Molecular Human Reproduction, Vol. 8, No. 8, 765-769,
August 2002
© 2002 European Society of Human Reproduction and Embryology
Uterine physiology |
Co-existence of heparin-binding epidermal growth factor-like growth factor and pinopodes in human endometrium at the time of implantation
1 Department of Woman and Child Health, Division of Reproductive Endocrinology, 2 Division of Pediatrics, Karolinska Hospital, 3 Department of Clinical Science, Division of Obstetrics and Gynaecology, Huddinge University Hospital, Karolinska Institutet, Stockholm, Sweden and 4 Yerevan State Medical University, Yerevan, Armenia
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Abstract |
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Pinopodes have been suggested to be markers of uterine receptivity. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is expressed in increasing amounts in the secretory phase endometrium and is considered to be important for the human implantation process. The aim of this study was to investigate a possible co-existence of pinopodes and HB-EGF in the normal human endometrium. Endometrial biopsies were obtained from women with normal menstrual cycles. The biopsies were examined by scanning electron microscopy for the detection of pinopodes, by immunohistochemistry for the expression of HB-EGF protein, and by confocal microscopy to determine if HB-EGF was present on the surface of the pinopodes. The expression of HB-EGF in luminal and glandular epithelium was highest when fully developed pinopodes were present. Using confocal microscopy it was shown that HB-EGF was present both inside the luminal epithelial cells and on the surface of pinopodes. These findings suggest that HB-EGF might play a role in both the attachment and penetration steps in the human implantation process. Furthermore, the immunohistochemical staining demonstrates that HB-EGF can be used as a marker for the implantation window.
endometrium/HB-EGF/pinopodes
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been suggested to have a role in the implantation process (Raab et al., 1996
; Yoo et al., 1997; Leach et al., 1999), and could therefore be a good marker for the receptive endometrium.
HB-EGF is a member of the EGF family of growth factors which exert their action via EGF receptors. HB-EGF binds to two members of the EGF receptor family, the HER1/ErbB1 and the HER4/ErbB4 receptors (Higashiyama et al., 1991; Elenius et al., 1997). HB-EGF exists in two biologically active forms, one transmembrane and one mature soluble form processed from a membrane-anchored precursor (Raab and Klagsbrun, 1997). The mature form of HB-EGF is a potent stimulator of cell proliferation, migration and cell motility, while the transmembrane form of HB-EGF acts as a juxtacrine growth and adhesion factor (Raab and Klagsbrun, 1997).
HB-EGF has been suggested to play several roles during the implantation process. HB-EGF promotes human blastocyst growth and differentiation (Martin et al., 1998). Additionally, the transmembrane form of HB-EGF promotes adhesion of the blastocyst to the uterine wall in a mouse in-vitro system (Raab et al., 1996). The EGF receptor HER4/ErbB4, specific for HB-EGF in the reproductive system, is expressed on the apical surface of mouse trophectoderm cells at the time of implantation (Raab et al., 1996). It has previously been shown that HB-EGF expression is elevated in the glandular and luminal epithelium during the secretory phase (Yoo et al., 1997; Leach et al., 1999).
Pinopodes on the endometrial surface have been suggested as ultrastructural markers of the implantation window (Martel, 1981; Nikas et al., 1995). The function of the pinopodes is not clear, and there are several possible explanations for their existence. Pinopodes were first discovered in rodents, and it was suggested that these structures were responsible for the uptake of uterine secretion (Enders and Nelson, 1973; Parr and Parr, 1974). This function of the pinopodes has only been observed in rodents. Uterine secretion is reduced in humans at the time of implantation (Gemzell-Danielsson and Hamberg, 1994). However, a connection between pinopodes and this reduction has not been established.
It is likely that adhesion factors present on the luminal epithelial surface are important for the initial steps of the implantation process. HB-EGF and integrin 
vß3 have been proposed as adhesion factors on the membrane of the pinopodes (Yoo et al., 1997; Lessey, 1998). Other factors, e.g. growth factors and cytokines, have been suggested as candidates important both for communication between the endometrium and the blastocyst and for the implantation process (Salamonsen et al., 2000).
To our knowledge, no data have been published showing the presence of biochemical markers restricted to pinopodes. In the present study, we have focused on the presence of pinopodes in the endometrium and the expression of HB-EGF in the luminal epithelial surface of the endometrium. To characterize the human endometrium at the time of implantation, the co-existence of HB-EGF and pinopode formation was determined by scanning electron microscopy (SEM), immunohistochemistry and confocal microscopy.
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Materials and methods |
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Clinical material
Twenty apparently healthy women with a history of regular menstrual periods (25–35 days) and with proven fertility volunteered for the study. The mean age of the women was 37 years (range 25–44). Seven women were >40 years old, but all of them had regular menstrual cycles. None of the women had used steroidal contraceptives or an intrauterine device for at least 3 months prior to the study. None of the women had a miscarriage or delivery within 1 year prior to the study, or had ever had a history of pelvic inflammatory disease. The study was approved by the ethics committees at the Karolinska Hospital (93–261, for normal volunteers) and at the Huddinge University Hospital (for sterilization patients, 294/01). Informed consent was obtained from all participating women.
Endometrial biopsies
Endometrial biopsies were obtained in the luteal phase from the anterior wall of the uterine cavity, without dilatation of cervix, using a Randall curette (Stille Werner AB, Stockholm, Sweden). The day the biopsy was obtained was randomly selected between days LH +4 to +13. Participants identified the day of the LH surge by testing their morning urine (Clearplan; Unipath Ltd, Bedford, UK). The specimens were divided into two pieces and immediately fixed. One of the pieces was processed for SEM and the other for immunohistochemistry and confocal microscopy. Samples for SEM were fixed in a solution containing 2.5% (w/v) glutaraldehyde, 0.5% paraformaldehyde, 0.1 mol/l sucrose, 0.1 mol/l sodium cacodylate and 3 mmol/l calcium chloride (pH 7.4). Samples for immunohistochemistry and confocal microscopy were fixed in 4% phosphate-buffered formaldehyde for a maximum of 24 h and stored in 70% ethanol until being embedded.
SEM
The samples were washed twice in a buffer containing sodium cacodylate (0.15 mol/l) and calcium chloride (3 mmol/l, pH 7.4) and once in distilled water. The specimens were dehydrated in increasing concentrations of ethanol (70, 95 and 99.5%), followed by acetone. They were dried in a critical-point dryer with CO2. The specimens were mounted, coated with platinum and examined using a Jeol 820 SEM.
Immunohistochemistry
The biopsy samples were embedded in paraffin and cut into 4 µm sections. The sections were dewaxed in Bioclear (Bio-Optica, Milan, Italy) and rehydrated in a decreasing concentrations of ethanol and finally in distilled water. Endogenous peroxidases were subsequently blocked by 3% hydrogen peroxide in methanol for 10 min. Sections were washed in phosphate-buffered saline (PBS), covered by 100 µl normal goat serum as blocking serum and incubated for 30 min. The sections were then incubated with primary antibody at room temperature for 1 h.
The HB-EGF antibody was a goat anti-human HB-EGF (AF-259-NA) purchased from R&D Systems Inc. (Abingdon, UK). Normal placenta was used as positive control and normal goat IgG was used as negative control.
The slides were washed in PBS with 0.01% Tween 20. As a secondary antibody, biotinylated rabbit anti-goat antibody (Vector Laboratories, Burlingame, CA, USA) was used. Following incubation for 30 min and washing, the sections were incubated with a freshly prepared solution of horseradish peroxidase–avidin–biotin complex (Vectastain ABC Elite; Vector) for 30 min. After washing, the site of bound enzyme was visualized by application of 3,3-diaminobenzidine in H2O2 (DAB kit; Vector), a chromogen that produced a brown insoluble precipitate when incubated with enzyme. The slides were washed in PBS 0.01% Tween 20. The sections were counterstained with haematoxylin and dehydrated before mounting with Pertex (Histolab, Gothenburg, Sweden).
Confocal microscopy
The same initial treatment was used for the immunostaining for confocal microscopy as for immunohistochemistry with the following exceptions. The peroxidase treatment was omitted. As second antibody, Cy3-conjugated rabbit anti-goat IgG (305-165-045; Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) was used. The slides were incubated for 1 h at room temperature with the secondary antibody, thereafter washed twice in PBS and then mounted in 90% glycerol in PBS. Confocal microscopy recordings were taken with the use of a Leica TCS SP inverted confocal scanning laser microscope using a 40X/1.25 NA objective with excitation at 543 nm and detection at 552–600 nm. Optical sections were recorded through the centre of the tissue to localize the fluorescence signal.
Evaluation of immunostaining
Two observers, blind to the identity of the slides, performed all assessments. The staining was evaluated semi-quantitatively using a grading system. The staining intensity and the number of stained cells were graded on a scale of 0 = no staining, +/– = few stained cells, + = faint staining, ++ = moderate staining and +++ = strong staining. Each sample was analysed twice, and the average value from the two observers was calculated. Statistical evaluations were performed using analysis of variance on ranks (Kruskal–Wallis) followed by Dunn's test.
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Results |
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SEM
The biopsies were grouped according to the developmental stage of the pinopodes (Stavreus-Evers et al., 2001
). Figure 1m shows a representative picture of pinopodes in the human endometrium. The number of biopsies in each group and the day of the cycle related to the LH surge are shown in Table I.
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Immunohistochemistry
The immunostaining of HB-EGF was seen in the cytoplasm of both the epithelial and stromal cells (Figure 1
). Prior to the appearance of pinopodes, the staining of HB-EGF was faint in the luminal epithelium and in the stroma (Figure 1a). Figure 1d shows the luminal epithelium at a higher magnification. The staining in the luminal epithelium increased and stayed intense when the pinopodes were present (Figure 1b and Figure 2A). A more detailed picture of intensely stained luminal epithelium, with pinopodes, is shown in Figure 1e. Following the disappearance of the pinopodes, the HB-EGF immunostaining in the luminal epithelium decreased and became faint (Figure 1c,f). The glandular epithelium showed the same time-dependent expression pattern as the luminal epithelium, but with a generally less intense staining. Figure 1g shows the faint immunostaining of glandular epithelium prior to pinopode appearance. Immunostaining was more intense in the glandular epithelium when the pinopodes were present, although the staining was not as intense as in the luminal epithelium (Figure 1h and 2B). The immunostaining in the glandular epithelium was again less intense following the disappearance of the pinopodes. The immunostaining in the stroma varied among different biopsies, but was not influenced by the day of the cycle. Immunostaining of HB-EGF was also present in blood vessels but without cyclic variation (Figure 1j,k)
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Confocal microscopy
Confocal microscopy demonstrated the presence of HB-EGF both in the luminal and glandular epithelial cells. HB-EGF was present in the cytoplasm and also on the surface of the pinopodes (Figure 1l
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Discussion |
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The most significant finding in this study was the relationship between pinopode development and the expression of HB-EGF. Although HB-EGF shows faint staining throughout the cycle (Leach et al., 1999
), the intense staining of HB-EGF, especially in the luminal epithelial cells at the time of maximum pinopode formation, suggests that both HB-EGF and the pinopodes play a role in the implantation process.
The increase in HB-EGF immunostaining at the time of implantation observed in this study confirms the findings from other groups (Yoo et al., 1997; Leach et al., 1999), showing that HB-EGF and HB-EGF mRNA increase in the mid- to late luteal phase. The scatter plot shows an individual variation, which leads to an overlap between the groups. However, it is clear that the intensity is more pronounced when the pinopodes are present. A de-novo synthesis of HB-EGF at the time of implantation has been suggested (Leach et al., 1999). Those studies were performed using endometrium from hysterectomy patients suffering from leiomyoma or cervical disorders. However, it has been shown that women with leiomyomas have a reduced production of HB-EGF in the endometrium during the luteal phase compared with normal controls (Ali et al., 2000). Therefore, regularly menstruating healthy fertile women were used as donors of endometrium in the present study.
A previous study demonstrated expression of HB-EGF in `ciliate-like projections' (Yoo et al., 1997). It was not further investigated whether these `ciliate-like' projections corresponded to pinopodes. The pinopodes on the epithelial surface are visible in light microscopy. However, other structures may be mistaken for pinopodes in light microscopy (Develioglu et al., 2000). Therefore, SEM was used both to confirm the presence of pinopodes in the endometrial tissue and to determine the developmental stage of the pinopodes.
The development of pinopodes in the endometrium seems to be synchronized. However, it is common to see both developing and fully developed pinopodes in the same sample (Martel, 1981; Kolb et al., 1997; Stavreus-Evers et al., 2001). Table I shows the appearance of the majority of the pinopodes.
The lifespan of pinopodes does not exceed 48 h (Nikas et al., 1995). Acosta et al. found projections resembling pinopodes late in the cycle, and suggested a lifespan of at least 7 days (Acosta et al., 2000). However, projections found late in the cycle differ in shape from pinopodes and are probably due to apoptotic or necrotic processes that precede menstruation.
Progesterone and progesterone receptors have been suggested to be involved both in the regulation of pinopode formation (Develioglu et al., 1999; Stavreus-Evers et al., 2001) and in the regulation of HB-EGF (Leach et al., 1999). In the baboon, it has been shown that HB-EGF accumulation is increased in the endometrium at the time of implantation, and that treatment with antiprogestins delays the HB-EGF increase. This suggests that progesterone is involved in the regulation of HB-EGF in the endometrium (Leach et al., 2001). Thus progesterone may be important for the regulation of HB-EGF and pinopodes and thereby also for the implantation process.
HB-EGF is a mitogen for human epithelial cells (Wilson et al., 1994; Kiso et al., 1995) and is also associated with cell migration and motility (Higashiyama et al., 1995; Faber-Elman et al., 1996). The transmembrane form of HB-EGF is associated with cell adhesion and cell migration (Iwamoto et al., 1994; Raab et al., 1996). In the present study, it was found that HB-EGF exists in the cytoplasm, and also on the surface of the pinopodes. It is possible that HB-EGF exists in the transmembrane form on the pinopodes. However, the possibility also exists that HB-EGF secreted from the endometrium is bound to a receptor on the endometrial surface. It is therefore possible that maternal HB-EGF binds to the embryo through a juxtacrine mechanism involving ErbB4. In both rats and humans, HB-EGF is involved in preimplantation embryo development (Martin et al., 1998; Tamada et al., 1999). In the mouse embryo the ErbB4 receptor, which binds HB-EGF, is translocated to the apical surface at the time of implantation (Paria et al., 1999; Wang et al., 2000). Consequently, HB-EGF, conceivably from maternal origin, may be important for the development of the embryo.
HB-EGF is involved in re-epithelialization during the healing process (Marikovsky et al., 1993, 1996). In addition, HB-EGF offers a protective role against cell hypoxia and ischaemia by down-regulation of the iNOS/NO pathway (Xia et al., 2001), and it has also been shown that addition of HB-EGF to an established apoptotic-inducing stimulus results in down-regulation of the relative levels of apoptosis (Michalsky et al., 2001). For this reason, it is also possible that this ability of HB-EGF is needed for healing of the endometrial surface at the site of implantation endometrium after penetration of the trophoblast cells through the epithelial surface. It is also possible that HB-EGF plays a regulatory role in trophoblast invasion by reducing apoptosis during implantation.
In summary, the intense expression of HB-EGF in the luminal epithelium and on the surface suggests that HB-EGF is important for implantation, and it is likely that HB-EGF plays several roles in this process. The first one could be that HB-EGF is proteolytically cleaved from the transmembrane form to communicate with the blastocyst, to promote the development of the blastocyst and/or to act as a chemoattractant for the blastocyst. The second role may be that HB-EGF can act as a transmembrane factor at the pinopodes during the blastocyst attachment phase to support adhesion of the blastocyst to the endometrial surface. The third role for HB-EGF may be to aid the healing of the epithelial surface after penetration of the blastocyst. In this study it was also shown that immunohistochemical demonstration of HB-EGF may be used as a marker of the implantation window.
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Acknowledgements |
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We would like to thank Dr Yorgos Nikas for valuable discussions. This work was supported by grants from the Swedish Medical Research Council (3972 and 12238), the Swedish Society for Medical Research, the Swedish Society of Medicine, Wallenbergstiftelsen, Recwood and Ragnhild and Elsa Lundströms minne.
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Notes |
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5 To whom correspondence should be addressed. E-mail: anneli.stavreus-evers{at}kbh.ki.se
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Submitted on November 30, 2001; resubmitted on February 14, 2002; accepted on April 25, 2002.
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