Effect of copper intrauterine device on the cyclooxygenase and inducible nitric oxide synthase expression in the luteal phase endometrium

Contraception 84 (2011) 637 – 641

Original research article

Effect of copper intrauterine device on the cyclooxygenase and inducible nitric oxide synthase expression in the luteal phase endometrium Ebru Coskuna , Yigit Cakiroglua,⁎, Banu Kumbak Aygunb , Bahar Muezzinogluc , Eray Caliskana a

Kocaeli University Department of Obstetrics and Gynecology, 41000 Kocaeli, Turkey b Firat University Department of Obstetrics and Gynecology, 23119 Elazig, Turkey c Kocaeli University Department of Pathology, 41000 Kocaeli, Turkey Received 10 October 2010; revised 28 March 2011; accepted 30 March 2011

Abstract Background: To evaluate the effect of copper intrauterine device (IUD) on the expression of cyclooxygenase (COX) and inducible nitric oxide synthase (iNOS) in the luteal phase endometrium. Study Design: A prospective clinical study was conducted on 30 women who were willing to use a copper IUD contraception. Endometrial biopsies and blood samples were taken before and 3 months after the insertion of the IUD on Day 3 and Days 20–24 of the cycle. Main outcome measures were to evaluate the effect of copper IUD on uterine artery blood flow using pulsed color Doppler ultrasonography and the relationship of bleeding abnormalities and menstrual pain level with the uterine blood flow, COX-2 and iNOS expression. Results: Only the left uterine artery pulsatility and resistance indices decreased statistically significantly (p=.005 and p=.039, respectively). Other Doppler parameters showed no change. Cyclooxygenase-2 expression of both endometrial luminal epithelium (p=.03) and gland epithelium (p=.03) increased significantly. Inducible NOS expression of the endometrial surface epithelium decreased significantly after IUD insertion (p=.01). Conclusions: Although COX-2 expression increased 3 months after copper IUD insertion, iNOS expression of the luminal epithelium decreased. Local hypoxia caused by copper and vasoconstrictor prostanoids may play a role in IUD-related menstrual abnormalities. © 2011 Elsevier Inc. All rights reserved. Keywords: COX-2; iNOS; TCu380A; Menstrual abnormalities; Dysmenorrhea

1. Introduction Intrauterine contraception's cumulative pregnancy rate in a 5-year period is reported to be 0.8 per 100 women [1,2]. Bleeding and pain are the most common reasons for removal, with rates of 10% in the first year and up to 50% within 5 years [3]. It is known that the copper intrauterine device (IUD) causes a sterile inflammation reaction in the endometrium [4]. The cyclooxygenase (COX) enzymes exist in three isoforms: COX-1, COX-2 and the recently identified COX-3. The different COX genes are regulated by two

⁎ Corresponding author. Department of Obstetrics and Gynecology, Faculty of Medicine, Kocaeli University, Umuttepe/Uctepeler 41000 Kocaeli, Turkey. Tel.: +90 262 3038433; fax: +90 262 3038003. E-mail address: [email protected] (Y. Cakiroglu). 0010-7824/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.contraception.2011.03.027

independent systems, although the enzymatic reactions they catalyze are identical [4,5]. Although endogenous COX enzyme is COX-1, the inducible enzyme is COX-2 [6]. Inflammatory factors, growing factors, platelet activating factor, and endothelin can induce the expression of COX-2 [7]. Nitric oxide can induce COX activity. The sterile inflammatory action of IUDs is spermicidal, but how the IUD causes side effects such as bleeding and pain is unknown. The morphological features of the endometrium exposed to an IUD are manifestations of localized mechanical trauma, foreign body response and impaired hemostasis [8]. As COX-2 and inducible nitric oxide synthase (iNOS) expression can be induced by inflammation, in the present study, we aimed to evaluate the effect of copper IUD on uterine artery blood flow using pulsed color Doppler ultrasonography, endometrial COX-2 and iNOS expression and the relationship of bleeding abnormalities and dysmenorrhea

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with the uterine blood flow, COX-2 and iNOS expression during the midluteal phase in regularly menstruating women.

2. Materials and methods The study population consisted of 30 women who were willing to use copper IUD for contraception. The women were menstruating regularly (i.e., menstrual cycle varying between 28 and 35 days) and were 18–40 years old. Exclusion criteria were pregnancy, acute or chronic pelvic inflammatory disease, metrorrhagia for unknown reason, cervicitis, dysplasia in the cervix, genital tumor, copper allergy, use of contraceptive pills within the previous 3 months, lactating women, hypothyroidism, hyperthyroidism, abnormalities in blood clotting and severe dysmenorrhea. Informed consent was taken from the women, and the study was approved by the ethical committee of the medical faculty of Kocaeli University. The main outcome measures of the study were to evaluate the effect of copper IUD on uterine artery blood flow using pulsed color Doppler ultrasonography and the relationship of bleeding abnormalities and menstrual pain level with the uterine blood flow, COX-2 and iNOS expression. All patients underwent gynecological examination and had a Papanicolaou smear taken during the previous 12 months. A menstrual calendar was used to evaluate the amount and length of the menstrual bleeding. Amount of menstrual bleeding was defined as light (less than one fourth of tampon or ped surface is stained with blood), moderate (one fourth to three fourths of tampon or ped surface is stained with blood) or heavy (more than three fourths of tampon or ped surface is stained with blood). Dysmenorrheic pain level was estimated using the visual analogue scale (VAS) pain score (0–10) at initial examination and after IUD insertion. The patients self-evaluated their pain levels at the night of the first 3 days of menstruation. The VAS score of each patient was calculated as an arithmetic mean of the first 3 days of the menstrual cycle. The women were first examined on Day 3 of their menstrual cycle, and blood samples were obtained between 7:00 and 9:00 a.m. to determine estradiol (E2), folliclestimulating hormone (FSH), luteinizing hormone (LH) levels, progesterone and thyroid-stimulating hormone (TSH). Hormone values were analyzed using a radioimmunoassay technique (IMMULITE® 2000; Diagnostic Products, Los Angeles, CA) via IMMULITE 2000 analyzer. The same hormone profile was repeated on Days 20–24 of their cycle. Transvaginal color Doppler examinations were performed by the same observer (E.C.) between 7:00 and 9:00 a.m. while patients were in the lithotomy position using Siemens Versa plus machine (Erlangen, Germany) equipped with a 5-MHz transvaginal probe for imaging and 6-MHz pulsed Doppler system for blood flow analysis [pulsatility index (PI), resistance index (RI), systole/diastole (S/D),

maximum velocity (Vmax) ] on Day 3 and Days 20–24 of the menstrual cycle before IUD insertion. After examination, a copper-containing IUD (TCu380A, Turkey) was inserted on the first day of the menstrual cycle. Three months after IUD insertion, the same examinations on Day 3 and Days 20–24 were repeated. The biopsy specimens were taken with Pipelle curette without dilatation of the cervix and without anesthesia. Endometrial biopsies were taken from the uterine cavity before and 3 months after the insertion of the IUD on Days 20–24 of the cycle. The biopsy specimens were fixed in 10% neutral buffered formalin for at least 6 h. After overnight tissue processing, they were embedded in paraffin. The histological sections were 5 μm thick and were stained with hematoxylin and eosin. Sections for immunohistochemical examination were 5 μm thick and were mounted onto adhesive-coated slides (Süperfrost® Plus, Menzel-Gläser, Braunschweig, Germany). For immunohistochemical staining, sections were kept at 56°C overnight and then soaked in xylene for 30 min. After washing with a decreasing series of ethanol, sections were washed with distilled water and phosphate-buffered saline (PBS) for 15 min. Coated slides were dried in an incubator at 56°C for 2 h. Antigen retrieval was performed for COX-2 antibody in a citrated buffer solution in the microwave oven and for iNOS antibody in a citrated buffer solution with high-pressure temperature. After the antigen retrieval step, slides were washed with PBS (pH 7.4), and then, to block endogenous peroxidase activity, the slides were incubated in 3% hydrogen peroxide for 20 min. Slides were washed with PBS for 5 min. Sections were then blocked with Super Block (ref. AAA 125, lot 12232) at room temperature for 15 min and then washed with PBS. Slides were immunostained at 1:100 dilution at room temperature (20°C–25°C) with commercially available rabbit monoclonal antibody COX-2 (cat no. RB-9072-P; Neo-Markers, Fremont, CA) for 1 h and with commercially available rabbit monoclonal antibody iNOS (no. RB-1605-P; Neo-Markers) for 2 h. Afterward, slides were washed with PBS and slides were incubated with UltraTek Anti-Polyvalent Biotinylated Antibody (ref. ABN 125, lot 11461; ScyTek Laboratories, Logan, UT) at room temperature for 25 min, were washed with PBS again and incubated with UltraTek HRP (ref. ABL 125, lot 11460). Slides were washed with PBS and incubated with Ultravision Detection System Large Volume AEC Substrate System (RTU) (ref. TA-125-HA, lot AHA60718; LabVision, Fremont, CA) at room temperature for 15 min. The sections were finally counterstained using Mayer's hematoxylin and mounted in an aqueous medium. Slides were analyzed with a BX50 conventional light microscope (Olympus, Tokyo, Japan) by a pathologist (BM) at ×100 and ×200 magnification. Staining intensity was graded as 0=no staining, +1 or b10% staining=weak staining, +2 or 10%–49% staining=mild staining and +3 or 50%–100% staining=strong staining. Immunohistochemical staining in luminal and glandular epithelium cytoplasm

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were graded in 60 sections counting 100 cells separately at ×400 magnification. The statistical analysis of data was performed using SPSS 11.5 for Windows packet program. McNemar–Bowker test was used for immunostaining grade and intensity before and 3 months following IUD insertion. Analysis of data was performed using χ2 test and Fisher's exact test. Correlation of data was determined using Pearson correlation test. Probability (p) values lower than .05 was considered significant. All values reported are mean±SD or percentage.

3. Results

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Table 2 Hormone profile on Day 3 and Days 20–24 of the menstrual cycle before and after IUD insertion Hormones

Day 3 FSH (mIU/mL) LH (mIU/mL) E2 (pmol/L) Days 20–24 LH (mIU/mL) E2 (pmol/L) Progesterone (ng/mL)

Before IUD replacement (mean±SD)

After IUD replacement (mean±SD)

p

8.17±2.5 5.11±3.1 39.1±23.3

8.65±2.6 4.66±1.8 41.3±19.5

.33 .39 .57

4.7±2.9 76.7±41.6 6.89±4.8

5.54±3.1 91.5±58.8 8±5.3

.28 .96 .31

p≤.05, statistically significant.

Patient characteristics and obstetric histories of the cases are presented on Table 1. Although the menstrual cycle length and quantity of bleeding was normal before IUD insertion, 13 (43%) of 30 women complained of heavy menstruation 3 months after IUD insertion (pb.001). The mean VAS scores at the cycle of IUD insertion and three cycles after the procedure were investigated for the first 3 days of menstrual cycle, and the results were calculated as 2.8±0.9 and 3.5±1.8, respectively (p=.003). Hormonal values are presented on Table 2. No differences were found between hormonal values both before and after IUD insertion. We found no correlation between hormonal values in the follicular and luteal phases of the menstrual cycle after IUD insertion or extended and/or prolonged menstruation and dysmenorrhea. Comparison of uterine artery Doppler indices before and after IUD insertion on Days 20–24 of the menstrual cycle are shown in Table 3. Only left uterine artery PI and RI showed statistically significant decrease (p=.005 and p=.039, respectively); other Doppler parameters showed no change. The expression grade of the COX-2 antibody is presented in Table 4. Cyclooxygenase-2 expression of both endometrial luminal epithelium (p=.03) and gland epithelium (p=.03) increased significantly. Before IUD insertion, COX-2 expression of the luminal epithelium was present in 51.3±27.8 cells, whereas after 3 months of IUD usage, it was present in 74.7±25.7 cells. Cyclooxygenase-2 expression of the luminal epithelium increased significantly

Table 1 Patient characteristics and obstetric histories of the cases Variable Age (years) Gravida (range) Para (range) Live birth (range) Dilatation and curettage (≤1) Abortus (≤1) Husband's age (years) Previous IUD usage Body mass index Data are mean±SD [range] or n (%).

32.4±6.5 [21–40] 2.7±1.43 [1–8] 2±0.69 [1–3] 1.93±0.64 [1–3] 8 (26.6) 7 (23.3) 37.2±5.9 [28–50] 12 (40) 24.7±2.97 [18.1–31.2]

3 months after IUD insertion (p=.001). Before IUD insertion, COX-2 expression of gland epithelium was present in 22.2±17.9 cells, whereas after IUD insertion, it was present in 35±25.4 cells. Cyclooxygenase-2 expression of the gland epithelium increased significantly after IUD insertion (p=.03). Dysmenorrheic women (n=9) showed staining with COX-2 in 81±29 epithelial cells, whereas asymptomatic women (n=21) had 52.9±27.9 cells stained in the luminal epithelium (p=.01). Dysmenorrheic women had more COX2-stained luminal cells than women who did not have this symptom (p=.01), but we found no relation between COX-2 expression and dysmenorrhea pain intensity. The expression grade of the iNOS antibody in the luminal epithelium and the stroma is presented in Table 5. Inducible NOS expression of the endometrial surface epithelium decreased significantly after IUD insertion (p=.01). The reduction of the stromal expression after IUD insertion was not found to be significant (p=.1). Before IUD replacement, iNOS expression of the luminal epithelium was present in 19.4±26.9 cells, whereas after IUD usage, it was present in 4.86±10.2 cells. Inducible NOS expression of the luminal epithelium decreased after IUD replacement significantly (p=.009). Before IUD replacement, iNOS expression of the Table 3 Comparison of uterine artery Doppler indices before and after IUD insertion on Days 20–24 of the menstrual cycle Doppler indices

Right uterine artery PI RI S/D Vmax Left uterine artery PI RI S/D Vmax

Before IUD replacement (mean±SD)

After IUD replacement (mean±SD)

2.24±0.51 0.86±0.08 10.3±8.23 0.12±0.05

2.5±0.75 0.87±0.08 10.1±8.1 0.13±0.06

2.49±0.7 0.89±0.08 10.6±9.55 0.148±0.07

2.42±0.58 0.88±0.07 11.9±10.9 0.133±0.06

⁎ pb.05, statistically significant.

p

.13 .10 .64 .14 .005⁎ .039⁎ .12 .32

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Table 4 Expression grade of the COX-2 antibody in the luminal epithelium and gland epithelium before and 3 months after the insertion of IUD on Days 20–24 of the cycle

Table 6 Expression grade of the COX-2 antibody in the luminal epithelium and gland epithelium compared with menstrual bleeding patterns before and 3 months after the insertion of IUD on Days 20–24 of the cycle

Antibody

Expression Before IUD After IUD P grade (n=30), n (%) (n=30), n (%)

Menstrual bleeding

COX-2 immunostaining in the luminal epithelium cytoplasm COX-2 immunostaining in the gland epithelium cytoplasm

0 +1 +2 +3 0 +1 +2 +3

1 (3.3) 4 (13.3) 9 (30) 16 (53.3) 2 (6.7) 13 (43.3) 15 (50) 0

0 1 (3.3) 6 (20) 23 (76.6) 0 11 (36.7) 15 (50) 4 (13.3)

.03

.03

stroma was present in 9.94±22.3 cells, whereas after IUD usage, it was present in 0.93±4.57 cells. The decrease in iNOS expression of the stroma was found significant after IUD usage (p=.03). The expression grade of the COX-2 antibody in the luminal epithelium and gland epithelium compared with menstrual bleeding patterns before and 3 months after the insertion of IUD on Days 20–24 of the cycle is presented in Table 6. The expression grade of the COX-2 antibody is found to be statistically significantly higher both in the luminal epithelium and the gland epithelium in patients with heavy bleeding compared with the patients with light and moderate bleedings (p=.03 and pb.001 respectively).

4. Discussion The IUD has been accepted as a highly preferable modern contraceptive method among reproductive-age women. It has been shown that menstrual abnormalities (spotting, light bleeding, prolonged and/or heavy menstruation periods) were frequent in the first 3–6 months after IUD insertion [9]. Although the IUD has a long-term effectiveness, its mode of action and how it causes side effects are not exactly known. We found no change in hormonal values after IUD insertion. Similar to our findings, Jarvela et al. [10] had Table 5 Expression grade of the iNOS antibody in the luminal epithelium and the stroma Antibody

Expression grade

Before IUD (n=30), n (%)

After IUD (n=30), n (%)

p

iNOS surface epithelium

0 +1 +2 +3 0 +1 +2 +3

11 (36.7) 10 (33.3) 6 (20) 3 (10) 23 (76) 4 (13.3) 2 (6.6) 1 (3.3)

19 (63.3) 8 (26.7) 3 (10) 0 28 (93.3) 1 (3.3) 1 (3.3) 0

.01

p≤.05, statistically significant.

+1

+2

+3

COX-2 immunostaining in the luminal epithelium cytoplasm Light and moderate 0 1 6 10 Heavy 0 0 0 13 COX-2 immunostaining in the gland epithelium cytoplasm Light and moderate 0 11 6 0 Heavy 0 0 9 4

p

.03⁎ b.001⁎

⁎ pb.05, statistically significant.

p≤.05, statistically significant.

iNOS stroma

0

.1

detected no change in hormonal values in their regularly menstruating patients after IUD insertion. We found no correlation between uterine artery Doppler parameters and extended and/or prolonged menstruation and dysmenorrhea after IUD insertion. Zalel et al. [11] had compared copper and levonorgestrel-releasing IUDs and found no change in the Doppler values, and they concluded that decreased menorrhagia in the levonorgestrel group was due to the local effects of the progesterone. When uterine artery Doppler indices were compared before and after IUD insertion on Days 20–24 of the menstrual cycle, only left uterine artery PI and RI showed statistically significant decrease (p=.005 and p=.039, respectively); other Doppler parameters showed no change. We believe that this finding could be a statistical anomaly and should be further investigated. We planned to investigate the effect of copper IUD on endometrial COX-2 and iNOS expression because these antibodies could be induced by inflammation. We found that COX-2 expression of both the endometrial luminal epithelium and the gland epithelium increased significantly after 3 months of IUD insertion. The important finding in long-term IUD usage was asynchrony between cyclic dating and endometrial histological dating (4 days of delay or advance) [12]. In our study, we did not find a correlation between histological dating and the COX-2 and iNOS expression intensity. This finding may show that the effect of the IUD on the antibody expression occurs irrespective of the histological endometrial day. In our study, we found that COX-2 expression of both the endometrial luminal epithelium and gland epithelium increased significantly after 3 months of IUD insertion. Our finding is in accordance with the literature that IUD causes inflammation, as COX-2 was reported to be an inducible enzyme [13]. Intrauterine device-related uterine bleeding and menstrual pain are thought to be caused by increased uterine secretion of prostaglandins, leading to abnormal uterine activity and vasoconstriction of the uterine arterioles [14]. In our study, the expression grade of the COX-2 antibody is found to be higher in patients with heavy bleeding compared with the patients with light and moderate bleeding. Heavy menstrual blood loss has been associated with aberrations in the synthesis and production

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of vasodilatory prostanoids from the uterus [15]. Intrauterine device-related COX-2 expression elevation was expected to be positively correlated with prolonged and/or extended menstruation, but this symptom was found to be decreased as the expression increased. This means that vasoconstrictor prostaglandins increase more than vasodilators due to the IUD. In our study, we found that when the local tissue nitric oxide decreased, the abnormal bleeding symptom related to IUD usage increased. We hoped to find an increased expression of iNOS due to IUD-related inflammation. We theorize that the decreased expression shows that IUD causes local tissue hypoxia due to released copper, and nitric oxide plays a mediator role in this hypoxia process. Copper ion was reported to increase vascular endothelial growth factor and hypoxia-inducible factor secretion due to hypoxia [9]. Three to 5 months after IUD insertion, the amount of released copper was reported to be decreased compared with the initial cycle [4]. Menstrual abnormalities were reported to be frequent in the first 3–6 months after IUD insertion [9]. After 3 or 6 months, the released copper decreases, resulting in reduced local hypoxia in the endometrium and vasodilator prostanoids increase in response to the initial local hypoxia. Eventually, tissue hypoxia and hypoxia-related side effects occur rarely 3–6 months after IUD insertion. In conclusion, COX-2 expression increases 3 months after copper IUD insertion. Patients with extended and/or prolonged menstruation and dysmenorrhea 3 months after insertion may benefit from selective COX inhibitors. Local hypoxia caused by copper and vasoconstrictor prostanoids may play a role in the IUD-related menstrual abnormalities. Acknowledgment This study was funded by the Kocaeli University Research Support Programme.

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