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Stromal cell–specific apoptotic and antiestrogenic mechanisms may explain uterine defects in humans after clomiphene citrate therapy

  • Magdalena Nutu
    Affiliations
    Department of Physiology/Endocrinology, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden
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  • Yi Feng
    Affiliations
    Department of Physiology/Endocrinology, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden

    Department of Neurobiology and Integrative Medicine, Shanghai Medical College of Fudan University, Shanghai, China
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  • Emil Egecioglu
    Affiliations
    Department of Physiology/Endocrinology, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden
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  • Birgitta Weijdegård
    Affiliations
    Department of Physiology/Endocrinology, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden
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  • Elisabet Stener-Victorin
    Affiliations
    Department of Physiology/Endocrinology, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden

    Department of Obstetrics and Gynecology, First Affiliated Hospital, Heilongjiang University of Chinese Medicine, Harbin, China
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  • Ruijin Shao
    Correspondence
    Reprints: Ruijin Shao, MD, PhD, Department of Physiology/Endocrinology, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at Gothenburg University, Medicinareg 11, PO Box 434, 413 90 Gothenburg, Sweden
    Affiliations
    Department of Physiology/Endocrinology, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden
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      Objective

      The purpose of this study was to investigate clomiphene citrate (CC)–induced modulation of uterine cell function in vivo.

      Study Design

      Prepubertal female Sprague-Dawley rats were treated intraperitoneally with CC for 6 or 24 hours or with a combination of CC and/or 17-β-estradiol (E2) for 4 days.

      Results

      Chronic CC treatment induced apoptosis in a fraction of uterine stromal cells by activating the caspase-3-mediated apoptotic pathway. The damage was prevented by successive E2 treatment; however, pretreatment or concomitant treatment with E2 did not protect against CC-induced uterine apoptosis. CC decreased the protein expression of estrogen receptor α and increased its phosphorylation but did not affect estrogen receptor β expression or phosphorylation. Furthermore, changes in Hoxa11, p27, and progesterone receptor protein levels and localization were associated with CC treatment.

      Conclusion

      We provide novel mechanistic insights into cellular and molecular events by which CC regulates uterine stromal cell function and hence the implantation process and pregnancy outcome.

      Key words

      Clomiphene citrate (CC), a nonsteroid tissue-selective estrogen receptor (ER) modulator,
      • Turner R.T.
      • Evans G.L.
      • Sluka J.P.
      • et al.
      Differential responses of estrogen target tissues in rats including bone to clomiphene, enclomiphene, and zuclomiphene.
      is used commonly to treat various infertilities in women, most notably polycystic ovary syndrome (PCOS) and unexplained infertility.
      • Homburg R.
      Clomiphene citrate: end of an era? A mini-review.
      • Palomba S.
      • Russo T.
      • Orio Jr, F.
      • et al.
      Uterine effects of clomiphene citrate in women with polycystic ovary syndrome: a prospective controlled study.
      Several clinical and epidemiologic studies have indicated that successful ovulation occurs in 70–99% of women who are treated with CC,
      • Homburg R.
      Clomiphene citrate: end of an era? A mini-review.
      • Benda J.A.
      Clomiphene's effect on endometrium in infertility.
      whereas ultimate pregnancy rates are only 27–40%.
      • Homburg R.
      Clomiphene citrate: end of an era? A mini-review.
      • Samani F.G.
      • Farzadi L.
      • Nezami N.
      • Tarzamni M.K.
      • Soleimani F.
      Endometrial and follicular development following letrozole intervention in unexplained infertile patients failed to get pregnant with clomiphene citrate.
      The total CC-treated pregnancy rates are approximately 10 times lower than “natural” rates.
      • Hosie M.
      • Adamson M.
      • Penny C.
      Actin binding protein expression is altered in uterine luminal epithelium by clomiphene citrate, a synthetic estrogen receptor modulator.
      Furthermore, spontaneous abortion and miscarriage occur frequently in women who receive CC therapy.
      • Homburg R.
      Clomiphene citrate: end of an era? A mini-review.
      • Benda J.A.
      Clomiphene's effect on endometrium in infertility.
      • Dickey R.P.
      • Taylor S.N.
      • Curole D.N.
      • Rye P.H.
      • Pyrzak R.
      Incidence of spontaneous abortion in clomiphene pregnancies.
      Although it has been proposed that the antiestrogenic effects of CC may lead to early pregnancy failure,
      • Benda J.A.
      Clomiphene's effect on endometrium in infertility.
      the precise molecular events underlying CC-induced uterine defects remain incompletely understood.
      For Editors' Commentary, see Table of Contents
      The mammalian uterus is a dynamic reproductive organ that undergoes cyclic changes in response to ovarian steroid hormones.
      • Diedrich K.
      • Fauser B.C.
      • Devroey P.
      • Griesinger G.
      The role of the endometrium and embryo in human implantation.
      The biologic effects of 17-β-estradiol (E2) and CC are mediated primarily by 2 nuclear receptors, ER-α and/or ER-β, which are encoded by 2 different genes and function as transcription factors.
      • DeMayo F.J.
      • Zhao B.
      • Takamoto N.
      • Tsai S.Y.
      Mechanisms of action of estrogen and progesterone.
      Although the distribution and relative levels of ER-α and ER-β expression are tissue specific and diverse,
      • Kuiper G.G.
      • Carlsson B.
      • Grandien K.
      • et al.
      Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta.
      both ER-α and ER-β are expressed in the rodent uterus and human endometrium.
      • Moutsatsou P.
      • Sekeris C.E.
      Steroid receptors in the uterus: implications in endometriosis.
      It has been reported that inappropriate activation or inhibition of ER subtypes may cause or contribute to a variety of uterine diseases, such as endometriosis and endometrial cancer.
      • Moutsatsou P.
      • Sekeris C.E.
      Steroid receptors in the uterus: implications in endometriosis.
      The protective effects of E2 in uterine homeostasis are evident both from in vivo and in vitro studies: the presence of E2 inhibits uterine apoptosis in vivo;
      • Yin Y.
      • Huang W.W.
      • Lin C.
      • Chen H.
      • MacKenzie A.
      • Ma L.
      Estrogen suppresses uterine epithelial apoptosis by inducing birc1 expression.
      however, human endometrial cells undergo apoptosis when E2 is withdrawn.
      • Song J.
      • Rutherford T.
      • Naftolin F.
      • Brown S.
      • Mor G.
      Hormonal regulation of apoptosis and the Fas and Fas ligand system in human endometrial cells.
      Previous studies from our laboratory and others have shown that CC enhances apoptotic processes in the ovaries, fallopian tubes, villi, and decidual tissues.
      • Chaube S.K.
      • Prasad P.V.
      • Tripathi V.
      • Shrivastav T.G.
      Clomiphene citrate inhibits gonadotropin-induced ovulation by reducing cyclic adenosine 3',5'-cyclic monophosphate and prostaglandin E2 levels in rat ovary.
      • Kokawa K.
      • Shikone T.
      • Nakano R.
      Apoptosis in human chorionic villi and decidua in normal and ectopic pregnancy.
      • Shao R.
      • Nutu M.
      • Weijdegard B.E.
      • et al.
      Clomiphene citrate causes aberrant tubal apoptosis and estrogen receptor activation in rat fallopian tube: implications for tubal ectopic pregnancy.
      Less is known, however, about the effects of CC on the apoptotic machinery in the uterus.
      The maternal endometrium shows prominent steroid-dependent cyclic changes in structure and function in preparation for the process of implantation.
      • Diedrich K.
      • Fauser B.C.
      • Devroey P.
      • Griesinger G.
      The role of the endometrium and embryo in human implantation.
      Successful implantation requires precise coordination between the embryo and uterus under the influence of ovarian steroids.
      • Dey S.K.
      • Lim H.
      • Das S.K.
      • et al.
      Molecular cues to implantation.
      After fertilization, specific uterine cell types undergo differentiation and proliferation to provide a suitable environment for embryo implantation and development.
      • Dey S.K.
      • Lim H.
      • Das S.K.
      • et al.
      Molecular cues to implantation.
      Genomic endometrial responses to estrogen are essential for the regulation of the “implantation window.”
      • Barkai U.
      • Kidron T.
      • Kraicer P.F.
      Inhibition of decidual induction in rats by clomiphene and tamoxifen.
      The transformation of endometrial stromal cells into decidual cells has been recognized as a fundamental step during the process of implantation.
      • Barkai U.
      • Kidron T.
      • Kraicer P.F.
      Inhibition of decidual induction in rats by clomiphene and tamoxifen.
      • Groothuis P.G.
      • Dassen H.H.
      • Romano A.
      • Punyadeera C.
      Estrogen and the endometrium: lessons learned from gene expression profiling in rodents and human.
      Previously, it has been shown that in vivo treatment with CC delays and/or inhibits implantation in rodents,
      • Barkai U.
      • Kidron T.
      • Kraicer P.F.
      Inhibition of decidual induction in rats by clomiphene and tamoxifen.
      • Gupta J.S.
      • Roy S.K.
      Effect of clomiphene on nuclear estrogen receptor of the fallopian tube during ovum transport in rabbits.
      probably because of abnormalities that are seen in the reproductive tract (including the uterus) after CC treatment in both rats
      • Shao R.
      • Nutu M.
      • Weijdegard B.E.
      • et al.
      Clomiphene citrate causes aberrant tubal apoptosis and estrogen receptor activation in rat fallopian tube: implications for tubal ectopic pregnancy.
      • Nagao T.
      • Yoshimura S.
      Oral administration of clomiphene to neonatal rats causes reproductive tract abnormalities.
      and humans.
      • Cunha G.R.
      • Taguchi O.
      • Namikawa R.
      • Nishizuka Y.
      • Robboy S.J.
      Teratogenic effects of clomiphene, tamoxifen, and diethylstilbestrol on the developing human female genital tract.
      Furthermore, a significant decrease in the implantation rate has also been observed in rabbits that were treated with CC before and after ovulation.
      • Birkenfeld A.
      • Mootz U.
      • Beier H.M.
      The effect of clomiphene citrate on blastocyst development and implantation in the rabbit.
      These observations, combined with clinical studies, have given rise to the hypothesis that CC may contribute to implantation-related complications through an unidentified regulatory process in the uterus. The goals of this study were (1) to determine whether CC treatment induces uterine cell apoptosis and (2) to investigate the molecular regulation of ERs and other potential implantation and cell cycle regulators
      • Dey S.K.
      • Lim H.
      • Das S.K.
      • et al.
      Molecular cues to implantation.
      • Franco H.L.
      • Jeong J.W.
      • Tsai S.Y.
      • Lydon J.P.
      • DeMayo F.J.
      In vivo analysis of progesterone receptor action in the uterus during embryo implantation.
      in the uteri of rats that were treated with CC.

      Materials and Methods

      Animals

      All experimental procedures and protocols were approved by the ethics committee at Gothenburg University. Prepubertal female Sprague-Dawley rats (20 days old) were obtained from Taconic M&B (Copenhagen, Denmark) and maintained in cages that contained wood chips under defined conditions (temperature, 21 ± 2°C; relative humidity, 45–55%; and 12-hour light/dark illumination schedule). Animals were acclimated to the animal facilities for 5 days before the initiation of the experiments. All animals had free access to tap water and were fed ad libitum with standard laboratory diet.

      Experimental design

      Three experiments were carried out (Figure 1) in 25-day-old rats. The rats were randomized to receive intraperitoneal injections of CC (Sigma-Aldrich, St. Louis, MO), E2 (Sigma-Aldrich), or both. Controls were treated with vehicle only. Bodyweight was recorded throughout the experiment. Rats were killed under light anesthesia with sodium pentobarbital (0.5 mL/kg bodyweight). Aliquots of serum were prepared from trunk blood after heart puncture and stored at –80°C until analysis. The uteri were dissected grossly with the removal of contaminating tissues (eg, adipose tissues), weighed, and immediately frozen in liquid nitrogen or fixed in formalin.
      Figure thumbnail gr1
      FIGURE 1Schematic of the experimental design
      CC, clomiphene citrate; E2, 17-β-estradiol; Exp, experiment.
      Nutu. Clomiphene citrate treatment and rat uterus. Am J Obstet Gynecol 2010.

      Experiment 1

      Rats received CC (1 or 10 mg/kg intraperitoneally) or an equivalent volume of vehicle (0.9% NaCl). Uteri were collected 6 and 24 hours (acute effect) after injection or on day 4 (chronic effect) after daily intraperitoneal injections (n = 8/group). For the selected doses and treatment schedule, CC is effective in rat fallopian tube and uterus in vivo.
      • Shao R.
      • Nutu M.
      • Weijdegard B.E.
      • et al.
      Clomiphene citrate causes aberrant tubal apoptosis and estrogen receptor activation in rat fallopian tube: implications for tubal ectopic pregnancy.
      • Young R.L.
      • Goldzieher J.W.
      • Chakraborty P.K.
      • Panko W.B.
      • Bridges C.N.
      Qualitative differences in estrogenic/antiestrogenic effects of clomiphene and zuclomiphene.

      Experiment 2

      Rats received injections of CC (10 mg/kg intraperitoneally) or vehicle once daily for 4 consecutive days. Twenty-four hours after the last injection, E2 (0.3 mg/kg in 100 μL sesame oil) or vehicle was injected for 4 consecutive days.

      Experiment 3

      Rats were injected with E2 or vehicle once daily for 4 consecutive days, as in experiment 2. Twenty-four hours after the last injection, CC (10 mg/kg), CC (10 mg/kg) and E2 (0.3 mg/kg), or vehicle was injected intraperitoneally for 4 consecutive days. In experiments 2 and 3, the rats were killed, and uteri were collected 24 hours after the final injection (n = 5/group).

      Antibodies

      The primary antibodies that were used for Western blot (WB) analysis and immunofluorescence were obtained from commercial sources (anticleaved caspase 3 and anticleaved caspase 9 [Cell Signaling Technology, Beverly, MA]; anti-ERá, antiphospho-ERâ [Ser87], anti-Hoxa10, anti-Hoxa11, anti-p27, and anti-p53 [Santa Cruz Biotechnology, Santa Cruz, CA]; antiphospho-ERá [Ser118] and anti-ERâ [Upstate Biotechnology, Lake Placid, NY]; anti-pan-cytokeratin, anti-á-smooth muscle actin and anti-â-actin [Sigma-Aldrich]; and antiprogesterone receptor [PR; Novocastra Laboratories, Newcastle Upon Tyne, UK] antibodies). The secondary antibodies for WB were goat-antimouse immunoglobulin G (Sigma-Aldrich), goat-antirabbit immunoglobulin G (AC31RL; Tropix, Bedford, MA), and donkey antigoat immunoglobulin G (Santa Cruz Biotechnology), which were all conjugated with alkaline phosphatase. The Cy3-conjugated antimouse antibody was from Jackson ImmunoResearch Laboratories (West Grove, PA).

      Histologic evaluation and immunofluorescence staining

      Uteri were fixed in neutral buffered 10% formalin, decalcified, dehydrated, and embedded in paraffin. Series sections (5 μm) were prepared and stained with hematoxylin/eosin to visualize cell nuclei (Histocenter, Västra Frolunda, Sweden). Uterine sections were subjected to immunofluorescence studies to test the localization of progesterone receptor A isoform, as described previously.
      • Shao R.
      • Weijdegard B.
      • Ljungstrom K.
      • et al.
      Nuclear progesterone receptor A and B isoforms in mouse fallopian tube and uterus: implications for expression, regulation, and cellular function.

      In situ detection of DNA fragmentation

      Tissue sections were dewaxed, protease digested, and incubated with terminal transferase mixture according to the instructions of the manufacturer, with the use of an in situ apoptosis detection kit (Roche Diagnostics GmbH, Mannheim, Germany). Briefly, sections were incubated in a permeabilization solution, which contained 0.1% Triton X-100 and 0.1% sodium citrate, and then were incubated with the terminal deoxynucleotidyl transferase mediated 2′-deoxyuridine, 5′-triphosphate nick-end-labeling (TUNEL) reaction mixture, which included the enzyme solution (terminal deoxynucleotidyl transferase) and label solution (tetramethylrhodamine isothiocyanate-labeled nucleotides), in a humidified chamber for 1 hour at 37°C. After being washed with phosphate-buffered saline solution, the sections were evaluated with a confocal laser microscopy. The enzyme solution was omitted in the negative control. Sections that were treated this way remained unstained.

      Protein extraction and WB analysis

      Whole-cell extracts from uterine tissues were analyzed by WB, as described previously.
      • Shao R.
      • Weijdegard B.
      • Ljungstrom K.
      • et al.
      Nuclear progesterone receptor A and B isoforms in mouse fallopian tube and uterus: implications for expression, regulation, and cellular function.

      Caspase activity assay

      For the measurement of CASP-3/7 activity in whole uterine tissues, frozen tissue was homogenized in lysis buffer (100 mmol/L HEPES, pH 7.4, 140 mmol/L NaCl, and protease inhibitors), and the crude homogenate was centrifuged at 12,000g for 30 minutes at 4°C. Cellular caspase activity was determined with the use of the Caspase-Glo-3/7 assay kit (Cell Signaling Technology), as previously described.
      • Shao R.
      • Nutu M.
      • Weijdegard B.E.
      • et al.
      Clomiphene citrate causes aberrant tubal apoptosis and estrogen receptor activation in rat fallopian tube: implications for tubal ectopic pregnancy.

      Statistical analysis

      Data were analyzed with SPSS software (version 13.0; SPSS, Inc, Chicago, IL). Two-way analysis of variance was used to assess the main effects of treatment and time and to identify interactions between them. If significant interactions between the fixed factors were observed, within-group analyses were performed with a 1-way analysis of variance followed by Bonferroni's multiple comparison test. Values are shown as means ± SEM. Significance was accepted at a probability value of < .05.

      Results

      Chronic, but not acute, CC treatment induces cell type-specific apoptosis in the uterus

      Rats were given 1 acute injection of CC (1 or 10 mg/kg) or vehicle, and the expression of cleaved caspase 9 and caspase 3 (Casp3) was measured in the uterus at 6 and 24 hours. Alternatively, CC was administered daily for 4 days (chronic treatment), and the expression of the same apoptotic molecules was measured in the uterus. WB analysis revealed elevated levels of cleaved caspase 9 and Casp3 in the uteri from rats that had been treated with CC for 4 days but not at 6 and 24 hours (Figure 2, A). Using the caspase-Glo 3/7 assay, we confirmed that elevated Casp3 activity was found only in uteri from rats that had been treated with CC for 4 days (Figure 2, B), which is consistent with the levels of cleaved Casp3 (Figure 2, A). Next, DNA fragmentation was detected in situ with TUNEL staining. TUNEL-positive cells were present specifically in the stromal cell layer in rats that had been treated with CC for 4 days (Figure 3, B) but not in control rats (Figure 3, A). The luminal (Figure 3, A and B) and glandular (data not shown) epithelial cells and the smooth muscle cell layers (Figure 3, A and B) in both control and CC-treated rats showed no staining. Ovarian sections (Figure 3, C and C1) were used as experimental controls.
      Figure thumbnail gr2
      FIGURE 2Chronic treatment with clomiphene citrate (CC) induces uterine apoptosis
      Uteri were dissected from rats that were treated with clomiphene citrate for 6 or 24 hours or 4 consecutive days. A, Western blot analysis of cleaved caspase-9 and -3 relative to whole protein. Gels were stained with Coomassie blue (n = 3 rats/group). In addition, β-actin served as a loading control. B, Caspase-3/7 activity was determined in whole uterine tissues from rats that were treated with clomiphene citrate for 6 or 24 hours or 4 consecutive days (n = 5 rats/group). Values are means ± SEM. The single asterisk denotes P < .05; the double asterisks denote P < .01; the triple asterisks denote P < .001 vs vehicle-treated controls.
      Nutu. Clomiphene citrate treatment and rat uterus. Am J Obstet Gynecol 2010.
      Figure thumbnail gr3
      FIGURE 3Clomiphene citrate (CC)–induced uterine apoptosis is cell type specific
      A, Vehicle treatment in the uterus compared with B, chronic treatment with clomiphene citrate (10 mg/kg)-induced DNA fragmentation (red) of the stromal cell layer. Apoptotic cells (B) in the stromal cell layer are shown in the inset at higher magnification. Sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, A1, and B1; blue) to visualize cell nuclei. Ovarian sections (C and C1) were used as experimental controls. The pictures in A-C are representative of stainings in tissues from different rats (n = 5/group). All photographs were taken with a ×10 or a ×40 magnification; the exact scale is given in the Figure.
      Le, luminal epithelial cells; M, muscle cells; S, stromal cells.
      Nutu. Clomiphene citrate treatment and rat uterus. Am J Obstet Gynecol 2010.

      E2 aids recovery from CC-induced uterine apoptosis but is not protective

      To test whether E2 might contribute to recovery from CC-induced uterine apoptosis, rats were treated sequentially with E2 or vehicle for 4 days after chronic treatment with CC. Cleaved Casp3 levels (Figure 4, A) and activity (Figure 4, B) were lower in uteri from the E2-treated rats. Neither pretreatment nor concomitant treatment with E2 significantly affected the ability of chronic CC treatment to decrease cleaved Casp3 expression (Figure 4, C) and activity (Figure 4, D).
      Figure thumbnail gr4
      FIGURE 4E2 treatment enables recovery from clomiphene citrate (CC)–induced uterine apoptosis but is not protective
      Rats were treated with 17-β-estradiol (E2) A, B, after or C, D, before 4 days of clomiphene citrate treatment. Western blot analysis (A and C) was used to measure cleaved caspase-3 protein level relative to whole protein. Gels were stained with Coomassie blue (n = 5 rats/group). β-actin served as a loading control. Caspase-3/7 activity (B and D) was determined in whole uterine tissues of rats (n = 5 rats/group). Values are means ± SEM (n = 5 rats/group). The double asterisks denote P < .01; the triple asterisks denote P < .001 vs 17-β-estradiol–treated rats.
      Nutu. Clomiphene citrate treatment and rat uterus. Am J Obstet Gynecol 2010.

      Chronic treatment with CC results in changes in uterine morphologic condition

      As shown in the Table, although bodyweight was unaffected by acute or chronic treatment with low-dose or high-dose CC, uterine weight increased after 24-hour and 4-day treatments at both doses. In control rats, individual epithelial cells contained large round or oval basal nuclei with very little surrounding cytoplasm (Figure 5, A1). Rats that were treated with CC for 4 days had increases in epithelial height, muscular thickness, and luminal space (Figure 5, C and C1) compared with control rats (Figure 5, A and A1). Essentially, the epithelium changed in character from cuboidal to columnar cells. In CC-treated rat uteri, the nuclei were less dark, and the long axis of the nuclei of the epithelium was parallel to the basement membrane (Figure 5, C1). Although the uterine morphologic condition of E2-treated rats (Figure 5, B and B1) was quite similar to that of CC-treated rats, treatment with E2 had less of an effect on the epithelium. Moreover, clear cells in the epithelium were occasionally present in CC-treated rats. This “bubbly” appearance may indicate the lipid and/or glycogen accumulation. There was less connective tissue between stromal cells in CC-treated rats (Figure 5, C1) than in E2-treated rats (Figure 5, B1). Histologic findings also showed a lack of integrity in the epithelium of CC-treated rats after an additional 4-day vehicle treatment (Figure 5, D and D1).
      TABLEEffects of clomiphene citrate (CC) on bodyweight and uterine tissue weights in rats
      TreatmentWeight
      Body, gUterus, mgUterus/body, mg
      Acute: 6 h
       Vehicle58.74 ± 0.5010.13 ± 0.420.17 ± 0.007
       1 mg/kg CC58.24 ± 0.8110.49 ± 0.390.18 ± 0.006
       10 mg/kg CC61.06 ± 1.9810.08 ± 0.320.17 ± 0.005
      Acute: 24 h
       Vehicle60.98 ± 0.6810.17 ± 0.160.17 ± 0.003
       1 mg/kg CC62.98 ± 0.4917.93 ± 0.98
      P < .05 vs vehicle (24 hours; Bonferroni's post hoc test after significant 2-way analysis of variance time and treatment interaction);
      0.28 ± 0.014
      P < .05 vs vehicle (24 hours; Bonferroni's post hoc test after significant 2-way analysis of variance time and treatment interaction);
       10 mg/kg CC60.58 ± 1.3818.32 ± 0.36
      P < .05 vs vehicle (24 hours; Bonferroni's post hoc test after significant 2-way analysis of variance time and treatment interaction);
      0.30 ± 0.005
      P < .05 vs vehicle (24 hours; Bonferroni's post hoc test after significant 2-way analysis of variance time and treatment interaction);
      Chronic: 4 days
       Vehicle79.92 ± 2.1610.68 ± 0.240.13 ± 0.005
       1 mg/kg CC73.68 ± 2.0730.90 ± 0.68
      P < .05 vs vehicle (4 days; Bonferroni's post hoc test after significant 2-way analysis of variance time and treatment interaction).
      0.42 ± 0.016
      P < .05 vs vehicle (4 days; Bonferroni's post hoc test after significant 2-way analysis of variance time and treatment interaction).
       10 mg/kg CC72.98 ± 2.1531.50 ± 0.60
      P < .05 vs vehicle (4 days; Bonferroni's post hoc test after significant 2-way analysis of variance time and treatment interaction).
      0.43 ± 0.013
      P < .05 vs vehicle (4 days; Bonferroni's post hoc test after significant 2-way analysis of variance time and treatment interaction).
      Values are means ± SEM (n = 5 in all groups).
      Nutu. Clomiphene citrate treatment and rat uterus. Am J Obstet Gynecol 2010.
      a P < .05 vs vehicle (24 hours; Bonferroni's post hoc test after significant 2-way analysis of variance time and treatment interaction);
      b P < .05 vs vehicle (4 days; Bonferroni's post hoc test after significant 2-way analysis of variance time and treatment interaction).
      Figure thumbnail gr5
      FIGURE 5Histologic condition of the rat uterus
      Hematoxylin/eosin staining of uterine tissues from rats treated with A, A1, vehicle, B, B1, 17-β-estradiol (E2; 0.3 mg/kg) or C, C1, clomiphene citrate (CC; 10 mg/kg) for 4 consecutive days plus 4 additional days of vehicle injections (D, D1). Note that, although treatment with both 17-β-estradiol (B1) and clomiphene citrate (C1) increases epithelial cell height, clear cells in the epithelial cell layer (C1, arrows) are present only in clomiphene citrate-treated rats. Incomplete epithelial cell layers were found in the 4-day clomiphene citrate plus 4-day vehicle-treated group (D1, arrow). Uterine tissues from different rats (n = 5/group) were evaluated. All photographs were taken with a ×10 or a ×40 magnification; the exact scale is given in the Figure.
      Ge, glandular epithelial cells; Le, luminal epithelial cells; Lu, lumen; S, stromal cells.
      Nutu. Clomiphene citrate treatment and rat uterus. Am J Obstet Gynecol 2010.

      Chronic treatment with CC activates ER-α but not ER-β in the uterus

      WB analysis revealed that chronic treatment with both low-dose and high-dose CC resulted in a significant decrease in ER-α expression (Figure 6). Because the stability and activity of ER subtypes are affected by ligand-dependent ER phosphorylation,
      • DeMayo F.J.
      • Zhao B.
      • Takamoto N.
      • Tsai S.Y.
      Mechanisms of action of estrogen and progesterone.
      it is important to note that chronic treatment with CC significantly enhanced the phosphorylation of ER-α, but not ER-β (Figure 6).
      Figure thumbnail gr6
      FIGURE 6Estrogen receptor (ER) subtype activation and cell markers for implantation during clomiphene citrate (CC)–induced uterine apoptosis
      Western blot analysis was used to measure estrogen receptor α, phospho-estrogen receptor α, estrogen receptor β, phospho-estrogen receptor β, Hoxa10, Hoxa11, p27, and p53 relative to whole protein; gels were stained with Coomassie blue (n = 5 rats/group). Expression of pan-cytokeratin and α-smooth muscle (SM) actin was evaluated in the same samples. Values are means ± SEM. The double asterisks denote P < .01; the triple asterisks denote P < .001 vs vehicle-treated controls.
      Nutu. Clomiphene citrate treatment and rat uterus. Am J Obstet Gynecol 2010.

      Chronic treatment with CC regulates Hoxa11, p27, and the progesterone receptor A isoform in the uterus

      WB analysis showed that, although the levels of Hoxa11 and p27 were decreased, neither Hoxa10 nor p53 levels were altered by chronic treatment with CC (Figure 6). Chronic treatment with low-dose or high-dose CC caused a mobility shift of pan-cytokeratin compared with controls (Figure 6, A), although the cause of this shift in the apparent molecular weight of cytokeratin remains unknown. There were no changes in the levels of α-smooth muscle actin in uteri after CC treatment (Figure 6, A). Because the PR antibody (NCL-L-PGR-312) recognizes only the progesterone receptor A (PRA) isoform in tissue sections with the immunohistochemical analysis,
      • Samalecos A.
      • Gellersen B.
      Systematic expression analysis and antibody screening do not support the existence of naturally occurring progesterone receptor (PR)-C, PR-M, or other truncated PR isoforms.
      only PRA expression was analyzed in successive sections of the uterus. Chronic treatment with CC (Figure 7, B) or E2 (Figure 7, C) increased the number of PRA-positive cells in the stromal cell layer compared with controls (Figure 7, A). Moreover, concomitant CC and E2 treatment for 4 days decreased the number of PRA-positive cells in the stromal cell layer (Figure 7, D) compared with E2 treatment alone (Figure 7, C).
      Figure thumbnail gr7
      FIGURE 7Expression of progesterone receptor A (PRA) isoform in the stromal cell layers of the rat uterus
      Uterine progesterone receptor A immunoreactivity is absent in the A, vehicle group but is seen in the B, clomiphene citrate (CC)–treated group. Note that the immunoreactivity of progesterone receptor A is less intense in the B, clomiphene citrate-treated and the D, clomiphene citrate + 17-β-estradiol (E2)-treated groups compared with the C, 17-β-estradiol–treated group. These stainings were repeated in 5 rats/group with similar results. All photographs were taken with a ×10 magnification; the exact scale is given in the Figure.
      Le, luminal epithelial cells; Lu, lumen; S, stromal cells.
      Nutu. Clomiphene citrate treatment and rat uterus. Am J Obstet Gynecol 2010.

      Comment

      To our knowledge, this is the first study to demonstrate that chronic treatment with CC can induce not only stromal cell apoptosis but also morphologic abnormalities in the uteri of rats in a dose-independent manner. The present study highlights the potential role of the ER-α signaling pathway in the uterus after chronic CC treatment. Furthermore, the high ovulation rates combined with low pregnancy rates in women who were treated with CC may be explained by the aberrant expression of Hoxa11, p27, and PRA because of repression of ER-α expression and consequent disruption of the implantation process.
      CC is a racemic mixture of zuclomiphene (38%) and enclomiphene (62%) that provides both estrogenic and antiestrogenic activities in a variety of E2-target tissues.
      • Turner R.T.
      • Evans G.L.
      • Sluka J.P.
      • et al.
      Differential responses of estrogen target tissues in rats including bone to clomiphene, enclomiphene, and zuclomiphene.
      • Homburg R.
      Clomiphene citrate: end of an era? A mini-review.
      The effects of CC in the rodent uterus and human endometrium are inconsistent, however. For example, CC has both antagonistic and agonistic activities in the rat uterus,
      • Young R.L.
      • Goldzieher J.W.
      • Chakraborty P.K.
      • Panko W.B.
      • Bridges C.N.
      Qualitative differences in estrogenic/antiestrogenic effects of clomiphene and zuclomiphene.
      whereas it displays a pure ER agonistic effect in the mouse uterus
      • Jordan V.C.
      • Rowsby L.
      • Dix C.J.
      • Prestwich G.
      Dose-related effects of non-steroidal antioestrogens and oestrogens on the measurement of cytoplasmic oestrogen receptors in the rat and mouse uterus.
      in contrast to the human endometrium.
      • Gonen Y.
      • Casper R.F.
      Sonographic determination of a possible adverse effect of clomiphene citrate on endometrial growth.
      • Homburg R.
      • Pap H.
      • Brandes M.
      • Huirne J.
      • Hompes P.
      • Lambalk C.B.
      Endometrial biopsy during induction of ovulation with clomiphene citrate in polycystic ovary syndrome.
      • Unfer V.
      • Costabile L.
      • Gerli S.
      • Papaleo E.
      • Marelli G.
      • Di Renzo G.C.
      Low dose of ethinyl estradiol can reverse the antiestrogenic effects of clomiphene citrate on endometrium.
      • Yagel S.
      • Ben-Chetrit A.
      • Anteby E.
      • Zacut D.
      • Hochner-Celnikier D.
      • Ron M.
      The effect of ethinyl estradiol on endometrial thickness and uterine volume during ovulation induction by clomiphene citrate.
      Our previous work has demonstrated that chronic treatment with CC induces epithelial cell apoptosis in rat fallopian tubes in a region-specific manner.
      • Shao R.
      • Nutu M.
      • Weijdegard B.E.
      • et al.
      Clomiphene citrate causes aberrant tubal apoptosis and estrogen receptor activation in rat fallopian tube: implications for tubal ectopic pregnancy.
      Here we show that, within the uterus, the stromal cells are the main cell type to be targeted by the apoptotic effects of chronic treatment with CC. In addition, in the rat uterus, CC displays agonistic activity at low concentration, although it functions as an ER antagonist at high concentration in vivo.
      • Young R.L.
      • Goldzieher J.W.
      • Chakraborty P.K.
      • Panko W.B.
      • Bridges C.N.
      Qualitative differences in estrogenic/antiestrogenic effects of clomiphene and zuclomiphene.
      Furthermore, only chronic treatment with CC induces uterine cell apoptosis, which indicates that the agonistic and/or antagonistic effects of CC are also dependent on the duration of treatment.
      • Nagao T.
      • Yoshimura S.
      Oral administration of clomiphene to neonatal rats causes reproductive tract abnormalities.
      • Markaverich B.M.
      • Upchurch S.
      • McCormack S.A.
      • Glasser S.R.
      • Clark J.H.
      Differential stimulation of uterine cells by nafoxidine and clomiphene: relationship between nuclear estrogen receptors and type II estrogen binding sites and cellular growth.
      These observations suggest that CC acts as an ER agonist and/or antagonist depending on the species, target tissue/cell specificity, dose range, and regimen.
      We show that the effects of CC in the uterus are time dependent rather than dose dependent and that the damage can be reversed with E2, which suggests that CC, as an apoptotic inducer, blocks the antiapoptotic effect of E2 in the uterus. The clear lack of protective effects of pretreatment or concomitant with E2 could be because CC exhibits much stronger antiestrogenic activity in the presence of an E2-stimulated rat uterus.
      • Shao R.
      • Nutu M.
      • Weijdegard B.E.
      • et al.
      Clomiphene citrate causes aberrant tubal apoptosis and estrogen receptor activation in rat fallopian tube: implications for tubal ectopic pregnancy.
      It is well accepted that the endometrium undergoes morphologic and biochemical changes that are required for successful implantation and pregnancy.
      • Diedrich K.
      • Fauser B.C.
      • Devroey P.
      • Griesinger G.
      The role of the endometrium and embryo in human implantation.
      • Dey S.K.
      • Lim H.
      • Das S.K.
      • et al.
      Molecular cues to implantation.
      Previous studies have shown that E2 is necessary for the induction of decidualization
      • Shelesnyak M.C.
      • Tic L.
      Studies on the mechanism of decidualization.
      and is inhibited by CC treatment in rats.
      • Barkai U.
      • Kidron T.
      • Kraicer P.F.
      Inhibition of decidual induction in rats by clomiphene and tamoxifen.
      Several clinical studies have demonstrated that luteal E2 action in the endometrium is blocked in women who have been treated with CC.
      • Fritz M.A.
      • Holmes R.T.
      • Keenan E.J.
      Effect of clomiphene citrate treatment on endometrial estrogen and progesterone receptor induction in women.
      • Fritz M.A.
      • Westfahl P.K.
      • Graham R.L.
      The effect of luteal phase estrogen antagonism on endometrial development and luteal function in women.
      Although increased stromal cell proliferation, but not the apoptosis, is considered an initiator of decidualization and inappropriate uterine cell apoptosis results in the failure of implantation,
      • Daftary G.S.
      • Taylor H.S.
      Endocrine regulation of HOX genes.
      our results suggest that aberrant apoptotic activities of uterine stromal cells after chronic CC treatment may be a mechanism whereby the implantation process is disrupted. In agreement with a previous study, Grunert et al
      • Grunert G.
      • Neumann G.
      • Porcia M.
      • Tchernitchin A.N.
      The estrogenic responses to clomiphene in the different cell types of the rat uterus: morphometrical evaluation.
      reported that chronic treatment with CC blocked the action of E2 only in stromal cells and not in epithelial cells. Although in vitro tissue recombinant studies have shown that stromal cells are able to regulate epithelial cell proliferation in response to E2 stimulation,
      • Groothuis P.G.
      • Dassen H.H.
      • Romano A.
      • Punyadeera C.
      Estrogen and the endometrium: lessons learned from gene expression profiling in rodents and human.
      our finding of massive damage to the epithelial cell layer in rats that are treated with CC when E2 is absent (Figure 5, D) indicates that stromal cells may fail to support and maintain their epithelial structure in response to CC when antagonizing E2 in vivo. Indeed, the addition of E2 after CC treatment in women has been shown to improve cervical mucus that results in increased endometrial thickness and conception rates.
      • Dickey R.P.
      • Holtkamp D.E.
      Development, pharmacology and clinical experience with clomiphene citrate.
      Collectively, our results suggest that negative effects of CC on the transformation of stromal cells (direct) and the disintegration of stromal-epithelial interactions that lead to epithelial cell destruction (indirect) may inhibit implantation in the uterus. Although chronic CC treatment increases endogenous E2 levels in rats,
      • Shao R.
      • Nutu M.
      • Weijdegard B.E.
      • et al.
      Clomiphene citrate causes aberrant tubal apoptosis and estrogen receptor activation in rat fallopian tube: implications for tubal ectopic pregnancy.
      it has been suggested that endometrial changes in women who are treated with CC are due to an antiestrogenic effect of CC itself rather than changes in CC-induced hormonal levels.
      • Massai M.R.
      • de Ziegler D.
      • Lesobre V.
      • Bergeron C.
      • Frydman R.
      • Bouchard P.
      Clomiphene citrate affects cervical mucus and endometrial morphology independently of the changes in plasma hormonal levels induced by multiple follicular recruitment.
      The molecular mechanism of E2 supplementation in the uterus of CC-treated rats suggests that estrogen status of patients who are appropriate candidates for CC therapy should be taken into consideration to increase efficacy. A better understanding of the CC-induced apoptotic mechanism in the uterus could reveal new therapeutic strategies for the improvement of implantation rates and reduction of the risk of miscarriage in humans who are treated with CC. For example, additional treatment with the antioxidant N-acetylated cysteine (an antiapoptotic agent) significantly improves endometrial function and leads to increases in ovulation and pregnancy rates, compared with CC treatment alone, in women with polycystic ovary syndrome.
      • Badawy A.
      • State O.
      • Abdelgawad S.
      N-Acetyl cysteine and clomiphene citrate for induction of ovulation in polycystic ovary syndrome: a cross-over trial.
      One of the most important findings of this study is that, although CC binds to both ER-α and ER-β with approximately the same affinity in vitro,
      • Paige L.A.
      • Christensen D.J.
      • Gron H.
      • et al.
      Estrogen receptor (ER) modulators each induce distinct conformational changes in ER alpha and ER beta.
      the cellular response to CC is dependent on ER-α expression and phosphorylation in the rat uterus in vivo, which indicates that ER-α and ER-β are not functionally equivalent in the response to CC treatment. Both ER subtypes are expressed in rodent uteri and human endometrium,
      • Moutsatsou P.
      • Sekeris C.E.
      Steroid receptors in the uterus: implications in endometriosis.
      and either subtype can display diverse transactivational properties in a ligand-dependent manner when ER-α and ER-β are coexpressed.
      • Hall J.M.
      • McDonnell D.P.
      The estrogen receptor beta-isoform (ERbeta) of the human estrogen receptor modulates ERalpha transcriptional activity and is a key regulator of the cellular response to estrogens and antiestrogens.
      • Pettersson K.
      • Delaunay F.
      • Gustafsson J.A.
      Estrogen receptor beta acts as a dominant regulator of estrogen signaling.
      Furthermore, ER-β has the capacity to regulate ER-α function in vitro
      • Hall J.M.
      • McDonnell D.P.
      The estrogen receptor beta-isoform (ERbeta) of the human estrogen receptor modulates ERalpha transcriptional activity and is a key regulator of the cellular response to estrogens and antiestrogens.
      • Pettersson K.
      • Delaunay F.
      • Gustafsson J.A.
      Estrogen receptor beta acts as a dominant regulator of estrogen signaling.
      through the formation of functional heterodimers (instead of homodimers) for ER-mediated transcription.
      • Pettersson K.
      • Grandien K.
      • Kuiper G.G.
      • Gustafsson J.A.
      Mouse estrogen receptor beta forms estrogen response element-binding heterodimers with estrogen receptor alpha.
      Our studies cannot eliminate the possibility that the agonist/antagonist activities of CC may also be influenced by the relative expression of the 2 receptors in the uterus in vivo.
      Successful implantation involves a complex sequence of signaling events.
      • Diedrich K.
      • Fauser B.C.
      • Devroey P.
      • Griesinger G.
      The role of the endometrium and embryo in human implantation.
      • Dey S.K.
      • Lim H.
      • Das S.K.
      • et al.
      Molecular cues to implantation.
      Several genetically modified mouse models with gene disruptions in ER, PR, Hoxa10, Hoxa11, p27, and p53 show implantation defects,
      • DeMayo F.J.
      • Zhao B.
      • Takamoto N.
      • Tsai S.Y.
      Mechanisms of action of estrogen and progesterone.
      • Dey S.K.
      • Lim H.
      • Das S.K.
      • et al.
      Molecular cues to implantation.
      • Franco H.L.
      • Jeong J.W.
      • Tsai S.Y.
      • Lydon J.P.
      • DeMayo F.J.
      In vivo analysis of progesterone receptor action in the uterus during embryo implantation.
      which suggests that the expression and activation of these genes are essential to the implantation process in vivo. In the mouse uterus, treatment with E2 increases ER-α expression in stromal cells but not in epithelial cells.
      • Moutsatsou P.
      • Sekeris C.E.
      Steroid receptors in the uterus: implications in endometriosis.
      It has been shown that the disruption of ER-α, but not ER-β, leads to failure of implantation in female mice,
      • DeMayo F.J.
      • Zhao B.
      • Takamoto N.
      • Tsai S.Y.
      Mechanisms of action of estrogen and progesterone.
      • Moutsatsou P.
      • Sekeris C.E.
      Steroid receptors in the uterus: implications in endometriosis.
      which provides evidence for a vital and distinctive role of the 2 ER subtypes in the uterus. Moreover, studies that have used ER-α knockout mice in vivo and uterine tissue recombinants in vitro have revealed that the estrogenic regulation of PR expression requires stromal ER-α but is independent of epithelial ER-α.
      • Kurita T.
      • Lee K.J.
      • Cooke P.S.
      • Taylor J.A.
      • Lubahn D.B.
      • Cunha G.R.
      Paracrine regulation of epithelial progesterone receptor by estradiol in the mouse female reproductive tract.
      In addition, the maintenance of stromal PR expression is required for the establishment of the “implantation window.”
      • Dey S.K.
      • Lim H.
      • Das S.K.
      • et al.
      Molecular cues to implantation.
      • Franco H.L.
      • Jeong J.W.
      • Tsai S.Y.
      • Lydon J.P.
      • DeMayo F.J.
      In vivo analysis of progesterone receptor action in the uterus during embryo implantation.
      Previous studies, however, could not distinguish fully which stromal PR isoform was responsive to E2 stimulation. Although PRA is the functional PR isoform that is responsible for the regulation of uterine stromal differentiation during implantation,
      • DeMayo F.J.
      • Zhao B.
      • Takamoto N.
      • Tsai S.Y.
      Mechanisms of action of estrogen and progesterone.
      • Moutsatsou P.
      • Sekeris C.E.
      Steroid receptors in the uterus: implications in endometriosis.
      it is interesting to note that CC is able not only to increase PRA expression when E2 is absent but is also able to block E2-induced PRA expression in rat uterine stromal cells, which suggests that CC converts from an estrogenic agonist to an estrogenic antagonist in the presence of E2. Previous studies from our laboratory and others have shown that p27 is a target gene of PR signaling in the uterus.
      • Shao R.
      • Weijdegard B.
      • Ljungstrom K.
      • et al.
      Nuclear progesterone receptor A and B isoforms in mouse fallopian tube and uterus: implications for expression, regulation, and cellular function.
      • Moutsatsou P.
      • Sekeris C.E.
      Estrogen and progesterone receptors in the endometrium.
      Because CC does not bind directly to the uterine PR
      • Barkai U.
      • Kidron T.
      • Kraicer P.F.
      Inhibition of decidual induction in rats by clomiphene and tamoxifen.
      and chronic treatment with CC fails to change endogenous progesterone levels,
      • Shao R.
      • Nutu M.
      • Weijdegard B.E.
      • et al.
      Clomiphene citrate causes aberrant tubal apoptosis and estrogen receptor activation in rat fallopian tube: implications for tubal ectopic pregnancy.
      we propose that the antiestrogenic effects of CC on the regulation of p27 expression seem to be mediated by ER-α and do not require the presence of progesterone directly. There is no change in the expression of p53 after CC treatment in the uterus, which suggests that divergent regulatory pathways between p27 and p53 are triggered by CC treatment. Although both Hoxa10 and Hoxa11 are downstream targets of the action of estrogens in the mouse uterus,
      • Daftary G.S.
      • Taylor H.S.
      Endocrine regulation of HOX genes.
      we observed only the down-regulation of Hoxa11 in the CC-treated rat uterus. Because Hoxa11 is expressed only in the stromal cells, our results support the idea that the CC-induced expression of stromal signals may control the epithelial fate. Together, these data highlight the major role of the ER-α signaling pathway in the antiestrogenic effect of CC on the uterine stromal cells. Whether these molecules are independent of 1 another in CC-induced implantation defects will require further investigation.
      In terms of the cell-type selection mechanism in the rat uterus in vivo, we have reported direct molecular mechanisms by which the antiestrogenic effects of CC induce stromal cell-specific apoptosis and regulate the expression of molecules that are important for the implantation process through the ER-α signaling pathway in the rat uterus. At least in part, the present study has resulted in a cellular and molecular mechanism-based explanation for the discrepancy between high ovulation rates and low successful pregnancy rates in women who are treated with CC.
      • Homburg R.
      Clomiphene citrate: end of an era? A mini-review.
      • Benda J.A.
      Clomiphene's effect on endometrium in infertility.
      • Samani F.G.
      • Farzadi L.
      • Nezami N.
      • Tarzamni M.K.
      • Soleimani F.
      Endometrial and follicular development following letrozole intervention in unexplained infertile patients failed to get pregnant with clomiphene citrate.
      Moving forward, it is noteworthy that there is also a need to evaluate such changes in women who undergo CC treatment. The use of endometrial biopsies in CC-treated women who do not become pregnant might help to explore our hypotheses that have been generated from this study.

      Acknowledgments

      We thank Prof Håkan Billig for his support with many aspects of this project and Profs Christina Bergh and Mats Brännström (Department of Obstetrics and Gynecology, Sahlgrenska University Hospital, Gothenburg, Sweden) for sharing their knowledge of clomiphene citrate.

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