REPRODUCTIVE ENDOCRINOLOGY

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1 REPRODUCTIVE ENDOCRINOLOGY Expression of heparin-binding epidermal growth factor like growth factor and its receptors in the human fallopian tube and endometrium after treatment with mifepristone Xiao Xi Sun, M.D., Ph.D., a Kristina Gemzell-Danielsson, M.D., Ph.D., a Hong Zhen Li, M.D., a Berit Stâbi, B.S., a and Anneli Stavreus-Evers, Ph.D. b a Department of Woman and Child Health and b Department of Clinical Science, Division of Obstetrics and Gynecology, Karolinska Institutet, Stockholm, Sweden Objective: To study the effect of mifepristone on heparin-binding epidermal growth factor (HB-EGF) and its receptors HER1 and HER4 in the fallopian tube and in the endometrium. Design: Prospective clinical study. Setting: Hospital-based unit for obstetrics and gynecology and research laboratories. Patient(s): Healthy women divided into two groups: controls and patients treated with a single dose of 200 mg mifepristone on day LH 2. Intervention(s): Endometrial biopsies from 30 women were obtained during one control cycle or one treatment cycle. Fallopian tubes from 14 women were collected during laparoscopic sterilizations. Main Outcome Measure(s): Immunohistochemistry and reverse transcriptase polymerase chain reaction. Result(s): The staining intensity of HB-EGF was not affected by mifepristone treatment. Treatment with mifepristone increased the immunostaining on HER1 in the epithelium and the stroma of the endometrium, which was not seen in the fallopian tube. The immunostaining of HER4 decreased in the stroma of the fallopian tube, while an increase was seen in the epithelial cells of the endometrium. Conclusion(s): Treatment with mifepristone has a limited effect on HB-EGF and its receptors in the fallopian tube, while the increase in HER1 and HER4 in the endometrium probably reflects defective endometrial maturation. (Fertil Steril 2006;85: by American Society for Reproductive Medicine.) Key Words: Mifepristone, HB-EGF, ErbB4, EGFR Received December 7, 2004; revised and accepted August 2, Supported by the Swedish Medical Research Council ( ), the Swedish Medical Society, the World Health Organization, the Goljes Foundation, the Magn. Bergvalls Foundation, and the Karolinska Institutet. Reprint requests: Anneli Stavreus-Evers, Ph.D., Division of Obstetrics and Gynecology, K57, Karolinska University Hospital-Huddinge, Stockholm, Sweden (FAX: ; Anneli.Stavreus- Evers@ki.se). Initiation of pregnancy includes fertilization, transport of the embryo through the fallopian tube, and implantation into the endometrium. The embryo must be able to communicate with the mother both during the transport through the fallopian tube and before and during implantation into the uterine wall. A number of endometrial factors, which may be important for a successful implantation and pregnancy, have been identified (1), but less is known about events in the fallopian tube. Heparin-binding epidermal growth factor (HB-EGF) is a member of the EGF family; HB-EGF exists in two biologically active forms: one transmembrane form and one soluble form, which is cleaved from the transmembrane form (2). The soluble form of HB-EGF is a potent stimulator of cell proliferation, migration, and cell motility, while the transmembrane form of HB-EGF acts as a juxtacrine growth and adhesion factor (2). HB-EGF acts through the binding and activating of two transmembrane receptors, HER1/ErbB-1/EGFR and HER4/ ErbB-4 (3, 4). Both receptors exist in the human embryo, fallopian tube, and endometrium (5 8). Ovarian steroid hormones are important regulators during all stages of female reproduction. Administration of the antiprogestin mifepristone in the early luteal phase is an effective contraceptive (9). The possible mechanisms include inhibition of embryo implantation due to the influence on endometrial development and function (9) and detriment to the peri-implantational milieu and preimplantational embryo development (10) /06/$32.00 Fertility and Sterility Vol. 85, No. 1, January 2006 doi: /j.fertnstert Copyright 2006 American Society for Reproductive Medicine, Published by Elsevier Inc. 171

2 TABLE 1 Primers used for PCR. Gene Sequence Product size (bp) HB-EGF Sense 5=-GGCTGAGTGAGCAAGACAAGAC-3= 439 Antisense 5=-TTGTGGCTTGGAGGATAAAGTG-3= HER1 Sence 5=-CAGCGCTACCTTGTCATTCAG-3= 727 Antisense 5=-TCATACTATCCTCCGTGGTCA-3= HER4 Sense 5=-AAG TAC AGT GCT GAC CCC ACC-3= 264 Antisense 5=-GTT TGC AAA GGT GTT GAG GT-3= HPRT Sense 5=-CCT GCT GGA TTA CAT CAA AGC ACT G-3= 380 Antisense 5=-GTC AAG GGC ATA TCC TAC AAC AAA C-3= The influence of steroid hormones on the expression of the receptors HER1 and HER4 in the fallopian tube has, according to our knowledge, not been extensively studied, but it is likely that estrogen is involved in their regulation (11). However, it is possible that P also is involved in the regulation of HB-EGF receptors. The expression of HB-EGF is elevated in the glandular and luminal epithelium during the secretory phase (12, 13), with the highest expression being when pinopodes, ultrastructural markers of uterine receptivity, are present on the endometrial surface (14). This corresponds to the time of the cycle when serum P is high. HB-EGF is present both inside the luminal epithelial cells and on the surface of the pinopodes (14). It is also known that HB-EGF can induce v 3- integrin, which is a suggested biochemical marker of endometrial receptivity, in the endometrial epithelium (15, 16). Therefore, it is likely that HB-EGF is involved in the embryo implantation process. TABLE 2 The immunostaining intensity of HB-EGF, HER1, and HER4 in the fallopian tube. Controls Mifepristone Isthmic Ampullary Isthmic Ampullary HB-EGF: Epithelum 1.2 (0 1), n (1 2), n (0 2), n (0 3), n 6 HER1: Epitheium 1.2 (1 2), n (0 2), n (0 1), n (0 1), n 7 Epithelial/stromal border region 1.5 (0 3), n (0 3), n (0 3), n (0 3), n 7 Stroma 1.0 (0 2), n (1), n (0 1), n (1), n 7 Vessels 0.7 (0 2), n (1 2), n (0 1), n (0 2), n 7 Surface epithelia 0.2 (0 1), n 6 0 (0), n 5 0 (0), n (0 1), n 7 HER4: Epitheium 2.5 (2 3), n (1 3), n (2 3), n (1 3), n 6 Epithelial/stromal border region 1.7 (1 3), n (0 2), n (0 2), n (1 2), n 6 Stroma 2.0 (1 3), n (2 3), n (1), n 5 a 1.5 (1 2), n 7 a Vessels 2.1 (0 3), n (1 3), n (0 3), n 6 3 (3), n 7 Surface epithelia 2.2 (1 3), n (0 3), n (0 3), n (0 3), n 6 Note: Values are given as average (from three observers) and range. The number of samples where the staining of the different cell types could be detected is shown. Statistical evaluation of controls compared with treatment groups according to Mann-Whitney rank sum test. a P.05 shows statistical significance compared with control. 172 Sun et al. Mifepristone, HB-EGF, and its receptors Vol. 85, No. 1, January 2006

3 It is possible that mifepristone, when used for contraceptive purposes, influences the function of the fallopian tube and thereby the embryo transport and development. It is likewise possible that mifepristone has a negative effect on the transition of the endometrium to a receptive stage, which could negatively affect pregnancy outcome. However, it is not known whether HB-EGF or its receptors are altered after mifepristone treatment. Therefore, in the present study, we investigated the influence of mifepristone on HB-EGF and its two receptors HER1 and HER4 in the human fallopian tube and endometrium. MATERIALS AND METHODS Subjects The study included healthy women (aged years) with regular menstrual cycles (25- to 35-day intervals) and proven fertility. Forty-four women were included in the study. Fallopian tube was obtained from 14 women, and endometrial tissue was obtained from 30 women. The endometrial biopsies were not obtained from the same women who had the tubal sterilization. None of the women had used hormonal contraceptives or an intrauterine device for a minimum of 3 months before the study. The women were randomly allocated into one control and one treatment group. In the treatment group, women were given a single oral dose of 200 mg mifepristone immediately after ovulation (day LH 2). Seven of the women donating fallopian tube and 16 of the women donating endometrium received mifepristone. Depending on the time for tissue collection, different groups of women were donating fallopian tube or endometrium. The number of samples used in each study is shown in Tables 2 and 3. The Karolinska Institute Ethics Committee approved the study, and all women gave written informed consent. Collection of Fallopian Tube Tissue Fallopian tubes were obtained from 14 women during tubal ligation surgery by laparoscopic technique. The women were divided into one control and one treatment group. The biopsies were obtained on day LH 3 tolh 5 from both sides of the fallopian tube. This corresponds to the time when a preembryo is transported through the fallopian tube. The biopsy from the right side was taken from the proximal isthmic part of the tube, while the left-side biopsy was taken from the distal, ampullary part of the tube. Collection of Endometrial Tissue Endometrial biopsies were obtained from 30 women once during the luteal phase on days LH 6 tolh 8, which corresponds to the time of embryo implantation. The women were divided into one control and one treatment group. The biopsies were obtained by curettage of the uterus using a Randall curette without prior cervical dilatation or anesthesia. All specimens were snap-frozen and stored in liquid TABLE 3 The immunostaining intensity of HB-EGF, HER1, and HER4 and the ratio between HB-EGF, HER1, and HER4 and the housekeeping gene using RT-PCR in the endometrium. HB-EGF immunostaining HER1 HER4 Control Mifepristone Control Mifepristone Control Mifepristone Location Luminal epithelium 2.3 (1 3), n (0 3), n (0 2), n (0 3), n 15 a 1.7 (1 3), n (1 3), n 16 a Glandular epithelium 1.6 (0 2), n (0 3), n (0 3), n (0 3), n 14 a 1.2 (0 2), n (1 3), n 16 a Stroma 1.0 (0 2), n (0 2), n (1 3), n (2 3), n 15 a 0 (0), n 14 0 (0), n 16 Ratio to HPRT: Total endometrium 0.896, n , n , n , n , n , n 9 Note: The endometrium was obtained from controls and after treatment with mifepristone. Glandular and luminal epithelium was not present in all biopsies. The number of biopsies shows the number of biopsies where the different cell types were detected. The average value (from three observers) and range is shown for the immunohistochemistry. Statistical evaluation of controls compared with treatment groups according to Mann-Whitney rank sum test. a P.05 shows statistical significance compared with the control. Fertility and Sterility 173

4 nitrogen until analyzed. All biopsies were used for immunohistochemistry of HER1 and HER4. Sixteen of the samples were also used for additional analysis of HB-EGF. Daily morning urine samples were collected throughout the cycle and analyzed for estrone- and pregnanediolglucuronide and LH using enzyme immunoassays (17). The hormones were expressed in nanomoles per millimoles creatinine for estrone-and pregnanediol-glucuronide and per mmoles creatinine for LH (18). For creatinine analysis, a commercial kit (Sigma Diagnostics, St. Louis) was used. In addition, all subjects determined the LH peak in urine samples collected twice daily from approximately cycle day 10 to LH 2 by using a rapid self-test (Clearplan, Searle Unipath Ltd, Bedford, UK). Immunohistochemistry The endometrium and the fallopian tube biopsies were mounted in an embedding medium (OTC Compound; Miles, Elkhart, IN) and serially sectioned to 9 m using a Reichert- Jung Cryocut 1800 (Cambridge Instruments GmbH, Nussloch, Germany) and then placed on poly-l-lysine-coated glass slides and immersed in acetone for 20 minutes for fixation. The slides were put into phosphate-buffered saline (PBS), incubated 30 minutes in H 2 O 2 (0.3% in methanol) to block endogenous peroxidase activity, and then washed 2 for 3 minutes with PBS/bovine serum albumin (BSA) (0.05%). The sections were incubated with the primary antibody overnight in a humidified chamber at 4 C. The antibody for HB-EGF is an affinity-purified goat polyclonal antibody (AF-259-NA) (R&D Systems, Abingdon, UK) raised against a peptide mapping at the carboxyterminus of HB-EGF of human origin. Antibodies for HER1 (sc-120) and HER4 (sc-8050) are mouse monoclonal antibody raised against HER1 and HER4 of human origin (Santa Cruz Biotechnology, Santa Cruz). The slides were washed in PBS/BSA three times for 3 minutes each and then incubated with 1.5% horse serum (in PBS/BSA). Thereafter the slides were incubated with the secondary antibody diluted 1:300 (horse anti-goat for HB- EGF, horse anti-mouse for HER1 and HER4) for 30 minutes at room temperature. The slides were then washed with PBS/BSA three times for 3 minutes each before incubation with ABC complex (Vectastain Elite ABC immunoperoxidase detection system; Vector Laboratories, Burlingame, CA, prepared according to the manufacturer s instructions). After washing three times with PBS/BSA, freshly prepared diaminobenzidine-hydrogen peroxide solution (DAB kit from Vector) was added to the slides, which were thereafter rinsed with distilled water. The slides were counterstained with 10% Mayer s hematoxylin (VWR, Stockholm, Sweden), then washed for 10 minutes in cold water and mounted with glycerol-gelatin. Three independent persons who were blinded to the identity of the samples evaluated the immunohistochemical staining. When their evaluation of the slides turned out to be different, the average value was used. The staining was graded on the following scale: 0 no staining of cells, 1 faint staining, 2 moderate staining, and 3 strong staining. To check for primary antibody specificity, the primary antibody was replaced with nonimmune serum of the equivalent concentration from the same species. RNA Preparation and Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) We used RT-PCR to study mrna levels of HB-EGF, HER1, and HER4 in the endometrium. Total RNA was extracted using TRIZOL Reagent (Gibco-BRL, InVitrogen, Stockholm, Sweden) according to the manufacture s protocol. Extracted RNA was dissolved in purified water and stored at 70 C. One milligram of the total RNA was reverse-transcribed using the Superscript II RNase H - Reverse Transcriptase Kit (Invitrogen) and cdna synthesis Kit (Pharmacia Biotech, Uppsala, Sweden). PCR was performed in a Mastercycler gradient (Eppendorf-Netheler-Hinz Gmbh, Hamburg, Germany) using the Master Taq Kit (Eppendorf, Germany). The cdna was coamplified in a reaction mix with defined primers and primers for the housekeeping gene HRPT. The primer sequences are shown in Table 1. All amplifications were carried out for 30 cycles. A 3-minute hot start at 95 C was performed. The amplification cycle for HB-EGF, HER1, and HER4 consisted of denaturation at 94 C for 1 minute, annealing at 58 C for 1 minute, and elongation at 72 C for 1 minute. To verify that the amplified products were from mrna and not genomic DNA contamination, RT was omitted from the reaction. A control PCR without template cdna was performed in each experiment. PCR products were separated by electrophoresis and visualized in a 1.5% agarose gel stained with ethidium bromide solution, which was identified in relation to a 100-bp ladder. The analysis of the ratio between the target cdna and HRPT was done using a ChemiDoc Gel Documentation System with the software Quantity One version from BioRad (Hercules, CA). Statistical Evaluation Nonparametric statistical evaluations were performed for differences in staining intensity of HB-EGF and its receptors. For evaluation of staining intensity before and after treatment, the Mann-Whitney rank sum test was used. P 0.5 was considered statistically significant. RESULTS Hormone Analysis All women had normal regulatory cycles including ovulation. There was no correlation between the hormonal levels 174 Sun et al. Mifepristone, HB-EGF, and its receptors Vol. 85, No. 1, January 2006

5 and the presence of HB-EGF and the receptors in the endometrium or in the fallopian tube. HB-EGF in the Fallopian Tube In the fallopian tube, the staining of HB-EGF was seen mainly in the epithelial cells, on the serosal surface, and in the endothelial cells of the artery (Fig. 1). There was no difference in staining intensity between the ampullary and isthmic regions of the fallopian tube. Treatment with mifepristone did not differ in staining intensity from controls (Table 2). HER1 in the Fallopian Tube The immunostaining of HER1 was most intense in the stromal tissue/cells adjacent to the luminal epithelial cells (Fig. 1). There was a tendency to a more intense staining in the ampullary region compared with the isthmic region (Table 2). HER4 in the Fallopian Tube The immunostaining of HER4 was most intense in the luminal epithelial cells, where the staining was concentrated on the basolateral side of the cells, and on the serosal surface (Fig. 1). Intense staining was also seen in the arteries after mifepristone treatment (Fig. 1 and Table 2). The stromal immunostaining was lower in fallopian tube from mifepristone-treated women compared with controls (Table 2). HB-EGF in the Endometrium Positive cytoplasmic staining could be seen in luminal and glandular epithelial cells and in stromal cells. The staining was more intense in the luminal epithelium compared with the glandular epithelium and the stroma (Fig. 1 and Table 3). The staining intensity did not differ between treated and nontreated women (Fig. 1 and Table 3). Likewise, there was no change in HB-EGF mrna after mifepristone treatment (Fig. 2 and Table 3). HER1 in the Endometrium The staining of HER1 was seen in epithelial and stromal cells (Fig. 1 and Table 3). ). HER1 showed relatively higher staining intensity in the luminal epithelium compared with the glandular epithelium (Fig. 1 and Table 3). Endometrium from women treated with mifepristone showed higher expression of HER1 in all cell types (Fig. 1 and Table 3). The expression of HER1 mrna did not differ between the groups (Fig. 2 and Table 3). HER4 in the Endometrium Positive staining was seen in glandular and luminal epithelium, while there was no staining in the stroma (Fig. 1 and Table 3). HER4 showed relatively higher staining intensity in the luminal epithelium compared with the glandular epithelium (Fig. 1 and Table 3). In biopsies from women treated with mifepristone, there was a significant increase in the expression of HER4 in both luminal and glandular epithelial cells (Fig. 1 and Table 3). The expression of HER1 mrna did not differ between the groups (Fig. 2 and Table 3). DISCUSSION To facilitate a normal pregnancy, the female reproductive tract undergoes a series of functional changes that are influenced directly or indirectly by the cyclic production of E 2 and P. Treatment with mifepristone has profound effects both on the microenvironment, which is important to ensure normal embryo development in the fallopian tube, and on the endometrial receptivity, which is important for implantation (9). A single dose of mifepristone immediately after ovulation increases the expression of P receptors in the fallopian tube and inhibits the normal down-regulation of P receptors in the endometrium (9). HB-EGF is a candidate molecule that can facilitate the communication between the embryo and the maternal side. The presence of HB-EGF and its receptors in the embryo, the fallopian tube, and the endometrium (11, 14, 19 21) suggests that this molecule is important for the initial stages of human pregnancy. HER1 is present on the cumulus cells surrounding the human embryo (8), and it is likely that there is a paracrine communication between the developing embryo and factors that belong to the EGF family, such as HB-EGF and TGF-, which are secreted from the fallopian tube. We did not see any change in the expression of HB-EGF in the fallopian tube after mifepristone treatment, which suggests that the early communication between the embryo and fallopian tube through HB-EGF and HER1 in the cumulus cells is not directly regulated by P. Mifepristone has an effect on other factors synthesized from the fallopian tube, such as TNF- and IL-8, which might affect the early embryo development (22). Our motive in not obtaining endometrium during the tubal sterilization was that the endometrium is not receptive at this time. The reason for obtaining tubal tissue on days LH 3 to LH 5 and endometrial biopsies on days LH 6 tolh 9 was to mirror the communication between the embryo and the maternal tract. The embryo is fertilized and thereafter transported through the fallopian tube. After approximately 5 days, the embryo will reach the uterus and implant into a receptive endometrium. In the present study, there was a decrease in HER1 in the luminal epithelium and a decrease in HER4 in the stroma of the fallopian tube. This decrease in the receptors for HB- EGF and members of the EGF family suggests that mifepristone alters the function of the fallopian tube. It has been shown that TNF- increases the levels of HER1 on endometrial stromal cells (21). Treatment with Fertility and Sterility 175

6 FIGURE 1 Immunostaining of HB-EGF, HER1, and HER4 in the human fallopian tube and endometrium. (A) HB-EGF in the isthmic part of the fallopian tube. (B) HB-EGF in the artery of the fallopian tube. (C) HB-EGF on the serosal surface of the fallopian tube. (D) Negative control for HB-EGF. (E) HB-EGF in the endometrium before mifepristone treatment. (F) HB-EGF in the endometrium after mifepristone treatment. (G) HER1 on the serosal surface of the fallopian tube. (H) HER4 on the serosal surface of the fallopian tube. (J) HER1 in the isthmic part of the fallopian tube. (K) HER4 in the isthmic part of the fallopian tube. (L) HER1 in the artery. (M) HER4 in the artery. (N) HER1 in the ampullary part of the fallopian tube. (O) HER4 in the ampullary part of the fallopian tube. (P) Negative control for HER1. (R) Negative control for HER4. (S) HER1 in the endometrium before mifepristone treatment. (T) HER1 in the endometrium after mifepristone treatment. (U) HER4 in the endometrium before mifepristone treatment. (V) HER4 in the endometrium after mifepristone treatment. 176 Sun et al. Mifepristone, HB-EGF, and its receptors Vol. 85, No. 1, January 2006

7 FIGURE 2 The mrna expression for four representative samples. Hypoxantine phosphoribosyltransferase was used as standard for the semiquantitative analysis of the relative amount of the transcripts for HB-EGF, HER1, and HER4. mifepristone increases the expression of TNF- in the fallopian tube (22), but in contrast to the endometrial stroma, this does not lead to an increase in HER1 levels. It was previously shown that the number of EGF receptor binding sites was higher in the late follicular and early luteal phase than in the late luteal phase and that estrogen treatment increased the expression of EGF receptors in both the human and baboon fallopian tube (11, 23). Our data did not show any changes in the expression of HER1 in the fallopian tube after mifepristone treatment, which supports the data that show that estrogen is a main regulator of this receptor in the fallopian tube. It is evident that HB-EGF plays an important role during the implantation of an embryo. HB-EGF exists both as a transmembrane and a secreted form that is produced by proteolytic cleavage of the transmembrane form (2). Both forms could be involved in the implantation process, and there are several possible roles of HB-EGF during this process. The soluble form can facilitate communication between the embryo and endometrium through the binding to HER4 on the surface of the embryo. Adhesion of the blastocyst to the endometrium could be facilitated either directly by the transmembrane form present on the pinopode surface (14)or by induction of v 3- integrin (16). The highest levels of HB-EGF, HER1, and HER4 are seen during the window of implantation, when the P levels are high (6, 14, 24). In the present study, neither HB-EGF nor HB-EGF mrna in the endometrium was affected by mifepristone treatment. Data from cell culture of human endometrial epithelial and stromal cells show that a combination of estrogen and P stimulate HB-EGF, while estrogen or P alone or in combination stimulated the secretion of HB-EGF from endometrial stromal cells (16). This shows that although a single oral dose of 200 mg mifepristone is enough to alter endometrial development and inhibit pregnancy (9), it does not alter the expression of EB-EGF in the endometrium. In contrast to the effect of mifepristone on HB-EGF, and in contrast to the effect in the fallopian tube, there was an increase in immunostaining of HER1 and HER4 in the endometrium after mifepristone treatment. This contradicts the finding that these receptors show the highest expression during the secretory phase in the endometrium, when serum P levels are high (6, 24). Endometrial maturation is mediated by the paracrine action of HB-EGF on HER1 and HER4 (16, 21). 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