{alpha}vß3 integrin expression and pinopod formation in normal and out-of-phase endometria of fertile and infertile women

Montserrat Creus1, Jaume Ordi2, Francisco Fábregues1, Roser Casamitjana3, Berta Ferrer2, Elisenda Coll4, Juan A. Vanrell1 and Juan Balasch1,5

1 Institut Clinic of Obstetrics and Gynaecology, 2 Department of Pathology, 3 Hormonal Laboratory and 4 Serveis Cientifico-Tècnics, Faculty of Medicine-University of Barcelona, Hospital Clínic-Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: There is scanty and contradictory information regarding the comparison of traditional histological dating criteria of the endometrium with the expression of the new markers of endometrial receptivity such as {alpha}vß3 integrin and pinopods. Also, definite data with respect to the potential correlation existing between these different new markers in defining the putative window of implantation are lacking. METHODS: The temporal relationship between {alpha}vß3 integrin expression and pinopod formation in normal and out-of-phase endometrial biopsies from normal healthy women (n = 12) and infertile patients (n = 36) was investigated. Two endometrial biopsies (postovulatory day +7 to +8 and 4 days later) were performed during a single menstrual cycle in each subject. Estradiol and progesterone serum concentrations were quantified on the same days as endometrial sampling. RESULTS: No statistically significant difference regarding {alpha}vß3 integrin expression, pinopod formation, and hormone concentrations was found between fertile controls and infertile patients irrespective of endometria being in-phase or out-of-phase. Although a coordinate high level of expression of {alpha}vß3 integrin and pinopod on postovulatory days 7–8 was observed, there was an evident lack of temporal co-expression of these markers over the luteal phase in the endometrial samples investigated. CONCLUSIONS: There is a clear dissociation in the temporal expression of the most cited markers postulated to frame the window of implantation. The functional significance (if any) of these new markers remains to be established.

Key words: endometrial histology/implantation/infertility/integrins/pinopod


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Throughout the menstrual cycle, changes that depend on the interplay between the gonadal steroids estrogen and progesterone take place that prepare the endometrium for implantation. Implantation is the rate-limiting event in reproduction and is still a poorly understood phenomenon. Similarly, establishment of uterine receptivity is still a biological mystery that remains unsolved despite the fact that, over recent years, our understanding of endometrial physiology has advanced markedly following extensive research into the molecules involved in its development and function (Giudice, 1999Go; Daftary and Taylor, 2001Go). It is now generally accepted that the endometrium is receptive to blastocyst implantation only during a short period in the luteal phase known as the implantation window. Based on pregnancy success rates after IVF and embryo transfer at different times after the LH peak, the presence of a probable implantation window of 4 days' duration, from approximately days 5.5–9.5 after ovulation could be inferred (Aplin, 2000Go).

The classic work of Noyes et al. on endometrial morphology and dating has been the basis for the evaluation of human endometrium in normal and abnormal circumstances for many years (Noyes et al., 1950Go). However, the relationship between histological changes and endometrial receptivity remains unknown (Balasch et al., 1992Go; Castelbaum et al., 1994Go). Thus, a reliable marker for uterine receptivity in women is urgently needed. In the last decade, an intensive search for specific markers for receptivity has been undertaken. Thus, a large number of physiological signals expressed in the endometrium during the luteal phase have been investigated, including secreted proteins, cell-surface receptors, nuclear transcription factors, and changes in cell-surface morphology (Giudice, 1999Go; Nikas, 1999aGo,bGo; Daftary and Taylor, 2001Go). Experimental work in animals and clinical studies have indicated that integrins may play a critical role in the process of implantation (Sueoka et al., 1997Go; González et al., 1999Go; Illera et al., 2000Go). Probably the two most cited markers framing the window of implantation are {alpha}vß3 integrin expression and pinopod formation in human endometrial epithelium, which are both estrogen and progesterone dependent as has been shown in natural and mock hormonal treatment cycles in the donor oocyte model (Lessey, 2000aGo,bGo; Nikas, 1999aGo,bGo, 2000Go; Damario et al., 2001Go).

However, there is scanty and contradictory information regarding the comparison of traditional histological dating criteria of the endometrium with the expression of the new markers of endometrial receptivity or with respect to the potential correlation existing between these different new markers in defining the putative window of implantation. Experimental work in vitro and in animals has recently shown that the maternal HOXA 10 homeobox gene directly regulates {alpha}3 integrin subunit and pinopod formation in endometrial cells (Bagot et al., 2001Go; Daftary et al., 2002Go). In the clinical setting, however, Lessey et al. concluded that endometrial integrin expression cannot yet replace traditional methods of endometrial assessment (Lessey et al., 2000Go), whereas another study (Acosta et al., 2000Go) stressed that the Noyes criteria do not seem to be accurate enough to enable the relating of the different events in the window of implantation. In addition, Acosta et al.(2000) reported asynchronous expression of pinopods and integrins in luteal phase endometrial biopsies of 14 healthy fertile women with normal endometria.

To gain further insight into the subject, the present study was undertaken to investigate the temporal relationship in {alpha}vß3 integrin expression and pinopod formation in normal and out-of-phase endometrial biopsies from normal healthy women and infertile patients. {alpha}vß3 integrin was selected because, as previously reported (Lessey et al., 1994Go; Creus et al., 1998Go), its expression is closely correlated with histological maturation of the endometrium and its abrupt appearance on day 20 of the menstrual cycle is coincident with the opening of the window of implantation, as traditionally described (Hertig et al., 1956Go).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patients and study cycle
We investigated the expression of {alpha}vß3 integrin and pinopod formation in the endometrium of infertile patients (n = 36) undergoing a routine work-up, and fertile controls (n = 12) who were undergoing tubal sterilization. The use of human tissue for research was based on informed consent and was approved by the Ethics Committee of our hospital. The mean age of infertility patients was 32.1 ± 1.3 (mean ± SEM) years (range 28–40). All had regular menstrual patterns every 27–32 days. The main causes of infertility in these women were: unexplained (n = 16); minimal to mild endometriosis (n = 8); non-absolute male factor (sperm density >5–<20x106/ml and/or <50% motile sperm grades a and b according to World Health Organization criteria) (Rowe et al., 2000Go) (n = 7); and tubal factor with no hydrosalpinges (n = 5). The control group included 12 fertile (mean parity 1.4, range 1–4) healthy women aged 29–41 years (mean age 33.8 ± 1.1). These control women had regular menstrual cycles (27–32 days) and were taking no medication. In all women, basal body temperature, luteal serum concentrations of estradiol and progesterone and endometrial biopsies were used in the same cycle to assess luteal function according to a scheme of evaluation previously reported (Creus et al., 1998Go).

Commencing on days 8–10 of the study cycle (depending on the cycle length of the woman) all patients underwent daily transvaginal ultrasonographic evaluation of follicular growth using a 5 MHz vaginal transducer attached to an Aloka scanner (Model SSD-620; Aloka Co. Ltd, Tokyo, Japan). The maximum follicular diameter was measured in all patients. Both ovaries were identified, and the largest diameter of all follicles was measured in both the longitudinal and transverse dimensions. The day of ovulation was designated as the day of maximum follicular enlargement, which was followed the next day by sudden disappearance or filling in of this follicle showing loss of clear demarcation of its walls and intrafollicular echoes (Shoupe et al., 1989Go; Peters et al., 1992Go). We used ultrasonographic monitoring of ovulation because previous studies have shown that the accuracy of histological endometrial dating is best determined when ovulation is detected by that method (Shoupe et al., 1989Go; Peters et al., 1992Go).

Two endometrial biopsies were performed during a single menstrual cycle in each subject. The patient's chronological day was determined by counting forward from the ovulation day as detected by ultrasonographic scans. The early biopsy (mid-luteal) was performed on ovulation day +7 to +8 whereas the second biopsy (late luteal) was always performed 4 days after the first biopsy.

Hormones in serum were quantified on the same days as endometrial sampling. All samples were obtained in the fasting state between 0800 and 1000 h which corresponded to the period of minimal progesterone variability, and added to the accuracy of the measurement (Filicori et al., 1984Go).

Endometrial samples
Biopsies were taken from the uterine fundus using a Pipelle (Laboratoire CCD, Paris, France). Endometrial samples were divided into three parts. One of them was fixed in 10% formalin and embedded in paraffin for light microscopy. The second portion of the tissue was snap frozen on methylbutane (Merck, Darmstadt, Germany) immersed in liquid nitrogen and stored at –70°C until immunolabelling for integrin determination. The remaining portion was fixed in glutaraldehyde for scanning electron microscopy (SEM) investigation. The use of separate endometrial portions for light microscopy study and SEM investigation was necessary considering a recent study (Develioglu et al., 2000Go) which concluded that SEM, and not light microscopy, remains the only conclusive tool for the evaluation of the stage of pinopod formation. One observer, an expert gynaecological pathologist, blinded to the identity of the slides as well as the ultrasonographically detected ovulatory day, performed all the assessments.

Endometrial dating
For endometrial dating 4 µm sections stained with haematoxylin and eosin and periodic acid Schiff (PAS) were evaluated. All endometrial biopsies were evaluated according to established histopathological criteria (Noyes et al., 1950Go) using a single-day evaluation whenever possible and when the traditional 2-day spread evaluation method (i.e., days 20–21) was provided, the later day was used for comparison in immunohistochemical assays. An out-of-phase biopsy was defined as >=3-day lag between the chronological and the histological day.

Immunohistochemistry
{alpha}vß3 integrin was detected in frozen sections using the EnVision system (Dako Co, Carpinteria, CA, USA) as previously reported (Creus et al., 1998Go, 2001Go). Briefly, 4 µm sections were fixed for 10 min in acetone at 4°C and dried. After washing in PBS for 5 min the peroxidase was blocked for 5 min in 0.03% H2O2 containing sodium azide. Then the slides were incubated with the primary antibody for 40 min and washed in TBS (Dako). The monoclonal antibody LM609 (Chemicon, Temecula, CA, USA, dilution 1:200), which recognizes the complete {alpha}vß3 heterodimer (Cheresh and Spiro, 1987Go) and has been widely applied by our group (Creus et al., 1998Go, 2001Go; Ordi et al., 2002Go) and others (Lessey et al., 1994Go; Vonlaufen et al., 2001Go;Sturn et al., 2002) was used. The peroxidase labelled polymer was then applied for 40 min. After washing in TBS, the slides were incubated with the diaminobenzidine substrate chromogen solution, washed in distilled water, counterstained with haematoxylin, washed, dehydrated and mounted. In every case a negative control was performed by omission of incubation with the primary specific antibody. As {alpha}vß3 is consistently expressed in vascular endothelia, positive staining of endometrial vessels was considered as the internal positive control (Ordi et al., 2002Go). The reactivity in the endometrial glands and luminal surface epithelium of the endometrium, stromal cells and vessels was assessed. The intensity of staining of the endometrial components was evaluated by a semi-quantitative scoring system (0–4) as follows (Creus et al., 1998Go): absent (–), weak or focal (+), moderate (++), and strong (+++). As in previous work it was found that the expression of {alpha}vß3 in the luminal surface epithelium starts abruptly on days 19–20 of the cycle, thus opening the window of implantation, and only staining in the glands seems to be clinically relevant (Lessey et al., 1992Go; Somkuti et al., 1995Go; Acosta et al., 2000Go), for the specific purpose of this study, endometrial samples were considered as expressing {alpha}vß3 integrin when this integrin was detected in both endometrial glands and luminal surface epithelium with any intensity of the reaction ranging from weak/focal to strong.

Scanning electron microscopy
Endometrial tissue was fixed for at least 24 h in phosphate buffered (0.1 mol/l, ph 7.4) 2.5% glutaraldehyde and post fixed for 1 h in 1% osmium tetroxide. The samples were dehydrated in a graded series of ethanol, critical-point-dried with a Polaron CPD 7501 system (VG Microtech, UK), mounted and coated with gold in a Bio-Rad SC510 sputter coater (VG Microtech, UK). All samples were observed under the same KV and electron beam current conditions in a Zeiss DSM940A SEM (Carl Zeiss, Oberkochen, Germany). For each biopsy 3–9 fragments of 2 mm each were evaluated and at least 4 mm2 of well-preserved epithelial luminal surface was required to be available for evaluation. A thorough examination of the complete surface was conducted. Digital micrographs were taken with the computer program Quartz PCI (Quartz Imaging Co, Vancouver, BC, Canada), and were evaluated independently by two observers. As previously reported (Nikas, 1999aGo,bGo; Acosta et al., 2000Go), pinopods were defined as spherical protrusions without microvilli on the apical surface of the luminal uterine endometrium and were semiquantitatively evaluated as absent (0), isolated pinopods (+), small groups of pinopods (++) and confluent pinopods (+++).

Hormone assays
Hormones in serum were measured using commercially available kits. Estradiol was measured by a competitive immunoenzymatic assay (Immuno 1; Bayer, Tarrytown, NY, USA). The sensitivity of the assay was 10 pg/ml and the interassay coefficients of variation 5%. Progesterone was determined by a competitive chemiluminiscent immunoassay (Immulite, DPC, Los Angeles, CA, USA). The sensitivity of the method was 0.2 ng/ml and the interassay coefficient of variation was 6.7%.

Statistics
Data were analysed by SPSS statistical software (Release 6.0, SPSS Inc., Chicago, IL, USA). The Mann–Whitney U-test and Fisher's exact test were used as appropriate. The Pearson correlation coefficient was used for correlative analyses. Results are expressed as means ± SEM. The level of significance was set at P <= 0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
All menstrual cycles included in the present study were ovulatory according to ultrasonographic criteria and mid-luteal serum progesterone concentration >10 ng/ml. A late luteal endometrial biopsy could not be carried out in four of the 36 infertile women and one of the controls because menses had commenced at the time of the second endometrial sampling. In all instances the endometrial specimens were noted to be clearly progestational fundal samples. No inflammatory or reactive change related to the first sampling was detected in any late luteal biopsy.

Tables I and IIGoGo summarize data relative to endometrial biopsy, {alpha}vß3 integrin expression and pinopod formation on the days of endometrial sampling, in the mid-luteal and late luteal phase biopsies performed in fertile controls and infertile patients, as well as hormone concentrations. No statistically significant difference between the two groups of women was observed for any parameter considered. Overall, at least weak or focal {alpha}vß3 integrin expression was detected in 22 out of the 48 (46%) mid-luteal endometrial biopsies and in 42 out of 43 (98%) late luteal biopsies. In contrast, whereas 81.2% (39/48) of the mid-luteal endometrial samples showed at least the presence of isolated well-formed pinopods, these structures could be seen in only 37% (16/43) of late luteal specimens. As reported in Table IIIGo, we observed differences in {alpha}vß3 integrin expression but not in pinopod formation between in-phase and out-of-phase mid-luteal biopsies for groups of both fertile and infertile women. The percentage of positive samples for {alpha}vß3 integrin expression and pinopod formation was higher, albeit not statistically different, in in-phase endometria of fertile women than in infertile patients. Ovarian steroid hormones were similar in the six groups studied (Table IIIGo).


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Table I. Endometrial biopsy, epithelial {alpha}vß3 integrin expression, pinopod formation and hormonal levels in fertile and infertile women studied in the mid-luteal phase
 

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Table II. Endometrial biopsy, epithelial {alpha}vß3 integrin expression, pinopod formation and hormonal levels in fertile and infertile women studied in the late luteal phase
 

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Table III. {alpha}vß3 integrin expression and pinopod formation in the endometrial epithelium of in-phase and out-of-phase mid-luteal endometrial biopsies in fertile and infertile women
 
{alpha}vß3 integrin expression was closely correlated with histological maturation of the endometrium appearing mainly at postovulatory days 6–7 and being expressed by almost all endometria dated as postovulatory day >=8. The intensity of its expression also increased from mid-luteal to late postovulatory days (Figure 1Go).



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Figure 1. Immunohistochemistry of {alpha}vß3 integrin in endometrial specimens. No expression of {alpha}vß3 integrin was detected in the epithelial cells at histological postovulatory day +3 (score 0, top left). At histological postovulatory day +6 (top right) focal immunostaining (score +) was detected in both the surface and the glandular epithelium. At histological postovulatory day +7 more extensive immunostaining is observed (score ++, bottom left) whereas a strong reaction was observed in all epithelial cells at postovulatory day +12 (score +++, bottom right). Original magnification 200x.

 
When examined by SEM the endometrial apical luminal surface was predominantly covered in all instances by ciliated and microvillous cells, with the last being the predominant component. Pinopods were semiquantitatively evaluated as detailed in the Materials and methods section. When present, pinopods appeared as bulbous protrusions with a smooth surface, which developed on the apical surface of microvillous cells (Figure 2Go). In several cases, pinopods appeared as small protrusions with a sea-anemone appearance (developing pinopods). Pinopods were usually focal and were frequently located around the opening of endometrial glands. Although large confluent areas covered by pinopods could be observed in some endometria, full coverage of the endometrial luminal surface by these structures was not observed in any sample.



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Figure 2. Scanning electron microscopy showing the apical surface of the luminal uterine epithelium in endometrial samples. Top left: postovulatory histological day +5; infertile woman: ciliated and microvillous cells are clearly seen (score 0). Top right: a few isolated pinopods with smooth surface are seen (score +) in a sample from a fertile woman on postovulatory histological day +12. Bottom left: several developing pinopods with bulging, villous, sea-anemone-like surface and several well-developed pinopods (score ++) (fertile patient, postovulatory histological day +4). Bottom right: fully developed pinopods cover the surface (score +++) (infertile patient, postovulatory histological day +4). Original magnification 2000x.

 
In contrast with the expression of {alpha}vß3 integrin, pinopods (already expressed in endometria dated as postovulatory day 3) were observed in 80–100% of endometrial biopsies dated as postovulatory days 4–8 and were much less frequently observed in endometria dated as postovulatory day >=10 (Figure 3Go). These changes in {alpha}vß3 integrin expression and pinopod formation occurred irrespective of endometria being in-phase or out-of phase. A coordinately high level of expression of both markers existed on postovulatory days 7–8 (Figure 3Go). However, the lack of temporal co-expression of {alpha}vß3 integrin and pinopod over the luteal phase in the endometrial samples studied is further evidenced in Table IVGo, which shows that the simultaneous presence or absence of both markers was observed in only 50% of cases. In addition, a lack of correlation (r = –0.228, P = 0.124, not significant) between staining intensity for {alpha}vß3 integrin and grading for pinopod formation in the mid-luteal phase biopsy was clearly evidenced in the whole study population (Figure 4Go). No differences either in mid-luteal or late luteal serum concentrations of estradiol and progesterone were detected among groups when stratified by the expression or not of {alpha}vß3 integrin, and by the presence or absence of pinopods (data not shown).



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Figure 3. Percentage of endometria showing pinopods and {alpha}vß3 integrin in epithelial cells for each histological day.

 

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Table IV. Correlation of {alpha}vß3 integrin expression with pinopod formation in the mid-luteal endometrial biopsies of fertile and infertile women
 


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Figure 4. Correlation between staining intensity for {alpha}vß3 integrin expression and pinopod formation in epithelial cells of mid-luteal endometrial biopsies in the whole group of patients (n = 48).

 
Ten infertile patients became spontaneously pregnant within 1 year after the study cycle. Of them, seven had in-phase endometria, nine evidenced pinopod formation, and four expressed {alpha}vß3 integrin in the mid-luteal phase biopsy. No differences were found between partners of women who became and did not become pregnant regarding sperm concentration (83x106/ml versus 65x106/ml), motility (57 versus 53%), and morphology (strict criteria) (20 versus 19%).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Implantation depends on the synchronization of two biological clocks—the embryonic clock and the maternal clock—and synchronous developmental progression is required between embryo and endometrium in most mammalian species (Aplin, 2000Go). The maternal clock effectively begins at ovulation when steroid production in the ovary switches from estrogen to progesterone plus estrogen production. Acting through receptors in epithelial, vascular and stromal cells, progesterone induces the endometrium to differentiate so that it reaches a state of readiness for implantation ~5.5 days after ovulation. Thus, implantation occurs in the mid-secretory phase of the menstrual cycle when the endometrium undergoes a defined period of receptivity. In other words, in the human, as in several other mammalian species, there appears to be a maternal steroidally controlled window during which implantation may occur (Aplin, 2000Go; Castelbaum and Lessey, 2001Go).

The above notwithstanding, there is little agreement that there is such a thing as endometrial receptivity. Several markers of the implantation window have been proposed in the recent literature, being integrins (mainly integrin {alpha}vß3) and pinopods among the most extensively characterized markers of endometrial receptivity (Lessey, 2000aGo,bGo; Nikas, 1999aGo,bGo, 2000Go; Castelbaum and Lessey, 2001Go). This report, where ovulation day was appropriately documented with daily ultrasonographic scans and both markers were identified as being present in the luteal phase, clearly showed a lack of temporal relationship in the expression of integrin {alpha}vß3 and pinopods in luteal phase endometria of both fertile women and infertile patients. Both the percentage of mid-luteal endometrial samples expressing integrin {alpha}vß3 in the epithelial cells, and the mean intensity of expression, increased progressively with the histological date of the endometrium after its appearance mainly on postovulatory day 7–8 and this occurred irrespective of in-phase or out-of-phase endometria. This may explain the apparent discrepancy with previous studies in parous women having high levels of expression of integrin {alpha}vß3 in endometria dated as postovulatory days 22–24 but where the period of postovulatory duration was not considered (Lessey et al., 1994Go). The reverse occurred with pinopod formation; in our material, pinopods were present mainly from postovulatory histological days 4–8 and their expression was markedly reduced afterwards, again irrespective of in-phase or out-of-phase endometria. Such asynchronous expression of the two markers may explain why the number of endometrial samples expressing {alpha}vß3 integrin but not pinopods was significantly lower among out-of-phase mid-luteal biopsies than for in-phase biopsies. Similarly, this fact may also explain why most infertile women becoming spontaneously pregnant after the study cycle had pinopods detected, but not expression of the {alpha}vß3 integrin in the mid-luteal biopsy. In fact, we have previously reported a lack of relationship between epithelial {alpha}vß3 integrin expression and fertility (Creus et al., 1998Go). The present report and previous studies by our group and others (Balasch and Vanrell, 1987Go; van der Linden et al., 1995Go; Creus et al., 1998Go, 2001Go) have also shown no relationship between the expression of endometrial integrins and the expression of estrogen and progesterone receptors in human endometrium or serum levels of ovarian steroids. Therefore, our data do not support the notion that analysis of {alpha}vß3 integrin expression and pinopod formation provides additional useful information beyond that derived from histological dating alone.

This is in agreement with a previous study (Lessey et al., 2000Go), concluding that the detection of integrins may reflect the endometrial function or receptivity, but cannot yet replace the traditional methods of endometrial assessment. Results in the present study are also in concordance with a previous report (Acosta et al., 2000Go) studying 14 healthy fertile women which concluded that the temporal patterns of expression of integrins and pinopods in that material were clearly asynchronous. It has been previously reported that the appearance of the pinopods is limited to a period of 24–48 h at the approximate time of blastocyst implantation, so their presence would indicate the `opening' of the window of implantation (Nikas, 1999aGo,bGo, 2000Go). Our results and those from others (Acosta et al., 2000Go) are somewhat different. In both studies, isolated or small groups of well-formed pinopods appeared on days 18–20 and persisted for the rest of the luteal phase, sometimes becoming more confluent but at no time covering the entire endometrial luminal surface. Therefore, integrins and pinopods as potential markers of implantation are defining different periods, if any, of endometrial receptivity. This notwithstanding, it should be noted that the cycle days on which pinopods develop may vary by up to 4 days between women (from days 19–22) (Nikas, 1999aGo, 2000Go) and thus a temporal relationship between {alpha}vß3 integrin expression and pinopod appearance can be found in some women (Lessey, 2000aGo).

In both the present report and a previous study (Acosta et al., 2000Go) two endometrial biopsies were taken during a single menstrual cycle in each woman. Thus, it could be argued that this fact might have affected the results. Although a mechanical effect of the first biopsy in inducing endometrial differentiation in the second biopsy cannot be completely excluded, this is unlikely. None of the late luteal biopsies revealed inflammatory or reactive changes consistent with a previous biopsy site and this is in keeping with previous findings by our group (Creus et al., 1998Go; Ordi et al., 2002Go) and others (Castelbaum et al., 1994Go) when using two endometrial biopsies performed during a single menstrual cycle for luteal phase investigation. In fact, the normal pattern of pinopod expression has been established on the basis of sequential endometrial biopsies performed in normal menstruating women (Nikas, 1999aGo,bGo).

Difficulties in establishing the concept of markers or biomarkers for the window of implantation have been previously emphasized (Coutifaris et al., 1998Go; Acosta et al., 2000Go). Because the initial phases of actual human implantation (apposition, adhesion, attachment and penetration through surface epithelium and basement membrane) have not been visualized, the real chronology of this period of endometrial receptivity is theoretical. Thus, most of the information is, at present, merely descriptive and correlative. On the other hand, whereas some of the biomarkers proposed in the literature may be related to apposition, adhesion and attachment and, therefore, they should be present at the level of the luminal surface epithelium, other markers may play no role at the early stages of implantation and be present and/or have a function at more advanced stages of nidation. Extracellular matrix components such as integrins, a widely expressed family of cell surface adhesion receptors, may promote embryo attachment (Sueoka et al., 1997Go; Giudice, 1999Go; Lessey et al., 2000aGo), whereas pinopods, specialized cell surface structures involved in endocytosis and pinocytosis, may be involved in trophoblast adhesion and/or facilitation of penetration to the stroma (Nikas, 1999aGo,bGo). Finally, it is noteworthy that the presence of the embryo in the uterine cavity could induce special local characteristics which are not present when we are investigating a non-conception cycle.

In conclusion, the present study and the few reports in the literature indicate that: (i) there is a clear dissociation in the temporal expression during the luteal phase of the most cited markers postulated to frame the window of implantation, and (ii) functional significance of the findings, apart from corroborating the significance (if any) of histological delay, is mostly hypothetical and potential application of these findings to clinical practice should await rigorous testing of these hypotheses in appropriately designed trials. This study, however, may have a type II statistical error because the number of patients included, mainly fertile controls, is limited. Considering the differences obtained in statistical comparisons conducted between different study groups in the present investigation, a sample size ranging between 25–1500 patients per group would be necessary in order to provide an 80% statistical power of avoiding a type II error, and a 5% chance of making a type I error. Thus, further studies are warranted to establish whether the investigation of endometrial integrin expression and pinopod formation may help in evaluating implantation potential in women.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors are grateful to Ms Elena Rull, Cristina Durana and Paquita Antonell for their technical assistance in the histological process and Dr Antonio Palacín, Ms Margarita Mainar and Ms Elena Gonzalvo for their help with the immunohistochemical studies. We thank Ms Teresa Roch for the preparation of the manuscript. This work was supported in part by grants 98/1193 and 00/0399 from the Fondo de Investigaciones Sanitarias to J.B.


    Notes
 
5 To whom correspondence should be addressed at: Institut Clinic of Obstetrics and Gynaecology. Hospital Clínic, c/ Casanova 143, 08036-Barcelona, Spain. E-mail: jbalasch{at}medicina.ub.es Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Acosta, A.A., Elberger, L., Borghi, M, Calamera, J.C., Chemes, H., Doncel, G.F., Kliman, H., Lema, B., Lustig, L. and Papier, S. (2000) Endometrial dating and determination of the window of implantation in healthy fertile women. Fertil. Steril., 73, 788–798[ISI][Medline]

Aplin, J.D. (2000) The cell biological basis of human implantation. Baillière's Clin. Obstet. Gynaecol., 14, 757–764.

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Balasch, J., Fábregues, F., Creus, M. and Vanrell, J.A. (1992) The usefulness of endometrial biopsy for luteal phase evaluation in infertility. Hum. Reprod., 7, 973–977.[Abstract]

Bagot, C.N., Kliman, H.K. and Taylor, H.S. (2001) Maternal HOXA10 is required for pinopod formation in the development of mouse uterine receptivity to embryo implantation. Dev. Dyn., 222, 538–544.[ISI][Medline]

Castelbaum, A.J. and Lessey, B.A. (2001) Infertility and implantation defects. Infertil. Reprod. Med. Clin. N. Am., 12, 427–446.

Castelbaum, A.J., Wheeler, J., Coutifaris, C., Matroianni, L.,Jr. and Lessey, B.A. (1994) Timing of the endometrial biopsy may be critical for the accurate diagnosis of luteal phase deficiency. Fertil. Steril., 61, 443–447.[ISI][Medline]

Cheresh, D.A. and Spiro, R.C. (1987) Biosynthetic and functional properties of an Arg-Gly-Asp-directed receptor involved in human melanoma cell attachment to vitronectin, fibrinogen and von Willebrand factor. J. Biol. Chem., 262, 17703–17711.[Abstract/Free Full Text]

Coutifaris, C., Omigbodun, A. and Coukos, G. (1998) Integrins, endometrial maturation, and human embryo implantation. Sem. Reprod. Endocrinol., 16, 219–229.[ISI][Medline]

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Submitted on December 13, 2001; resubmitted on March 20, 2002; accepted on May 16, 2002.