1 Institut Clinic of Obstetrics and Gynaecology, 2 Department of Pathology and 3 Hormonal Laboratory, Faculty of Medicine University of Barcelona, Hospital Clínic Institut dInvestigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain
4 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
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Abstract |
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Key words: endometrium/hormone treatment/implantation/integrins/pinopodes
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Introduction |
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Investigation of endometrial function has been traditionally made by dating pre-menstrual endometrial biopsy according to the morphological criteria reported 50 years ago by Noyes et al. (1950). Using this diagnostic approach, adverse effects on endometrial secretory patterns have been reported in patients receiving clomiphene citrate or gonadotrophins for ovarian stimulation, in those given HRT, and in women using oral contraceptives (Balasch et al., 1983, 1991; Birkenfeld et al., 1986
; Balasch and Vanrell, 1987
; Lee, 1987
; Habiba et al., 1998
; ESHRE Capri Workshop Group, 2001). Similarly, the administration of progesterone in the follicular phase of an artificial cycle (as a model of premature luteinization) has been reported to impair endometrial development (Ezra et al., 1994
). In contrast, luteal estradiol depletion in HRT cycles does not seem to adversely affect the morphological developmental capacity of the endometrium (Younis et al., 1994
). Dehydrogesterone can be successfully used to induce normal endometrial maturation in patients diagnosed as having luteal phase deficiency (Balasch et al., 1982
; Jacobs et al., 1987
) but an association between the treatment for delayed maturation endometria and pregnancy in infertile patients is lacking (Balasch et al., 1986
, 1992; Balasch and Vanrell, 1987
).
Until recently, pre-menstrual endometrial dating was considered as the gold standard for endometrial function evaluation. However, over the past decade the relationship between histological changes and endometrial receptivity has been seriously questioned (Balasch et al., 1992; Castelbaum et al., 1994
; Somkuti et al., 1995
; Murray et al., 2002
; Reproductive Medicine Network, 2002). Recently, several reports have indicated that midluteal endometrial evaluation can provide more valuable information on endometrial receptivity. Thus, both studies based on the detection of hCG in healthy women and studies in hormonally prepared recipients from assisted reproduction technology cycles have documented a period of receptivity for the transfer of human embryos during the early to midluteal phase (Navot et al., 1991
; Wilcox et al., 1999
). In addition, it has been shown that earlier sampling is more sensitive for identifying delayed or other altered patterns of endometrial maturation (Castelbaum et al., 1994
; Creus et al., 1998
; Ordi et al., 2002
). On the other hand, a few markers have been shown to appear in the endometrial mucosa coinciding with this period, suggesting that they may be involved in receptivity. In this regard,
v
3 integrin expression and pinopode formation, the two most cited markers postulated to frame the window of implantation, have been proposed as a means of distinguishing receptive endometrium from non-receptive in clinical practice, thus offering new directions for the development of a novel contraceptive approach targeted to the endometrium as well as a better understanding of occult causes of infertility in women (Lessey et al., 1996
; Nikas, 1999
).
On the above evidence, the aim of the present study was to investigate the effect of ovulation induction in clomiphene citrate and IVF treatment cycles, oral contraception, treatment with dehydrogesterone, and different regimens of HRT on endometrial v
3 integrin expression and pinopode formation using a prospective, controlled study design. Recently, we have reported (Creus et al., 2002
) that there is a clear dissociation in the temporal expression of
v
3 integrin and pinopode during the luteal phase. Thus, a feature of the present study is that we investigated both markers in the same endometrial sample.
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Materials and methods |
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The use of human tissue for research was based on informed consent and was approved by the Ethics Committee of our hospital. According to the above study design, each woman acted as her own control for endometrial markers of implantation. However, considering that experimental subjects were mostly infertile or menopausal women, we also included an additional group of 12 fertile (mean parity 1.4, range 14) healthy women (group F) aged 2941 years (mean ± SEM age 33.8 ± 1.1 years) who were undergoing tubal sterilization and served as a general control group. These control women had regular menstrual cycles (2732 days) and were taking no medication. The mean age of study patients was 31.9 ± 1.4 years (range 2539) and all had regular ovulatory menstrual patterns every 2733 days.
In all groups of recruited, normally ovulating, 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., 1998, 2002). Commencing on days 810 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, 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 (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 two menstrual cycles (control and treated) in each experimental subject and in a single menstrual cycle in control fertile women. The patients chronological day was determined by counting forward from the ovulation day as detected by ultrasonographic scans. The early biopsy (midluteal) was performed on ovulation day +7 to +8 whereas the second biopsy (late luteal) was always performed 4 days after the first biopsy. The day of oocyte retrieval was designated day 14 in ovarian stimulation cycles (Develioglu et al., 1999; Nikas et al., 1999
), while HRT cycles were studied on days 78 after the commencement of progesterone treatment and 4 days later. In OC-treated cycles, endometrial samples were obtained on cycle days 2122 and 4 days later.
Hormones in serum were quantified on the same days as endometrial sampling. All 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 (Filicori et al., 1984). In patients receiving premature progesterone administration (group E+P+P) during HRT cycles, additional blood samples were obtained 46 h after each dose of vaginal progesterone administered during the artificial follicular phase in order to evaluate serum progesterone at the steady-state levels previously reported in pharmacokinetic studies (Miles et al., 1994
).
Endometrial samples
Biopsies were taken from the uterine fundus using the Pipelle (Laboratoire CCD, 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 in methylbutane (Merck, 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 investigation. The use of separate endometrial portions for light microscopy study and scanning electron microscopy investigation was necessary considering a recent study (Develioglu et al., 2000) concluding that scanning electron microscopy but not light microscopy remains the only conclusive tool for the evaluation of the stage of pinopode formation. One observer, an expert gynaecological pathologist (J.O.), who was blinded to the identity of the slides as well as with regard to the ultrasonographically detected ovulatory day, performed all the assessments.
Endometrial dating
For endometrial dating, 4 µm sections stained with haematoxylin and eosin and periodic acidSchiff stain were evaluated. All endometrial biopsies were evaluated according to the histopathological criteria of Noyes et al. (1950) using a single-day evaluation whenever possible and when the traditional 2 day spread evaluation method (i.e. day 20 to day 21) was provided, the later day was used for comparison with immunohistochemical assays. An out-of-phase biopsy was defined as
3-day lag between the chronological and the histological day.
Immunohistochemistry
v
3 integrin was detected in frozen sections using the EnVision system (Dako Co., USA) as previously reported (Creus et al., 1998, 2001, 2002). 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 Tris-buffered saline (TBS; Dako). The monoclonal antibody LM609 (Chemicon, USA; dilution 1:200), which recognizes the complete
v
3 heterodimer (Cheresh and Spiro, 1987
) and is being widely applied by us (Creus et al., 1998
, 2001, 2002; Ordi et al., 2002
) and others (Lessey et al., 1994
; Vonlaufen et al., 2001
; Sturm 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
v
3 is consistently expressed in vascular endothelia, positive staining of endometrial vessels was considered as internal positive control (Ordi et al., 2002
).
The reactivity in the endometrial gland 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 semi-quantitative scoring system (0 to 3) as follows (Creus et al., 1998, 2002; Ordi et al., 2002
): absent (0), weak or focal (+), moderate (++), and strong (+++). As in previous work it was found that the expression of
v
3 in the luminal surface epithelium starts abruptly on day 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
v
3 integrin when this integrin was detected in endometrial glands and luminal surface epithelium with any intensity of the reaction ranging from weak/focal to strong.
Scanning electron microscopy
As previously reported (Creus et al., 2002), endometrial tissue was fixed for
24 h in phosphate-buffered (0.1 mol/l, 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, UK), mounted and coated with gold in a Bio-Rad SC510 sputter coater (VG Microtech). All samples were observed under the same KV and electron beam current conditions in a Zeiss DSM940A scanning electron microscope (Carl Zeiss, Germany). For each biopsy, three to nine fragments 2 mm each were evaluated and
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., Canada), and were evaluated independently by two observers. As previously reported by others and ourselves (Nikas, 1999
; Acosta et al., 2000
; Creus et al., 2002
), pinopodes were defined as spherical protrusions without microvilli on the apical surface of the luminal uterine endometrium and were semiquantitatively evaluated as absent (0), isolated pinopodes (+), small groups of pinopodes (++) and confluent pinopodes (+++).
Hormone assays
Hormones in serum were measured using commercially available kits. Estradiol was measured by a competitive immunoenzymatic assay (Immuno 1; Bayer, USA). The sensitivity of the assay was 10 pg/ml and the interassay coefficients of variation 5%. Progesterone was determined by a competitive chemiluminescent immunoassay (Immulite, DPC, USA). 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 analysed in a single assay.
Statistics
Data were analysed by SPSS statistical software (Release 10.0, SPSS Inc., USA). The MannWhitney U-test and Wilcoxon matched-pairs signed-ranks test were used as appropriate with Bonferroni correction for multiple comparisons. 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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Discussion |
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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 day 5.59.5 after ovulation, could be inferred (Aplin, 2000). Traditionally, research to understand endometrial function and to define the window of implantation has focused on morphological features of the endometrium (Noyes et al., 1950
; Balasch and Vanrell, 1987
). However, the relationship between histological changes and endometrial receptivity has not been definitely established (Balasch et al., 1992
; Castelbaum et al., 1994
; Somkuti et al., 1995
). Thus, more recent attempts have focused on the characterization of molecular features believed to regulate endometrial receptivity and further characterization of the cyclic morphological changes occurring in the human endometrium by means of scanning electron microscopy (Lessey 1992
, 1994, 1996; Somkuti et al., 1995
; Nikas, 1999
; Nikas et al., 1999
). At present, the two most cited markers framing the window of implantation are the
v
3 integrin expression and pinopode formation in human endometrial epithelium, which are both estrogen and progesterone dependent (Nikas, 1999
, 2000; Lessey, 2000
; Damario et al., 2001
). Remarkably, the uterine surface protrusions observed in the human (pinopodes or uterodomes) are not pinocytotic and therefore probably perform a function different from similar structures (pinopods) observed in rats and mice (Murphy, 2000
; Adams et al., 2002
).
Several authors have reported that ovulation induction with gonadotrophins may disrupt luteal phase function by shifts in the window of receptivity as defined by v
3 integrin or pinopode expression in the endometrium resulting in ovo-endometrial asynchrony and limiting implantation success in assisted reproduction (Kolb et al., 1997
; Develioglu et al., 1999
; Nikas et al., 1999
; Tavaniotou et al., 2001
; Thomas et al., 2002
). In addition, a potential deleterious effect of clomiphene citrate on endometrial pinopode formation (Martel et al., 1987
) but not
v
3 integrin expression (Lacin et al., 2001
) has been reported. However, others were unable to determine whether controlled ovarian hyperstimulation was associated with any functional uncoupling of histological development and endometrial integrin expression (Meyer et al., 1999
); in those previous studies women did not act as their own control, only
v
3 integrin or pinopode formation but not both markers simultaneously were investigated, and temporal patterns of
v
3 integrin and pinopode expression according to endometrial maturation evaluated by histological dating were not considered.
The present study investigated the effect of different hormone treatments on the endometrial expression of those two markers of implantation using a prospective, controlled study design where patients were investigated during spontaneous and ensuing treated cycles. We found that a coordinately high level of expression of v
3 integrin and pinopodes existed on postovulatory days 6 to 7 but there was a lack of temporal co-expression of these markers over the luteal phase in the endometrial samples studied. Interestingly, this was true irrespective of endometria being in-phase or out-of-phase and coming from spontaneous or treated cycles. This is in keeping with previous studies by us (Creus et al., 2002
) investigating spontaneous cycles in fertile and infertile women. The temporal patterns of
v
3 integrin expression and pinopode formation reported in Figure 7 are the key to understanding the results obtained in the present study as discussed below.
In the clomiphene citrate group, mean histological dating in spontaneous and ensuing treated cycles was similar and thus no effect on v
3 integrin expression was observed, which is in agreement with data previously reported by others (Lacin et al., 2001
). However, in keeping with previous studies (Martel et al., 1987
) a significant reduction in pinopode formation was found in clomiphene citrate-treated cycles. This has been explained on the basis of the antiestrogenic action on the endometrium of the clomiphene citrate given during the first part of the menstrual cycle (Birkenfeld et al., 1986
; Martel et al., 1987
; Bonhoff et al., 1993
). In the IVF group, endometrial dating was advanced from
day 6 in spontaneous cycles to
day 8 during IVF cycles and this implies a marked increment in
v
3 integrin expression according to the temporal pattern for this endometrial marker reported in Figure 7. In contrast, pinopode formation is similar in endometria dated as postovulatory days 6 to 8 (see Figure 7).
OC treatment induced a dramatic advance in histological dating from days 56 in spontaneous cycles to days 1213 in treated cycles. Accordingly with the temporal patterns of expression depicted in Figure 7, a significant increase in integrin staining and marked reduction in pinopode formation were observed during OC treatment cycles. The pattern of integrin expression over the luteal phase would explain why in previous reports (Taskin et al., 1994) it was reported that there is no apparent change in the level of
v
3 integrin in the human endometrium when high-dose oral contraceptives are given on days 2425 of the menstrual cycle. Similarly, no differences were found in the present study between spontaneous and treated cycles in the 6 experimental groups included with respect to
v
3 integrin and pinopode expression in the late luteal phase when all endometrial specimens were dated as being postovulatory day
10. Dehydrogesterone treatment in patients having out-of-phase endometria caused significant advance in histological dating from
day 4 in spontaneous cycles to
day 6 in treatment cycles which, according to temporal patterns shown (Figure 7), is associated with a significant increase in
v
3 integrin expression but not pinopode formation.
In artificial cycles where estrogen was given for 28 days and progesterone was started on day 15 of the cycle, concomitantly with the same dosage of estrogen, the three parameters of endometrial maturation analysed, i.e. histological dating, v
3 integrin expression and pinopode formation, were similar to those found in control fertile women during the midluteal phase. However, as occurred with OC treatment and accordingly with temporal patterns of
v
3 integrin and pinopode expression (Figure 7), the precocious endometrial luteinization induced by premature progesterone administration (group E+P+EP) was associated with significant advanced histological dating (
day 6 versus
day 12) and increased staining for
v
3 integrin but reduced pinopode formation. In contrast and in keeping with previous studies (Younis et al., 1994
), luteal estradiol depletion (group E+P) did not adversely affect the morphological developmental capacity of the endometrium as evidenced by histological dating. Accordingly, no significant changes either in
v
3 integrin expression or pinopode formation were detected in our study in group E+P as compared with groups E+EP or control fertile women.
In groups OC and E+P+EP, dyssynchrony in maturation between the stromal and the glandular components of the endometrium was a common feature typically with a marked decidualized stroma but underdeveloped glands. However, as clearly shown in Figures 4 and 6, epithelial v
3 integrin and pinopode expression was markedly increased in the face of an evident delayed glandular maturation as compared with endometrial stroma. Therefore, traditional morphological changes did not run in parallel with the expression of the new endometrial markers of implantation.
In conclusion, v
3 integrin expression and pinopode formation in the human endometrium are processes closely related to endometrial maturation and this is irrespective of endometria being in-phase or out-of-phase and the hormonal treatment received. Only for clomiphene citrate did a direct effect with reduction in pinopode formation in the midluteal phase seem to exist. Therefore, the potential usefulness of those two so-called endometrial markers of implantation as targets for contraceptive approaches or fertility-promoting strategies seems unlikely.
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Acknowledgements |
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Submitted on October 18, 2002; accepted on January 7, 2003.