Eeva-Marja Rutanen, M.D., Ph.D.,* Ritva Hurskainen, M.D.,* Patrik Finne, M.D., and Kristiina Nokelainen*

Transcription

1 FERTILITY AND STERILITY VOL. 73, NO. 5, MAY 2000 Copyright 2000 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Induction of endometrial plasminogen activator inhibitor 1: a possible mechanism contributing to the effect of intrauterine levonorgestrel in the treatment of menorrhagia Eeva-Marja Rutanen, M.D., Ph.D.,* Ritva Hurskainen, M.D.,* Patrik Finne, M.D., and Kristiina Nokelainen* Helsinki University Central Hospital, Helsinki, Finland Received September 2, 1999; revised and accepted November 23, This work was supported by grants from Helsinki University Central Hospital, Helsinki, Finland. Reprint requests: Eeva- Marja Rutanen, M.D., Department of Obstetrics and Gynecology, Helsinki University Central Hospital, P.O. Box 140, Huch, Finland (FAX: ; eeva-marja.rutanen@huch.fi). * Department of Obstetrics and Gynecology. Department of Clinical Chemistry /00/$20.00 PII S (00) Objective: To elucidate molecular mechanisms accounting for excessive menstrual blood loss, and to present the therapeutic effect of an intrauterine levonorgestrel system (LNG IUS) in menorrhagia. Design: A multicenter study comparing hysterectomy with the LNG IUS in treating menorrhagia. Setting: A university hospital. Patient(s): Women with (n 27) and without (n 14) menorrhagia, and women with uterine fibroids but undetermined menstrual blood loss (n 35). Intervention(s): An LNG IUS was inserted into the uterine cavity in 11 women with menorrhagia and six women experiencing normal menstrual blood loss. Main Outcome Measure(s): Expression of the messenger ribonucleic acid (mrna) of tissue-type plasminogen activator (t-pa) and that of a specific PA inhibitor type 1 (PAI-1) in endometrial tissue samples, as evaluated with the use of Northern blot analysis. Result(s): t-pa mrna was expressed in the endometrium throughout the menstrual cycle, with no statistically significant difference between a proliferative (n 30) and a secretory endometrium (n 40), or between women experiencing normal menstrual blood loss (n 14) and those with menorrhagia (n 27). The levels of t-pa mrna in menstrual phase samples (n 6) were significantly higher than those in proliferative or secretory endometrium. PAI-1 mrna was detected in the endometrium during menstruation only. Both t-pa mrna and PAI-1 mrna were expressed in all endometrial samples (n 17) obtained 6 months after an LNG-IUS was inserted, regardless of the menstrual cycle phase. The relative levels of both types of mrna were significantly higher in LNG endometrium than in proliferative or secretory endometrium, but levels did not differ from those in menstrual-phase endometrium. Conclusion(s): The mean ( SD) levels of t-pa mrna and PAI-1 mrna in the endometrium of women with and without menorrhagia did not differ, suggesting that the PA system is not the major determinant of menstrual blood loss. However, continuous induction of PAI-1 may contribute to the therapeutic effect of LNG IUS in treating menorrhagia. (Fertil Steril 2000;73: by American Society for Reproductive Medicine.) Key Words: t-pa mrna, PAI-1 mrna, endometrium, menorrhagia, intrauterine levonorgestrel system Many women who complain of menorrhagia have no detectable disease and are classified as cases of dysfunctional uterine bleeding. The molecular mechanisms underlying excessive menstrual blood loss are poorly understood. Recent research into these molecular mechanisms has focused on endometrial proteinases and their inhibitors. The plasminogen activator (PA) system, which converts plasminogen into active plasmin, is one of the mediators controlling extracellular matrix degradation and hemostasis as well as tissue remodeling (1, 2). Tissue-type PA (t-pa) is the primary protease in plasmin generation and, therefore, is the key factor in local fibrinolysis. Tissue-type PA and the fastacting and specific PA inhibitor type 1 (PAI-1) are regulated by hormones and local factors at 1020

2 a transcriptional level and in a tissue-specific manner (3, 4). In vitro, decidualization of endometrial stromal cells is associated with enhanced PAI-1 protein and messenger ribonucleic acid (mrna) expression as well as decreased PA activity (5). Little is known about the expression and regulation of the PA system in the human endometrium in vivo. The PA system s role in controlling endometrial hemostasis and menstruation also is unclear. The levonorgestrel intrauterine system (LNG IUS) (Levonova; Leiras Oy, Turku, Finland) has recently emerged as a highly effective nonsurgical method for reducing menstrual blood loss in women with menorrhagia (6 8). The LNG IUS works by releasing 20 g levonorgestrel into the uterine cavity every 24 hours. Atrophy of endometrial epithelium associated with stromal decidualization has been thought to explain the therapeutic effect of the LNG IUS. To elucidate mechanisms that account for both idiopathic menorrhagia and the therapeutic effect of the LNG IUS, we examined the expression of t-pa mrna and PAI-1 mrna in the endometrium. Examination took place at different phases of the menstrual cycle and during use of the LNG IUS. Patients were women with normal menstrual blood loss and those experiencing menorrhagia. MATERIALS AND METHODS Tissue Samples We obtained endometrial tissue samples in connection with a multicenter study in which 76 women (aged 35 to 49 years) with objective menorrhagia were randomly chosen to be treated by hysterectomy or with the use of an LNG-IUS. Before any treatment began, endometrial samples were obtained from 27 women with ovulatory menorrhagia (nine proliferative, 17 secretory, one menstrual) and from 14 women who had regular menstrual cycles and normal menstrual blood loss (seven proliferative, five secretory, two menstrual). Endometrial tissue samples also were collected from 35 women in the same age range who had uterine fibroids and varying amounts of menstrual blood loss (14 proliferative, 18 secretory, three menstrual). An LNG IUS, which releases 20 g levonorgestrel every 24 hours, was inserted into the uterine cavity in 11 women with menorrhagia and six who were experiencing normal menstrual blood loss. Endometrial samples were obtained from these 17 women 6 months after the LNG IUS was inserted. The tissue samples were collected using a disposable biopsy curette (Pipelle; Laboratoire CCD, Paris, France). The samples were frozen in liquid nitrogen and stored at 70 C until they could be processed for Northern blot analysis. A piece of tissue was prepared for routine histopathology. We determined the menstrual cycle phase on the basis of each woman s menstrual history, and then confirmed our assessments by performing histological dating according to Noyes et al. (9). Menstrual blood loss was measured using an alkaline hematin method as described previously (10). True menorrhagia was defined as menstrual blood loss of 80 ml or more per period. All clinical data were collected from hospital records. The use of human tissues for this study was approved by the local ethics committee, and all patients gave their informed consent for the collection of tissue samples. Northern Blot Analysis and Probes Guanidium isothiocyanate was extracted from the frozen endometrial tissues to isolate RNA (11). RNA (25 g total) was denatured, separated by 1% formaldehyde agarose gel electrophoresis, and transferred onto a nylon transfer membrane (MSI, Westboro, MA). RNA was fixed at 80 C for 2 hours. Blots of t-pa and PAI-1 were prehybridized at 60 C for 3 6 hours in ExpressHyb Hybridization Solution (Clontech Laboratories, Inc., Palo Alto, CA). cdnas (25 ng) for t-pa and PAI-1 were labeled with [ - 32 P]dCTP (6,000 Ci/ mmol, Amersham, Bucks, United Kingdom) with the use of a Redi prime II labeling kit (Amersham). The labeled cd- NAs were added to hybridization solution (to cpm/ml). Hybridization was performed at 60 C for 24 hours. Thereafter, the filters were washed as follows: twice with a solution of 2 SSC, 0.1% SDS for 10 minutes; once with a solution of 1 SSC, 0.1% SDS for 15 minutes at 50 C; and once with a solution of 0.1 SSC, 0.1% SDS at room temperature. A control probe was 3 end-labeled with the use of terminal transferase (12). For autoradiography, the filters were exposed to Hyperfilm MP (Amersham) for 1 6 days at 70 C. The intensities of autoradiographic signals were measured in a densitometric scanner (X-Rite 331 Densitometer, X-Rite, Grandville, MI). To compare the relative levels of the t-pa and PAI-1 mrna types in different samples, we normalized the hybridization signal from each sample to its respective S28 ribonucleic RNA level to correct for uneven loading. The 290-bp t-pa cdna probe (1) and the 357-bp PAI-1 cdna probe (13) were sequenced in an ABI BRISM 310 genetic analyzer with the use of DNA sequencing kits (PE Applied Biosystems, Foster City, CA). The control was the human 28 S antisense oligonucleotide probe 5 -AACGATCAGAGTAGTGGTATTTCAAA-3 (14). Statistical Analysis The Mann-Whitney U test was used to compare the levels of t-pa mrna and PAI-1 mrna and their ratio. Comparisons were made between women with normal menstrual blood loss and those with menorrhagia, and between women in various phases of the menstrual cycle. The possible confounding effect of blood loss status was investigated by logistic regression analysis; the phase of the menstrual cycle (menstrual vs. other) was the binary outcome variable. The FERTILITY & STERILITY 1021

3 FIGURE 1 Representative Northern blot analysis of tissue-type plasminogen activator (t-pa) mrna and plasminogen activator inhibitor 1 (PAI-1) mrna in the human endometrium. Total RNA was extracted from endometrial tissue samples obtained from women with and without objective menorrhagia. Samples were collected before any treatment and 6 months after insertion of a levonorgestrel-releasing intrauterine system (LNG IUS). The phase of the menstrual cycle is indicated above each lane. P, proliferative; S, secretory; M, menstrual; L, LNG IUS endometrium. The P and S lanes before L lanes come from the same women before the LNG IUS was inserted. A human 28S rrna probe was used to normalize the amount of RNA loaded in each lane. TABLE 1 Relative levels of t-pa mrna and PAI-1 mrna in the endometrial samples grouped according to menstrual cycle phase and bleeding pattern. Menstrual phase Bleeding status 80 ml 80 ml Undetermined Proliferative n 9 n 7 n 14 t-pa PAI Secretory n 17 n 5 n 18 t-pa PAI Menstrual n 1 n 2 n 3 t-pa 4, PAI-1 1, Note: All values are arbitrary units and expressed as mean SD. Rutanen. Intrauterine levonorgestrel system. Fertil Steril Rutanen. Intrauterine levonorgestrel system. Fertil Steril level of t-pa mrna was log-normally distributed and was therefore logarithmically transformed in logistic regression analysis. The t-pa mrna level 6 months after LNG IUS insertion was compared with the level before treatment in a paired manner, with the use of the sign test. RESULTS In Northern blot analysis, a 2.8 kilobase t-pa mrna was detected in all endometrial tissue samples obtained at various menstrual cycle phases and at 6 months after an LNG IUS was inserted. A 3.2 kilobase PAI-1 mrna was detected in the same samples during the menstrual phase but not in the proliferative or secretory phase. PAI-1 mrna was constantly expressed in all endometrial samples obtained during use of the LNG IUS. A representative Northern blot is shown in Figure 1. Atrophy of the epithelium and widespread decidualization were evident in all endometrial samples obtained during use of the LNG IUS. Menstrual blood loss decreased significantly in all women, regardless of the amount of menstrual blood loss before the LNG IUS was inserted (unpublished observations). Table 1 shows the relative levels (mean SD) of t-pa mrna and PAI-1 mrna in the endometrial samples. No differences were found between the tissue samples obtained from women with normal menstrual blood loss and those from women with objective menorrhagia. Similar t-pa mrna levels were found in the endometria of women with uterine leiomyomas, in whom menstrual blood loss was not measured. The mean ( SD) level of t-pa mrna in proliferative endometria (n 30) ( ) did not differ from that in secretory endometria (n 40) ( ). However, this level was significantly higher during the menstrual phase (n 6) ( ; P.0003) than during the other phases of the cycle, when tissue samples from women with different bleeding patterns were analyzed together (Mann Whitney U test). When adjustment for blood loss status was made by logistic regression analysis, the t-pa mrna level remained a significant discriminator of the menstrual phase (menstrual vs. other) (P.0088). The mean ( SD) t-pa mrna level in the endometrium 6 months after LNG IUS insertion was (n 17). A significant elevation in t-pa mrna levels was detected in a comparison between endometrial samples obtained during LNG IUS use and those obtained from the same women before treatment (P.0042) (sign test). No difference in PAI-1 mrna levels was apparent when endometrial samples obtained during menstrual bleeding ( ) (n 6) were compared with those obtained during LNG IUS use ( ) (n 17). The ratio of t-pa mrna to PAI-1 mrna in menstruating endometria was During use of the LNG IUS, the ratio was ; P DISCUSSION Menorrhagia is a common gynecologic disorder, but the mechanisms that account for excessive menstrual blood loss remain unclear. Apparently, factors involved in endometrial breakdown, remodeling, and hemostasis are all important contributors in regulating endometrial bleeding. Hemostatic 1022 Rutanen et al. t-pa and PAI-1 mrna in the endometrium Vol. 73, No. 5, May 2000

4 balance is influenced by several anticoagulant and procoagulant factors in a tissue-specific manner (15). In this study, we examined expression of the mrnas of t-pa and PAI-1, the two major components in fibrinolysis, in endometrial tissue samples collected from women experiencing normal menstrual blood loss and from women with objective menorrhagia. Our results show that the mrna of t-pa the primary factor in plasmin generation, and thus the key protease in fibrinolysis is expressed in the human endometrium throughout the menstrual cycle. In contrast, the mrna of the primary fibrinolysis inhibitor, PAI-1, was detected by Northern blotting in menstrual phase endometria only. Our study found no differences in either endometrial t-pa or PAI-1 mrna expression among women with different bleeding patterns. Nevertheless, several factors suggest that the local fibrinolytic pathway is important in maintaining endometrial hemostasis and that disorders in this system may contribute to menorrhagia. These factors are as follows: [1] Estrogen has been shown to increase PA concentrations in endometrial tissue and luminal fluid (16). [2] Progestins stimulate PAI-1 production by cultured endometrial stromal cells during decidualization (5, 17 19). Elevated PAI-1 levels in decidualized and, consequently, in menstrual phase endometrium would tend to inhibit local plasminogen/ plasmin. As a result, fibrinolysis and hemorrhage also would be inhibited (5). [3] Antifibrinolytic agents, such as tranexamic acid, reduce menstrual blood loss in women with menorrhagia (20 22). The LNG IUS was originally designed for contraception. Currently, however, it is used increasingly to protect the endometrium during estrogen replacement therapy and also to treat menorrhagia (6 8). Women using the LNG IUS have reported reductions in menstrual blood loss up to 96% (6 8, 20). In this respect, the blood loss results in our study (unpublished observations) agreed with the data from previous studies. The LNG IUS alters endometrial morphology by maintaining epithelial atrophy and decidualization in the stroma regardless of ovarian cycle phase (23). The LNG IUS also alters endometrial function (e.g., it enhances and maintains production of decidualization-related proteins, such as insulin-like growth factor-binding protein 1) (24, 25). Here we show that both t-pa mrna and PAI-1 mrna are constantly expressed in endometria exposed to the LNG IUS, regardless of the ovarian cycle phase. The steady-state levels of both mrnas were significantly higher in LNG IUS-exposed endometria than in proliferative and secretory phase endometria, but similar to those in menstruating endometrium. Our finding of increased t-pa expression in menstruating endometrium agrees with data published by Schatz et al. (5), which showed a rise in endometrial PA activity just before menstruation. Regarding PA expression, an increase in t-pa mrna levels in LNG-exposed endometrium contradicts data from studies carried out in vitro, which have suggested that progesterone inhibits endometrial PA production (26). One explanation for this discrepancy may be that in vivo the regulatory system is more complex than in experiments carried out in vitro. Increased PAI-1 mrna expression in LNG-exposed endometrium and, paradoxically, in menstrual endometrium supports the hypothesis made by Lockwood et al. (19). These investigators suggested that decidualization, rather than progesterone itself, regulates endometrial hemostasis and that PAI-1 may be a determining factor in regulation of the fibrinolytic cascade in the endometrium. It is possible that even small disturbances in the endometrial t-pa/pai-1 balance, undetectable by Northern blot analysis, may result in disorders of bleeding. Tranexamic acid and prostaglandin synthetase inhibitors have been reported to reduce menstrual bleeding by 44% 54% and by 12% 21%, respectively (20 22, 27). The efficacy of systemic tranexamic acid lies in its antifibrinolytic action in the endometrium. Prostaglandin synthetase inhibitors correct alterations in the uterine prostaglandin balance, another factor thought to account for excess menstrual blood loss. Our results indicate that, at the molecular level, the LNG IUS affects the endometrium in at least partly the same manner as tranexamic acid. Both act as inhibitors of fibrinolysis. Tranexamic acid, as a systemic agent, has potential side effects in the whole body, whereas LNG released from the IUS acts mainly in the uterus. The concentrations of LNG and those of endometrial products induced by the LNG IUS have been reported to be low in the circulation (24). Plasma levels of tpa and PAI-1 during LNG IUS use remain to be studied. In conclusion, we have shown that t-pa mrna is constantly expressed in the endometrium during the proliferative and secretory phases as well as during menstrual bleeding. In contrast, the mrna of the specific inhibitor of t-pa, PAI-1, is expressed in the menstruating endometrium only. These findings suggest that the balance in the local plasminogen system plays a role in endometrial hemostasis and remodeling, and, consequently, in the regulation of endometrial bleeding. However, the plasminogen system might not be the major regulator of menstrual blood loss. Our study found no differences between women with and without menorrhagia. Use of the LNG IUS maintains continuous decidualization and a similar balance in endometrial PAI-1/t-PA mrna as detected during menstrual bleeding, suggesting that the inhibition of fibrinolysis contributes to the therapeutic effect of the LNG IUS in treating menorrhagia. FERTILITY & STERILITY 1023

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