Biology and Medicine
Editor-in-Chief: Eytan R. Barnea MD, FACOG
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EARLY
PREGNANCY: Biology and Medicine Editor-in-Chief: Eytan R. Barnea MD, FACOG |
April 2001
Volume V, Number 2
ISSN: 1537-6583
Pages:
Abnormal Pattern Of Integrin Expression At The Implantation Window In Endometrium From Fertile Women Treated With Clomiphene Citrate And Users Of Intrauterine Device.*
1,4 R.R. González, Ph.D. , 2A. Palomino, M.D. , 2 D. Vantman, M.D., 3 F. Gabler, M.D. , 2L. Devoto, M.D.
1Boston Biomedical Research Institute (BBRI), 64 Grove St, Watertown, MA 02472, USA, 2 Institute of Maternal and Child Research (IDIMI), School of Medicine, University of Chile and Department of Gynecology and Obstetrics, Hospital "San Borja Arriarán", Santiago, Chile, P.O.Box 226-3., 3 Department of Pathology, Hospital "San Borja Arriarán", Santiago, Chile
Capsule
The under-expression of endometrial epithelial ß3 integrins was assessed only in CC-patients. However, CC-patients and intrauterine device users showed an abnormal pattern of expression for other integrins in endometrial epithelial and stromal cells at the implantation window.Abstract
ObjectivesResults
Biopsies from CC-treated women had a high incidence of out-of-phase endometria.
Interestingly, CC-treated women over-expressed a1, aV and b3-ESC
integrins and under-expressed b3-EEC
subunit (P<0.05). IUD users over-expressed the a1-EEC and under-expressed a4-ESC (P<0.05) at the time of the implantation window.
Conclusions
CC treatment in fertile women provokes a high frequency of out-of-phase endometrium
and desynchronises the expression of endometrial integrins at the implantation window. The
epithelial b3 integrin was under-expressed in all CC-treated
patients. The T-Cu intrauterine device alters endometrial receptivity by a different
mechanism independent of the expression of the epithelial b3 integrin. However, both CC and IUD use alter the expression of
some epithelial and stromal integrins during the implantation window.
Introduction
The molecular mechanism involved in human blastocyst implantation is only partly elucidated (1). Maximal endometrial receptivity " implantation window" is found at mid-luteal phase (2-4). During this period a unique expression pattern of endometrial adhesion molecules has been described. Integrins a1, a4, aV and b3 are co-expressed cyclically in the endometrium during the implantation window (5-7). Altered patterns of integrin expression have been correlated with diverse fertility disorders and endometrial receptivity (7-9).
Clomiphene citrate (CC), a non-steroidal drug effectively induces ovulation and has both estrogen agonist/antagonist effects (10). High ovulation rates by CC treatment are not followed by similar implantation and pregnancy rates (11-14). CC increases the rate of spontaneous abortions (15,16), negatively affects oocyte and embryo development (17,18) and alters endometrial morphology, cervical mucus and uterine blood flow (19-21). Adverse effect of CC on expression of integrin markers of endometrial receptivity may be related to the observed low implantation rates.
The TCu380A IUD is the most widely used device today in the world (22). Copper IUD might seriously interfere with implantation (23,24) and alter expression of estrogen, and progesterone receptors (25) as well as endometrial cytokines (26). Differential expression of steroid receptors (27) and production of cytokines (28,29) in endometria from IUD users might be related to altered integrin expression.
The aim of the present investigation was to assess by flow cytometry (7) quantitative expression of integrins a1, a4, aV and b3 in stromal and epithelial cells of endometria at the implantation window, in fertile women treated with CC, IUD users as compared to fertile controls.
Materials And Methods
A research protocol was approved by the Ethics Committee of the "San Borja Arriaran" Clinical Hospital and School of Medicine of the University of Chile. All subjects involved gave signed an informed consent form.Study subjects: Ten proven fertile and ovulatory women with regular menstrual cycles who had laparoscopic tubal ligation. Patient's age was 33-41 years (mean 36.3). These patients had not used any contraception within the last 6 months. Patients were treated with 50 mg of CC (Serofen, Serono) from 5th to the 9th day of the cycle. Ovulation time was assessed by estradiol and progesterone measurements at days 14-17 of the cycle and in the same days by transvaginal ultrasound (ALOKA, 630 5 MHz vaginal probe, Tokyo, Japan) to document follicular size. Follicle of 18> mm was considered as mature. Ovulation day was assessed by repeated transvaginal ultrasonography. Follicular rupture was considered to have occurred when diameter of preovulatory follicles decreased by more than 3 mm and free liquid was observed in the cul-de-sac. Hormonal patterns and sonographic profile were similar in all treated patients.
Six fertile normally cycling women users of T-Cu-380 A (Population Council, USA) for more than two years were included in this study. Women age ranged from 33-41 years old (mean 36.6). Nine fertile women, aged 29-40 (mean 37), with regular ovulatory cycles, that did not use contraception for the past six months were used as controls.
Endometrial biopsies
Tissue samples were taken in the mid-secretory phase or implantation window (6-10 days
after ovulation) using a flexible sampling device (Pipelle de Cornier, Prodimed, Neully-en
Thelle, France).
Endometrial dating was evaluated in a blinded fashion in formalin-paraffin embedded sections according to Noyes' criteria (30). Samples were considered out of phase when they were >+/-3 days from expected criteria (see Table I).
Hormonal determinations
Serum samples were taken from patients and controls at the same the time as the
endometrial biopsy. Progesterone (P4) and estradiol (E2) serum concentrations were
measured by specific RIA (31).
Expression of endometrial integrins
Flow cytometry and immunohistochemistry were used to assess the expression of integrins
sub-units a1, a4, aV and b3 in endometria.
Flow cytometric measurements: To isolate ESC and EEC, endometrial samples were digested in a two-step protocol as earlier described (7). Briefly, endometrial tissue was minced and treated with collagenase (0.1%)-deoxyribonuclease I (DNase, 0.005%) solution at 37ºC for 1 h. Further, endometrial glands were separated and disrupted by treatment with trypsin (0.25%)-EDTA (0.03%)-DNase (0.1 %) for 10 min at 37 ºC. Specific antibodies to vimentin, cytokeratin and CD45 were used to assess homogeneity of ESC and EEC preparations. The following specific monoclonal antibodies were used to determine integrins, anti-a1 (clone TS 2/7), anti-a4 (clone B5 G10) and anti-b3 (clone SS A6) all kindly donated by Dr. B.A. Lessey, University of North Carolina, NC, USA (for references see 5). Anti aV (clone VNR 147) was purchased from GIBCO BRL Products, Gaithersburg, MD, USA. Concentrations of integrins were measured and the results were expressed in relative fluorescence units (RFU) as earlier described (7).
Immunohistochemistry: Cryostat sections of endometrial biopsies were fixed with acetone and treated with anti-integrin antibodies. Specific staining was developed using the Dako LSAB kit system. Negative controls were performed with irrelevant mouse monoclonal antibodies instead of with primary antibodies. Staining of amine-ethyl-carbazol (AEC) enzymatic product on cryostat sections counter-stained with hematoxylin was evaluated on a Nikon microscope (7). Staining intensity was assigned using a semiquantitative HSCORE. The HSCORE was calculated using the following equation: HSCORE = ? Pi (i +1) as previously described (5).
Statistical analysis
The comparison of integrin concentrations in endometria was assessed by the non-parametric
Mann-Whitney test. Statistical significance was considered to be P<0.05.
Results
Endometrial development and integrin expression in fertile women treated with clomiphene citrateDespite the fact that all endometrial biopsies from CC-treated women were taken during the mid-secretory phase of the menstrual cycle, a high proportion (4 of 10) of these endometria were found to be out-of-phase as assessed by histological evaluation according to the Noyes' criteria (30).
In addition, in some cases (2 of 10) a desynchronised development between stroma and glands was observed. Interestingly, no differences in serum E2 or P4 levels or E2/P4 ratio was found among CC-treated women with in-phase or out-of-phase endometria (data not shown).
CC-treatment affected the expression of integrins in the endometrium of fertile women as compared to fertile controls at the implantation window. Flow cytometric measurements of ESC-integrins revealed that of a1, aV and b3 integrin subunit concentrations were significantly (P<0.05) increased in both out-of-phase and in- phase endometria from CC-treated women (Fig 1A). However, higher a1 and b3-ESC integrin concentrations were found in out-of-phase endometria (Fig.1A).
Endometrial epithelial cells from CC-treated women had similar concentrations of a1, a4 and av integrins compared to controls (Fig 1B). Nevertheless, CC-treated women exhibited a significant down-regulation of b3 integrin subunit in EEC. b3-EEC concentration was lower in out-of-phase endometria from CC-treated patients (Fig 1B).
Integrin concentrations in endometria from IUD users
Endometrial dating from IUD users was similar to mid-secretory endometria from controls.
Nevertheless, all endometria from users of the T-Cu IUD showed strong signs of acute
inflammation. A similar pattern of a1, aV and
b3 stromal integrin concentrations was found in
endometria from IUD users and controls (Fig 2A). Only the a4-ESC
integrin from endometria of IUD users was found at lower concentrations during the
implantation window. No differences were observed between fertile controls and IUD users
for the expression of a4, aV and b3 epithelial integrin subunits during the implantation window (Fig 2B). Nevertheless, flow cytometric measurements revealed higher
concentration for a1-EEC in IUD users at the time of the
implantation window (see Fig 2B).
Immunohistochemical integrin detection
Immunohistochemical results of endometria from the CC-treated patients, IUD
users and control women correlated well with the flow cytometric quantitative measurements
of integrin molecules (Fig 3).
A comparative staining for b3 integrin in endometria from patients and controls is shown in Figure 4. A similar positive staining for b3 integrin was found in EEC and ESC from controls (Fig 4A) and IUD users (Fig 4B). However, b3 integrin staining in the endometrial glands from CC-patients showed a non-homogenous distribution. Indeed, some glands were moderately stained for b3 integrin in in-phase endometria from CC-patients (Fig 4C). However, the majority of glands from out-of-phase endometria of CC-patients showed a very slight staining for this integrin (Fig 4D). In contrast, endometrial stromal cells from all CC-patients were always positively stained for b3 integrin (see Fig 4 C and D).
Discussion
The present investigation represents the first quantitative appraisal for describing the effects of CC-treatment and Cu-IUD effects on the expression of integrins in the endometrium. To compare these data, we also assessed the expression pattern of integrins in endometria from fertile controls by flow cytometry and immunohistochemistry.
Flow cytometric measurement of endometrial integrin is a useful methodology to study the expression of uterine receptivity markers (7), it has also been used to assess the dating of the endometrium (32).
Despite the fact that CC is a proven effective and low cost ovulation inducer, it yields only low pregnancy rates. Detrimental effects of CC on endometrial development have been underlined in several investigations, despite higher values of estrogen and progesterone that was found in these patients (20, 33, 34). Recently, Sereepapong et al (35) reported that CC reduced endometrial glandular density, increases number of vacuolated cells and is associated with a significantly thin peri-implantatory endometrial thickness. However, other investigators reported normal ultrasonographic appearance and thickness (36), morphology (37) and pregnancy rates (38) in endometria exposed to CC. Interference with endometrial estrogen and progesterone receptor induction has been also reported in association with CC treatment (36, 37).
The present investigation confirmed that CC-treatment provoked a high frequency of out-of-phase endometrium (11). Furthermore, we found that CC leads to alterations in the normal expression of integrins in endometria of fertile patients.
In-phase or out-of-phase endometria from CC-treated women have under-expressed epithelial b3 integrin at the time of the implantation window.
In addition, CC-treatment also affected the normal pattern of integrin expression in stromal cells. Up-regulation of a1- (a component of a1b1 laminin/collagen receptor), aV- and b3-stromal integrin subunits found in CC-treated endometria may be relevant for further invasion of trophoblast cells. It seems that under CC-effects, stromal cells could achieve advanced development in their repertoire of adhesion molecules before the blastocyst attaches to the endometrial lining and invades the decidua. Also, down-regulation of epithelial b3 and up-regulation of stromal integrins at the time of the implantation window might be interpreted as a non-synchronous development of epithelial glands and stroma, which could impair uterine receptivity and contribute to higher rates of miscarriages found in CC-treated women (15,16,38).
On the other hand, the precise mechanism contraceptive action of Cu-IUD remains uncertain though likely due to a local inflammatory reaction (40,41). However, it is not related to blocking of P4 effect on the endometrium (42,43). Although prolonged use of IUD causes a proportional decrease in estrogen and progesterone receptors (25).
We found that b3-EEC was normally expressed during the implantation window in the endometria of IUD users. These results could indicate that Cu-IUD alters endometrial receptivity by a different mechanism that is independent of altered epithelial b3 integrin expression, considered the most reliable marker of endometrial receptivity. The present data is in contrast with the recent report that showed an under-expression of aVb3 integrin in endometria of IUD-users. This is due probably to the semi-quantitative measurement of integrin staining used by these authors (44). However, we found that the a4-ESC (a component of a4b1 fibronectin receptor) integrin was under-expressed and the a1-EEC was over-expressed at the time of the implantation window in the endometria from Cu-IUD users.
The expression of an abnormal pattern of other integrins different from the epithelial b3 integrin found in IUD users might also have a biological significance for the implantation process.
In conclusion, these investigations demonstrate that CC treatment can impair uterine receptivity by down-regulating b3 epithelial integrin. Consequently, CC negative effects on expression of endometrial estrogen (36) and progesterone receptors (39), could be related to under expressed b3-EEC integrin. To establish the relationship between these two observations, it remains to be determined whether in EEC with b3-deficient integrin also under-expresses progesterone receptors in CC-treated patients. A normal pattern for the expression of b3-EEC integrin was observed in IUD users. However, these patients showed an abnormal quantitative pattern for the expression of other endometrial integrin molecules, which may impair implantation.
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Concentrations of a1, a4, aV and b3 integrin subunits determined by flow cytometry at the implantation window in endometrial stromal (A) and epithelial cells (B) from fertile women treated with clomiphene citrate (CC) and fertile controls. Forty percent of endometrial biopsies from CC-treated women were found out-of phase (CC-out phase) according to Noyes' criteria. Stromal cells (ESC) from CC-treated women (CC-all group) showed higher concentrations for a1, av and b3 integrins than fertile untreated controls. The over-expression of the b3-ESC-integrin was higher in the CC-out phase endometria group. Epithelial cells (EEC) from CC-treated women showed lower concentrations of the b3-integrin subunit than fertile untreated controls. The under-expression of b3-EEC was most evident in CC-out phase endometria. (*): P<0.05
Integrin expression by endometrial stromal (ESC) and epithelial
cells (EEC) from CC-treated women, users of T-Cu-380ª intrauterine
device (IUD-users) and fertile controls was quantitatively determined by flow cytometry
and expressed in relative fluorescence units (RFU), for reference see Gonzalez et al.,
1999 (7). Integrin expression was semiquantitatively determined by immunohistochemistry
and HSCORE values were calculated (5). Both methods showed a good correlation (r = 0.74;
n= 193).
