Human Reproduction, Vol. 16, No. 10, 2103-2108,
October 2001
© 2001 European Society of Human Reproduction and Embryology
Effects of the levonorgestrel-releasing intrauterine system on proliferation and apoptosis in the endometrium
1 Department of Obstetrics and Gynecology, Kobe University Graduate School of Medicine, Kobe, Japan, 2 Department of Obstetrics and Gynecology, University of Helsinki, Finland, 3 Institute of Hormone Research, Jerusalem, Israel and 4 Population Council, New York, USA
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Abstract |
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BACKGROUND: The levonorgestrel-releasing intrauterine system (LNg-IUS) has been shown to be effective in the management of menorrhagia. In order to evaluate the effects of LNg-IUS on endometrial proliferation and apoptosis, proliferating cell nuclear antigen (PCNA) expression, apoptosis, Fas and Bcl-2 protein expression in the endometrium were determined at the early proliferative phase of the menstrual cycle before and 3 months after LNg-IUS insertion. METHODS: PCNA, Fas and Bcl-2 protein expression were analysed using an avidin–biotin immunoperoxidase method. Apoptosis was assessed by the terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick-end labelling (TUNEL) method. RESULTS: PCNA, immunolocalized both in the nuclei of endometrial glands and stroma was less abundant 3 months after insertion (P < 0.05). Bcl-2 protein, immunolocalized in the cytoplasm of endometrial glands but not in the stroma, became scanty 3 months after insertion. Fas antigen, immunolocalized only in endometrial glands before insertion, became prominent in both endometrial glands and stroma 3 months after insertion. The apoptosis-positive rate of the nuclei in both endometrial glands and stroma was significantly higher 3 months after insertion relative to that before insertion (P < 0.05). CONCLUSIONS: LNg-IUS resulted in a decrease in endometrial proliferation and an increase in apoptosis in endometrial glands and stroma. The increase in apoptosis associated with increased Fas antigen expression and decreased Bcl-2 protein expression in the endometrium may be one of the underlying molecular mechanisms by which LNg-IUS insertion causes the atrophic change of the endometrium.
Key words: apoptosis/endometrium/levonorgestrel-releasing intrauterine system/proliferation
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Introduction |
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The use of a levonorgestrel-releasing intrauterine system (LNg-IUS) has gained popularity not only as a contraceptive device (Sturridge and Guillebaud, 1997
) but also as a mode in the management of menorrhagia (Kerstin Andersson and Rybo, 1990; Barrington and Bowen-Simpkins, 1997; Ikomi and Gupta, 1998; Fedele et al., 1999). The efficacy of LNg-IUS has been studied as an alternative to hysteroscopic endometrial resection (Crosignani et al., 1997) in the treatment of dysfunctional uterine bleeding. LNg-IUS has been found to be beneficial among women with menorrhagia associated with adenomyosis (Fedele et al., 1997). The molecular mechanism by which LNg-IUS affects the endometrium to control uterine bleeding is, however, still unknown.
Uterine endometrium in mammals is regulated by apoptosis (Suganuma et al., 1997). Fas antigen was indicated as a mediator of apoptotic signal in haematopoietic cells (Itoh et al., 1991; Itoh and Nagata, 1993; Suda et al., 1993), and is known to be a receptor of Fas ligand, a death factor. The Fas/Fas ligand system is involved in early events mediating apoptosis, not only in a variety of tumour cells (Nagata and Golstein, 1995) but also in the endometrium (Harada et al., 1996; Watanabe et al., 1997; Yamashita et al., 1999). In contrast, Bcl-2 and its family have been implicated in controlling the survival and death of cells (Nunez et al., 1990; Korsmeyer, 1992). The Bcl-2 gene product, when elevated in cells either in vivo or in vitro, prevents the apoptotic cell death in normal cell populations that is induced by trophic factor deprivation or other stimuli, without altering proliferation (Reed, 1994; Matsuo et al., 1997). The role of Bcl-2 protein in promoting survival and blocking the apoptotic pathway in the endometrium has been described (Otsuki et al., 1994; Tao et al., 1997; Jones et al., 1998; Matsumoto et al., 1999).
Although it has been reported recently (Rogers et al., 2000) that there were no differences in the levels of expression of Bcl-2, Fas and caspase 3 in the endometrium of levonorgestrel implant (Norplant) users with and without breakthrough bleeding, few data are available regarding the molecular mechanisms by which LNg-IUS affects the endometrium to control menorrhagia. Thus, the present study was conducted to determine the changes in the proliferative activity, apoptosis, Fas antigen and Bcl-2 protein expression in the endometrium in relation to LNg-IUS usage. This is the first study to demonstrate the effects of LNg-IUS on the proliferation and apoptosis in the endometrium.
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Materials and methods |
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Endometrial curettage specimens
Fifteen women of mean age 32.5 years (range 26–39) and with recurrent menorrhagia associated with adenomyosis, participated in this study. A clinical diagnosis of adenomyosis was determined using magnetic resonance imaging. The LNg-IUS used (Oy Leiras, Turku, Finland) has a T-shaped polyethylene skeleton, the LNg (50% by weight) being located in the cylindrical sleeve and dispersed homogeneously from a polydimethylsiloxane reservoir which is covered by a rate-limiting membrane on the vertical arm of the device. The total amount of LNg in the device was 46 mg, and the rate of release was 20 µg/day. The IUS was inserted in each subject within 7 days of the start of menstrual flow. After having obtained the informed consent of each subject, endometrial curettage specimens were obtained before and 3 months after LNg-IUS insertion. Both endometrial specimens were taken from day 4 to day 7 at late menstrual phase, concomitant with the early proliferative phase of the endometrium. The endometrial specimens obtained were fixed in 4% buffered neutral formalin, dehydrated and embedded in paraffin. Sections (4 µm in diameter) were deparaffinized and taken for immunohistochemical analysis.
Immunohistochemical analysis
Immunohistochemical staining was carried out using the avidin–biotin immunoperoxidase method, with the polyvalent immunoperoxidase kit (Omnitag, Lipshaw, MI, USA) as described previously (Maruo and Mochizuki, 1987). Mouse monoclonal antibodies against human proliferating cell nuclear antigen (PCNA) (Miles, Elkhart, IN, USA), human Fas antigen (UB2, MBL, Nagoya, Japan) and Bcl-2 protein (Miles) were used as primary antibodies. In order to improve the immunostaining efficacy for Bcl-2 protein, the antigen retrieval method was used (Quenby et al., 1998). The specimens for immunostaining of Bcl-2 protein were heated at 95–99°C in 10 mmol/l citrate buffer, pH 6.0. The anti-PCNA and the anti-Bcl-2 protein antibodies were diluted 1:100 before use, whereas the anti-Fas antigen antibody was diluted 1:200. The first incubation was carried out with the primary antibody, followed by the second incubation with biotinylated polyvalent antibody. The third incubation was carried out with avidin–horseradish peroxidase. Thereafter, the chromogenic reaction was developed by incubating with a freshly prepared solution of 3-amino-9-ethylcarbazole and hydrogen peroxide. The sections were counterstained with Harris' haematoxylin, mounted in glycerine phosphate buffer solution, and examined microscopically.
The following control procedures were undertaken to assure the specificity of the immunological reactions: adjacent control sections were subjected to the same immunoperoxidase method, except that the primary antibodies to PCNA, Fas antigen and Bcl-2 protein were replaced by non-immune murine IgG (Miles) at the same dilution as the specific primary antibody. The replacement of the specific primary antibody with non-immune murine IgG resulted in a lack of positive immunostaining.
The intensity of immunostaining for Fas and Bcl-2 were evaluated by more than two observers, and was graded as: (–) for no immunostaining; (+) for weak but definitely detectable immunostaining; (++) for moderate immunostaining; and (+++) for intense immunostaining. The mean percentage of PCNA-positive nuclei of endometrial tissue samples was determined by counting the number of nuclei of both the endometrial glands and endometrial stroma (>1000 for each group).
Terminal deoxynucleotidyl transferase (TdT) deoxy-UTP nick end labelling (TUNEL)
In order to detect apoptotic cells by direct immunoperoxidase detection of digoxigenin-labelled genomic DNA, an Apoptag kit (Oncor, Gaithersburg, MD, USA) was used in accordance with the manufacturer's instructions. Formalin-fixed, paraffin-embedded tissue sections were placed on coated slides for a molecular biological–histochemical system. Sections were deparaffinized and proteins in the specimens were digested with proteinase K. The endogenous peroxidase activity was quenched with 2% hydrogen peroxide in phosphate-buffered saline. The slides were then placed in equilibration buffer and in TdT enzyme, followed by a stop-wash buffer. Two drops of anti-digoxigenin peroxidase were then applied to the slides, and peroxidase was detected using a 3,3-diaminobenzidine substrate working solution for 5 min at room temperature. In the negative control, distilled water replaced the TdT enzyme. A section of rat mammary gland obtained on the fourth day after weaning was used as a positive control. The mean percentage of apoptosis-positive nuclei for each group of endometrial tissue samples was determined by counting the number of nuclei of both the endometrial glands and stroma (>1000 for each group).
Statistical analysis
The PCNA-positive rate and the apoptosis-positive rate were expressed as mean ± SD. A paired Student's t-test was used to determine the statistical significance of the difference between the sample means before and after LNg-IUS insertion. A P-value < 0.05 was considered to be statistically significant.
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Results |
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PCNA was immunolocalized both in the endometrial glands and endometrial stroma. PCNA expression in the endometrium was less abundant at 3 months after LNg-IUS insertion than before insertion (Figure 1A and B
). Determination of the mean percentages of PCNA-positive nuclei in the endometrial glands and endometrial stroma showed that the PCNA-positive rate, both in the endometrial glands and endometrial stroma, was significantly lower after 3 months of IUS use (P < 0.05) compared with before insertion (Table I).
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Apoptotic nuclei assessed by TUNEL were noted on the endometrial surface, and in the endometrial glands and stroma. The apoptotic nuclei in the endometrium seemed more abundant 3 months after IUS insertion (Figure 2B
) than before insertion (Figure 2A). The quantitative analysis of apoptosis on the basis of TUNEL analysis showed the apoptosis-positive rate in the nuclei of the endometrial glands and endometrial stroma obtained 3 months after IUS insertion to be significantly (P < 0.05) higher than before insertion (Table I
).
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Before LNg-IUS insertion, immunohistochemical staining of Fas antigen in the endometrium was scarcely apparent, being only slightly visible in the endometrial glands (Figure 3A
). The immunostaining intensity of Fas antigen became predominant in both the endometrial glands and endometrial stroma 3 months after IUS insertion (Figure 3B).
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Bcl-2 protein was immunolocalized in the cytoplasm of the endometrial glands, but not in the endometrial stroma. Immunostaining of Bcl-2 protein in the endometrial glands was moderate before LNg-IUS insertion (Figure 4A
), but became scanty 3 months after insertion (Figure 4B).
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The PCNA-positive rate, apoptosis-positive rate, Fas antigen and Bcl-2 protein expression levels in the endometrium before and 3 months after LNg-IUS insertion are summarized in Table I
. The PCNA-positive rate was significantly lower after LNg-IUS insertion, but the reverse was true for the apoptosis-positive rate. At 3 months after LNg-IUS insertion, Fas antigen expression was remarkably increased, whilst Bcl-2 protein expression was decreased when compared with pre-insertion values. |
Discussion |
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The LNg-IUS, which was originally developed as an `ideal' contraceptive, has been shown in recent years also to be effective in achieving a significant reduction of menstrual blood loss (Kerstin Andersson and Rybo, 1990
; Sivin and Stern, 1994; Barrington and Bowen-Simpkins, 1997; Crosignani et al., 1997; Fedele et al., 1997, 1999; Sturridge and Guillebaud, 1997; Ikomi and Gupta, 1998). This non-contraceptive benefit of LNg-IUS use implies that the device may be a safe, non-surgical alternative in the management of menorrhagia.
LNg released locally from the IUS has been found to inhibit the expression of both oestrogen and progesterone receptors in the human endometrium (Zhu et al., 1999). Such decrease in receptors in the endometrium was suggested as a contributory mechanism for amenorrhoea associated with LNg-IUS use. By contrast, others (Pakarinen et al., 1995) reported that local release of LNg abolished cyclic changes in the endometrium in relation to the menstrual cycle and also reduced the endometrial thickness, as evidenced by ultrasonography. LNg also caused atrophy of the endometrial glands and decidualization of the stroma (Silverberg et al., 1986; Jones and Critchley, 2000); thinning of the mucosa was evident, and the stroma became swollen during LNg-IUS use (Luukkainen et al., 1990).
The role of apoptosis in the endometrium in the regulation of menstrual cycle has also been studied (Kokawa et al., 1996). Apoptotic cells were reported to be rare in the endometrium, regardless of it being normal, ectopic, or associated with adenomyosis (Suganuma et al., 1997; Jones et al., 1998). Apoptosis was detected in the early proliferative phase, mainly in the glandular epithelium with few positive cells in the stroma, and decreased during the secretory phase (Vaskivuo et al., 2000). In the current study, samples were obtained from day 4 to day 7 at late menstrual phase, concomitant with the early proliferative phase of the menstrual cycle, before and 3 months after LNg-IUS insertion. The current findings revealed that, although minimal apoptosis was detected in the endometrial samples taken in the early proliferative phase before LNg-IUS insertion, there was a significant increase in apoptosis-positive nuclei in the endometrium 3 months after LNg-IUS insertion. Apoptosis-positive nuclei were apparent not only in the endometrial glandular and surface epithelia but also in the stroma.
Several studies on Fas expression in the endometrium have demonstrated its expression in the glandular cells, but not in the stromal cells (Harada et al., 1996; Watanabe et al., 1997; Yamashita et al., 1999). In the current study, such congruent findings were also obtained in the endometrial curettage samples taken before LNg-IUS insertion. In accordance with the increased apoptosis in the endometrium, Fas expression in the endometrium became prominent 3 months after LNg-IUS insertion, not only in the glandular cells but also in the stromal cells. This finding was of significant interest in understanding the molecular basis of the increased apoptosis that occurs in the endometrium during LNg-IUS use, relative to that before IUS insertion.
Bcl-2 protein expression in the endometrium has been reported to have cyclic changes in relation to the menstrual cycle, with peak levels in the proliferative phase (Otsuki et al., 1994; Harada et al., 1996; Suganuma et al., 1997; Tao et al., 1997; Watanabe et al., 1997; Jones et al., 1998; Yamashita et al., 1999). One group (Jones et al., 1998) further noted that Bcl-2 protein expression in ectopic endometrium in adenomyosis remained at low levels, and did not vary with menstrual cycle phase. In the current study, minimal Bcl-2 protein expression was detected in the endometrial curettage samples obtained during the early proliferative phase before LNg-IUS insertion, but became less detectable 3 months after insertion. The decreased Bcl-2 protein expression in the endometrium after 3 months use of IUS might also be linked to the increased apoptosis that occurs in the endometrium during LNg-IUS use.
Although no differences in the levels of expression of Bcl-2, Fas and caspase 3 in the endometrium were noted between levonorgestrel implant (Norplant) users with and without breakthrough bleeding (Rogers et al., 2000), the lack of the effect of LNg on the endometrium in Norplant users might be due to much lower concentrations of LNg in the endometrial tissues compared with those in LNg-IUS users. Indeed, although one group (Rogers et al., 2000) reported no difference in the functional layer of the endometrium between 3 and 12 months of Norplant use, others (Jones and Critchley, 2000) noted a general and rapid thinning of the functional layer 1 month after LNg-IUS insertion.
The current study demonstrates a decline in proliferative activity, associated with an increase in apoptosis, in both the endometrial glands and stroma after LNg-IUS insertion. The increased apoptosis in the endometrium after 3 months use of LNg-IUS was congruent with a remarkable increase in Fas antigen expression, together with a decline in Bcl-2 protein expression in the endometrium. As Fas antigen is a mediator of apoptosis, and Bcl-2 protein is an apoptosis-inhibiting gene product, the increased apoptosis in the endometrial glands and stroma that occurs after LNg-IUS insertion may represent an underlying molecular mechanism that causes atrophic changes of the endometrium, leading in turn to improved management of menorrhagia.
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Acknowledgements |
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This study was supported in part by a grant from the International Committee of Contraceptive Research of the Population Council, New York, USA. The authors with to thank Dr Pekka Lahteenmaki for support and kind donation of the LNg-IUS.
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5 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E-mail: maruo{at}kobe-u.ac.jp
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Submitted on March 1, 2001; accepted on June 28, 2001.
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