Human Reproduction, Vol. 14, No. 10, 2485-2492,
October 1999
© 1999 European Society of Human Reproduction and Embryology
A quantitative evaluation of 
1, 4, V and ß3 endometrial integrins of fertile and unexplained infertile women during the menstrual cycle. A flow cytometric appraisal
1 Institute of Maternal and Child Research (IDIMI), School Of Medicine, University Of Chile, `San Borja Arriaran' Clinical Hospital, and 2 Department Of Obstetrics and Gynecology, School of Medicine, University of Chile, Santiago, Chile
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Abstract |
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The expression of integrin molecules
1ß1, 4ß1 and Vß3 within endometrial tissue has been proposed as a marker of uterine receptivity during the implantation window. The present investigation examines by flow cytometric analysis the concentrations of 1, 4, V and ß3 integrin subunits in endometrial stromal (ESC) and epithelial cells (EEC) in two groups of women throughout the menstrual cycle: normal fertile women (n = 27) and women with unexplained infertility (n = 26). Integrin concentrations in endometrial cells were calculated in relative fluorescence units against a negative cellular control. The assessment of integrin subunits detected the protein in ESC and EEC from the late proliferative to the late secretory phase. In both groups of women, the 1 was the highest integrin expressed in ESC and EEC throughout the menstrual cycle. All women exhibited low concentrations of 4-EEC at the time of the implantation window. Infertile women expressed lower concentrations of the 4-ESC during the proliferative and early secretory phase while lower concentrations of the 1-ESC were seen during the late secretory phase. Interestingly, the infertile women expressed lower concentrations of ß3-EEC in the early, mid-secretory and late secretory phases (P < 0.05). Infertile women also expressed lower concentrations of 1-EEC and V-EEC during the late secretory phase (P < 0.05). It can be concluded that the quantitative determination of ß3-EEC by flow cytometry confirmed its potential feature as a marker of endometrial receptivity at the time of the implantation window. In addition, the defective expression of the 1-ESC found in the late secretory phase might be associated with the poor fertility outcome of women with unexplained infertility.
Key words: endometrial receptivity/endometrial stromal– epithelial cells/flow cytometry/integrins/unexplained infertility
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Many investigators have been searching intensively for markers of endometrial receptivity (Fay and Grudzinskas, 1991
; Lessey and Arnold, 1998). Ultrasonography alone or combined with Doppler analysis have also been employed. Nevertheless, their value as prognostic indicators for successful implantation is uncertain (Herman and Lewin, 1996).
The 1, 4, V and ß3 integrin molecules are cyclically expressed in the endometrium during the menstrual cycle (Lessey et al., 1992; Tabibzadeh, 1992). These molecules have been proposed as in-situ biochemical markers of endometrial receptivity during the implantation window (Lessey et al., 1994, 1995, 1996a). The regulation of the expression of these markers and their role in the molecular mechanism of human implantation are virtually unknown (Creus et al., 1998; Marions et al., 1998; Simón et al., 1998; Sulz et al., 1998). Inside endometrial tissue, the actions of ovarian steroids, modulated by a variety of growth factors and cytokines, lead the expression of the cycle-dependent integrins (Grosskinsky et al., 1996; Lessey et al., 1996b; Simón et al., 1996).
In vitro, the Ishikawa cells do express most of the normal endometrial epithelial integrins and are a useful model in studying steroid-mediated events in human endometrial epithelium (Lessey et al., 1996c; Somkuti et al., 1997). The same steroidogenic controlling signals could up-regulate the simultaneous expression of the ß3 integrin in the human Fallopian tube epithelium and in the endometrium (Sulz et al., 1998). In addition, the up-regulation of the ß3 expression by endometrial epithelial cells co-cultured with competent human embryos could be mediated by the interleukin 1ß (IL-1ß) system, which is present in both the embryo and the endometrium (Simón et al., 1997).
In vivo, the expression of 4 and ß3 integrins has been positively correlated with endometrial development and could be down-regulated by antiprogestin treatment, while the expression of the Vß3 integrin dimer was found to be independent of these factors and could be triggered by cytokines (Marions et al., 1998). However, other investigators reported that 1, 4 and ß3 integrins were not dependent on endometrial maturation in infertile women. The expression of Vß3 was closely related to the endometrial dating but its relationship to fertility was not definitely established (Creus et al., 1998). Contradictory data have also been published for the expression of the Vß3 integrin in the endometrium of women with and without endometriosis (Lessey and Young, 1997; Creus et al., 1998; Hii and Rogers, 1998; Lessey and Castelbaum, 1998).
The present study describes the flow cytometric assessment of the integrins within the endometrium, including the concentrations of 1, 4, V and ß3 throughout the menstrual cycle in fertile women and patients with unexplained infertility. Here we describe for the first time the quantitative expression of endometrial stromal and epithelial integrins in both groups of women throughout the menstrual cycle.
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Materials and methods |
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Patients
Twenty-seven proven fertile women (control) and 26 patients with unexplained infertility participated in this study, aged 31–44 (mean 35.6) and 21–41 (mean 30.5) years old respectively. The research protocol was approved by the Ethics Committee of the School of Medicine of the University of Chile. All patients involved gave a written informed consent. The inclusion criteria for fertile women participating in this investigation included: (i) healthy and proven fertile, (ii) who had regular ovulatory cycles, (iii) and had not used any type of contraceptive drugs or intrauterine devices within the last 6 months. Infertile women with unexplained infertility were characterized according to the American College of Obstetrics and Gynecology (Endometriosis, 1993
; Kim and Hornstein, 1997). The inclusion criteria of women with unexplained infertility encompassed: infertility of a duration exceeding 1 year; normal quality sperm (WHO, 1988), ovulation determined by serial ultrasonographic scans and serum progesterone higher than 4 ng/ml during the mid-luteal phase and tubal patency assessed by histerosalpingography and/or laparoscopy. Endometrial biopsies were obtained by using a flexible-sampling device (Pipelle de Cornier; Prodimed, Neully-en-Thelle, France). Tissue samples were taken from fertile women in the late proliferative phase (n = 5), 1–2 days before ovulation; in the early secretory phase (n = 9), 2–4 days after ovulation; in the mid-secretory phase or implantation window (n = 9), 6–10 days after ovulation; and in the late secretory phase (n = 4), 12–14 days post-ovulation. In the group of infertile patients, biopsies were taken in the late proliferative (n = 4), in the early secretory (n = 4), in the mid-secretory (n = 13) and in the late secretory (n = 5) phases. The histological dating of endometrial biopsies was assessed according to Noyes' criteria (Noyes et al., 1950). Endometrial dating correlated well with the date of ovulation as assessed by repeated vaginal ultrasonography (ALOKA, 630 5 MHz vaginal probe, Tokyo, Japan). Follicular rupture was considered to have occurred when the diameter of the dominant follicles was decreased by more than 3 mm and free liquid was observed in the cul de-sac.
Homogeneous dispersions of endometrial cells
All chemicals and culture media were obtained from Sigma Chemical Co. (St Louis, MO, USA) and from Worthington Biochemical Corporation, NJ, USA. Endometrial biopsies were digested in a two-step design to prepare cellular dispersions for flow cytometry. The procedure to isolate endometrial stromal cells (ESC) utilized 100–150 mg of wet endometrial tissue that was treated with collagenase I (0.1%)–deoxyribonuclease I (DNase, 0.005%) dissolved in Dulbecco's modified Eagle's medium (DMEM) during 90 min at 37°C (Satyaswaroop et al., 1979; Simón et al., 1993). Following sedimentation of glandular structures and separation of ESC, the glands were purified of ESC and macrophage contaminants by incubation at 37°C in a Falcon flask. Thus, a second enzymatic digestion was performed to isolate the individual endometrial epithelial cells (EEC) from the glands for flow cytometric measurements. Briefly, the glands were digested with trypsin (0.25%)–EDTA (0.03%)–DNase (0.1%) for 10 min at 37°C. Subsequently EEC were washed with DMEM–fetal bovine serum (FBS, 2%) and filtered out through a 37 µm mesh sieve which retained undigested tissue. Stromal and epithelial cell dispersions were counted in a haemocytometer and cell viability was assessed using the Trypan Blue exclusion method. Means of viability for ESC and EEC were 85 and 80% respectively. Polyclonal antibodies to vimentin (Vm) and cytokeratin (Ck) were used to examine the homogeneity of ESC (Vm+) and EEC (Ck+) in cell smears of cellular dispersions. The maximum cross-contamination found between the endometrial cells (ESC–EEC) was <1%. Preliminary experiments were conducted to test whether collagenase or trypsin treatment provoked an important loss of membrane-bound integrins in EEC and ESC. For these purposes, the digestion conditions were mimicked on ESC and EEC cultured in vitro. Following the initial separation, the endometrial cells were cultured for 4 days in a conditioned medium (Simón et al., 1993). Therefore, cells were detached with trypsin (0.025 %)-EDTA (1 mM) for 5 min at room temperature. After washing, the cell suspensions were incubated with enzymatic solutions as described above. Integrins were determined by flow cytometry. The control experiment was represented by the suspensions of endometrial cells detached in routine conditions without further enzymatic treatment. No differences of integrin concentrations were found between controls and protease-treated cells.
Flow cytometric measurements of endometrial integrins
Specific monoclonal antibodies were used to determine the expression of integrins in ESC and EEC by flow cytometry: anti 1 (anti CD49a, clone TS 2/7), anti 1 (anti CD49d, clone B5 G10) and anti ß3 (anti CD61, clone SS A6) were kindly donated by Dr B.A.Lessey, University of North Carolina, NC, USA (for references see Lessey et al., 1992). Anti V (anti CD51, clone VNR 147) was purchased from GIBCO BRL Products, Gaithersburg, MD, USA. Anti-human leukocitary antigen-I (anti HLA-I, clone w6/32), anti CD45-phycoeritryn (PE) conjugated and fluorescein isothiocyanate (FITC)-conjugated rabbit-antimouse IgG [F(ab')2] were obtained from Dako Corporation, Carpinteria, CA, USA. All the following incubation steps were performed in an ice-bath during 30 min. A total of 2x105 to 5x105 cells (ESC or EEC) dispersed in 100 µl of phosphate-buffered saline (PBS)–2% FBS were incubated with the primary antibodies directed towards integrins. After washing with 500 µl of PBS-FBS, the cells were treated with the FITC-conjugate. Subsequently, the cells were incubated with the anti-CD45-PE to eliminate the leukocytes from the cytometric analysis. Red blood cells were eliminated by incubation with a lysing solution (Lysing Solution, Becton Dickinson, San Diego, CA, USA) for 5 min. A negative control with unspecific immunoglobulin G (IgG)-isotype-matched monoclonal antibody for each label and a positive control with anti-human leukocyte antigen (HLA were introduced in each determination. Flow cytometric measurements were taken in a FASCcan Flow Cytometer (Becton Dickinson) using the CONSORT 32 II Version 2.0 software for data acquisition and analysis. Concentrations of integrins were expressed in relative fluorescence units (RFU). One RFU was defined as the ratio (F/Fo) of the mean of fluorescence (F) obtained from the labelled integrins and the background of the mean of fluorescence of the negative control (Fo).
Immunohistochemistry
Acetone-fixed cryostat sections from endometrial biopsies were immunostained for integrin detection. Samples were incubated for 1 h at room temperature with anti-integrin monoclonal antibodies (described above), and stained with streptavidin–biotin–horse radish peroxidase system (Dako LSAB kit system). Negative controls were incubated with irrelevant mouse monoclonal antibodies instead of primary antibodies. Cryostat sections stained by amine-ethyl-carbazol (AEC) enzymatic product and counter-stained with haematoxylin were evaluated on a Nikon microscope.
Statistical analysis
The comparison of integrin concentrations in the endometria of fertile and infertile patients throughout the menstrual cycle was assessed by the non-parametric Mann–Whitney test. All data are presented as mean ±SEM. Statistical significance was considered to be P < 0.05.
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Results |
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Profile of endometrial integrin subunits in ESC of fertile women
The number of fertile endometria evaluated in each phase was as follows: late proliferative (n = 5); early secretory (n = 9); mid-secretory phase or implantation window (n = 9); and late secretory phase (n = 4).
Figure 1A shows the concentration of stromal integrins in fertile women. The stromal 1 integrin subunit was the highest expressed throughout the menstrual cycle (P < 0.05). The maximum concentration of 1 integrin was during the mid-secretory phase (6–10 days after ovulation), reaching a plateau in the late secretory phase (12–14 days after ovulation). Among the stromal integrin subunits, 1 exhibited the highest variability of concentration.
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The patterns of expression for the ß3-ESC and for
V-ESC were similar during the menstrual cycle. A slight increase in V-ESC and ß3-ESC was observed after ovulation and higher concentrations were found in the mid-secretory phase. However, it did not achieve significance. Concentrations of V and ß3 declined after the day +10 post-ovulatory (see Figure 1A
). In two fertile women (2 of 9), the stromal ß3 was absent during the mid-secretory phase.
The 4-ESC exhibited a different expression pattern as it was higher in the early secretory phase as compared to the mid-secretory phase (P < 0.05; Figure 1A).
Profile of endometrial integrin subunits in EEC of fertile women
The number of fertile endometria evaluated in each phase was as follows: late proliferative (n = 5; except 1 and ß3 which were n = 3 and n = 4 respectively); early secretory (n = 9); mid-secretory phase or implantation window (n = 9); and late secretory phase (n = 4).
Figure 1B shows the concentrations of the integrin subunits in the epithelium of fertile women throughout the menstrual cycle. The epithelial 1 integrin was the highest expressed. A significant increase was observed in 1-EEC during the late secretory phase (P < 0.05; see Figure 1B). This finding could be related to the over-expression of 1 by epithelial glands found in the inner mass of the functionalis endometrium.
The ß3- and V-EEC subunits were expressed either during the late proliferative or the secretory phase (see Figure 1B). The concentrations were similar for V and ß3 until the early secretory phase. Epithelial V and ß3 (P < 0.05) increased significantly at the time of the implantation window (6 to 10 days after ovulation; P < 0.05). However, from the implantation window to the late secretory phase, the ß3-EEC concentrations were higher than V-EEC (P < 0.05).
The 4-EEC was poorly detected during the proliferative phase. Slightly higher concentrations were noted after ovulation but were almost undetectable at the time of the implantation window (P < 0.05; see Figure 1B).
Overall, the pattern of integrin expression at the time of the implantation window in fertile women was characterized by an over-expression of epithelial 1 and ß3, a slight increase in V expression and an under-expression of 4 integrin subunits.
Profile of endometrial integrin subunits in ESC of women with unexplained infertility
The number of infertile women evaluated in each phase of the menstrual cycle was as follows: late proliferative (n = 4); early secretory (n = 4); mid-secretory or implantation window (n = 13); and late secretory phase (n = 5).
Figure 2 shows the comparison between the concentrations of the stromal integrin subunits in infertile and fertile women throughout the menstrual cycle. The stromal 1 integrin was detected in the endometria of infertile women throughout the menstrual cycle (see Figure 2A). A trend towards a slightly lower concentration of 1-ESC was observed in infertile women during the late proliferative and mid-secretory phases. However, 1-ESC concentrations in infertile women were significantly lower (P < 0.05) during the late secretory phase (Figure 2A).
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The
4-ESC concentration was lower in infertile patients than in fertile women during the late proliferative and early secretory phases (P < 0.05; see Figure 2B). However, no significant differences were found in 4-ESC concentrations at the time of the implantation window and a trend towards a higher 4-ESC concentration was observed in infertile women during the late secretory phase.
The V-ESC (Figure 2C) and ß3-ESC (Figure 2D) showed expression patterns similar to those determined for fertile women.
The immunohistochemical staining of stromal integrins was similar for fertile and infertile women. A weak to moderate signal was found for 4-ESC and was consistent with the flow cytometric measurements. Interestingly, the higher sensitivity of flow cytometric analysis led to the discovery of significant differences in the 4-ESC expression between fertile and infertile women during the peri-ovulatory days. Stromal 1 and ß3 in fertile and infertile women showed a similar immunohistochemical staining intensity, contrary to the findings in the immunohistochemical evaluation of ß3-EEC that showed evident differences between stained fertile and non-stained infertile endometria (see Figure 3).
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Profile of endometrial integrin subunits in EEC of women with unexplained infertility
The number of infertile women studied in each phase of the menstrual cycle was as follows: late proliferative (n = 4); early secretory (n = 4); mid-secretory (n = 13); and late secretory phases (n = 5).
Figure 4 depicts the pattern of the concentrations of epithelial integrin subunits in infertile and fertile women throughout the menstrual cycle. The concentrations of 1-, 4- and V-EEC subunits (Figure 4, panels A, B and C respectively) in infertile patients were not significantly different from those calculated in EEC of fertile women from the proliferative to the mid-secretory phases. However, a trend was found in infertile women towards lower 1-EEC concentrations in post-ovulatory days. The concentrations of epithelial 1 were significantly lower in infertile women in the late secretory phase (P < 0.05; see Figure 4A).
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The concentrations of
4-EEC exhibited no significant differences between infertile and fertile women throughout the menstrual cycle although the concentration of epithelial 4 in infertile women showed a tendency to increase from the mid-secretory to the late secretory phase (see Figure 4B).
Slightly lower V-EEC concentrations were observed during the peri-ovulatory days in infertile patients as compared to fertile women (see Figure 4C). Interestingly, similar concentrations for the epithelial V were observed in both groups of women at the time of the implantation window. However, during the late secretory phase, the V-EEC in infertile patients has a significantly lower expression (P < 0.05).
It is interesting to note that the main differences between infertile and fertile women were observed in regard to the epithelial-ß3 subunit. During the late proliferative phase, the concentration of ß3-EEC in infertile women was slightly higher than in fertile women. On the contrary, during the secretory phase the concentrations of the epithelial ß3 were always lower in infertile women than in fertile women. Significantly lower concentrations of the ß3-EEC (P < 0.05) were observed in infertile women at the time of the implantation window and in the late secretory phase (Figure 4D). However, at the time of the implantation window, two infertile patients (2 of 13) expressed ß3 concentrations similar to those of fertile women.
The principal differences in the expression of epithelial integrin subunits between infertile and fertile women were observed during the implantation window for the ß3 and during the late secretory phase for the 1, V and ß3.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Examination of endometrial receptivity is an attractive research subject in the human reproductive field (Coutifaris et al., 1998
; Lessey and Arnold, 1998). Integrin molecules are widespread and have common characteristics in all the mammalian cells (Hynes, 1992; Lessey and Young, 1997; Suekoda et al., 1997). In addition, a cyclic pattern of expression has also been described for 1, 4, V and ß3 integrins in endometrial stroma and epithelium (Lessey et al., 1992; Tabibzadeh, 1992). However, other investigators have not been able to demonstrate the cyclic expression of the stromal 1 (Beliard et al., 1997) or the glandular Vß3 (Hii and Rogers, 1998). It has also been suggested that the 6ß4 integrin is a cycle-dependent endometrial molecule (Lanteri et al., 1998).
Most of the data for the pattern of integrin expression in endometria have been determined by immunohistochemical studies, and integrin concentrations have been assessed semi-quantitatively according to the degree of positive staining or the HSCORE approach (Lessey et al., 1992, 1994; Tabibzadeh, 1992). Immunochemistry is an essential technique in determining the tissue and cellular localization of integrins in the endometrium (Klentzeris et al., 1993; Lessey et al., 1994, 1995). However, the expression of cellular antigens in whole organs and tissue could be quantitatively examined by flow cytometry (Serna et al., 1998). Flow cytometry has been used to assess integrin expression in endometrial stromal cells in vitro (Grosskinsky et al., 1996), Ishikawa cells (Castelbaum et al., 1997) and to evaluate the expression of the ß3-integrin subunit in endometrial epithelial cells co-cultured with human blastocyst (Simón et al., 1997).
In the present investigation, flow cytometry was used to determine the concentration of integrin molecules in homogeneous cellular dispersions isolated directly from endometrial biopsies. The main differences found in the expression of the 1, 4, V and ß3 integrin subunits in the endometria of fertile and infertile women throughout the menstrual cycle are schematically represented in Figure 5. The patients have a wide range of age, but eligible subjects were homogeneous in both groups. Consequently, the variations in integrin expression observed in these groups should be related to infertility and not to age difference.
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The
1 integrin subunit, a component of the collagen/laminin receptor (1ß1), had the highest expression in both types of cells (ESC and EEC). Fertile and infertile women showed similar expressions of this integrin during the time of the implantation window. However, infertile women expressed lower concentrations of the 1 in epithelium and stroma during the late secretory phase (see Figure 5). A lack of stromal 1 expression in infertile women might be related to the absence of laminin and collagen IV (Bilalis et al., 1996). The molecular significance of these changes in infertile patients is unknown. The low expression of both the receptor and ligands could influence trophoblast invasion. From this point of view, the stromal 1ß1 integrin might be a potential marker of endometrial receptivity in the late secretory phase related to the trophoblast invasion.
The epithelial 4-integrin subunit (4ß1, a fibronectin receptor) had a low expression in fertile and infertile women throughout the menstrual cycle. Flow cytometric analysis detected a tendency for the expression of the epithelial 4 to increase in infertile women during the implantation window (see Figure 5). A similar increase in the epithelial glandular 4ß1 expression has been observed in women treated with oral contraceptives (Somkuti et al., 1996).
A moderate expression of the stromal 4 integrin in fertile women during the different phases of the menstrual cycle has been described (Tabibzadeh, 1992; Lessey et al., 1994; Marions et al., 1998). The flow cytometric measurements in our study for the stromal 4 integrin demonstrated the under-expression of this molecule in infertile patients during the late proliferative and early secretory phases. Although the immunohistochemical staining of this integrin was moderate, it related closely to the flow cytometry determinations (data not shown). These observations suggest changes in the pattern of expression of this adhesion molecule in the stroma of women with unexplained infertility.
The expression of the V integrin subunit, a component of the vitronectin receptor (Vß3), was different from the expression of its molecular partner, the ß3 integrin subunit. The V integrin was under-expressed only in the endometrial epithelium of infertile women during the late secretory phase. This integrin could be constitutively expressed in the endometrial epithelium, as was found in the Ishikawa cells (Widra et al., 1997). In addition to ß3, the V subunit could also be the molecular partner for diverse ß subunits, i.e. ß1 and ß5–ß8 (Lessey and Young, 1997). An investigation is pending of the implications of the under-expression of the V and 1 endometrial epithelial integrins in infertile women during the late secretory phase (see Figure 5).
The epithelial ß3 integrin subunit has been proposed as the more reliable biochemical marker for failure in endometrial receptivity (Lessey et al., 1995). Oral contraceptive use has been associated with a decrease in glandular Vß3 expression (Somkuti et al., 1996). Nevertheless, the expression of the glandular Vß3 did not vary throughout the menstrual cycle in fertile women or in patients with endometriosis (Creus et al., 1998; Hii and Rogers, 1998). The expression of the ß3 integrin subunit was not affected in normal and out-phase endometria of infertile women, contrary to the Vß3 integrin dimer (Creus et al., 1998). The epithelial ß3 integrin was under-expressed in fertile women treated with antiprogestin, but expression of the epithelial vß3 molecule was constant (Marions et al., 1998). Diverse methodologies or the specificity of antibodies used might be responsible for these differences (Lessey and Castelbaum, 1998).
Using flow cytometry, it has been confirmed that the epithelial ß3 subunit was significantly higher in fertile women at the time of the implantation window (see Figure 5). Infertile women expressed lower epithelial ß3 from the mid-secretory phase. Defects in epithelial ß3 expression have been postulated as a cause of infertility (Lessey et al., 1995).
In conclusion, temporal and spatial differences of expression of the cycle-dependent integrins were found between fertile and infertile endometria. We underline the importance of the quantitative measurement of the endometrial integrins, particularly the epithelial ß3 subunit, during the implantation window. In addition, the lower concentration of the stromal 1ß1 integrin detected in women with unexplained infertility suggests that this adhesion molecule may be involved in the interaction between stromal and trophoblast cells. The pattern of integrin concentrations in the endometrium, quantified by flow cytometry, could contribute to infertility or fertility regulation assessment.
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Acknowledgments |
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This study was funded by a grant provided by the Rockefeller Foundation to L.D. (RF 94025). R.R.G. is a post-doctorate research scientist at the University of Chile supported by the Rockefeller Foundation. We wish to thank Professor Bruce A.Lessey (University of North Carolina, NC) for providing us with the monoclonal antibodies anti
1, 4 and ß3 integrin subunits. We also wish to thank Professor Paul Bischof (University of Geneva, Switzerland) for reviewing the manuscript. |
Notes |
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3 To whom correspondence should be addressed at: PO Box 226–3, IDIMI, University of Chile, Santiago, Chile.
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Submitted on February 3, 1999; accepted on June 25, 1999.
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