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
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Abstract |
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Key words: endometrial histology/implantation/infertility/integrins/pinopod
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Introduction |
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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., 1950). However, the relationship between histological changes and endometrial receptivity remains unknown (Balasch et al., 1992
; Castelbaum et al., 1994
). 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, 1999
; Nikas, 1999a
,b
; Daftary and Taylor, 2001
). Experimental work in animals and clinical studies have indicated that integrins may play a critical role in the process of implantation (Sueoka et al., 1997
; González et al., 1999
; Illera et al., 2000
). Probably the two most cited markers framing the window of implantation are
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, 2000a
,b
; Nikas, 1999a
,b
, 2000
; Damario et al., 2001
).
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 3 integrin subunit and pinopod formation in endometrial cells (Bagot et al., 2001
; Daftary et al., 2002
). In the clinical setting, however, Lessey et al. concluded that endometrial integrin expression cannot yet replace traditional methods of endometrial assessment (Lessey et al., 2000
), whereas another study (Acosta et al., 2000
) 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 vß3 integrin expression and pinopod formation in normal and out-of-phase endometrial biopsies from normal healthy women and infertile patients.
vß3 integrin was selected because, as previously reported (Lessey et al., 1994
; Creus et al., 1998
), 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., 1956
).
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Materials and methods |
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Commencing on days 810 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., 1989; Peters et al., 1992
). 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., 1989
; Peters et al., 1992
).
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., 1984).
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., 2000) 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., 1950) using a single-day evaluation whenever possible and when the traditional 2-day spread evaluation method (i.e., days 2021) 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
vß3 integrin was detected in frozen sections using the EnVision system (Dako Co, Carpinteria, CA, USA) as previously reported (Creus et al., 1998
, 2001
). 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
vß3 heterodimer (Cheresh and Spiro, 1987
) and has been widely applied by our group (Creus et al., 1998
, 2001
; Ordi et al., 2002
) and others (Lessey et al., 1994
; Vonlaufen et al., 2001
;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
vß3 is consistently expressed in vascular endothelia, positive staining of endometrial vessels was considered as the internal positive control (Ordi et al., 2002
). 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 (04) as follows (Creus et al., 1998
): absent (), weak or focal (+), moderate (++), and strong (+++). As in previous work it was found that the expression of
vß3 in the luminal surface epithelium starts abruptly on days 1920 of the cycle, thus opening the window of implantation, and only staining in the glands seems to be clinically relevant (Lessey et al., 1992
; Somkuti et al., 1995
; Acosta et al., 2000
), for the specific purpose of this study, endometrial samples were considered as expressing
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 39 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, 1999a,b
; Acosta et al., 2000
), 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 MannWhitney 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.
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Results |
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Tables I and II summarize data relative to endometrial biopsy,
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
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 III
, we observed differences in
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
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 III
).
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Discussion |
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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 vß3) and pinopods among the most extensively characterized markers of endometrial receptivity (Lessey, 2000a
,b
; Nikas, 1999a
,b
, 2000
; Castelbaum and Lessey, 2001
). 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
vß3 and pinopods in luteal phase endometria of both fertile women and infertile patients. Both the percentage of mid-luteal endometrial samples expressing integrin
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 78 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
vß3 in endometria dated as postovulatory days 2224 but where the period of postovulatory duration was not considered (Lessey et al., 1994
). The reverse occurred with pinopod formation; in our material, pinopods were present mainly from postovulatory histological days 48 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
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
vß3 integrin in the mid-luteal biopsy. In fact, we have previously reported a lack of relationship between epithelial
vß3 integrin expression and fertility (Creus et al., 1998
). The present report and previous studies by our group and others (Balasch and Vanrell, 1987
; van der Linden et al., 1995
; Creus et al., 1998
, 2001
) 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
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., 2000), 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., 2000
) 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 2448 h at the approximate time of blastocyst implantation, so their presence would indicate the `opening' of the window of implantation (Nikas, 1999a
,b
, 2000
). Our results and those from others (Acosta et al., 2000
) are somewhat different. In both studies, isolated or small groups of well-formed pinopods appeared on days 1820 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 1922) (Nikas, 1999a
, 2000
) and thus a temporal relationship between
vß3 integrin expression and pinopod appearance can be found in some women (Lessey, 2000a
).
In both the present report and a previous study (Acosta et al., 2000) 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., 1998
; Ordi et al., 2002
) and others (Castelbaum et al., 1994
) 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, 1999a
,b
).
Difficulties in establishing the concept of markers or biomarkers for the window of implantation have been previously emphasized (Coutifaris et al., 1998; Acosta et al., 2000
). 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., 1997
; Giudice, 1999
; Lessey et al., 2000a
), 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, 1999a
,b
). 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 251500 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.
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Acknowledgements |
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Notes |
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References |
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Submitted on December 13, 2001; resubmitted on March 20, 2002; accepted on May 16, 2002.