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Within-Subject Between-Cycle Variability of Histological Dating, vß3 Integrin Expression, and Pinopod Formation in the Human Endometrium

Department of Pathology (J.O., A.C.); Institut Clinic of Gynaecology, Obstetrics and Neonatology (M.C., J.B.); Epidemiology and Biostatistics Unit (L.Q.); and Hormonal Laboratory (R.C.), Faculty of Medicine-University of Barcelona, Hospital Clínic, Institut d’Investigacions Biomèdiques August Pi i Sunyer, Barcelona 08036, Spain

Address all correspondence and requests for reprints to: Juan Balasch, M.D., Institut Clinic of Obstetrics and Gynaecology, Hospital Clínic, c/Casanova 143, 08036 Barcelona, Spain. E-mail: jbalasch{at}medicina.ub.es.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
vß3 integrin expression and pinopod formation have been proposed as a means of distinguishing receptive endometrium from nonreceptive in clinical practice. However, one of the most intriguing facts in infertility is whether one cyclic event may be representative of all patients’ cycles, and no study has evaluated the cycle-to-cycle variability of the expression of any of these new proposed markers in human endometrium. We investigated histological dating, vß3 integrin expression, and pinopod formation in 45 endometrial biopsy specimens obtained in 15 primary infertility patients. All patients underwent three endometrial biopsies in consecutive spontaneous cycles, regardless of the previous histological findings. All endometrial samples were obtained on postovulatory d 7 as determined by ultrasonography. Agreement between the repeated observations (first vs. second, and first vs. third biopsies) with regard to histological dating and the presence or absence of vß3 integrin and pinopods in the endometrium was analyzed using the 2 x 2 frequency tables and Cohen’s coefficient. The values ranged from a low of -0.39 (level of agreement, poor) for vß3 integrin expression to a high of 0.32 (level of agreement, fair) for biopsy dating by anticipated window of implantation. Overall, these results demonstrate that all endometrial variables investigated had poor reproducibility and high variability cycle to cycle.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
A NORMAL ENDOMETRIAL milieu is essential in embryo implantation, and understanding the factors that contribute to a receptive endometrium is, at present, a pivotal area of research. Traditionally, this has been accomplished by histological dating of the endometrial biopsy specimen obtained in the late secretory phase (1, 2). It has been accepted that the endometrial biopsy, properly obtained and properly diagnosed, can serve as a bioassay. If the biopsy is obtained as close to the expected menses as possible, almost the entire steroidogenic function of the corpus luteum is reflected in the endometrial histological pattern. Also, it was considered that the biopsy reflects the response of the target organ to the hormone (1). The basis for the use of the endometrial biopsy as a bioassay of the steroidogenic function of the corpus luteum has been the traditional morphological endometrial dating according to the criteria of Noyes et al. (2). A biopsy specimen was considered abnormal when there was a lack of expected endometrial development, which represented a lag of 2 or more days between endometrial date and calendar dating from the subsequent menstrual period (1, 2). If the biopsy was out of phase, a second biopsy should be done during a subsequent cycle, and the original finding should be confirmed. Unless this finding was consistent and repetitive, it could not be considered as a factor responsible for infertility (1).

More recently, endometrial biopsy for assessment of the luteal phase has been reviewed, and this includes when to perform the biopsy, the number of lag days that should be required to consider a sample abnormal, and the chronological reference point. Thus, recent evidence suggests that earlier sampling during the window of implantation (i.e. 6–8 d after ovulation) is more sensitive for identifying altered patterns of endometrial maturation (3, 4, 5). Also, it has been stressed that chronological dating should be assigned prospectively rather than retrospectively to avoid inaccuracies caused by the variable length of the luteal phase and the accelerated onset of menses induced by the procedure. Thus, ultrasonographic studies have shown that the accuracy of histological endometrial dating is best determined when ovulation is detected by that method (6, 7). Also it has been reported that a defective luteal phase should be suspected only when endometrial histology lags at least 3 d behind the prospective chronological dating of the biopsy (8). Finally, our sequential study of endometrial biopsy specimens in infertile patients showed that at least two endometrial biopsies from separate cycles are needed to evaluate endometrial function, and a third biopsy must be taken in patients with divergent findings in the first two endometrial biopsies (9).

Despite those attempts to refine the technique of endometrial biopsy, the definitive study to validate this diagnostic approach has never been done, and thus the relationship between histological changes and endometrial receptivity remains unknown (3, 10, 11). In recent years, an intensive search for specific markers for receptivity has been undertaken. It has been proposed that the in phase endometrium may exhibit aberrant behavior, not correlated with histological delay, yet still be associated with decreased cycle fecundity (11). Markers of normal endometrial development are being uncovered that, according to some authors, will allow us to go beyond merely histological criteria in the evaluation of endometrial function and receptivity. In this regard, vß3 integrin expression and pinopod formation, the two most cited markers postulated to frame the window of implantation, have been proposed as a means of distinguishing receptive endometrium from nonreceptive in clinical practice, thus offering new directions for assessing uterine function in various pathological and physiological conditions (11, 12, 13, 14).

However, one of the most intriguing facts in infertility is whether one cyclic event may be representative of all patients’ cycles; it has been stressed that no study has evaluated the cycle-to-cycle variability of the expression of any of these new proposed markers in human endometrium, and thus their application in clinical medicine should proceed with caution (15). Therefore, the present study was undertaken to investigate the within-subject, between-cycle reproducibility of histological dating, vß3 integrin expression, and pinopod formation in the human endometrium. Remarkably, endometrial biopsies were performed according to the more recently proposed technique refinements as discussed above.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subjects and study cycles

We investigated histological dating, vß3 integrin expression, and pinopod formation in 45 endometrial biopsy specimens obtained in 15 primary infertility patients (mean duration of infertility, 4.6 yr; range, 2–8 yr) undergoing a routine work-up. All patients underwent three endometrial biopsies in consecutive spontaneous cycles, regardless of the previous histological findings. All women involved gave informed consent to participate in the present study, which was approved by the Ethics Committee of our hospital. The mean age of these women was 31.2 ± 3.1 (mean ± SD) yr (range, 25–36 d), and all of them had a history of normal ovulatory menstrual cycles (27–32 d) and normal body mass index (19–28 kg/m²), were nonsmoking, and were not involved in intensive exercise. No woman had taken hormones during the previous 12 months, and prolactin serum levels were normal in all patients. Infertility work-up included, in addition to the endometrial biopsy, a semen analysis, a midluteal serum progesterone determination, and a hysterosalpingogram (and/or laparoscopy when appropriate). The main causes of infertility in these women were: unexplained (n = 6), male factor (n = 4), minimal endometriosis (n = 3), and tubal factor (n = 2). In all study cycles, basal body temperature, midluteal serum concentrations of estradiol and progesterone, and endometrial biopsy were used to assess luteal function according to a scheme of evaluation previously reported (5, 16, 17).

Commencing on d 8–10 of the study cycle (depending on the cycle length of the woman), patients underwent daily transvaginal ultrasonographic evaluation of the 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 was measured in both the longitudinal and transverse dimensions in all follicles. 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 (6, 7). 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 (6, 7). The patient’s chronological day was determined by counting forward from the ovulation day as detected by ultrasonographic scans. Endometrial sampling was always performed on ovulation d 7 as the best corresponding to the window of implantation (4). Biopsies were taken from the uterine fundus using the Pipelle (Laboratoire CCD, Paris, France), a plastic instrument that causes less discomfort than a metal curette and when used in patients undergoing endometrial biopsy for the evaluation of luteal phase inadequacy, provides adequate tissue with preservation of glandular, stromal, and capsular architecture (4, 18).

Hormones in serum were quantified on the same day as endometrial sampling. All blood samples were obtained in the fasted state between 0800 and 1000 h, which corresponded to the period of minimal progesterone variability in spontaneous menstrual cycles and added to the accuracy of the measurement (19).

Endometrial samples

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 immunolabeling for integrin determination. The remaining portion was fixed in glutaraldehyde for scanning electron microscopy investigation. The use of separate endometrial portions for light microscopy study and scanning electron microscopy investigation was necessary, considering a recent study (20) concluding that scanning electron microscopy but not light microscopy remains the only conclusive tool for the evaluation of the stage of pinopod formation. One observer, an expert gynecological pathologist who was blinded with regard to patient identification and the ultrasonographically detected ovulatory day, performed all of the assessments, including endometrial dating, immunohistochemistry, and pinopod evaluation.

Endometrial dating

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

Immunohistochemistry

vß3 integrin was detected in frozen sections using the EnVision system (DAKO Corp., Carpinteria, CA) as previously reported (16, 17, 21). Briefly, 4-µm sections were fixed 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 Tris-buffered saline (DAKO Corp.). The monoclonal antibody LM609 (Chemicon, Temecula CA; dilution, 1:200), which recognizes the complete vß3 heterodimer (22) and is being widely applied by us (5, 16, 17, 21) and others (23, 24, 25), was used. The peroxidase-labeled polymer was then applied for 40 min. After washing in Tris-buffered saline, the slides were incubated with the diaminobenzidine substrate chromogen solution, washed in distilled water, counterstained with hematoxylin, washed, dehydrated, and mounted. Because vß3 is consistently expressed in vascular endothelia, positive staining of endometrial vessels was considered as internal positive control (5). In every case, a negative control was performed. The staining procedure for negative controls was the same, except for the replacement of the primary antibody with a preimmunized mouse serum.

The reactivity in the endometrial glands epithelium 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 semiquantitative scoring system (0 to 3) as follows (5, 16, 17): absent (0), weak or focal (+), moderate (++), and strong (+++). In previous work, it was found that the expression of vß3 in the luminal surface epithelium starts abruptly on d 19–20 of the cycle, thus opening the window of implantation, and only staining in the glands seems to be clinically relevant (11, 26, 27). Therefore, for the specific purpose of this study and as previously reported (5, 17, 28, 29), endometrial positive staining for vß3 integrin was defined as immunoreactivity detected in both endometrial glands and luminal surface epithelium, with any intensity of the reaction ranging from weak/focal to strong.

Scanning electron microscopy

As previously reported (17), endometrial tissue was fixed for at least 24 h in phosphate buffered [0.1 mol/liter (pH 7.4)] 2.5% glutaraldehyde and postfixed 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, East Sussex, UK), mounted, and coated with gold in a Bio-Rad SC510 sputter coater (VG Microtech). All samples were observed under the same kilovolt and electron beam current conditions in a Zeiss DSM940A scanning electron microscope (Carl Zeiss, Oberkochen, Germany). For each biopsy, three to nine 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, British Columbia, Canada) and were evaluated independently by two observers. As previously reported by others and ourselves (13, 17, 27), 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 (+++). For the specific purpose of the present investigation and as previously reported (17, 27), endometrial samples were considered as expressing pinopods when these protrusions were detected at any stage of development and density ranging from isolated to confluent.

Hormone assays

Hormones in serum were measured using commercially available kits. Estradiol was measured by a competitive immunoenzymatic assay (Immuno 1, Bayer Corp., Tarrytown, NY). The sensitivity of the assay was 10 pg/ml, and the interassay coefficient of variation was 5%. Progesterone was determined by a competitive chemiluminescent immunoassay (Immulite, Diagnostic Products Corp., Los Angeles, CA). The sensitivity of the method was 0.2 ng/ml, and the interassay coefficient of variation was 6.7%. Blood was allowed to clot, and serum was separated and stored at -20 C until assayed. Samples from each subject were analyzed in a single assay.

Statistics

Data were entered and analyzed using SPSS statistical software (release 10.0, SPSS, Inc., Chicago, IL) and Stata (release 7.0, Stata Corp., College Station, TX). Agreement between the repeated observations (first vs. second, and first vs. third biopsies) with regard to histological dating and the presence or absence of vß3 integrin and pinopods in the endometrium was analyzed using the 2 x 2 frequency tables and Cohen’s coefficient. Statistic is a measure of agreement above or below what is expected by chance alone. The coefficient can range from -1.0 to 1.0, with negative values indicating agreement worse than chance, a value of zero indicating agreement no better than chance agreement, and a value of 1.0 indicating perfect agreement. Values were interpreted as previously suggested (30, 31): a less than zero indicates poor agreement, 0–0.2 indicates slight agreement, 0.2–0.4 indicates fair agreement, 0.4–0.6 indicates moderate agreement, 0.6–0.8 indicates substantial agreement, and 0.8–1.0 indicates almost perfect agreement. Hormonal levels were compared between groups using the Mann-Whitney U test, assuming independence of observations. Results are presented as mean ± SEM.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
All menstrual cycles included in the present study were ovulatory according to ultrasonographic criteria, midluteal serum progesterone concentration greater than 10 ng/ml, and clearly progestational characteristics of the endometrium. Table 1 summarizes results obtained in the 45 cycles studied in 15 patients. Women with male factor infertility were patients 2, 3, 6, and 8 (Table 1). All endometrial samples were obtained on postovulatory d 7, but the three biopsies occurred within the anticipated window of implantation (6–8 d after ovulation, according to histological dating) in only four patients (26.6%), and in four additional women (26.6%) were early. In the remaining seven patients (46.7%), there was a discrepancy between the three biopsies with respect to their relation to the implantation window. Only seven patients (46.7%) had in phase endometria in the three study cycles, whereas divergent findings were obtained in the remaining eight women (53.3%). vß3 integrin was expressed in the three endometrial specimens in only 1 of the 15 patients (6.7%), staining of this integrin was negative in the 3 cycles studied in 3 patients (20%), and divergent results were observed for the remaining 11 women (73.3%). Pinopods were observed in the three endometrial specimens investigated in nine patients (60%), and in the remaining six women (40%) findings for the three study cycles disagreed. No inflammatory or reactive change related to previous samplings was detected in any subsequent biopsy.

View this table: [in this window] [in a new window]   Table 1. Histological dating and its timing to the implantation window (postovulatory d 6–8), vß3 integrin expression, and pinopod formation in the 45 cycles studied in 15 patients

View larger version (41K): [in this window] [in a new window]   Figure 1. Percentage of endometria showing vß3 integrin and pinopods for each histological day in the 45 endometrial samples investigated.

View this table: [in this window] [in a new window]   Table 3. Midluteal serum concentration of estradiol and progesterone according to histological dating and expression of vß3 integrin and pinopods in the 45 cycles studied

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

Histological endometrial dating according to the criteria of Noyes et al. (2) has long been considered as the gold standard for evaluating the endometrium and its receptivity. For endometrial dating to be clinically valuable in the diagnosis of luteal phase defects, it should be accurate and reproducible. This seems a difficult goal, given that a major pitfall of histological dating is subjectivity (35). The within-subject, between-cycle variability indicates that the endometrial dating from one cycle cannot be relied on to predict that of another cycle (9, 10) and, based on analysis of 1492 endometrial biopsies from 1055 infertile patients, we reported that the histological endometrial adequacy or inadequacy in the cycle of conception, or in previous cycles, was not to be related to the pregnancy outcome (10). Thus, at present, the accuracy of morphological criteria to diagnose receptive and nonreceptive endometrium is in question. Moreover, histological dating is a relatively crude and inferential measure of endometrial function, this being the specific area of interest. It cannot identify clinically important functional abnormalities that have no morphological manifestation (36). In contrast, it has been proposed recently that potential new markers of endometrial receptivity, mainly vß3 integrin expression and pinopod formation, would provide more objective information beyond that derived from histological dating alone (12, 13, 14, 36). Ideally, the precision of the measurement should be so accurate that a diagnosis will be possible from a single endometrial biopsy.

However, despite claims of improved reliability for those new markers of endometrial receptivity, no previous report has investigated the cycle-to-cycle variability of their expression in the human endometrium. To the best of our knowledge, this is the first study dealing with this subject ever conducted. In this study, we found that both vß3 integrin expression and pinopod formation in the endometrium of infertile patients were poorly reproducible and highly variable from one cycle to another, as indicated by agreement levels using values. Even more, reproducibility for those suggested new markers of implantation was similar to that observed for traditional histological dating. In fact, we have previously reported that expression of vß3 integrin, in both infertile and fertile women, is closely related to endometrial maturation evaluated by histological dating consistently appearing at postovulatory d 6–7 and being expressed by all endometria dated as postovulatory d 8 or later (16, 17). Remarkably, this was true irrespective of endometria being in phase or out of phase. The reverse occurred with pinopod formation that was present mainly from postovulatory histological d 4–8, and their expression was markedly reduced afterward, again irrespective of in phase or out of phase endometria (17). The above patterns of temporal expression of vß3 integrin and pinopods over the luteal phase are in keeping with results reported in Fig. 1 in the present study. However, pinopods seem to have a more transient nature than integrins, and they are a more chronologically variable event within each patient. Thus, the cycle days on which pinopods develop may vary up to 4 d between normally cycling women, and some women show very few or no pinopods, despite regular ovulation and menstruation (13). This might be due to the sporadic nature of the abnormality of the luteal phase endometrium (heterogeneity of cycles), but may well be due to a failure in detecting pinopods in the small tissue samples analyzed.

Our study has several strengths. Chronological dating was assigned prospectively, and the day of ovulation was detected using serial ultrasonographic scans, which is the best method for determining the day of ovulation and improving the accuracy of histological endometrial dating (6, 7). All endometrial biopsies were performed on postovulatory d 7, thus fitting in with the anticipated window of implantation (i.e. 6–8 d after ovulation; Ref. 4). The 15 women included in our study underwent serial endometrial biopsies in three consecutive cycles. Finally, all endometrial specimens were evaluated by the same expert gynecological pathologist, thus avoiding interobserver variability (the greatest source of error in reading endometria) and adding to the accuracy and reproducibility of assessments (35).

There were several potential limitations to this study. The number of women included was relatively small, limiting the precision of our results. However, one cannot emphasize enough the difficulty of recruiting patients willing to undergo three consecutive endometrial biopsies. The subjects included were infertile patients but not healthy fertile women. Descriptions of the histology of the endometrium in the literature are generally made from observations on material obtained at biopsy. The descriptions do not clearly define the limits of normality, but it seems that variability from one subject to another is considerable. Differences between individuals may contribute to this apparently high variability. The expected variation between normal fertile subjects could be spuriously increased by the inclusion of subjects who are not fertile and may not be strictly normal (37). However, we have recently reported that no statistically significant difference regarding vß3 integrin expression and pinopod formation was found between fertile controls and infertile patients irrespective of endometria being in phase or out of phase (17). In addition, most of the biopsies included in the original paper by Noyes et al. (2) were taken during routine infertility studies, and infertile patients are the target for endometrial functional studies. Finally, it could be argued that repetitive endometrial biopsies may have an impact on subsequent endometrial findings. Although this possibility cannot be completely excluded, it is unlikely. Thus, we and others (3, 5, 16) have found no inflammatory or reactive changes consistent with a previous biopsy site when performing two endometrial biopsies during a single menstrual cycle for luteal phase evaluation. This is in keeping with results in the present study. On the other hand, in the clinical setting, it has been common for infertility patients to undergo sequential biopsies in different cycles in an attempt to diagnose and correct endometrial luteal phase defects, and clinicians have assessed each of these biopsies as though they were independent events.

In conclusion, histological dating, vß3 integrin expression, and pinopod formation in the human endometrium have poor reproducibility and high variability from cycle to cycle. Therefore, their potential usefulness as targets for both infertility and antifertility treatment seems scanty. In fact, as previously stressed (15), the evidence to date is not convincing as to a cause-and-effect relationship between the presence or absence of these markers and their effect on fertility. It should be kept in mind that the observations may be an epiphenomenon of abnormal endometrial development during the particular cycle of study. Findings in the present report, which are in keeping with previous studies by us (16, 17) and show that vß3 integrin expression and pinopod formation in the human endometrium are phenomena closely related to endometrial maturation irrespective of endometria being in phase or out of phase, further support this contention. However, further studies in fertile women are warranted before our findings may have general applicability. Also, reproducibility among several observers of the subjective parameters evaluated in the present study remains to be elucidated.

	Acknowledgments

 
We are grateful to Ms. Elena Rull, Ms. Cristina Durana, and Ms. Elisenda Coll for their help in the electron microscopy studies; Ms. Paquita Antonell for her technical assistance; and Dr. Antonio Palacín, Ms. Margarita Mainar, and Ms. Elena Gonzalvo for their help with the immunohistochemical studies. We thank Ms. Olga Ten for her help in the preparation of this manuscript.

	Footnotes

 
This work was supported in part by Grants 00/0399 and PI020036 from the Fondo de Investigaciones Sanitarias (to J.B.) and Grant RCMN (C03/08) from the Instituto de Salud Carlos III.

Received October 24, 2002.

Accepted February 14, 2003.

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