Expression of inducible nitric oxide synthase in trophoblasts and deciduas in early medical abortion

Abstract

The purpose of this study was to investigate the expression of inducible nitric oxide synthase in trophoblasts and deciduas in early medical abortion, and study the relationship of medical abortion through mifepristone and inducible nitric oxide synthase (iNOS) in early pregnancy. Expression of iNOS in trophoblasts and deciduas was detected by both in situ hybridization and immunohistochemical assay in 40 patients (experimental group); the positive expression of iNOS was represented by number density (N/S) and positive unit (Pu) using computer color magic image analysis system (CMIAS). All results were compared with that obtained from vacuum aspiration. In the experimental group, N/S and Pu in trophoblasts were 0.120 ± 0.010 and 15.3 ± 2.6, respectively, while in the control group, they were 0.021 ± 0.003 and 3.1 ± 0.5, respectively, and there were significant differences between the two groups. By immunohistochemical assay, N/S and Pu were 0.090 ± 0.010, 10.24 ± 1.55 vs 0.016 ± 0.002, 1.26 ± 0.33 in the trophoblasts of the two groups; there were also significant differences between the two groups. There were lower iNOS expressions in deciduas by in situ hybridization and immunohistochemical assay, and the difference between the two groups was not significant.It was concluded that mifepristone induced medical abortion through the expression of iNOS in trophoblasts but not in deciduas.

Keywords

mifepristone / inducible nitric oxide synthase / early pregnancy / placental immunity

Introduction

Placental immunity is a hot spot in gestational immunity, while inducible nitric oxide synthase (iNOS) has a tight relationship with gestational immunity. Expression of iNOS can directly impact the function of the placental barrier. Mifepristone has been widely used in medical abortion for many years. The mechanism of its anti-early pregnancy function is related to its effect as an antiprogestin, but more and more researchers are paying attention to its immune regulation functions and its interaction with immune factors [1,2]. In our present study, we mainly tried to investigate whether mifepristone plays an important role in the expression of iNOS in villi and deciduas.

Materials and methods

Subjects

The experimental group included 40 patients who voluntarily requested medical abortion. Forty patients in the control group underwent artificially induced abortion. All the patients were characterized as follows: age 19-35 years old, 5-7 weeks pregnant, natural pregnancy, never took any steroidal hormones in the past 6 months, not pregnant or with other complications, positive urine pregnancy test, and intrauterine early pregnancy diagnosed by ultrasound. All patients were outpatients from our hospital.

Obtaining samples

In the experimental group, mifepristone (Shanghai Hualian Pharmaceutical Co., Ltd., China) was administered orally. The total amount of mifepristone was 150 mg, and the first dose was 50 mg; 12 hours later, it was administered nocte et mane on the second day, and at 7 o’clock on the third day, in an amount of 25 mg at each time. One hour after having taken the last 25 mg mifepristone, 600 µg of misoprostol (Searle, USA) was added. Patients were then asked to go to the hospital for further observation. If gravid tissues could be completely discharged within 2 hours, they were collected immediately. If not, curettage was to be performed immediately. The villi and deciduas were immersed into 4% paraformaldehyde and fixed for 2-4 hours. Finally, samples were immersed in 20% glucose. On the next day, frozen sections were obtained on a freezing microtome at -20°C.

Reagents

Digoxin-labeled iNOS cDNA probe was purchased from R&D Company (USA), Strept Actividin-Biotin Complex (SABC) immunohistochemistry kit was procured from Wuhan Boster Biological Technology, LTD. (Wuhan, China), with the titer being 1∶100.

Methods

To investigate the expression of iNOS mRNA in trophoblast, we employed the method of hybridization in situ assay. The frozen sections were soaked in 0.01 mol/L phosphate buffered saline (PBS), treated by 0.2 mol/L HCL and digested with 1 µg/mL protease K; then all of these slides were immersed in 0.1 mol/L triethanolamine (TEA) buffer solution, and non-specific sites were blocked with prehybridization solution. Then about 20 µL hybridization solution was added to each slide (40% deionized formamide, 10% dextran sulfate, 1 mg/mL salmon sperm DNA, 4 × SSC, Digoxin labeled probes). Hybridization was performed in wet boxes at 42°C for 16-24 h, and then the slides were washed in 2 × SSC 3 times and stained with diaminobenzidine (DAB). A positive result was the cytoplasm presenting a brown color. Immunohistochemical SABC method was employed according to the kit procedure.

Statistical analysis

Positive unit (Pu) and number density (N/S) was obtained by using CMIAS computer image analysis. The statistical area was 877.96 µm². All data are presented as \overline{x}\pm s. T test was employed, and a P value that was less than 0.05 was considered to be statistically significant [3].

Results

General information

The average age of pregnant women in the experimental group and the control group were 27.8 and 29.2 (27.8 ± 4.6 vs 29.2 ± 5.0) years old, respectively; the average gestational age of the experimental group and the control group were (8 ± 2.8) and (9 ± 3.5) weeks, respectively, and there were no significant differences between the two groups.

The expression of iNOS in villi

In situ hybridization assay showed that the expression of iNOS mRNA in trophoblast cells was positive. A significant difference existed between the two groups. Immunohistochemistry further proved that with the augmented expression of iNOS mRNA, the amount of iNOS protein in villi increased. iNOS activity in the experimental group was significantly increased compared with that in the control group ( P < 0.05, Table 1).

The expression of iNOS in deciduas

Both hybridization in situ and immunohistochemistry showed that dispersive positive cells existed in deciduas. There was no significant difference between the two groups (P > 0.05, Table 2).

Discussion

Mifepristone and misoprostol have been used for medical abortion in early pregnancy for many years. Misoprostol, which promotes uterine smooth muscle contraction, can facilitate the discharge of necrotic embryo and placenta, but is not related to the death of embryos. Therefore, mifepristone, through various channels that induce the death of early pregnant embryos, is the key in medical abortion [4,5]. Our recent research revealed that mifepristone not only bound to the progesterone receptor and had an anti-progesterone effect, but also had a tight relationship with a number of immune-related molecules. Mifepristone has a direct effect on trophoblast cells and is involved in the immune response of the placental barrier. These actions lead to degeneration and/or necrosis of the deciduas and trophoblast cells. Eventually the embryos die and the pregnancy is terminated [6-9].

The placental barrier and placental immunity form the basis of pathological pregnancy, and many immune factors have been found to contribute to placental immunity. As a new member of the immunity family, NO has a wide range of immunological significance, plays an important role in placental immunity and has become a novel hot spot for research in recent years. NO synthesis depends on its key enzyme — nitric oxide synthase (NOS). There are three types of NOS, among which iNOS is related to immunity. With the in-depth study of the placental immune barrier, it has been found that both normal deciduas and chorionic villi express iNOS, suggesting the importance of iNOS in the maintenance of a normal maternal-fetal barrier and development of the embryos [10-13]. The results of our study indicated that in the control group, the expression of iNOS in the villi and deciduas was positive, which was consistent with several foreign scholars’ studies [11-13]. The abnormal expression of iNOS may lead to an abnormal placental barrier and induce pathological pregnancy, such as abortion, pregnancy-induced hypertension, and other diseases. The research of Eis et al supports the above point of view [14,15]. Ogando et al found that in lipopolysaccharide (LPS) induced mouse abortion model, LPS might mediate the augmented expression of iNOS and increase the production of NO. An NO inhibitor could significantly decrease the abortion rate [16]. NO has multi-cytotoxicity, including the combination of cellular cation superoxide or the suppression of Bcl-2 expression which leads to the relatively high expression of Fas, resulting in cell apoptosis, and eventually abortion occurs. In our study about spontaneous abortion, we have verified this correlation [3,17-19]. In the experimental group of our present study, the expression of iNOS in the villi increased, which manifested that mifepristone could induce iNOS expression in trophoblast cells. In turn, NO synthesis increased. Through the cell toxicity caused by NO the immune barrier was damaged, the cell permeability increased, and then abortion occurred. Compared with the control group, there was no significant difference in iNOS expression of deciduas in the experimental group. The immunomodulatory effect of mifepristone was mainly involved in the villi. However, deciduas were insensitive regions. These mechanisms are different from spontaneous abortion. The non-response of deciduas may account for decidual residuals and long-term bleeding in medical abortion [20-22]. Whether there is a more complex regulatory mechanism which leads to the non-synchronization response of the villi and decidua to mifepristone awaits further research.

Since mifepristone is applied in early medical abortion, its adverse effects, such as decidual residual and long-term bleeding, are always a nettlesome trouble in clinical practice. This study showed that mifepristone can significantly increase the expression of NO in the villi, while having no effect on the decidua. The results revealed that the immune regulation of mifepristone in the villi and decidua presents a significant difference. The difference may also exist in other immune factors of the placental immune network. The discovery provides a new direction to resolve the common adverse effects of medical abortion (mifepristone), such as decidual residual [23-25].

References

[1] SrivastavaM D, ThomasA, SrivastavaB I, CheckJ H. Expression and modulation of progesterone induced blocking factor (PIBF) and innate immune factors in human leukemia cell lines by progesterone and mifepristone. Leuk Lymphoma, 2007, 48(8): 1610-1617

[2] KleiverdaG. First trimester medical abortion: a good method that is seldom used in The Netherlands. Ned Tijdschr Geneeskd, 2006, 150(14): 766-769

[3] XiaG Q, SunY Y. Expression of apoptosis and inducible nitric oxide synthase in chorion in early pregnancy. Reprod Contracept, 2001, 12(1): 56-60

[4] HausknechtR. Mifepristone and misoprostol for early medical abortion: 18 months experience in the United States. Contraception, 2003, 67(6): 463-465

[5] De GrandiP, GiudiciG. Oral administration of an antiprogesterone (Mifepristone, RU 468) for preparing the cervix uteri for pregnancy interruption during the first trimester. J Gynecol Obstet Biol Reprod, 1989, 18(6): 801-808

[6] XiaG Q, XiongY L, SunY Y. Effect of mifepristone on the telomerase activity in chorion and decidua during early gestation. Reprod Contracept, 2004, 15(4): 245-248

[7] LidegaardO, LarsenJ F, BlaabjergJ, LarsenE. The first 100 early medical abortions. Ugeskr Laeger, 1999, 161(22): 3278-3281

[8] VervestH A, HaspelsA A. Initial Dutch experiences with early pregnancy interruption using the antiprogesterone agent mifepriston. Ned Tijdschr Geneeskd, 1985, 129(35): 1680-1683

[9] PappC, SchatzF, KrikunG, HausknechtV, LockwoodC J. Biological mechanisms underlying the clinical effects of mifepristone (RU 486) on the endometrium. Early Pregnancy, 2000, 4(4): 230-239

[10] ParadisiR, FabbriR, BattagliaC, FacchinettiF, VenturoliS. Nitric oxide levels in women with missed and threatened abortion: results of a pilot study. Fertil Steril, 2007, 88(3): 744-748

[11] MarinoniE, Di IorioR, LucchiniC, Di NettaT, LetiziaC, CosmiE V. Adrenomedullin and nitric oxide synthase at the maternal-decidual interface in early spontaneous abortion. J Reprod Med, 2004, 49(3): 153-161

[12] HämäläinenM, LiljaR, KankaanrantaH, MoilanenE. Inhibition of iNOS expression and NO production by anti-inflammatory steroids. Reversal by histone deacetylase inhibitors. Pulm Pharmacol Ther, 2008, 21(2): 331-339

[13] UrbanG, MarinoniE, Di IorioR, LucchiniC, AloP, Di TondoU. New placental factors: Between implantation and inflammatory reaction. Early Pregnancy, 2001, 5(1): 70-71

[14] EisA L, BrockmanD E, MyattL, Immunolocalization of the inducible nitric oxide synthase isoform in human fetal membranes. Am J Reprod Immunol, 1997, 38(4): 289-294

[15] ZenclussenA C, SollwedelA, BertojaA Z, GerlofK, ZenclussenM L, WoiciechowskyC, VolkH D. Heme oxygenase as a therapeutic target in immunological pregnancy complications. Int Immunopharmacol, 2005, 5(1): 41-51

[16] OgandoD G, PazD, CellaM, FranchiA M. The fundamental role of increased production of nitric oxide in lipopolysaccharide-induced embryonicresorption in mice. Reproduction, 2003, 125(1): 95-110

[17] MatsumuraM, KakishitaH, SuzukiM, BanbaN, HattoriY. Dexamethasone suppresses iNOS gene expression by inhibiting NF-kappaB in vascular smooth muscle cells. Life Sci, 2001, 69(9): 1067-1077

[18] ZhangD, KishiharaK, WangB, MizobeK, KuboC, NomotoK. Restraint stress-induced immunosuppression by inhibiting leukocyte migration and Th1 cytokine expression during the intraperitoneal infection of Listeria monocytogenes. J Neuroimmunol, 1998, 92(1,2): 139-151

[19] MillerL, AlleyE W, MurphyW J, RussellS W, HuntJ S. Progesterone inhibits inducible nitric oxide synthase gene expression and nitric oxide production in murine macrophages. J Leukoc Biol, 1996, 59(3): 442-450

[20] GoldeS, ColesA, LindquistJ A, CompstonA. Decreased iNOS synthesis mediates dexamethasone-induced protection of neurons from inflammatory injury in vitro. Eur J Neurosci, 2003, 18(9): 2527-2537

[21] XiaG, SunY. Expression of the inducible nitric oxide synthase isoform in chorionic villi in the early spontaneous abortion. J Tongji Med Univ, 2000, 20(4): 338-339

[22] HaddadE K, DuclosA J, BainesM G. Early embryo loss is associated with local production of nitric oxide by decidual mononuclear cells. J Exp Med, 1995, 182(4): 1143-1151

[23] MiechR P, Pathopharmacology of excessive hemorrhage in mifepristone abortions. Ann Pharmacother, 2007, 41(12): 2002-2007

[24] Väisänen-TommiskaM, ButzowR, YlikorkalaO, MikkolaT S. Mifepristone-induced nitric oxide release and expression of nitric oxide synthases in the human cervix during early pregnancy. Hum Reprod, 2006, 21(8): 2180-2184

[25] De GrandiP, GiudiciG. Oral administration of an antiprogesterone (Mifepristone, RU 486) for preparing the cervix uteri for pregnancy interruption during the first trimester. J Gynecol Obstet Biol Reprod, 1989, 18(6): 801-808

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