1 Department of Obstetrics and Gynaecology, 2 Department of Pathology, Herlev University Hospital, Herlev Ringvej 75, DK2730 Herlev, Denmark, 3 Department of Obstetrics and Gynaecology, Centre for Reproductive Medicine, Sahlgrenska Hospital, University of Gothenburg and 4 Karolinska Institutet, Stockholm, Sweden
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Abstract |
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Key words: embryo/human/implantation/in vitro/morphology
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Introduction |
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Animal models have shown that the endometrium is only receptive for blastocyst implantation during a short time period, known as the `implantation window' (Psychoyos, 1976, 1986
; Yoshinaga, 1988
). In the rat, the implantation window is preceded by a neutral state and is followed by a refractory state (Psychoyos, 1976
, 1986
). Blastocysts remain viable during the neutral state, but undergo degeneration and are expelled from the uterus when transferred during the refractory phase.
In the human, oocyte donation programmes have provided an excellent model to investigate the influence of asynchronous embryo transfer. Transfer into an advanced endometrium impaired embryo implantation (Rosenwaks, 1987; Navot, 1991). The expected window of implantation is assumed to coincide with cycle days 2022 in a standardized cycle (Davis and Rosenwaks, 1993). This period corresponds well to the presence of endometrial pinopodes on the apical surface of endometrial epithelial cells (Martel et al., 1981
). In animal studies, pinopodes extract uterine fluid in order to bring the blastocyst into intimate contact with the uterine epithelium (Vokaer and Leroy, 1962
; Enders and Nelson, 1973
), but pinopodes are furthermore supposed to be an ultrastructural marker of endometrial receptivity (Martel et al., 1981
, 1987
, 1991
; Psychoyos and Martel, 1985
; Massai et al., 1993
; Psychoyos and Nikas, 1994
; Nikas et al., 1995
; Edwards, 1995
).
We have developed an in-vitro model of the human endometrium (Bentin-Ley et al., 1994, 1995
) in which human blastocysts adhere to the endometrial surface. The aim of this study was to investigate the ultrastructure of blastocystendometrial interactions at in-vitro implantation sites by scanning electron microscopy (SEM) with focus on endometrial changes in the presence of a blastocyst.
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Materials and methods |
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Preparation of cell cultures
Endometrial cell cultures were prepared as published earlier (Bentin-Ley et al., 1994) with minor modifications. Endometrial biopsies were obtained by curettage (Cunell biopsy). The culture medium was an alpha modification of Eagle's medium; 100 ml were supplemented with 2000 IU penicillinstreptomycin, 0.2 ml L-glutamine (200 mmol/l), 4 ml Amniomax (Life Technologies, Roskilde, Denmark), 5 ml fetal calf serum, 0.5 g bovine serum albumin (Hoechst, Marburg, Germany), 10 µg retinoic acid and progesterone was added to physiologic concentrations. The fetal calf serum provided oestrogen in sufficient amounts. All cultures were placed in an incubator (Forma Scientific, Ohio, USA) at 37°C with 5% CO2 and culture medium was changed every second day. After 5 days of culture, the epithelial layer was confluent and ready for implantation studies. Implantation studies were performed at Herlev Hospital, Denmark, and Sahlgrenska Hospital, Sweden. Cell cultures for implantation studies were transported from Denmark to Sweden in a transport incubator (K-systems; Henning Knudsen Engeneering, Birkerød, Denmark).
Embryo culture and implantation
Surplus embryos from IVF treatments were cultured in S2 medium (Scandinavian IVF Science Ltd, Gothenburg, Sweden) to the expanded or hatching blastocyst stage. Three blastocysts (see Table I) were placed on three endometrial cell cultures and were examined for implantation sites every morning through a ZeissTM stereo microscope (Göttingen, Germany). Since the cell cultures were translucent, identification of the blastocysts was easy. The attachment of the blastocysts was checked by gentle shaking of the culture dish. When the blastocysts were attached to the endometrial cell cultures, the implantation sites were fixed ~48 h later in order to allow adhesion to progress. The study was approved by the local ethical committees in Sweden and Denmark. All patients, both endometrial donors and embryo donors, gave informed consent.
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Results |
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Discussion |
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The definition of endometrial pinopodes at various stages is somewhat uncertain when different studies are compared. In this study, pinopodes were classified according to the definition given by Nikas et al. (1998); fully developed pinopodes were defined as a protruding naked surface with many foldings. A protruding smooth surface with or without short microvilli represents developing pinopodes, whereas a wrinkled surface with microvilli characterizes regressing pinopodes. Transmission electron microscopy (TEM) has shown that pinopodes are apical cytoplasmic protrusions containing organelles, vesicles and glycogen granules (Ferenczy and Richart, 1973; Nilsson and Nygren, 1974
). The time of pinopode formation coincides with the period of decrease of cell polarity as reported by Denker (1993), which might be essential for the induction of endometrial receptivity (Thie et al., 1996
). Functional analyses of the cytoskeleton by Thie et al. (1997) have shown that receptivity of the apical cell pole in vitro depends on reorganization and not destruction of the actin filament system.
The most striking finding in this study was the attachment of blastocysts to the top of endometrial pinopodes, which was found in all three cases. Since three implantation sites is a small number, we further studied serial sectioned implantation sites prepared for light microscopy (LM) and TEM (unpublished results) that also demonstrated pinopodes close to the blastocysts. These findings support our detection by SEM.
Since blastocysts seemed to be attached to pinopode presenting epithelial cells, and although the possibility that trophoblast may intrude between endometrial epithelial cells without adhesion formation cannot be excluded (Lopata, 1996), one can speculate whether the naked membrane of pinopodes could contain receptors necessary for blastocyst adhesion. Immunohistochemical studies have in fact shown that luminal endometrial epithelial cells do present the interleukin-1 receptor type 1 (IL-1R t1) and the integrin subunit ß3, which are supposed to participate in embryoendometrial interactions during adhesion (Lessey et al., 1992
, 1996
; Simon et al., 1993
Simon et al., 1997
; Lessey, 1997
; Meyer et al., 1997
). Both the IL-1R tI and the receptor antagonist present on luminal epithelium are actually expressed as patches (Simon et al., 1995b
). The upcoming of the integrin ß3 subunit coincides with the presence of endometrial pinopodes (Lessey et al., 1992
). But expression of these receptors has not been correlated to the presence of pinopodes. The question whether the blastocyst can induce endometrial receptivity at the implantation site remains unclear at the moment. Simon et al. (1997) noted enhancement of integrin expression when blastocyst conditioned media were used for culture of endometrial epithelial cells grown on plastic. These studies indicate that blastocysts can influence the up-regulation of possible receptors on the uterine epithelium in vitro. Further studies are needed to define to what extent they reflect the in-vivo situation.
Nikas et al. (1995) showed that the timing of the appearance of fully developed pinopodes varied from patient to patient in women receiving exogenous oestradiol and progesterone, but fully developed pinopodes were only present for 1 day. Furthermore, endometrial biopsies from women undergoing controlled ovarian hyperstimulation with clomiphene citrate and human menopausal gonadotrophin (HMG) showed early formation of pinopodes (Martel et al., 1987; Psychoyos and Nikas, 1994
). Paulson et al. (1997) and Kolb et al. (1997) investigated endometrial biopsies from oocyte donors undergoing ovarian stimulation with leuprolide acetate and HMG and also found that these were histologically advanced. When studied by SEM, pinopodes were absent in most biopsies 7 days after oocyte aspiration, whereas they were present in recipients undergoing hormone replacement therapy cycles (HRT) with exogenous oestradiol and progesterone. Advanced endometrial maturation in ovarian hyperstimulation cycles might result in early closure of the implantation window, whereas in HRT cycles, the opening and closure of the implantation window, as indicated by pinopode formation, seems to be delayed (Psychoyos and Nikas, 1994
; Nikas et al., 1995
). The extra time could allow more embryos to develop to the hatched blastocyst stage before the implantation window closes. This could explain why embryo implantation rates are often higher in oocyte recipients than in oocyte donors (Paulson et al., 1990
; Edwards et al., 1991
).
The endometrial surface on cell cultures did not reveal any other changes in the region of the blastocyst except pinopode formation. Lindenberg (1991) published observations on in-vitro implantation of human blastocysts. SEM revealed that the endometrial epithelial cells in that culture system lost their microvilli in the attachment area. We were not able to confirm these observations in our culture system. A possible explanation could be that the observations made by Lindenberg (1991) represented rudimentary pinopode formations. Loss of microvilli was only found extremely rarely close to what we think represents trophoblast intrusion (Figure 8) whereas all other cells did not reveal these changes. No gaps were present at the presumed trophoblasticendometrial interface, indicating an efficient sealing of the invasion site as demonstrated by TEM (unpublished results). These findings are consistent with those in the rabbit and the skunk (Enders et al., 1995
). We must conclude that implantation in vivo could very well happen in the way this in-vitro model demonstrates.
Although great care should be taken when extrapolating from in-vitro to in-vivo situations, the selective adhesion of blastocysts to pinopode presenting areas in this study emphasizes the importance of endometrial pinopodes as indicators of endometrial receptivity. We suggest that the apical surface of endometrial pinopodes participates directly in the adhesion process of the human blastocyst, but further studies on endometrial pinopodes are necessary.
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Acknowledgments |
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Notes |
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References |
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Submitted on July 8, 1998; accepted on November 11, 1998.