30 cycles of PCR amplification with oligonucleotides from the human DPPIV gene as primers (sense primer 5'-TACTCTGCTCTGTGGTGGTC-3': positions 706725; antisense primer 5'-AATACTTCGCCTCTTTACTG-3': positions 14391458) (Tanaka et al., 1992), or with human S26 primers (sense primer 5'-GGTCCGTGCCTCCAAGATGA-3': positions 827; antisense primer 5'-TAAATCGGGGTGGGGGTGTT-3': positions 308327) (Vincent et al., 1993
). After PCR amplification, 10 µl of each PCR product was electrophoresed on a 1.0% agarose gel, and the amplified bands were detected by ethidium bromide staining. The identity of these PCR products was confirmed by determining the sequence. The cloned DPPIV and S26 cDNAs were then labelled with 32P using a commercial labelling kit (Megaprime DNA labelling system; Amersham Pharmacia, Inc.) and used as probes for the subsequent Northern blot analysis.
Northern blot analysis
A sample (8 µg) of total RNA from EECs co-cultured with or without PBMCs for 3 days was separated by electrophoresis on a 1% agarose-formaldehyde gel and transferred to a nylon membrane (Hybond-N+; Amersham, Arlington, IL, USA). The membrane was incubated with prehybridization solution (Rapid Hybridization Buffer; Amersham) for 30 min at 65°C, and then hybridized with the 32P-labelled DPPIV cDNA probe for 2 h at 65°C in the same solution. After hybridization, the membrane was washed in 2x standard saline citrate (SSC: 2 mmol/l sodium citrate and 20 mmol/l sodium chloride in distilled water, pH7.0) with 0.1% sodium dodecyl sulphate (SDS) at room temperature for 15 min and in 0.2x SSC with 0.1% SDS at 65°C for 30 min, and then subjected to autoradiography. The membrane was washed and rehybridized with the S26 probe. Levels of DPPIV mRNA were determined by densitometric scanning of the autoradiograph, and the mRNA level from each condition was corrected with S26 mRNA expression.
Statistical analysis
The data for attachment analysis were expressed as means ± SEM. Statistical analyses were performed by one-way analysis of variance, followed by Scheffes F-test for multiple comparisons of results. The differences in DPPIV enzyme activity of EEC cultures between the late proliferative and early secretory phases were analysed using unpaired t-tests, and the effects of PBMCs on DPPIV activity were analysed using paired t-tests. Differences were considered significant at the 5% level.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Attachment of BeWo-cell spheroids to EEC cultures
The rates (percentage) of spheroids attached to EECs are shown in Figure 2. In the EEC cultures obtained from women in the mid-secretory phase, almost all the BeWo-cell spheroids (90 ± 2.9%) adhered to EECs. In contrast, in the EEC cultures prepared from women in the mid- and late proliferative phases and early and late secretory phases, the spheroids were barely adhered to the EECs (5.8 ± 3.7, 5.0 ± 2.0, 4.4 ± 1.9 and 0% respectively).
|
|
Effects of PBMCs on expression of DPPIV mRNA in cultured EECs
EECs derived from women in the late proliferative phase (n = 3) and early secretory phase (n = 3) cultured with or without PBMCs were analysed by Northern blot analysis. There was no significant difference of DPPIV mRNA expression between the PBMC-treated group and non-treated group (data not shown).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To exclude bias based on the purity of the EECs, the cell populations of EEC cultures were analysed for CD9 and CD13 expression. Flow cytometry analysis revealed that the purity of EECs in monolayer cultures was high, and contamination by endometrial stromal cells or immune cells was very low. As no apparent difference in purity was observed among the EEC cultures derived from each menstrual phase, changes in the receptivity of EEC cultures to BeWo-cell spheroids were considered to reflect functional changes of EECs. Thus, it is suggested that this assay might represent a new method by which to assess endometrial receptivity.
This assay is also considered likely to contribute to an analysis of the mechanism used for the initial attachment of the embryo onto the uterine luminal epithelium. To clarify any possible involvement of the immune system in the regulation of embryo attachment, the effects of PBMCs on endometrial receptivity were examined using this assay. The number of attached BeWo-cell spheroids was remarkably increased after PBMC treatment in cultures of EECs derived from women in the early secretory phase and, with the exception of one patient, a similar increase was also observed in EEC cultures from women in the late proliferative phase. Flow cytometry measurements showed that there was no apparent change in the CD9 expression rate in EECs cultured with PBMC. In contrast, the number of attached spheroids did not increase in EEC cultures from late-secretory phase women, even when they were incubated with PBMCs. These results indicate that human PBMCs promote the adhesiveness of cultured EECs for BeWo-cell spheroids, and suggest that the endometrium which has not yet reached the implantation window can be induced by PBMCs to enter the receptive phase. As the membrane filter used in this assay prevented direct interaction between the PBMCs and EECs, some soluble factor(s) secreted from PBMCs and/or EECs may be responsible for the effects observed.
In order to investigate the effects of PBMCs on EEC differentiation, the expression of DPPIV, which has been suggested to play a role in embryo implantation (Denker, 1977; Classen-Linke et al., 1987
) was also examined. Immunohistochemical analysis showed that DDPIV expression on human EECs increased gradually from the proliferative phase to mid- secretory phases (Imai et al., 1992b
), and this was confirmed by the DPPIV enzyme assay used. In contrast to expectation, neither DPPIV enzyme activity nor the mRNA expression of DPPIV was affected by PBMC treatment. Thus, these investigations failed to provide direct evidence that PBMCs can promote EEC differentiation in vitro.
In the assay used in the present study, BeWo-cell spheroids could barely adhere to monolayer-cultured EECs derived from women in the proliferative phase and early and late secretory phases. Although BeWo-cell spheroids have certain characteristics that resemble those of blastocysts, BeWo cells were established from carcinoma cells and have potent adhesive ability. Recently, the existence has been suggested of macromolecules which can interfere with the binding between embryo and EECs such that embryo/EEC interaction is regulated (Aplin, 1999). Thus, it is of interest to consider why BeWo-cell spheroids were unable to attach to EECs, except for the cultured EECs derived from women in mid-secretory phase. It is theoretically reasonable to speculate that soluble factor(s) secreted from PBMCs suppress the function or expression of certain interfering molecules. However, it is also possible that PBMC treatment enhanced the expression of cell adhesion molecules such as integrins, which have been proposed to mediate embryo/EEC interaction (Lessey, 2000
). The administration of RDG peptides or neutralizing antibodies for some integrins using this system may help to identify the molecule involved in embryo attachment. In addition, structural changes of EEC cellsincluding pinopode formationshould be examined in the culture (Denker, 1993
; Nikas et al., 1999
).
The presence of immune cells around EECs and intraepithelial lymphocytes has been observed in the endometrium (Pace et al., 1991; Bulmer, 1996
), and the induction of HLA-DR on EECs by endometrial T-lymphocytes was demonstrated in vitro (Tabibzadeh, 1991
). Although the physiological significance of the effects of PBMCs on EEC function remains unclear, the present findings support the concept that immune cells might play a role in the local regulation of endometrial functions, including receptivity for the embryo.
In recent times, attention has been focused on patients who fail to achieve a successful implantation in spite of repeated intrauterine transfers of morphologically good embryos (Edwards, 1995). Preliminary experiments have shown that dilatation and curettage procedures for patients in the mid-secretory phase can provide sufficient endometrial tissue to conduct this assay, and so this approach could be used relatively easily to screen infertile patients at out-patient clinics. The clinical value of this assayespecially to assess endometrial receptivity and predict those patients suffering implantation failurecan be evaluated by accumulating evidence in the future. To treat these patients, attempts have been made worldwide to improve the quality of the transferred embryo or endometrium. Although ovarian sex steroid hormones are mainly used to ameliorate endometrial receptivity, there is not yet any effective therapy for those patients who do not respond to hormonal controls. It has been shown previously that T-lymphocytes promote endometrial differentiation and induce embryo implantation in mice, and the proposal has been made that local administration of autologous PBMCs into the endometrium is a possible approach to treat patients suffering from implantation failure (Takabatake et al., 1997
; Fujita et al., 1998
). The results of the present study provide important evidence suggesting that administration of PBMCs into the uterine lumen may change EEC function and promote endometrial receptivity for embryo implantation. These findings support the above concept that not only an endocrine but also an immunological approach is one of several attractive therapies for implantation failure. It should be also possible to apply, both easily and safely, this knowledge to the treatment of infertile patients at out-patient clinics. For example, PBMCs isolated from the patient could be transferred into the uterine lumen 2 or 3 days after intrauterine insemination, when the embryo is still within the Fallopian tube. In IVF and embryo transfer treatment, PBMC administration could be safely performed a few days before blastocyst transfer. Thus, although the possibility of harmful actions of PBMCs on human embryos has not yet been excluded, the intra-luminal administration of PBMCs during the early secretory phase represents a possible new candidate for infertility treatment.
In conclusion, the present results have shown that an attachment assay using BeWo-cell spheroids and EECs would be useful to assess the receptivity of the endometrium. As PBMCs promoted the attachment of BeWo-cell spheroids to EECs derived from women in the late proliferative and early secretory phases, it may be speculated that PBMCs are able to induce EECs to become receptive to the embryo in vivo. Further studies should help to clarify the mechanism of human embryo implantation, to develop methods for the evaluation of endometrial receptivity, and to establish effective therapies for patients suffering from implantation failure.
![]() |
Acknowledgements |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bischof, P. and Campana A. (1996) A model for implantation of the human blastocyst and early placentation. Hum. Reprod. Update, 2, 262270.
Bulmer, J.N. (1996) Cellular constituents of human endometrium in the menstrual cycle and early pregnancy. In: Bronson, R.A., Alexander, N.J., Anderson, D., Branch, D.W., Kutteh, W.H. (eds), Reproductive Immunology. Blackwell Science, Inc., Cambridge, p. 212.
Classen-Linke, I, Denker, H.W. and Winterhager, E. (1987) Apical plasma membrane-bound enzymes of rabbit uterine epithelium. Pattern changes during the periimplantation phase. Histochemistry, 87, 517529.[ISI][Medline]
Denker, H.W. (1977) Implantation. The role of proteinases, and blockage of implantation by proteinase inhibitors. Adv. Anat. Embryol. Cell Biol., 53, 3123.
Denker, H.W. (1993) Implantation: a cell biological paradox. J. Exp. Zool., 266, 541558.[ISI][Medline]
Edwards, R.G. (1995) Clinical approaches to increasing uterine receptivity during human implantation. Hum. Reprod., 10 (Suppl. 2), 6066.
Fujita, K., Nakayama, T., Takabatake, K., Higuchi, T., Fujita, J., Maeda, M., Fujiwara, H. and Mori, T. (1998) Administration of thymocytes derived from non-pregnant mice induce an endometrial receptive stage and leukemia inhibitory factor expression in the uterus. Hum. Reprod., 13, 28882894.
Fujiwara, H., Fukuoka, M., Yasuda, K., Ueda, M., Imai, K., Goto, Y., Suginami, H., Kanzaki, H., Maeda, M. and Mori, T. (1994) Cytokines stimulate dipeptidyl peptidase-IV expression on human luteinizing granulosa cells. J. Clin. Endocrinol. Metab., 79, 10071011.[Abstract]
Grummer, R., Hohn, H.P., Mareel, M.M. and Denker, H.W. (1994) Adhesion and invasion of three human choriocarcinoma cell lines into human endometrium in a three-dimensional organ culture system. Placenta, 15, 411429.[ISI][Medline]
Hashii, K., Fujiwara, H., Yoshioka, S., Hashii, K., Fujiwara, H., Yoshioka, S., Kataoka, N., Yamada, S., Hirano, T., Mori, T. et al. (1998) Peripheral blood mononuclear cells stimulate progesterone production by luteal cells derived from pregnant and non-pregnant women: possible involvement of interleukin-4 and 10 in corpus luteum function and differentiation. Hum. Reprod., 13, 27382744.
Hohn, H.P., Linke, M. and Denker, H.W. (2000) Adhesion of trophoblast to uterine epithelium as related to the state of trophoblast differentiation: in vitro studies using cell lines. Mol. Reprod. Dev., 57, 135145.[CrossRef][ISI][Medline]
Imai, K., Maeda, M., Fujiwara, H., Okamoto, N., Kariya, M., Emi, N., Takakura, K., Kanzaki, H. and Mori, T. (1992a) Human endometrial stromal cells and decidual cells express cluster of differentiation (CD) 13 antigen/ aminopeptidase N and CD10 antigen/neutral endopeptidase. Biol. Reprod., 46, 328334.[Abstract]
Imai, K., Maeda, M., Fujiwara, H., Kariya, M., Takakura, K., Kanzaki, H. and Mori, T. (1992b) Dipeptidyl peptidase IV as a differentiation marker of the human endometrial glandular cells. Hum. Reprod., 7, 11891194.[Abstract]
Imai, K., Kanzaki, H., Fujiwara, H., Kariya, M., Okamoto, N., Takakura, K., Maeda, M. and Mori T. (1992c) Expression of aminopeptidase N and neutral endopeptidase on the endometrial stromal cells in endometriosis and adenomyosis. Hum. Reprod., 7, 13261328.[Abstract]
John, N.J., Linke, M. and Denker, H.W. (1993) Quantitation of human choriocarcinoma spheroid attachment to uterine epithelial cell monolayers. In Vitro Cell. Dev. Biol. Anim., 29A, 461468.
Kariya, M., Kanzaki, H., Takakura, K., Imai, K., Okamoto, N., Emi, N., Kariya, Y. and Mori T. (1991) Interleukin-1 inhibits in vitro decidualization of human endometrial stromal cells. J. Clin. Endocrinol. Metab., 73, 11701174.[Abstract]
Lessey, B.A. (2000) The role of the endometrium during embryo implantation. Hum. Reprod., 15 (Suppl. 6), 3950.
Nakayama, T., Fujiwara, H., Maeda, M., Inoue, T., Yoshioka, S., Mori, T. and Fujii, S. (2002) Human peripheral blood mononuclear cells (PBMC) in early pregnancy promote embryo invasion in vitro: hCG enhances the effects of PBMC. Hum. Reprod., 17, 207212.
Nikas, G., Develioglu, O.H., Toner, J.P. and Jones, H.W., Jr (1999) Endometrial pinopodes indicate a shift in the window of receptivity in IVF cycles. Hum. Reprod., 14, 787792.
Noyes, R.W., Hertig, A.T. and Rock, J. (1950) Dating the endometrial biopsy. Fertil. Steril., 1, 325.[ISI][Medline]
Pace, D., Longfellow, M. and Bulmer, J.N. (1991) Characterization of intraepithelial lymphocytes in human endometrium. J. Reprod. Fertil., 91, 165174.[Abstract]
Park, K.R., Inoue, T., Ueda, M., Hirano, T., Higuchi, T., Maeda, M., Konishi, I., Fujiwara, H. and Fujii, S. (2000a) CD9 is expressed on human endometrial epithelial cells in association with integrins 6,
3 and ß1. Mol. Hum. Reprod., 6, 252257.
Park, K.R., Inoue, T., Ueda, M., Hirano, T., Higuchi, T., Konishi, I., Fujiwara, H. and Fujii, S. (2000b) Anti-CD9 monoclonal antibody-stimulated invasion of endometrial cancer cell lines in vitro: possible inhibitory effect of CD9 in endometrial cancer invasion. Mol. Hum. Reprod., 6, 719725.
Patillo, R.A. and Gey, G.O. (1968) The establishment of a cell line of human hormone-synthesizing trophoblast cells in vitro. Cancer Res., 28, 12311236.[ISI][Medline]
Psychoyos, A. (1993) The implantation window: basic and clinical aspects. In: Mori, T., Aono, T., Tominaga, T. and Hiroi, M. (eds), Perspectives in Assisted Reproduction. Ares Serono Symposia 4, pp. 5762.
Simon, C., Gimeno, M.J., Mercader, A., OConnor, J.E., Remohi, J., Polan, M.L. and Pellicer, A. (1997) Embryonic regulation of integrins ß3, 4, and
1 in human endometrial epithelial cells in vitro. J. Clin. Endocrinol. Metab., 82, 26072616.
Simon, C., Moreno, C., Remohi, J. and Pellicer, A. (1998) Molecular interactions between embryo and uterus in the adhesion phase of human implantation. Hum. Reprod., 13 (Suppl. 3), 219232.
Tabibzadeh, S. (1991) Induction of HLA-DR expression in endometrial epithelial cells by endometrial T-cells: potential regulatory role of endometrial T-cells in vivo. J. Clin. Endocrinol. Metab., 73, 13521359.[Abstract]
Tabibzadeh, S. and Babaknia, A. (1995) The signal and molecular pathways involved in implantation, a symbiotic interaction between blastocyst and endometrium involving adhesion and tissue invasion. Mol. Hum. Reprod., 1, 15791602.
Takabatake, K., Fujiwara, H., Goto, Y., Nakayama, T., Higuchi, T., Fujita, J., Maeda, M. and Mori, T. (1997) Splenocytes in early pregnancy promote embryo implantation by regulating endometrial differentiation in mice. Hum. Reprod., 12, 21022107.[Abstract]
Tanaka, T., Camerini, D., Seed, B., Torimoto, Y., Dang, N.H., Kameoka, J., Dahlberg, H.N., Schlossman, S.F. and Morimoto C. (1992) Cloning and functional expression of the T cell activation antigen CD26. J. Immunol., 149, 481486.
Thie, M., Fuchs, P., Butz, S., Sieckmann, F., Hoschutzky, H., Kemler, R. and Denker, H.W. (1996) Adhesiveness of the apical surface of uterine epithelial cells: the role of junctional complex integrity. Eur. J. Cell Biol., 70, 221232.[ISI][Medline]
Thie, M., Herter, P., Pommerenke, H., Durr, F., Sieckmann, F., Nebe, B., Rychly, J. and Denker, H.W. (1997) Adhesiveness of the free surface of a human endometrial monolayer for trophoblast as related to actin cytoskeleton. Mol. Hum. Reprod., 3, 275283.[Abstract]
Thie, M., Rospel, R., Dettmann, W., Benoit, M., Ludwig, M., Gaub, H.E. and Denker, H.W. (1998) Interactions between trophoblast and uterine epithelium: monitoring of adhesive forces. Hum. Reprod., 13, 32113219.[Abstract]
Tsujimura, K., Park, Y., Miyama-Inaba, M., Meguro, T., Ohno, T., Ueda, M. and Masuda, T. (1990) Comparative studies on FcR (FcRII, FcRIII and FcR) functions of murine B cells. J. Immunol., 144, 45714578.
Vigano, P., Di Blasio, A. M., DellAntonio, G. and Vignali, M. (1993) Culture of human endometrial cells; a new simple technique to completely separate epithelial glands. Acta Obstet. Gynecol. Scand., 72, 8792.[ISI][Medline]
Vincent, S., Marty, L. and Fort, P. (1993) S26 ribosomal protein RNA: an invariant control for gene regulation experiments in eukaryotic cells and tissues. Nucleic Acids Res., 21, 1498.
Submitted on January 4, 2002; resubmitted on August 9, 2002. accepted on September 9, 2002