Clomiphene citrate does not affect the secretion of {alpha}3, {alpha}V and ß1 integrin molecules during the implantation window in patients with unexplained infertility

S. Lacin1,3, S. Vatansever2, N.K. Kuscu1, F. Koyuncu1, K. Ozbilgin2 and E. Ceylan1

1 Department of Obstetrics and Gynaecology and 2 Department of Histology and Embryology, School of Medicine, Celal Bayar University, Manisa, Turkey


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The expression of integrin molecules on the endometrium suggests that certain integrins may participate in the cascade of molecular events leading to successful implantation. A prospective, controlled study was carried out to investigate the effect of clomiphene citrate (CC) on secretions of ß1, {alpha}3 and {alpha}V integrin molecules in the endometrium of patients with unexplained infertility during the implantation window. METHODS: A total of 40 endometrial samples was evaluated in both spontaneous (n = 13) and ensuing clomiphene-treated cycles (100 mg on days 5–9) and also from fertile women serving as controls (n = 14) during postovulatory 7th or 8th day of menstrual cycle. A semiquantitative grading system (H-score) was used to compare the immunohistochemical staining intensities. Endometrial thickness and serum oestradiol and progesterone concentrations were also measured on the day of sampling. RESULTS: Staining of {alpha}v but not ß1 and {alpha}3 integrins was significantly less intense in infertile cases than fertile control cases (1.42 ± 0.12 versus 2.21 ± 0.13 respectively, P = 0.012) and this was not restored to normal concentrations with treatment. CONCLUSIONS: Our study indicated that cc treatment significantly decreased the endometrial thickness and increased oestradiol and progesterone concentrations. However, secretion of {alpha}v, ß1 and {alpha}3 integrin molecules, which might play a role in implantation, was not affected.

Key words: clomiphene/implantation/integrins/unexplained infertility


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Integrins belong to a family of cell adhesion molecules that are present on virtually all cells participating in cell–cell and cell–matrix recognition (Sheppard, 1996Go). The expression of these proteins on the endometrium suggests that certain integrins may participate in the cascade of molecular events leading to successful implantation. Different authors have proposed that special integrins have emerged as reliable markers of implantation and fertility (Ilensanmi et al., 1993Go; Sutherland et al., 1993Go; Damsky et al., 1994Go; Lessey et al., 1994Go; Sueoka et al., 1997Go) and their absence in the endometrium were thought to be associated with special aetiologies of infertility such as luteal phase deficiency (Lessey et al., 1992Go), endometriosis (Ota and Tanaka, 1997Go) and unexplained infertility (Lessey et al., 1995Go).

Clomiphene citrate (CC) is a widely used drug in the induction or enhancement of ovulation for infertility of different aetiologies mentioned above. Since it is a mixture of cis and trans stereo-isomers, it has both oestrogenic and anti-oestrogenic properties. The discrepancy between ovulation and pregnancy rates after CC therapy in anovulatory patients is thought to be the result of anti-oestrogenic effects of CC on both cervical mucus and endometrium (Randall and Templeton, 1991Go; Yeko et al., 1992Go). The popular use of intrauterine insemination (IUI) has largely overcome the anti-oestrogenic effects of CC on cervical mucus, but endometrium remains an inevitable target for these potential adverse effects. For the patients with unexplained infertility, ovulation induction with CC has been shown to be an efficacious and reasonable initial therapy (Glazener et al., 1990Go; Fisch et al., 1998Go; Guzick et al., 1998Go).

Detrimental effects of CC on the endometrium have been described by different investigators using ultrasonographic, histological and biochemical markers (Nelson et al., 1990Go; Wolman et al., 1994Go; Bonhoff et al., 1996Go; Nakamura et al., 1997Go). It has also been shown that endometrial secretion of E-cadherin, which is another important adhesion molecule, was not significantly affected by CC treatment (Dawood et al., 1998Go). To the best of our knowledge, there is no study in the literature investigating the effect of CC on immunohistochemical uterine receptivity markers, namely integrin molecules.

In this study, we investigated the effect of CC on secretions of specific integrins, ß1, {alpha}3 and {alpha}v, in endometrium of patients with unexplained infertility using a prospective, controlled study design.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Subjects and treatment
Forty endometrial samples were obtained in the Department of Obstetrics and Gynecology at the University of Celal Bayar, Manisa, Turkey between June and December 2000. We studied endometrial and venous blood samples taken from patients with primary unexplained infertility (n = 13) in both spontaneous and ensuing CC-stimulated cycles (100 mg CC orally from day 5 to day 9 of the cycle) and also from fertile women serving as controls (n = 14) during postovulatory 7th or 8th day of menstrual cycle, i.e. at the implantation window. The control population was chosen among fertile women admitted to our Hospital for surgical sterilization.

Unexplained infertility was defined as the absence of an apparent cause for infertility after a series of examinations which included hormone profiles, histopathological evaluation of endometrium, ultrasonography, semen analysis, hysterosalpingography and laparoscopy. All recruited cases had regular cycles, were free from any chronic illnesses and were not taking any medications nor using an intrauterine device. The onset of menstruations and the cycle lengths in all cycles studied were similar to those in the subject's previous cycles. Anovulatory patients or cases with male or tubal problems or showing inflammation in their endometrial samples were excluded. Biopsy day was judged according to the ultrasonographic follow-up. Beginning at the 11th day of the cycle, patients were evaluated daily. Ovulation was defined ultrasonographically as the disappearance of the dominant follicle and the collection of fluid in the pouch of Douglas, and was later confirmed by progesterone concentration.

We also measured endometrial thickness in the sagittal plane in all subjects before sampling and investigated the correlation with integrins and hormone concentrations. We used transvaginal ultrasonography (Hitachi, EUB 420) with a 7.5 MHz transducer. Endometrial thickness was defined as the distance from the hyperechogenic interface between the endometrium and myometrium to the opposite interface including the midline echo.

The Research Ethics Committee of the Medical School of Celal Bayar University approved our study protocol, and all subjects provided informed consent.

Tissue collection and laboratory work
Endometrial biopsies were obtained by using a sampling device (Pipelle de Cornier, Paris, France). A small portion of samples was also evaluated for histological dating. All samples were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for a maximum of 24 h. Samples were then washed with PBS and soaked in a series of 50, 60, 70, 80 and 90% ethanol (30 min each solution) and then in 95 and 100% ethanol for 1 h. They were then held in a solution of 100% ethanol and xylene at a 1:1 ratio for 30 min and embedded in paraffin at 60°C for 1 h to form paraffin blocks. Cross-sections of 5 µm thickness were cut from the blocks and prepared for immunohistochemical staining. The samples were first incubated at 60°C overnight and then held in xylene for 30 min. After washing with serial concentrations of ethanol (95, 80, 70 and 60% for 2 min for each), the sections were washed with distilled water followed by PBS for 10 min. They were then held in 2% trypsin in Tris buffer at 37°C for 15 min and washed with PBS three times for 5 min. The limits of sections were drawn with a Dako pen (Dako, Carpinteria, CA, USA) and incubated with 3% hydrogen peroxide for 15 min to inhibit endogenous peroxidase activity. Then the tissues were washed with PBS three times for 5 min each and stained with primary antibodies; polyclonal anti-integrin {alpha}v (1/100; Calbiochem, San Diego, CA, USA), monoclonal anti-integrin {alpha}3 (1/100; Oncogene Research, Cambridge, MA, USA) and monoclonal anti-integrin ß1 (1/100; Oncogene Research) for 18 h. They were washed with PBS three times for 5 min each, followed by incubation with biotinylated anti-rabbit IgG, then streptavidin–peroxsidase was administered (Universal Dako LSAB2 kit). Incubation steps were interspersed by three washing steps. After washing the secondary antibody with PBS three times for 5 min, the sections were washed with Dako AEC Substrate system containing 3-amino-9-ethylcarbazole (5 min) to detect immunoreactivity and then washed with Mayer's haematoxylin. They were covered with Dako medium and were observed with an Olympus BX 50 light microscope. Control samples were processed in an identical manner, but in the absence of primary antibody. Two observers blinded to the clinical information of endometrial samples independently evaluated the staining scores. The staining of integrins was graded semiquantitatively and the H-score was calculated using the following equation: H-score = {Sigma} Pi (i + 1), where i = intensity of staining with a value of 1, 2 or 3 (weak, moderate or strong respectively) and Pi is the percentage of stained epithelial cells for each intensity, varying from 0 to 100% (Lessey et al., 1992Go).

Peripheral blood was drawn on the same day of endometrial sampling for determination of oestradiol and progesterone concentrations (Chiron Diagnostic Corporation, East Walpole, MA, USA). The total assay variations were 8.3% for oestradiol and 11.5% for progesterone. Results were expressed as mean ± SEM. Differences between groups were statistically analysed using the Mann–Whitney U-test and one-way analysis of variance as appropriate. Scheffé's test was used for post-hoc analysis. P < 0.05 was considered significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The mean duration of infertility was 2.4 ± 3.1 years. The mean ages and the cycle lengths were not significantly different between the three groups. All cases except two samples in CC-treated group showed a normal luteal function judged by normal progesterone concentrations and normal endometrial histology in biopsy specimens. Oestradiol in CC-treated cycles was significantly higher than those in spontaneous cycles (P < 0.05). Progesterone was also significantly higher in CC-treated cycles than both natural cycles and cycles in fertile controls (P < 0.05). The patient and cycle characteristics are summarized in Table IGo.


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Table I. Demographic and cycle characteristics of subjects with unexplained infertility and fertile controls
 
Endometrial thickness, measured by transvaginal ultrasonography during the implantation window, was significantly thinner (P < 0.05) in CC-stimulated cycles (8.9 ± 0.4 mm) than in either spontaneous cycles (10.7 ± 0.4 mm) or fertile controls (10.6 ± 0.3 mm) (Figure 1Go).



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Figure 1. Box and whisker plot of endometrial thicknesses in the three groups. Values are median (horizontal bar), 25th/75th interquartile ranges (box) and maximum/minimum values (whiskers). *P < 0.05 versus unexplained infertile and fertile control cases (one-way analysis of variance with Scheffé's post-hoc analysis).

 
Immunohistochemically, {alpha}v integrins were detected in the epithelium and also in stroma (Figure 2AGo–C) in both fertile and infertile endometrial samples. Staining of {alpha}v integrin was significantly less intense in infertile cases than fertile control cases (H-score: 1.42 ± 0.12 versus 2.21 ± 0.13, P = 0.012) and showed an increase in epithelium after treatment with CC to an intensity which was not as high as fertile cases. Immunoreactivity of {alpha}3 integrin was only detected weakly in epithelial cells of endometrium of both fertile and infertile, non-treated group, and showed a non-significant increase after CC treatment. ß1 integrin immunoreactivities were detected at minimal levels in all groups. Staining intensities are summarized in Table IIGo.



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Figure 2. Staining of {alpha}v integrin in infertile cases (A) which was significantly less intense than fertile control cases (C) (H-score: 1.42 ± 0.12 versus 2.21 ± 0.13, P = 0.012). (B) Staining of {alpha}v integrin in infertile cases after clomiphene citrate treatment. Original magnification x400.

 

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Table II. Staining intensities of ß1, {alpha}3 and {alpha}v integrin molecules in the three groups
 
Clomiphene treatment did not affect the secretion of {alpha}v, {alpha}3 and ß1 integrin molecules compared with spontaneous cycles in patients with unexplained infertility.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Many physiological processes involving implantation depend on timely expression of cell adhesion molecules which maintain tissue architecture by mediating cell–cell and cell–matrix attachments. One family of cell adhesion molecules is the integrins: cell surface glycoproteins composed of heterodimeric {alpha} and ß subunits that serve as receptors for the extracellular matrix. While the role of integrins in endometrial function is not yet clearly defined, these interesting molecules seem to function as markers of normal and abnormal states of receptivity and may provide clues to the physiology of implantation in general.

CC or gonadotrophins in combination with IUI are still chosen by many physicians, reasonably, as the initial treatment modality for helping patients with unexplained infertility (Glazener et al., 1990Go; Fisch et al., 1998Go; Guzick et al., 1998Go). For cost and convenience, CC is generally the initially preferred drug before using gonadotrophins.

In this study, we performed endometrial biopsies in patients with unexplained infertility in spontaneous cycles and after stimulation with CC at a period of the luteal phase during which the implantation is expected. We observed that the CC therapy did not affect the secretion of some special integrin molecules contrary to our hypothesis at the beginning of the study.

The integrins we investigated in this study certainly do not represent the complete family of adhesion molecules, which play a role during implantation. However, the subunits {alpha}v, {alpha}3 and ß1 join in different combinations forming receptors on the cell surface and appear to be important for implantation. The integrin {alpha}v subunit is known to associate with a number of ß subunits forming integrins capable of binding to fibronectin, fibrinogen, vitronectin and thrombospondin (Cheresh et al., 1989Go). Secretion of {alpha}v and ß1 has been demonstrated to increase during the secretory phase of the menstrual cycle, probably suggesting a role in implantation (Dou et al., 1999Go). In addition, impaired blastocyst implantation has been shown with down-regulation of {alpha}v by using interleukin-1 receptor antagonist (Simon et al., 1998Go). Similarly, it has been demonstrated that {alpha}3ß1 complex mediated trophoblast adhesion in vitro (Yelian et al., 1995Go).

The integrin subunits ß1, {alpha}4 and {alpha}3 have been previously investigated in endometrium of patients with unexplained infertility. In these patients, neither ß1 and {alpha}4 or {alpha}3 subunit expression was found to be defective (Lessey et al., 1995Go). On the other hand, it has been found that the {alpha}4ß1 complex in both endometrial glands and surface epithelial cells of unexplained infertile women was persistently absent throughout the luteal phase (Klentzeris et al., 1993Go). The latter authors suggested that abnormal integrin expression was a frequent finding in women with unexplained infertility. In this study, we showed that the {alpha}v subunit was also secreted less in endometrium of these group of patients compared to fertile controls.

Another important conclusion from our study was that although the endometrial thickness decreased significantly after CC therapy, the expression of specific integrin molecules was not affected. Conversely, it has been found that the mid-secretory echogenic pattern but not endometrial thickness was significantly different in CC-stimulated cycles (Nakamura et al., 1997Go). A possible explanation for this difference may be the lower dose (50 mg) used in their study.

Luteal phase defect is a clinical entity in which the window of implantation is shifted or lost. This situation, although not universally accepted, was reported to appear in some CC cycles (Cook et al., 1984Go). Studies investigating the effect of CC on endometrial oestrogen and progesterone receptor contents also gave different results (Rönnberg et al., 1985Go; Aksel et al., 1986Go; Fritz et al., 1991Go). We did not include patients with luteal phase defects in our study and luteal phase defect has appeared only in two samples (15.3%) in the CC-treated group. Our results have supported the theory that the effect of CC given in mid-proliferative days still existed during implantation window.

If we had found that CC significantly reduced the secretion of integrins, the question of whether this specific biological change interferes with implantation and pregnancy rates in ovulation induction cycles would still be a matter of debate. With these results, we cannot rule out the possibility that CC does not alter the milieu of the implantation process since the integrins ß1, {alpha}3 and {alpha}v constitute only a small portion of the adhesion molecules that play a role in implantation. However, one can speculate that the increased oestradiol and progesterone secretion by the corpus luteum compensated for the anti-oestrogenic effects of CC and subsequently led to a balance, causing no significant change in secretion of integrin molecules that we studied.

Various authors have studied the integrin molecules in different groups of infertile populations, but this is the first study in the literature investigating the effect of CC on specific integrin molecules. The optimal conditions required for implantation of human blastocysts need further investigation with regard to these specific markers.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This study was supported by a grant (AFS-1999/26) from Celal Bayan University, Manisa, Turkey.


    Notes
 
3 To whom correspondence should be addressed at: Sakarya Mahallesi Uzunyol Caddesi, No: 122/2 Unlu-II Apt., 45020 Manisa, Turkey. E-mail: lacins{at}egenet.com.tr Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on February 16, 2001; accepted on July 23, 2001.