Farhana B. Lockhat, Mb.Ch.B., a Joseph E. Emembolu, F.R.C.O.G., b and Justin C. Konje, M.D. a

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1 Serum and peritoneal fluid levels of levonorgestrel in women with endometriosis who were treated with an intrauterine contraceptive device containing levonorgestrel Farhana B. Lockhat, Mb.Ch.B., a Joseph E. Emembolu, F.R.C.O.G., b and Justin C. Konje, M.D. a Reproductive Sciences Section, a Department of Cancer Studies and Molecular Medicine and b Women s, Perinatal and Sexual Health Services, Leicester Royal Infirmary and University of Leicester, Leicester, United Kingdom Objective: To determine and compare levels of levonorgestrel (Lng) in serum and peritoneal fluid (PF) of patients on the Lng intrauterine system Mirena (Schering Health, Berlin, Germany) for endometriosis and to relate these to symptoms. Design: Prospective clinical trial. Setting: Gynecology unit of a teaching hospital. Patient(s): Women with minimal to moderate endometriosis at diagnostic laparoscopy. Intervention(s): Mirena was inserted at diagnostic laparoscopy and blood and PF collected for Lng levels. Levonorgestrel was again quantified in serum at 1, 3, and 6 months and PF at 6 months. Main Outcome Measure(s): Serum and PF Lng levels during 6 months, differences in levels before and 6 months after Mirena insertion, and the relationship between these levels and symptoms of endometriosis. Result(s): There was significant improvement in symptoms after 6 months on Mirena. The mean (SD) serum Lng levels were (100.2), (51.8), and (53.0) pg/ml at 1, 3, and 6 months, respectively. The PF levels at 6 months were approximately two-thirds the serum levels in patients showing improvement in symptoms. Conclusion(s): Mirena delivers significant amounts of Lng into the PF and serum. The relationship between Lng levels in these compartments is linear. (Fertil Steril 2005;83: by American Society for Reproductive Medicine.) Key Words: Endometriosis, mirena, serum, peritoneal fluid, levonorgestrel Endometriosis is a chronic debilitating condition afflicting between 5% and 10% of women in the reproductive years (1). The treatment, which depends on the stage of the disease and the fertility needs of the patient, can be hormonal, conservative, or radical surgery. Hormonal therapy is associated with a 70% 80% alleviation of symptoms and in most cases a recurrence rate of 20% 40% within 1 2 years of discontinuation of therapy (1). The side effects of hormonal treatment often affect compliance and therefore continuation rates. This is more so with androgens (e.g., danazol) and antiestrogens (e.g., GnRH analogues). Progestogens, on the other hand, are theoretically better tolerated as they have fewer antiestrogenic or androgenic side effects. However, when administered orally or as implants, they are associated with side effects such as weight gain, acne, breast tenderness, menstrual irregularity, and mood changes, which are thought to be secondary to high systemic levels of the progestogens (2). These side Received December 2, 2003; revised and accepted July 20, Supported by Schering Health Care, UK. Reprints requests: Justin C. Konje MD, Reproductive Sciences Section, Department of Cancer Studies and Molecular Medicine, Robert Kilpatrick Clinical Sciences Building, Leicester Royal Infirmary, Leicester LE2 7LX, UK (FAX: 144(116) ; jck4@le.ac.uk). effects and the need for repeated administration or ingestion significantly affect compliance. Any effective treatment, which therefore delivers the progestogen locally and is associated with a lower systemic level, is likely to be more acceptable to the patient as the side effects would be more tolerable. The Mirena intrauterine system (Schering Health, Berlin, Germany) delivers levonorgestrel (Lng) locally at a steady rate of 20 g/24 hours during a 5-year period and appears to alleviate symptoms in a significant proportion of patients (3 5). It offers an added advantage of a single administration for a possible duration of 5 years. The systemic levels are lower than those after oral or depot administration. The precise mechanism by which Lng delivered this way alleviates symptoms of endometriosis is uncertain but it may include action on the endometrium, inhibition of ovulation, and a local effect on the endometriotic implants (possibly depending on the peritoneal fluid [PF] levels). Such local action will depend on Lng delivered either through the bloodstream or directly in the PF. There have been no reported studies on concurrently measured Lng levels in the blood and PF, even in those on Mirena for contraception. We therefore undertook a pro- 398 Fertility and Sterility Vol. 83, No. 2, February /05/$30.00 Copyright 2005 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 spective study to measure the levels of Lng in the blood and PF of patients with endometriosis and to relate the levels to symptoms of the disease with the hope of suggesting possible mechanisms of action of Mirena when used this way. MATERIALS AND METHODS Women with symptoms suspicious of endometriosis were recruited into a 6-month prospective cohort study of the efficacy of Mirena on the symptomatic relief of endometriosis. The inclusion criteria were minimal to moderate endometriosis (based on the Revised American Fertility Society score) (6) as diagnosed at laparoscopy, consent to have a second-look laparoscopy 6 months after the Mirena insertion, and no desire for pregnancy within at least 6 months of the diagnosis of endometriosis. The exclusion criteria included hormonal treatment within 3 months of the diagnosis, severe endometriosis, desire to become pregnant during the ensuing 6 months after the diagnosis, history or laparoscopic findings indicative of pelvic inflammatory disease, and unwillingness to undergo a second-look laparoscopy. Severe endometriosis was excluded as most of these cases are treated by a combination of surgery and hormonal treatment in our center. Each subject gave a signed informed consent before the initial laparoscopy. The local Ethics Committee (Institutional Review Board of the University Hospitals of Leicester NHS Trust) approved the study. At the time of the laparoscopy, a 20-mL blood sample was collected from the antecubital vein and PF was aspirated from the pouch of Douglas (cul-de-sac) with the patient in a 15-degree Trendelenburg position (7). Blood and PF were collected from 8 and 6 patients, respectively, for between 5 and 120 minutes after insertion of the intrauterine device (IUD) to determine how quickly Lng reached the blood and PF after the insertion. In the remaining cases, sampling was done before insertion. The endometriosis was then staged according to the Revised American Fertility Society classification (6) and those meeting the inclusion criteria had the Mirena inserted. A peritoneal biopsy was taken with laparoscopic punch biopsy forceps from one of the endometriosis sites for histological confirmation of the disease and for subsequent studies on estrogen (E) and progesterone (P) receptors (not reported here). A month before the laparoscopy and at 1, 3, and 6 months after insertion of the Mirena, each patient kept a symptom diary for pelvic pain using a 10-cm visual analogue scale (VAS) and a 4-point (0 3; where 0 no pain, 1 mild pain, 2 moderate pain, and 3 severe pain) verbal rating score (VRS). The visual analogue scale was one of increasing pain severity where 0 was for no pain and 10 was for the most severe pain experienced. The patient marked on this scale the point equivalent to her perception/assessment of pain. The verbal rating score, on the other hand, was used to calculate the monthly pain score. Each day was given a score of 0 3 and the sum of the daily scores for 28 consecutive days was used as the monthly score. Menstrual blood loss was assessed using the pictorial blood loss assessment chart devised by Higham et al. (8). Briefly, each subject was given a diary consisting of a series of diagrams representing lightly, moderately, and heavily soiled tampons or towels. A mark was made in an appropriate box at the time that each item of sanitary protection was being discarded. In addition, the passage of blood clots (size equated with that of coinage) and episodes of flooding were recorded. A score was generated from this information to determine the total blood loss. This semiquantitative method of assessing blood loss has been demonstrated to closely reflect loss measured by the hematin dilution technique (8). Satisfaction with treatment was based on the patient s assessment of her symptoms at initial presentation. This was classified as very satisfied (symptoms completely resolved and with either minimal or no side effects of the treatment), satisfied (mild symptoms, not requiring any attention and associated with either mild or no side effects of the treatment), uncertain (symptoms considered to either be better than before treatment but associated with side effects, which are severe enough for the patient to consider them as the main indication for requesting discontinuation or not better enough to regard treatment as effective), and dissatisfied (symptoms either same or worse than before treatment or associated side effects severe enough to warrant a request for removal of the device). The symptoms used to assess response to treatment included dysmenorrhea, quantified menstrual loss, and the VAS and VRS. Dysmenorrhea was assessed independently at each of the follow-up visits as mild if no medication was required, moderate if regular analgesia was required, and severe if in addition to regular analgesia, the patient was incapacitated or took time off from work. At the follow-up visits 1 and 3 months after the insertion of Mirena, blood was again collected for estimation of serum Lng levels. After 6 months on Mirena a second-look laparoscopy was performed during which blood and PF were again collected for Lng levels. The blood and peritoneal samples were stored on ice until processed. Serum was obtained after the blood was centrifuged at 3,000 g at 4 C for 5 minutes, aliquoted, and stored at 80 C until Lng assays were performed. The volume of the PF was measured before it was aliquoted and stored at 80 C until Lng assays were performed. The Lng levels were measured by published RIA procedures with WHO-matched reagents and methods (9). The assays, which were in duplicate, were blinded to patients and Fertility and Sterility 399

3 symptoms. The sensitivity (limit of detection) of the RIA was 25 pg/ml. The interassay and intra-assay variations were, respectively, 10% and 6%. Assays were only undertaken in those with histologically confirmed endometriosis. The SPSS version 11.0 (SPSS, Inc., Chicago, IL) was used for data analyses. The volume of PF and PF Lng levels at time zero (insertion of the Mirena ) and 6 months after insertion were compared using a paired t test or Wilcoxon rank analysis as appropriate. The changes in the levels of Lng during the 6 months of treatment were also compared. Serum Lng levels at 3 and 6 months were related to symptoms at these time points. RESULTS During the 2 years of the study, 42 women with symptoms of endometriosis who underwent the initial laparoscopy were recruited but only 37 (81%) of those with histologically confirmed endometriosis formed the subjects of this study. These symptoms included dysmenorrhea, deep dyspareunia, noncyclical abdominal pain, and menorrhagia. Three were excluded because of severe endometriosis (2) and Fitz- Hugh-Curtis syndrome taken as evidence of previous Chlamydia trachomatis infection (1). The mean (SD) age of the 34 patients was 31 (7.2) years. Five patients requested for the device to be removed before the end of the trial for the following reasons: pelvic pain (3), worsening acne (1), and personal reasons (1). The voluntary discontinuation rate before the end of the 6 months was therefore 14.7%. Another patient was excluded as she was on prolonged potent analgesics after a road traffic accident (although she retained the device). Another 3 women did not undergo the second-look laparoscopy because of relocation to another country (1), personal reasons (1), and failure to attend the laparoscopy (1). This latter group of 4 women have, however, continued to provide follow-up diaries demonstrating satisfaction with the device (now in their fourth year). The effects of this device on clinical symptoms, which we have already published (5) are summarized. There was a significant improvement in pain during the period of treatment. The visual analogue scale fell from cm pretherapy to cm at 3 months and then to cm at 6 months. These changes were all statistically significant (P.01). The proportion of patients experiencing moderate or severe dysmenorrhea fell from 96% (27 patients) before therapy to 68% (19) after 3 months (P.001) on the device and to 50% (14 patients) after 6 months (P.001). The number of pain days experienced per month (i.e., 28 days) fell from days to days after 6 months (P.05). The mean verbal rating score (from a total of 84 per month) dropped from pretherapy to at 6 months (P.08). The mean quantified menstrual loss from the pictorial blood loss assessment chart fell from pretherapy to after 6 months on the device (P.001). The mean score in the 26 patients who had the second-look laparoscopy improved in 16 patients (61.5%), remained the same in 2 (7.7%), and got worse in 8 patients (30.8%). A second-look laparoscopy was performed on 25 patients and 6 of these requested for the device to be removed for the following reasons: migraine, weight gain, irregular bleeding, and decreased libido (1), weight gain and acne (1) and minimal/no improvement in pain and irregular bleeding (4). Satisfactory PF was obtained from all patients at insertion but 5 were excluded because the fluid was heavily blood stained. At the second-look laparoscopy, satisfactory fluid was collected from 16 cases. In the remaining 9 cases, the samples were either too small (5) or were heavily blood stained (4). The mean volume of PF from the paired sampled (16) cases at the first laparoscopy was significantly higher than 6 months after Mirena insertion ( ml vs ml; P.001). Although paired PF was only obtained from 16 patients, the serum levels for the other patients are presented [1] to demonstrate the changes in the serum levels with time and [2] to relate these to the symptoms of the patients after 6 months of treatment. The baseline serum and PF Lng values (at insertion of Mirena ) were undetectable in 21 and 18 women, respectively, in whom the sampling was before the IUD insertion. In those in whom the sampling was after the IUD insertion, the levels were above the detection value of 25 pg/ml. Table 1 shows these levels in relation to the timing of sampling with respect to Mirena insertion. The mean SD serum Lng levels after 1, 3, and 6 months on Mirena are shown in Table 2. The levels after 1 month were significantly (P.05) higher than those after 3 and 6 months. There were no statistically significant differences (P.05) between the levels after 3 and 6 months. Three of the 16 cases in which paired PF was measured had been sterilized. The mean SD level of Lng in the serum of the sterilized women was similar to that in those who were not sterilized after 1, 3, and 6 months. The mean SD PF levels of Lng after 6 months on Mirena were pg/ml. These levels were similar in the 3 sterilized women ( pg/ml) and the 13 who were not sterilized ( pg/ml; P.05). The relationship between PF and serum Lng levels, as shown in Figure 1, was linear (R ). The mean PF Lng level was approximately 58% of the serum level. Table 3 shows the mean serum and PF Lng levels, Revised American Fertility Society score, quantified menstrual loss, and number of bleeding days per month after 6 months on Mirena in four groups of patients stratified according to response to treatment as dissatisfied, uncertain, satisfied, and very satisfied. Those who were dissatisfied with the treat- 400 Lockhat et al. Mirena levonorgestrel and endometriosis Vol. 83, No. 2, February 2005

4 TABLE 1 Serum and peritoneal fluid levonorgestrel concentration in women where the sampling was undertaken after insertion of Mirena. Study number Time (min) from insertion to blood sampling Serum Lng Time (min) from insertion to PF sampling PF Lng Note: Lng levonorgestrel; PF peritoneal fluid. ment had serum Lng levels that were significantly higher than the levels in those who were satisfied, very satisfied, or uncertain of the effect of the device on their symptoms (P.05). The PF levels were lowest in those who were very satisfied with the device but the differences in levels in the four groups were not statistically significant (P.05). As there was no definite relationship between the serum and PF levels of Lng and response to treatment, we examined the ratio of PF to serum levels of Lng. Although it was lowest in those who were dissatisfied, there was once again no obvious pattern identified for the other three groups. The mean PFto-serum Lng ratio was, however, significantly lower in the group reporting dissatisfaction with the treatment compared to those who were satisfied and very satisfied (0.56 vs. 0.77; P.05). The semiquantified menstrual flow in the last month of the trial was significantly lower in patients where the PF-toserum Lng ratio was between 0.63 and 0.86 (i.e., the satisfied group) compared to the ratio of 0.56 in the dissatisfied group. Similarly, the mean Revised American Fertility Society FIGURE 1 The relationship between peritoneal fluid and serum levonorgestrel levels after 6 months of therapy with Mirena. TABLE 2 Mean (SD) serum levonorgestrel levels (pg/ ml) during 6 months on Mirena. Time (months) No. of subjects Mean (SD) (100.2)* (51.8) (53.0) *P.05 when compared to levels at 3 and 6 months. Fertility and Sterility 401

5 TABLE 3 Mean serum and peritoneal fluid (PF) levonorgestrel (Lng) levels, revised AFS score, quantified menstrual loss, and number of bleeding days/month after 6 months on Mirena stratified according to patients response to treatment. Patients classification of response to treatment Mean revised AFS score Quantified menstrual loss No. of bleeding months Serum Lng Peritoneal fluid Lng Mean PF:serum Lng ratio Dissatisfied Uncertain Satisfied Very satisfied score was higher in those who were dissatisfied compared to the satisfied group (21 vs. 9; P.05). DISCUSSION Our findings of an improvement in symptoms of endometriosis after treatment with Mirena are in keeping with those of Vercellini et al. (3) and Fedele et al. (4). The continuation rate of 68% (22 of 34) after 6 months is similar to that of most other forms of treatment. Although not reported here, this has remained steady for the first 3 years of follow-up after the insertion. The levels of Lng demonstrated in the serum are similar to those previously reported in women using this device for contraception (10). Those levels after 3 months were not significantly different from those after 6 months, although the latter were lower. The significantly higher Lng levels during the first 3 months may be responsible for the high anovulation rate (71% 85%) reported during the first months on Mirena (11, 12). None of our subjects had serum levels below 200 pg/ml, a critical value below which ovulation occurs (13). The high and fluctuating, albeit falling, serum Lng levels during the first few months may account for the bleeding irregularities associated with the device. Once the levels are stabilized, the bleeding pattern becomes more predictable. It is perhaps during this initial period that most of the systemic effects of progestogens are severe. In this small cohort, the discontinuation as a result of systemic side effects was mainly due to those experienced during the first 3 months after insertion. These side effects included worsening acne, weight gain, migraine, and loss of libido. Most of the women, however, persisted with the device to the end of the study. The PF levels of Lng in this study were closely related to the levels in the serum. They were as high as values reported in the serum of some patients using the device for contraception (11). Although the number of patients who had been sterilized was small, the level of Lng in their PF was similar to that in patients who were not sterilized. An important question is how does Lng reach the peritoneal cavity, and more importantly how does it affect the symptoms of endometriosis? Three possible mechanisms by which Lng reaches the peritoneal cavity can be postulated. The first is direct diffusion across the uterus and peritoneum. Although this is possible, we do not believe it to be the main route. Nilsson et al. (14) demonstrated in women undergoing a hysterectomy shortly after insertion of Mirena that Lng concentrations (in nanograms per gram of wet tissue) were much lower in the myometrium ( ) and fallopian tube (1.8) compared to the endometrium ( ). It is therefore likely that only a small amount of Lng reaches the peritoneum through this route. Second, it could be through the fallopian tubes depending on their patency and peristalsis. The fact that the levels were not different in those who were sterilized and those who were not will suggest that this is unlikely to be an important route. In addition, the concentration differentials demonstrated in the various tissues by Nilsson et al. (14) will again affirm that this is not a primary route. The third and probably the dominant possible mechanism is hematogenous. The speed with which Lng reached the PF and the high serum levels in the patients where sampling was performed soon after insertion of Mirena suggests a very rapid transfer mechanism. This is supported by the high concentration gradient between the endometrium and the myometrium (808 vs. 2.43) (14), suggesting that the subendometrial vascular and lymphatic network quickly take up Lng for a rapid transfer to PF and other sites. The exact mechanism of action of Mirena in the alleviation of symptoms of endometriosis is equally unclear. In women with menorrhagia, this is secondary to its local action 402 Lockhat et al. Mirena levonorgestrel and endometriosis Vol. 83, No. 2, February 2005

6 on the endometrium with resultant hypomenorrhea or amenorrhea (15, 16) in addition to inhibition of ovulation, which potentiates these effects. Because most of the anovulatory effects occur during the first 3 months after insertion, additional mechanisms must be involved after this period. We believe that this involves a local action on the endometriotic implants a mechanism similar to that of the progestogen on the endometrium in women with menorrhagia. The high level of Lng in the PF has direct access to the E receptors on the implants where it induces decidualization. A third mechanism is probably through the inhibition of various mediators of inflammation. Several studies have demonstrated an increased inflammatory process in the peritoneal cavity of women with endometriosis, for example, an increase in PF volume (7, 17, 18), macrophages (19), prostaglandins and cytokines (18, 19), growth factors (20), and RANTES, the selective chemoattractant for human blood monocytes and memory T lymphocytes (18). Medroxyprogesterone acetate (MPA), a progestogen, has not only been shown to reduce the PF volume but also the concentration of macrophages and some of the inflammatory markers (21). Although we did not measure the inflammatory markers in this study, the reduction in the volume of PF indicates that Lng released by the device may mediate it actions in a way similar to MPA. The final possible mechanism is by a reduction in the vascular supply to the pelvis. Such a reduction will reduce pelvic congestion associated with endometriosis. There is, however, no robust evidence to support this. Although Jarvela et al. (12) concluded that the Lng device increased impedance to blood flow in the uterine arteries during the midluteal phase, they failed to demonstrate a correlation with serum concentration of Lng. Pakarinen at al. (22), on the other hand, failed to demonstrate any alterations in the pulsatility index in treated and control groups and Zalel et al. (23) also failed to demonstrate any changes in the Doppler flow in the cervical branch of the uterine artery in treated and control groups. The fact that there was no obvious relationship between PF and serum Lng levels and symptom improvement suggests multiple mechanisms of action. If, as we believe, local concentrations significantly influence response to treatment, then it would be expected that the higher the PF levels the better the response. Our findings, however, do not support this argument. It is perhaps the relative availability of Lng as determined by the proportion of Lng in the PF compared to serum that influences response to treatment. Because the dissatisfied patients had the highest serum levels but a relatively lower PF level, it could be speculated that the serum levels were responsible for the systemic side effects, which contributed to discontinuation; however, a larger number of patients is needed to draw such conclusions. Whatever the systemic or local mechanisms by which Lng acts to reduce the symptoms of endometriosis, it is likely that these actions are mediated through E and progestogen receptors on the endometriotic implants. Levonorgestrel is known to down-regulate the P and E receptors in the endometrium (24, 25) and to inhibit endometrial production of E 2 -induced growth factors or production of growth factor-binding protein from the endometrium (26). 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