Human Reproduction

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Human Reproduction, Vol. 14, No. 2, 515-520, February 1999
© 1999 European Society of Human Reproduction and Embryology

Presence of uterine pinopodes at the embryo–endometrial interface during human implantation in vitro

U. Bentin-Ley^1,5, A. Sjögren³, L. Nilsson⁴, L. Hamberger³, J.F. Larsen¹ and T. Horn²

¹ Department of Obstetrics and Gynaecology, ² Department of Pathology, Herlev University Hospital, Herlev Ringvej 75, DK–2730 Herlev, Denmark, ³ Department of Obstetrics and Gynaecology, Centre for Reproductive Medicine, Sahlgrenska Hospital, University of Gothenburg and ⁴ Karolinska Institutet, Stockholm, Sweden

	Abstract

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In order to study changes occurring on the surfaces of human endometrial epithelial cells in the presence of an implanted blastocyst, we used scanning electron microscopy for investigation of five endometrial biopsies and three human implantation sites obtained in vitro. All specimens showed areas with endometrial pinopodes, separated by cells displaying microvilli or cilia at the apical surface. Pinopode formation was more pronounced in endometrial biopsies than in cell cultures. All blastocysts adhered to pinopode presenting cells. Endometrial surface changes were not seen around the blastocysts. The results of this study demonstrate that cultured endometrial epithelial cells are capable of pinopode formation. Furthermore, endometrial epithelial pinopodes, generally considered as a marker of endometrial receptivity, seem to be directly involved in the adhesion of the blastocyst to the endometrial surface.

Key words: embryo/human/implantation/in vitro/morphology

	Introduction

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The introduction of new protocols for ovarian hyperstimulation in human in-vitro fertilization (IVF) and application of new embryo culture media have improved implantation rates. But implantation failure remains a major problem in infertility treatment. One of the reasons for this failure is suspected to be due to impaired uterine receptivity (Edwards, 1988; Paulson et al., 1990; Edwards et al., 1991) because of high serum oestradiol concentrations induced by ovulation induction treatments (Simón et al., 1995a, 1998; Pellicer et al., 1996).

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 20–22 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 blastocyst–endometrial interactions at in-vitro implantation sites by scanning electron microscopy (SEM) with focus on endometrial changes in the presence of a blastocyst.

	Materials and methods

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Endometrial biopsy material
The biopsies were obtained from five regularly menstruating women with proven fertility, applying for tubal sterilization. All patients used barrier methods for contraception. The biopsies were obtained from the upper part of the endometrial cavity at cycle days luteinizing hormone (LH) + 5 to LH + 7. Morphological dating was in accordance with occurrence of the endogenous LH peak. Endometrial tissue from one of these patients was also used for preparation of cell cultures.

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 penicillin–streptomycin, 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 Zeiss^TM 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.

View this table: [in this window] [in a new window]   Table I. Timing of observations on three blastocysts placed in three endometrial cell cultures

 
Preparation for scanning electron microscopy
The endometrial biopsies and cell cultures were fixed with 2.5% glutaraldehyde in a sodium cacodylate buffer (0.1 M, pH 7.4; Merck, Darmstadt, Germany) for 24 h at 5°C. For cell cultures, the fixative was diluted with 20% distilled water in order to reduce osmolarity. After rinsing in sodium cacodylate buffer (0.1 M, pH 7.4) 4x15 min, the cultures and biopsy material were post-fixed with 1% OsO₄ in 0.1 M sodium cacodylate buffer for 1 h at room temperature and the implantation sites were cut out with razor blades. Afterwards, the specimens were taken through series of graded ethanols before they were dried in a critical point dryer with liquid CO₂, sputter coated with gold and visualized in a Jeol JSM35 scanning electron microscope.

	Results

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By SEM, all biopsies showed uniformly distributed pinopode presenting cells, separated by single ciliated cells (Figure 1). Most pinopodes still presented short microvilli whereas others were almost naked. Pinopodes with highly wrinkled surfaces were generally not seen. In a few areas pinopodes were not present (Figure 2) and cells presented microvilli or cilia at the apical surfaces. There were no marked differences between individual biopsies.

View larger version (230K): [in this window] [in a new window]   Figure 1. Scanning electron microscopy (SEM) of endometrial biopsy day LH + 6. The endometrial surface is covered by pinopode presenting cells, separated by single ciliated cells. The pinopodes are at the developing stage, most with smooth regular surfaces, whereas some cells still present short microvilli at the surfaces (scale bar = 40 µm).

 

View larger version (212K): [in this window] [in a new window]   Figure 2. Scanning electron microscopy (SEM) of endometrial biopsy day LH + 6. In this area no pinopodes were present. Single ciliated cells separated endometrial cells with flattened surfaces and short microvilli. Right at the bottom (arrow) pinopode formation has started (scale bar = 40 µm).

 
Cultured cells demonstrated a confluent epithelial surface that was slightly folded into ridges. At low magnifications most cell surfaces seemed flattened, but interspersed clusters of cells with bulging epithelial surfaces were seen (Figure 3). Higher magnification revealed presence of pinopodes in these areas (Figure 4). Some pinopodes presented a smooth naked surface (developing pinopodes) (Nikas et al., 1995Nikas et al., 1998), others presented a smooth surface with short microvilli (developing pinopodes) or a wrinkled naked surface (fully developed pinopodes). Endometrial cells without pinopode formation, presenting dense short microvilli or cilia, separated the pinopode accumulations (Figure 5). Single pinopode formations were observed in these areas.

View larger version (175K): [in this window] [in a new window]   Figure 3. Scanning electron microscopy (SEM) of endometrial cell culture, demonstrating a confluent endometrial surface that has been folded in ridges. In some areas the surface has broken during the preparation procedure. Generally, the endometrial surface is smooth, but clusters with bulging surfaces are seen in a patchy pattern (arrows). The curved arrow indicates a human blastocyst that adhered to one of the bulging areas (scale bar = 550 µm).

 

View larger version (233K): [in this window] [in a new window]   Figure 4. Scanning electron microscopy (SEM) of one of the bulging areas on the endometrial cell culture, demonstrating presence of endometrial pinopodes in these areas. Some pinopodes are fully developed with smooth wrinkled surfaces (arrows), others are in the developing stage with smooth surfaces still presenting microvilli (scale bar = 40 µm).

 

View larger version (246K): [in this window] [in a new window]   Figure 5. Scanning electron microscopy (SEM) of endometrial cell culture. In this area only two single pinopodes are present (arrows), whereas the other cells demonstrate many short microvilli at the apical surface. Single ciliated cells are also shown (scale bar = 40 µm).

 
All three blastocysts were fully hatched and adhered to the endometrial surface. The blastocysts were not evenly rounded but more or less oblong and flattened (Figures 6 and 8). Dense short microvilli covered the surface of the trophectodermal cells. A few cells also had long microvilli. All three blastocysts had attached to areas where epithelial cells displayed pinopodes (Figures 6 and 8), whereas surrounding areas were devoid of pinopodes, comprising cells with microvilli or ciliated cells. Higher magnification (Figure 7) showed that many pinopodes were fully developed or developing right at the embryo–endometrial interface and were in contact with the surface of the trophectodermal cells (Figure 7). A few cells projected from the blastocyst surface in between the endometrial cells (Figure 8). All cells displayed many microvilli at the apical surfaces. There were no gaps present between presumed epithelial cells and penetrating trophoblastic cells in this region.

View larger version (214K): [in this window] [in a new window]   Figure 6. Scanning electron microscopy (SEM) of a human blastocyst (no. 2, Table I) that adhered to the cell culture (also shown in Figure 3). The blastocyst is oblong and flattened, presenting many short microvilli at the apical surface. Many trophoblastic cells have bulging surfaces, thereby illustrating cell borders. The blastocyst adhered to a pinopode presenting area on the endometrial cells. Flattened endometrial cells dominate the surrounding area with microvilli at the apical surface. Single ciliated cells are interspersed (scale bar = 130 µm).

 

View larger version (226K): [in this window] [in a new window]   Figure 8. Scanning electron microscopy (SEM) of a human blastocyst (no. 1, Table I) that adhered to the endometrial cell culture. The surfaces of the trophoblastic cells show many microvilli with slightly varying length. To the left, several cells are in contact with the endometrial surface (arrows). The cell stretching from the blastocyst surface and into the endometrium could possibly illustrate early penetration. Just to the left of this cell (asterisk), a naked area without microvilli is seen (scale bar = 60 µm).

 

View larger version (223K): [in this window] [in a new window]   Figure 7. Scanning electron microscopy (SEM) of the contact area between trophoblastic cells (T) and endometrial epithelial cells (E), demonstrating several pinopodes in contact with the trophoblastic surface of blastocyst no. 3 (Table I) (scale bar = 15 µm).

 

	Discussion

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Comparison between endometrial cell cultures and biopsy material from five different patients did not show any marked differences except that there seemed to be more pinopodes and ciliated cells on endometrial biopsies. Previous studies have shown that the number of ciliated cells is subject to wide regional variations within and between biopsies from the same patients (Sundström et al., 1983). Pinopodes were also present on endometrial cell cultures without blastocysts (Bentin-Ley et al., 1995). The maturation of pinopodes in biopsies appeared generally synchronized. This finding is in accordance with previous studies (Martel et al., 1981, 1987; Sundström et al., 1983; Kolb et al., 1997); however, some other papers do not demonstrate or comment on regional differences in pinopode development (Johannisson and Nilsson, 1972; Ferenczy et al., 1972; Ferenczy and Richart, 1973; Ferenczy, 1977; Hafez and Ludwig, 1977; Massai et al., 1993; Nikas et al., 1995). In endometrial cell cultures, pinopode development seemed to vary more than in biopsies. These variations are possibly due to in-vitro circumstances with loss of controlled growth and development. But generally, the morphology of the endometrial cell cultures corresponded very well to that found in biopsy material. Therefore, this model seems to be usable for human implantation studies.

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 embryo–endometrial 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 trophoblastic–endometrial 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.

	Acknowledgments

 
The authors want to thank Susanne Sørensen and Hanne Kruse for excellent preparation of the specimens and photographs. We also want to thank the staff at the Fertility Clinics at Brædstrup, Herlev and Sahlgrenska Hospitals and the Triangle Fertility Clinic in Copenhagen for their assistance in collecting surplus embryos for the study. This study has been supported by research grants from the Michaelsen Foundation, Mimi and Victor Larsens Foundation, the Danish Medical Research Council (no. 28809), the Swedish Medical Research Council (no. 2893) and unrestricted research grants from Organon Denmark and Serono Nordic AB.

	Notes

 
⁵ To whom correspondence should be addressed

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Submitted on July 8, 1998; accepted on November 11, 1998.

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