Human Reproduction, Vol. 18, No. 4, 683-693,
April 2003
© 2003 European Society of Human Reproduction and Embryology
The effect of different hormone therapies on integrin expression and pinopode formation in the human endometrium: a controlled study
1 Institut Clinic of Obstetrics and Gynaecology, 2 Department of Pathology and 3 Hormonal Laboratory, Faculty of Medicine – University of Barcelona, Hospital Clínic – Institut d’Investigacions 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
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
BACKGROUND: Integrin expression and pinopode formation have been proposed as a means of distinguishing receptive endometrium from non-receptive in clinical practice, thus offering new directions for the development of contraceptive approaches targeted to the endometrium as well as a better understanding of occult causes of infertility in women. The aim of the present study was to investigate the effect of ovulation induction, oral contraception, treatment with dehydrogesterone, and different regimens of hormone replacement therapy on endometrial
v
3 integrin expression and pinopode formation using a prospective, controlled study design. METHODS: Histological dating, v3 integrin expression and pinopode formation were evaluated in control and treated cycles in six groups of women including eight subjects per group and who received clomiphene citrate, ovarian stimulation for IVF, oral contraception, dehydrogesterone for endometrial luteal phase defect, or two different regimens of hormone replacement therapy. Twelve healthy fertile women served as a general control group. RESULTS: v3 integrin expression and pinopode formation in the human endometrium were closely related to endometrial maturation as defined by histological dating and this was 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. CONCLUSION: v3 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. The potential usefulness of those two so-called endometrial markers of implantation as targets for contraceptive approaches or fertility-promoting strategies seems unlikely.
Key words: endometrium/hormone treatment/implantation/integrins/pinopodes
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
A normal endometrial milieu is essential for implantation and thus evaluation of endometrial receptivity (the period during which successful implantation can occur) has been considered a basic goal in the evaluation of the infertile patient (Wentz, 1988
). However, the assessment of endometrial function under physiological conditions is a highly controversial area in fertility, and the situation is further complicated during different types of hormone therapies such as ovulation induction and assisted reproduction cycles or hormonal contraception (Balasch and Vanrell, 1987). Inducing follicular development may improve the hormonal status of the patient but this is not necessarily associated with a receptive endometrium suitable for implantation (Bonhoff et al., 1993). In contrast, agonadal and menopausal women given hormone replacement therapy (HRT) and donor oocytes achieve higher implantation rates than cyclic women, even if respective donors for both groups have equal pregnancy rates (Edwards et al., 1991). Also, it is well known that the oral contraceptive pill, which is the most popular method of hormonal contraception, has marked effects on the endometrium (ESHRE Capri Workshop Group, 2001).
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, v3 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 v3 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 v3 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.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Patients, treatments and study cycles
We investigated the expression of
v3 integrin and pinopode formation in the endometrium of the following groups of patients. (i) Eight infertile patients receiving clomiphene citrate (50 mg/day from cycle day 5 to day 9) and who were studied in both spontaneous and ensuing clomiphene citrate-stimulated cycles. (ii) Eight oocyte donors undergoing ovarian stimulation with highly purified FSH (Neofertinorm; Serono S.A., Spain) under pituitary suppression (triptorelin depot; Decapeptyl 3.75 mg; Ipsen-Pharma, Spain) for IVF (group IVF). For the specific purpose of this investigation the luteal phase was supported with micronized vaginal progesterone (Progeffik; Laboratorios Effik, Spain) (600 mg/day for 12 days and commencing on the day after oocyte retrieval). Three to 4 months after the IVF cycle these patients underwent endometrial and blood samplings during a spontaneous ovulatory control cycle. (iii) Eight normal healthy women requesting contraception and receiving a low-dose oral contraceptive containing 30 µg of ethinyl estradiol plus 150 µg of desogestrel (Microdiol; Organon Española, S.A., Barcelona, Spain) (group OC) and undergoing endometrial samplings in both spontaneous and ensuing OC-treated cycles. OC treatment was commenced on cycle day 3. (iv) Eight infertile patients with endometrial luteal phase defect as defined by delayed endometrial maturation (
3 days) in two separate cycles and treated with dehydrogesterone (Duphaston; Duphar Nezel, S.L., Spain) (20 mg/day for 12 days, starting 2 days after ovulation) (group DHG). The second spontaneous menstrual cycle showing delayed endometrial maturation was used for comparative purposes in this study. (v) Sixteen women with primary or secondary ovarian failure (serum FSH and LH >40 IU/l and serum estradiol <30 pg/ml) and receiving standard HRT in the form of estradiol valerate (Progynova; Schering España S.A., Spain) (6 mg daily during 28 days per month) and micronized vaginal progesterone (Progeffik; Laboratorios Effik) (600 mg/day administered from day 15 to day 28 of the estrogenic treatment) (Group E+EP). Treatment cycle E+EP represented the control cycle for those 16 menopausal women who were divided randomly and prospectively into two study subgroups, each group including eight patients. In an ensuing cycle, patients in group E+P (n = 8) received estradiol valerate treatment for 14 days but estrogen treatment was stopped during the artificial luteal phase and only micronized vaginal progesterone was administered for another 14 days. The remaining eight patients (group E+P+EP) were given the same standard HRT as that in the control cycle (E+EP) but now including episodic progesterone administration during the artificial follicular phase on days 8 and 11 (200 of micronized vaginal progesterone) to mimic premature luteinization. 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 1–4) healthy women (group F) aged 29–41 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 (27–32 days) and were taking no medication. The mean age of study patients was 31.9 ± 1.4 years (range 25–39) and all had regular ovulatory menstrual patterns every 27–33 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 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, 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 patient’s 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 7–8 after the commencement of progesterone treatment and 4 days later. In OC-treated cycles, endometrial samples were obtained on cycle days 21–22 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 4–6 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 acid–Schiff 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
v3 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 v3 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 v3 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 v3 in the luminal surface epithelium starts abruptly on day 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., 1992; Somkuti et al., 1995; Acosta et al., 2000), for the specific purpose of this study, endometrial samples were considered as expressing v3 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 Mann–Whitney 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.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
All spontaneous menstrual cycles included in the present study were ovulatory according to ultrasonographic criteria and midluteal serum progesterone concentration >10 ng/ml. A late luteal endometrial biopsy could not be done in 11 of the 96 cycles in experimental subjects 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. When dyssynchronous glandular and stromal endometrial development was found, endometrial maturation was defined according to the most advanced elements in either the glands or stroma (Develioglu et al., 1999
; Meyer et al., 1999). However, the glands were never more advanced than the stroma. This was especially evident in OC- and E+P+EP-treated groups which showed a markedly decidualized stroma with glands with an underdeveloped or atrophic appearance. However, the apparent delayed glandular development did not preclude the expression of potential markers of endometrial receptivity (Figure 1) and thus, histological dating according to glandular differentiation would not change the results with respect to markers of implantation in the present study. No inflammatory or reactive change related to the first sampling was detected in any late luteal biopsy.
|
Midluteal hormonal levels are presented in Table I. As expected, estradiol and progesterone serum concentrations in clomiphene citrate and IVF treatment cycles were significantly higher than those in spontaneous cycles and cycles in fertile controls while they were significantly lower in OC-treated cycles. Among patients receiving HRT estradiol was significantly higher in groups E+EP and E+P+EP than in fertile controls and group E+P. As expected, midluteal estradiol serum levels were significantly lower in group E+P as compared with fertile control cycles. For patients in group E+P+EP, serum progesterone levels after premature progesterone administration on cycle days 8 and 11 were 8.1 ± 0.8 and 9.2 ± 1.1 ng/ml respectively. In all cases, serum progesterone concentration was >5 ng/ml 4–6 h after each single dose administration which is in keeping with previously reported pharmacokinetic studies of vaginally administered micronized progesterone (Miles et al., 1994
).
|
As expected, in all study groups chronological dating of endometrial biopsies (day of sampling) was the same in control and treated cycles (data not shown). Histological dating,
v3 integrin expression, and pinopode formation in spontaneous and treatment endometrium specimens of experimental subjects and control ovulatory cycles in fertile women are presented in Figures 2–6. Histological dating (mean ± SEM) was similar in spontaneous (postovulatory day 5.9 ± 0.5) and treatment (postovulatory day 5.1 ± 0.5) cycles in the clomiphene citrate group. However, pinopode formation but not v3 integrin expression was significantly reduced in clomiphene citrate (CC)-treated cycles (Figure 2). Ovarian stimulation for IVF caused advanced histological dating (postovulatory day 5.5 ± 0.4 versus 7.7 ± 0.4) and increased v3 integrin expression but not significant changes in pinopode formation (Figure 3). OC treatment was associated with advanced histological dating (postovulatory day 5.4 ± 0.6 versus 12.5 ± 0.2) and v3 integrin expression but reduced pinopode formation (Figure 4). Dehydrogesterone treatment in patients having retarded endometrial maturation in spontaneous cycles was associated with advanced endometrial dating (postovulatory day 3.8 ± 0.2 versus 5.8 ± 0.6) and increased v3 integrin expression but not pinopode formation (Figure 5). Parameters of endometrial morphology and function in group E+EP were similar to those in fertile women (Figure 6), thus stressing the validity of E+EP-treated cycles as controls for the two study subgroups of patients receiving different forms of HRT in ensuing cycles. No differences were found between control and treatment cycles in group E+P with respect to the three endometrial variables investigated. However, premature administration of progesterone during the follicular phase (group E+P+EP) was associated with advanced histological dating (postovulatory day 5.9 ± 0.6 versus 12.1 ± 0.1) and v3 integrin expression but not pinopode formation (Figure 6).
|
|
|
|
|
v3 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 (Figure 7). In contrast with the expression of v3 integrin, pinopodes were already expressed in 50% of endometria dated as postovulatory day 3, were observed in 70–90% of endometrial biopsies dated as postovulatory days 4–9, and were much less frequently observed in endometria dated as postovulatory day 10 (Figure 7). These changes in v3 integrin expression and pinopode formation occurred irrespective of endometria being in-phase or out-of-phase and corresponding to spontaneous or treated cycles. A coordinate high level of expression of both markers existed on postovulatory days 6 to 7 (Figure 7). However, a lack of temporal coexpression of v3 integrin and pinopode over the luteal phase in the endometrial samples studied was seen (Figure 7).
|
No significant differences were found between spontaneous and treatment cycles in the five experimental groups studied with respect to the expression of endometrial markers in the late luteal phase biopsy (data not shown). Steroid levels reached the threshold necessary for endometrial priming and differentiation (Develioglu et al., 1999
) in all study groups and controls but estradiol and progesterone levels in the mid or late luteal phase could not distinguish between cases with expression or not of v3 integrin and the presence or absence of pinopodes (data not shown). |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Endometrial receptivity is an essential factor for successful embryo implantation and it is thus widely accepted that viable embryos cannot implant on an inhospitable endometrium. In fact, postcoital contraception is based on this principle. Over the past decade the issue of endometrial receptivity has become more prominent with an appreciation for its impact on the overall success of assisted reproduction (Paulson et al., 1990
; Yaron et al., 1994). Embryo implantation remains the most inefficient process in the complex series of events that culminates in the establishment of an IVF pregnancy.
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 5.5–9.5 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 v3 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 v3 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 v3 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 v3 integrin or pinopode formation but not both markers simultaneously were investigated, and temporal patterns of v3 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 v3 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 v3 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 v3 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 v3 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 5–6 in spontaneous cycles to days 12–13 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 v3 integrin in the human endometrium when high-dose oral contraceptives are given on days 24–25 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 v3 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 v3 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, v3 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 v3 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 v3 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 v3 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 v3 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, v3 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.
|
Acknowledgements |
|---|
The authors are grateful to Ms Elena Rull, Cristina Durana and 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 Gemma Laguna for the preparation of the manuscript. This work was supported in part by grants 00/0399 and PI020036 from the Fondo de Investigaciones Sanitarias to J.B.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
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[CrossRef][Web of Science][Medline]
Adams, S.M., Gayer, N., Hosie, M.J. and Murphy, C.R. (2002) Human uterodomes (pinopods) do not display pinocytotic function. Hum. Reprod., 17, 1980–1986.
Aplin, J.D. (2000) The cell biological basis of human implantation. Baillière’s Clin. Obstet. Gynecol., 14, 757–764.
Balasch, J. and Vanrell, J.A. (1987) Corpus luteum insufficiency and fertility: a matter of controversy. Hum. Reprod., 2, 557–567.
Balasch, J., Vanrell, J.A., Márquez, M., Burzaco, I. and González-Merlo, J. (1982) Dehydrogesterone versus vaginal progesterone in the treatment of the endometrial luteal phase deficiency. Fertil. Steril., 37, 751–754.[Web of Science][Medline]
Balasch, J., Vanrell, J.A., Durán, M. and González-Merlo, J. (1983) Luteal phase evaluation after clomiphene–chorionic gonadotrophin-induced ovulation. Int. J. Fertil., 28, 104–106.[Web of Science][Medline]
Balasch, J., Creus, M., Márquez, M., Burzaco, I. and Vanrell, J.A. (1986) The significance of luteal phase deficiency on fertility: a diagnostic and therapeutic approach. Hum. Reprod., 1, 145–147.
Balasch, J., Jové, I., Márquez, M. and Vanrell, J.A. (1991) Hormonal and histological evaluation of the luteal phase after combined GnRH-agonist/gonadotrophin treatment for superovulation and luteal phase support in in-vitro fertilization. Hum. Reprod., 6, 914–917.
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.
Balasch, J., Vidal, E., Peñarrubia, J., Casamitjana, R., Carmona, F., Creus, M., Fábregues, F. and Vanrell, J.A. (2001) Suppression of LH during ovarian stimulation: analysing threshold values and effects on ovarian response and the outcome of assisted reproduction in down-regulated women stimulated with recombinant FSH. Hum. Reprod., 16, 1636–1643.
Birkenfeld, A., Beier, H.M. and Schenker, J.G. (1986) The effect of clomiphene citrate on early embryonic development, endometrium and implantation. Hum. Reprod., 1, 387–395.
Bonhoff, A., Naether, O., Johanisson, E. and Bohnet, H.G. (1993) Morphometric characteristics of endometrial biopsies after different types of ovarian stimulation for infertility treatment. Fertil. Steril., 59, 560–566.[Web of Science][Medline]
Castelbaum, A.J., Wheeler, J., Coutifaris, C., Mastroianni, 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.[Web of Science][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.
Creus, M., Balasch, J., Ordi, J., Fábregues, F., Casamitjana, R., Quintó, L., Coutifaris, C. and Vanrell, J.A. (1998) Integrin expression in normal and out-of-phase endometria. Hum. Reprod., 13, 3460–3468.
Creus, M., Ordi, J., Fábregues, F., Casamitjana, R., Vanrell, J.A. and Balasch, J. (2001) Mid-luteal serum inhibin -A concentration as a marker of endometrial differentiation. Hum. Reprod., 16, 1347–1352.
Creus, M., Ordi, J., Fábregues, F., Casamitjana, R., Ferrer, B., Coll, E., Vanrell, J.A. and Balasch, J. (2002) v3 integrin expression and pinopod formation in normal and out-of-phase endometria of fertile and infertile women. Hum. Reprod., 17, 2279–2286.
Damario, M.A., Lesnick, T.G., Lessey, B.A., Kowalik, A., Mandelin, E., Seppala, M. and Rosenwaks, Z. (2001) Endometrial markers of uterine receptivity utilizing the donor oocyte model. Hum. Reprod., 16, 1893–1899.
Develioglu, O.H., Hsiu, J.G., Nikas, G., Hsiu, J.G., Toner, J.P., Oehninger, S. and Jones, H.W. Jr (1999) Endometrial estrogen and progesterone receptor and pinopode expression in stimulated cycles of oocyte donors. Fertil. Steril., 71, 1040–1047.[CrossRef][Web of Science][Medline]
Develioglu, O.H., Nikas, G., Hsiu, J.G., Torner, J.P. and Jones, H.W. Jr (2000) Detection of endometrial pinopodes by light microscopy. Fertil. Steril., 74, 767–770.[CrossRef][Web of Science][Medline]
Edwards, R.G., Marcus, S., Macnamee, M., Balmaceda, J.P., Walters, D.E. and Asch, R. (1991) High fecundity of amenorrheic women in embryo-transfer programmes. Lancet, 338, 292–294.[CrossRef][Web of Science][Medline]
ESHRE Capri Workshop Group (2001) Ovarian and endometrial function during hormonal contraception. Hum. Reprod., 16, 1527–1535.
Ezra, Y., Simon, A., Sherman, Y., Benshushan, A., Younis, J.S. and Laufer, N. (1994) The effect of progesterone administration in the follicular phase of an artificial cycle on endometrial morphology: a model of premature luteinization. Fertil. Steril., 62, 108–112.[Web of Science][Medline]
Filicori, M., Butler, J.P. and Crowley, W.F. (1984) Neuroendocrine regulation of corpus luteum in the human: evidence for pulsatile progesterone secretion. J. Clin. Invest., 73, 1638–1647.[Web of Science][Medline]
Habiba, M.A., Bell, S.C. and Al-Azzawi, E. (1998) Endometrial responses to hormone replacement therapy: histological features compared with those of late luteal phase endometrium. Hum. Reprod., 13, 1674–1682.
Jacobs, M.H., Balasch, J., González-Merlo, J., Vanrell, J.A., Wheeler, C., Strauss, J.F., Blasco, L., Wheeler, J.E. and Lyttle, C.R. (1987) Endometrial cytosolic and nuclear progesterone receptors in the luteal phase defect. J. Clin. Endocrinol. Metab., 64, 472–475.
Kolb, B.A., Najmabadi, S. and Paulson, R.J. (1997) Ultrastructural characteristics of the luteal phase endometrium in patients undergoing controlled ovarian hyperstimulation. Fertil. Steril., 67, 625–630.[CrossRef][Web of Science][Medline]
Lacin, S., Vatansever, S., Kuscu, N.K., Koyuncu, F., Ozbilgin, K. and Ceylan, E. (2001) Clomiphene citrate does not affect the secretion of 3, v and 1 integrin molecules during the implantation window in patients with unexplained infertility. Hum. Reprod., 16, 2305–2309.
Lee, C.S. (1987) Luteal phase defects. Obstet. Gynecol. Surv., 42, 267–274.
Lessey, B.A. (2000) The role of the endometrium during embryo implantation. Hum. Reprod., 15 (Suppl. 6), 39–50.
Lessey, B.A., Danjanovich, L., Coutifaris, C., Castelbaum, A, Albelda, S.M. and Buck, C.A. (1992) Integrin adhesion molecules in the human endometrium. Correlation with the normal and abnormal menstrual cycle. J. Clin. Invest., 90, 188–195.[Web of Science][Medline]
Lessey, B.A., Castelbaum, A.J., Buck, C.A., Lei, Y., Yowell, C.W. and Sun, J. (1994) Furhter characterization of endometrial integrins during the menstrual cycle and pregnancy. Fertil. Steril., 62, 497–506.[Web of Science][Medline]
Lessey, B.A., Ilesanmi, A.O., Lessey, M.A., Riben, M., Harris, J.E. and Chwalisz, K. (1996) Luminal and glandular endometrial epithelium express integrins differentially throughout the menstrual cycle: implications for implantation, contraception, and infertility. Am. J. Reprod. Immunol., 35, 195–204.[Medline]
Martel, D., Frydman, R., Glissant, M., Maggioni, C., Roche, D. and Psychoyos, A. (1987) Scanning electron microscopy of postovulatory human endometrium in spontaneous cycles and cycles stimulated by hormone treatment. J. Endocrinol., 114, 319–324.
Meyer, W.R., Novotny, D.B., Fritz, M.A., Beyler, S.A., Wolf, L.J. and Lessey, B.A. (1999) Effect of exogenous gonadotropins on endometrial maturation in oocyte donors. Fertil. Steril., 71, 109–114.[CrossRef][Web of Science][Medline]
Miles, R.A., Paulson, R.J., Lobo, R.A., Press, M.F., Dahmoush, L. and Sauer, M.V. (1994) Pharmacokinetics and endometrial tissue levels of progesterone after administration by intramuscular and vaginal routes: a comparative study. Fertil. Steril., 62, 485–490.[Web of Science][Medline]
Murphy, C.R. (2000) Understanding the apical surface markers of uterine receptivity. Pinopods or uterodomes? Hum. Reprod., 15, 2451–2454.
Murray, M.J., Meyer, W.R., Lessey, B.A., Zaino R.J., Novotny, D.B. and Fritz, M.A. (2002) Endometrial dating revisited: a randomized systematic study of secretory phase histologic characteristics in normally cycling fertile women. Fertil. Steril., 78 (Suppl. 1), S67.
Navot, D., Scott, R.T., Droesch, K., Veeck, L.L., Liu, H.C. and Rosenwaks, Z. (1991) The window of embryo transfer and efficiency of human conception in vitro. Fertil. Steril., 55, 114–118.[Web of Science][Medline]
Nikas, G. (1999) Pinopodes as markers of endometrial receptivity in clinical practice. Hum. Reprod., 14 (Suppl. 2), 99–106.
Nikas, G. (2000) Endometrial receptivity: changes in cell-surface morphology. Semin. Reprod. Med., 18, 229–235.[CrossRef][Web of Science][Medline]
Nikas, G., Develioglu, O.H., Torner, J.P. and Jones, H.W. Jr (1999) Endometrial pinopodes indicate a shift in the window of receptivity in IVF cycles. Hum. Reprod., 14, 787–792.
Noyes, R.W., Hertig, A.T. and Rock, J. (1950) Dating the endometrial biopsy. Fertil. Steril., 1, 3–25.[Medline]
Ordi, J., Creus, M., Ferrer, B., Fábregues, F., Carmona, F., Casamitjana, R., Vanrell, J.A. and Balasch, J. (2002) Midluteal endometrial biopsy and v3 integrin expression in the evalution of the endometrium in infertility: implications for fecundity. Int. J. Gynecol. Pathol., 21, 231–238.[Web of Science][Medline]
Paulson, R.J., Sauer, M.V. and Lobo, R.A. (1990) Embryo implantation after human in vitro fertilization: importance of endometrial receptivity. Fertil. Steril., 53, 870–874.[Web of Science][Medline]
Peters, A.J., Lloyd, R.P. and Coulam, C.B. (1992) Prevalence of out-of-phase endometrial biopsy specimens. Am. J. Obstet. Gynecol., 166, 1738–1746.[Web of Science][Medline]
Reproductive Medicine Network (2002) The endometrial biopsy as a diagnostic tool in the evaluation of the infertile patient. Fertil. Steril., 78 (Suppl. 1), S2.
Shoupe, D., Mishell, D.R., Lacarra, M., Lobo R.A., Horenstein, J., d’Ablaing, G. and Moyer, D. (1989) Correlation of endometrial maturation with four methods of estimating day of ovulation. Obstet. Gynecol., 73, 88–92.[Web of Science][Medline]
Somkuti, S.G., Appenzeller, M.F. and Lessey, B.A. (1995) Advances in the assessement of endometrial function. Infertil. Reprod. Med. Clin. N. Am., 6, 303–328.
Sturm, R.A., Satyamoorthy, K., Meier, F., Gardiner, B.B., Smit, D.J., Vaidya, B. and Herlyn, M. (2002) Osteonectin/SPARC induction by ectopic beta(3) integrin in human radial growth phase primary melanoma cells. Cancer Res., 62, 226–32.
Taskin, O., Brown, R.W., Young, D.C., Poindexter, A.N. and Wiehle, R.D. (1994) High doses of oral contraceptives do not alter endometrial 1 and v3 integrins in the late implantation window. Fertil. Steril., 61, 850–855.[Web of Science][Medline]
Tavaniotou, A., Smitz, J., Bourgain, C. and Devroey, P. (2001) Ovulation induction disrupts luteal phase function. Ann. NY Acad. Sci., 943, 55–63.[Web of Science][Medline]
Thomas, K., Thomson, A.J., Sephton, V., Cowan, C., Wood, S., Vince, G., Kingsland, C.R. and Lewis-Jones, D.I. (2002) The effect of gonadotrophic stimulation on integrin expression in the endometrium. Hum. Reprod., 17, 63–82.
Vonlaufen, A., Wiedle, G., Borisch, B., Birrer, S., Luder, P. and Imhof, B.A. (2001) Integrin alpha(v)beta(3) expression in colon carcinoma correlates with survival. Mod. Pathol. 14, 1126–1132.[CrossRef][Web of Science][Medline]
Wentz, A.C. (1988) Luteal phase inadequacy. In Behrman, S.J., Kistner, R.W. and Patton, G.W. (eds), Progess in Infertility, 3rd edn. Little, Brown & Co., Boston, pp. 405–462.
Wilcox, A.J., Baird, D.D. and Weinberg, C.R. (1999) Time of implantation of the conceptus and loss of pregnancy. N. Engl. J. Med., 340, 1796–1799.
Yaron, Y., Botchan, A., Amit, A., Peyser, M.R., David, M.P. and Lessing, J.B. (1994) Endometrial receptivity in the light of modern assisted reproductive technologies. Fertil. Steril., 62, 225–232.[Web of Science][Medline]
Younis, Y.S., Ezra, Y., Sherman, Y., Simon, A., Schenker, J.G. and Laufer, N. (1994) The effect of estradiol depletion during the luteal phase on endometrial development. Fertil. Steril., 62, 103–107.[Web of Science][Medline]
Submitted on October 18, 2002; accepted on January 7, 2003.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  C.E. Quinn and R.F. Casper Pinopodes: a questionable role in endometrial receptivity Hum. Reprod. Update, March 1, 2009; 15(2): 229 - 236. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Bedaiwy, N. A. Mousa, N. Esfandiari, R. Forman, and R. F. Casper Follicular Phase Dynamics with Combined Aromatase Inhibitor and Follicle Stimulating Hormone Treatment J. Clin. Endocrinol. Metab., March 1, 2007; 92(3): 825 - 833. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H.-C. Ruan, X.-M. Zhu, Q. Luo, A.-X. Liu, Y.-L. Qian, C.-Y. Zhou, F. Jin, H.-F. Huang, and J.-Z. Sheng Ovarian stimulation with GnRH agonist, but not GnRH antagonist, partially restores the expression of endometrial integrin beta3 and leukaemia-inhibitory factor and improves uterine receptivity in mice Hum. Reprod., October 1, 2006; 21(10): 2521 - 2529. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Strowitzki, A. Germeyer, R. Popovici, and M. von Wolff The human endometrium as a fertility-determining factor Hum. Reprod. Update, September 1, 2006; 12(5): 617 - 630. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Mirkin, G. Nikas, J.-G. Hsiu, J. Diaz, and S. Oehninger Gene Expression Profiles and Structural/Functional Features of the Peri-Implantation Endometrium in Natural and Gonadotropin-Stimulated Cycles J. Clin. Endocrinol. Metab., November 1, 2004; 89(11): 5742 - 5752. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||