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The anti-inflammatory action of danazol and leuprorelin acetate depot on endometriosis is crh independent

In ammopharmacology, Vol. 9, No. 3, pp. 249–255 (2002) The anti-in ammatory action of danazol and leuprorelin
acetate depot on endometriosis is CRH independent

IOANNIS M. MATALLIOTAKIS1, IRENE ATHANASSAKIS2,ANASTASIA G. GOUMENOU1, SIMON VASSILIADIS1;2;¤,GEORGIOS E. KOUMANTAKIS1, MARIA A. NEONAKI1and EVGENIOS E. KOUMANTAKIS11 Department of Obstetrics and Gynecology, University of Crete, Greece 2 Department of Biology, University of Crete, Greece Received 20 December 2000; revised 13 March 2001; accepted 4 May 2001 Abstract—Corticotropin-releasing hormone (CRH) is a hypothalamic neuropeptide involved in the
neuroendocrine response to stress, also playing a role in cell mediated immune functions. The aim of
this study was to determine the circulating in the serum CRH levels in women with endometriosis and
investigatethe effect of the routinely 6-month administered treatment of danazol or leuprorelin acetate
depot on these hormonal levels. Serum CRH levels were not signi cantly different in women with
endometriosis and in the control group. The 6-month danazol or leuprorelin treatment had no effect
on the levels of CRH. Three months after danazol treatment CRH levels were signi cantly lower
(p < 0:005) than those before treatment. In contrast, after treatment with leuprorelin, CRH levels
were signi cantly higher (p < 0:001). Our results suggest that endometriosis is not associated with
CRH and that danazol as well as leuprorelin acetate depot have no effect on these levels during the
treatment-speci c period. However, they both showed signi cant uctuations after the administration
of these compounds ceased.
Key words: Endometriosis; danazol; leuprorelin acetate depot; CRH.
Endometriosis as de ned by the American Fertility Society (1985) is an enigmaticdisease characterized by an acute local in ammation (Ho et al., 1997). It iswell known that endometriosis is often associated with infertility and miscarriage(Panidis and Matalliotakis, 1998). Yet the precise mechanism leading to infertilityis still obscure. Until recently, the majority of studies have focused on the ¤To whom correspondenceshould be addressed at Department of Biology, University of Crete, P.O.
Box 2208, Heraklion 714-09, Crete, Greece. E-mail: immunological factors of peritoneal uid in reproductive failure (Clifton et al.,1998; Dmowski et al., 1981; Panidis and Matalliotakis, 1998), while little attentionhas focused on the possible role of the intrauterine endometrium in the pathogenesisof endometriosis.
It is generally believed that endometriosis is provoked by the presence of ectopic endometrium. The endometrium is unique among adult tissues because it undergoesintense proliferation, secretion, regression, and regeneration during each menstrualcycle. It is plausible that subtle alterations in this complex series of events couldlead to pathological proliferation of endometrial tissue (Koninckx et al., 1998; Wuet al., 1998).
Corticotropin-releasing hormone (CRH) is a 41-amino acid neurohormone in- volved in the neuroendocrine response to stress, also playing a role in cell-mediatedimmune functions and in in ammation (DiBlasio et al., 1997; Florio et al., 1998).
Its major role is the regulation of the hypothalamus– pituitary – adrenal (HPA) axisthrough induction of the proopiomelanocortin (POMC) gene and the secretion ofcorticotropin (ACTH) from anterior pituitary (Sakata et al., 1994; Sizonenko, 1986).
Moreover, the CRH gene is also expressed in other intracranial sites and in an in-creasing number of peripheral tissues, many of which are unrelated to the HPA axis.
As an example, and in addition to the hypothalamic origin of CRH, this hormonehas been identi ed in the uterus of pregnant and non-pregnant women (Athanassakiset al., 1999; Zoumakis et al., 1997).
The distribution of CRH receptors in the pregnant and non-pregnant uterus indicates that CRH is an active peptide in the reproductive cycle and it has beenpostulated to participate in the processes of embryo implantation, endometrialvascularization and labour by modulating myometrial contractibility (Matalliotakiset al., 1997; Zoumakis et al., 1997) In the light of recent evidence showing that endometriosis is associated with various autoimmune phenomena (Badawy et al., 1984; Ho et al., 1997), the purposeof this work was to determine the serum corticotropin releasing hormone (CRH)levels in women with endometriosis and compare those to already reported levelsin the peritoneal uid (Florio et al., 1998). We also evaluated the effects of danazoland leuprorelin acetate depot on the levels of CRH.
The study population included 68 women with infertility that attended the Depart-ment at Obstetrics and Gynaecology of the University Hospital of Crete from 1991to 1999. All the women underwent laparoscopic examination because of suspectedendometriosis. The University Committee approved speci c and/ or future proto-cols and a written informed consent was obtained from each subject before partici-pation.
Thirty-eight of the women had endometriosis (mean 27:7 § 5:8 years) and thirty had no pelvic disease (mean 29:2 § 5:7 years). After their case histories were compiled, clinical examinations were done; their husbands’ semen was normal in allcases. Thereafter, ovulation was tested, cervical mucus evaluated, and tubal potencyascertained by hystero-salpingography. All examinations showed normal results forall women.
Subjects were classi ed into two groups; 30 with pelvic pain who had no disease on laparoscopy were considered as controls and 38 women with laparoscopicallydiagnosed and staged endometriosis (15 cases) were at stage I, 11 at stage II, 6 atstage III, and 6 at stage IV (American Fertility Society 1985). Due to the diversityof the above-described endometriotic stages, the patients underwent different extentof surgery. No residues of endometriotic tissue could be detected macroscopicallyafter completion of surgery. This observation assures that our results are free ofany possible bias that would jeopardize the interpretation of the results that follow.
The study was double blind and the women with endometriosis were assignedto the two groups (danazol and leuprorelin) by opening one of 22 sealed opaqueenvelopes.
The rst 11 women were given danazol capsules, an isoxazolic derivative of synthetic steroid, 17a-ethynyletestosterone, 200 mg by mouth every 8 hours for 6months. The stage of endometriosis of this speci c group of study was in favour ofstage I since 5 out of the 11 patients were in this category. The group also consistedof 2 women in each of the II, III and IV stages. The second group of 11 women wastreated with leuprorelin acetate depot 3.75 mg every 28 days for 6 months. Thisset contained 3 patients in stage I, 4 in stage II, and 2 in each of the III and IVstages. Finally, the distribution stage of endometriotic women who did not receivetreatment (16 patients) was 7 in stage I, 5 in II, and 2 in each of the III and IV stages.
Blood samples were taken from all women before laparoscopy for the evaluation ofCRH levels.
Serum CRH levels were also measured in the 22 endometriotic women, before treatment, during the last 15 days at a 6-month course of danazol and leuprorelinacetate, and 3 months after treatment. Blood samples from women with endometrio-sis were collected between the fth and seventh days of their menstrual cycles be-fore and after treatment. Blood samples were centrifuged immediately at 3000 rpm(400 £ g) and serum was kept at ¡80±C until assayed.
The rabbit anti-CRH polyclonal antibody (Peninsula Labs, Baltimore, MD) wasused at a concentration of 1 : 1000. The antiserum cross-reacts with human CRHand has a minimal af nity for human ACTH, LHRH and AVP. Rabbit pre-immuneserum was used as negative control in our experiments.
2.3. Enzyme-linked immunoassay (ELISA) The ELISA experiments were performed as previously described (Athanassakis andIconomidou, 1996; Athanassakis et al., 1999). Brie y, sera collected as mentionedabove were used at a concentration of 1 : 100 in carbonate buffer pH 9.6. These werecoated in 96-well at bottom plates (Sarstedt, Numbrecht, Germany), incubatedovernight at 4±C and washed four times in 5% Tween-20 (Sigma, St Louis, MO).
The remaining protein- free sites in the plate were blocked by 2% PBS-BSA solutionafter an incubation of 2 hours at room temperature. After washing four times, 100¹l of test antibody diluted in 0.1% PBS-BSA were added and incubated for 1 hourat room temperature. Extensive washing of the plate was followed by addition of100 ¹l of goat anti-rabbit IgG coupled to horseradish peroxidase (1 : 1000 dilution;Sigma) and incubated for 1 hour at room temperature, in the dark. Finally, thereaction was developed by adding 100 ¹l/ well of tetramethyl benzidine-H2O2(Sigma) for 20 minutes. The enzymatic reaction was stopped with 50 ¹l H2SO4(2 M). Optical density (OD) was measured at 450 nm using a Titertec ELISAphotometer (Digiscan, ASYS Hitech GmbH, Engendorf, Austria). The results areexpressed as OD § SEM and percent of increase over background § SEM.
For statistical analysis we used independent t test for among-group comparisons,and paired t test for within-group comparisons.
The present study was designed to quantify the serum levels of CRH in patients withendometriosis and to correlate these levels in response to treatment with danazol orleuprorelin acetate depot (during and after therapy).
Serum CRH levels of women with and without endometriosis are presented in Table 1 (A). As shown in part A of this Table, CRH (optical density § SEM and percent of increase over background § SEM) did not differ in women with endometriosis and in the control group. These data suggest that endometriosis mustnot be classi ed among other diseases that cause a change in CRH levels.
The mean age in both groups studied was similar (28:4 § 5:3; 27:9 § 5:7 years for the danazol and leuprorelin treatments respectively) and not signi cantly differentfrom the mean age of the normal subjects (29:2 § 5:7 years). The mean age of subjects with endometriosis who did not receive therapy (16 patients; see Materialsand Methods) was 27:5 § 5:6 years. The before-treatment menstrual cycles of all women studied were normal and, as expected, pain related to endometriosis wasgradually alleviated and menses disappeared in all 22 women with both treatmentsoffered. After cessation of treatment, menstrual bleeding returned to normal in 1–2months. All women treated developed amenorrhea during the study and displayedsigns or symptoms of hypoestrogenism.
Table 1.
Serum CRH levels in women with and without endometriosis (A) and in women with endometriosis
before, during and after treatment with danazol and leuprorelin acetate depot respectively (B). The
results are expressed as net optical density § SEM (% of increase over background § SEM) from
* Not statistically signi cant variation between healthy and non-healthy subjects.
** No statistic signi cance between the two therapies of choice in the before and during treatment groups as compared to their controls shown in part A.
*** Signi cant decrease after danazol therapy, p < 0:005.
**** Signi cant increase after leuprorelin treatment, p < 0:001.
The optical density and the percent of modulation of the serum CRH in the women with endometriosis before and during treatment with danazol or leuprorelinrespectively are also presented in Table 1 (part B). As seen, danazol or leuprorelinadministration had no effect on the levels of CRH.
Recent work by Florio et al. (1998) support that peritoneal uid levels of CRH and CRH-BP were similar in healthy patients and women with pelvic adhesions or withendometriosis and, when patients with endometriosis were considered as a singlegroup, no difference in CRH and CRH-BP levels was noted in comparison to thecontrol group. This nding points to a limited role of CRH in the immunologicalchanges related to endometriosis and to the in ammated site. Previous work byWilson et al. (1993) has shown that the chronic use of GnRH agonists suppressesthe hypothalamic– pituitary – ovarian axis and is associated with GH and PRLsuppression as well, whilst it does not alter the hypothalamic– pituitary – adrenalaxis. Moreover, Sakata et al. (1994) showed that danazol has very little effect on thehypothalamic– pituitary – ovarian axis. Although both treatments employed in thisstudy do bring the disease to a controlled state, they do not affect the biosyntheticpathways of CRH. This observation suggests that the bene cial effect of danazoland leuprorelin are CRH independent and that their anti-in ammatory action is regulated by other mechanisms that are being activated during the treatment periodwhere their clinical action is exerted.
Three months after treatment with danazol, CRH values were statistically signif- icantly lower (p < 0:005) than those before treatment. In contrast, after treatmentwith leuprorelin acetate depot CRH levels were signi cantly higher (p < 0:001)than those before treatment (Table 1; part B). These data suggest that the mecha-nisms of action of GnRH-a and danazol on endometriosis are different from eachother. However, one should be cautious also about the mode of action of these twomedications. It is known that medical therapy or even conservative surgery does notcure endometriosis, which recurs in most patients once treatment is stopped (onlytotal ablation of ovarian function prevents recurrence of the disease). Danazol isan anti-gonadotropin-type drug that induces a state of pseudo-menopause by sup-pressing estrogen levels. Such action appears to be indirect via hormones producedin the brain (hypothalamus and pituitary) that cause ovulation. On the other hand,leuprorelin is associated with bone loss. In general, GnRH agonists may effec-tively suppress levels of FSH, LH, estradiol and estrone. Since thus the hormonalinteraction creates a complex system of cross-talk, one must consider any kind ofregulation that may lead to increased or decreased hormonal levels. Taking alsointo account the presence of numerous cytokines that are being produced in thislocal in ammatory microenvironment, the complexity of the system becomes ap-parent. Although no valid explanation can be offered at the present time (i.e. whydanazol and leuprorelin have opposite effects after the cessation of treatment), webelieve that the hormonal/ cytokinic network that de nes CRH production is largelyin uenced by unknown yet parameters that need to be clari ed.
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