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Hysteroscopic polypectomy decreases NF-κB1 expression in the mid-secretory endometrium of women with endometrial polyp
European Journal of Obstetrics & Gynecology and Reproductive Biology 189 (2015) 96–100
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Hysteroscopic polypectomy decreases NF-kB1 expression in the mid-secretory endometrium of women with endometrial polyp Murat Bozkurt a,*, Levent S¸ahin a, Mustafa Ulas¸ b a b
Kafkas University School of Medicine, Department of Obstetrics and Gynecology, Kars, Turkey Fırat University School of Medicine, Department of Physiology, Elazıg˘, Turkey
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 July 2014 Received in revised form 8 March 2015 Accepted 31 March 2015
Objectives: Endometrial polyps have been identified as a possible factor for sub-fertility, their implication on endometrial NF-kB1 activity; the central regulator of the immune system gene expression involved in implantation has yet been little studied. We evaluated the NF-kB1 level and NF-kB p65 expression in the endometrium before and after hysteroscopic removal of the endometrial polyp during the mid-secretory phase. Study design: Infertile women with polyp (n = 15) were enrolled. Unexplained infertile patients with normal endometrium (n = 5) and healthy fertile women (n = 5) were recruited as controls. Endometrial samples were obtained before and 4 months after the hysteroscopic polypectomy. NF-kB1 levels were analyzed by ELISA in the endometrium of all groups before and after polypectomy. The H-Score method was used to evaluate immunohistochemical NF-kB p65 expression in the endometrium. Results: NF-kB1 expression and NF-kB p65 immunoreactivity in the endometrium were significantly higher in women with polyp compared to unexplained infertile and fertile controls. Hysteroscopic polypectomy resulted in a significant decrease in endometrium NF-kB1 and NF-kB p65 activity. Conclusions: Endometrium of women with endometrial polyp has abnormalities in expression of NFkB1 and NF-kB p65 which may contribute to endometrial receptivity and so polyp related sub-fertility. Hysteroscopic polypectomy may help to the normalization of endometrial NF-kB concentrations. ß 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: NF-kB1 Endometrial polyps Hysteroscopic polypectomy
Introduction Endometrial polyps, which are benign, hyperplastic-localized overgrowths of glands and stroma have been reported in 15–25% of infertile women and in about 1.4% of women undergoing in vitro fertilization (IVF) [1–3]. The mechanism by which endometrial polyp influences fertility is not clearly understood yet; however, it has been suggested the endometrial polyps may be associated with mechanical interference with sperm transport, embryo implantation or aberrant expression of endometrial markers consisting of HOXA, insulin growth factor binding protein 1 (IGFBP-1), tumor necrotizing factor alpha (TNF alpha) [4–6]. Nuclear factor-kappa B (NF-kB) is a transcription factor involved in endometrial pathologies and known to be a proinflammatory, mitogenic and antiapoptotic factor in many cell types [7–9]. Its function was recognized in TNF and IL1B receptors signal transduction pathways [10,11].
¨ niversitesi Kampu¨su¨ Sag˘lık Aras¸tırma ve * Corresponding author at: Kafkas U Uygulama Hastanesi, Kars, Turkey. Tel.: +90 5322279072/5056330044; fax: +90 0474 225 14 30. E-mail address: [email protected] (M. Bozkurt). http://dx.doi.org/10.1016/j.ejogrb.2015.03.032 0301-2115/ß 2015 Elsevier Ireland Ltd. All rights reserved.
Little is known about endometrial NF-kB activity in women with endometrial polyp. Basal NF-kB activity has been demonstrated in normal human endometrium and endometrial cells [8,10]. Recent clinical study has demonstrated that physiologic endometrial NF-kB activation in the three different phases of the menstrual cycle [7]. During menstruation, NF-kB is activated in the glandular epithelium and endothelium of the endometrium [12]. Estrogen and progesterone receptors may interact with the NF-kB protein in a repressive way [13]. Alterations of the NF-kB expression were shown in eutopic endometrium of endometriosis patients [8,14]. The NF-kB peptides construct homodimers or heterodimers from five subunits: p50/p105 (NF-kB1), p52/p100 (NF-kB2), p65 (RelA), c-Rel, and RelB. Rel-A/NF-kB1 heterodimer is the more commonly found in vivo and Rel-A is the subunit that most frequently participates in the active NF-kB complex [15,16] NFkB1 (p105) and NF-kB2 (p100) are converted to p50 and p52, respectively, which can pass to the nucleus and bind to DNA for activation of gene transcription. However, p65 (Rel A) can readily translocate to the nucleus without any conversion. NF-kB dimers are located in the cell cytoplasm bound to its inhibitory protein IkBa, which blocks the nuclear translocation of NF-kB. In response
M. Bozkurt et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 189 (2015) 96–100
to an extracellular signal, NF-kB cascade is activated and IkBa is phosphorylated allowing the release of NF-kB [16,17] dimers capable of binding to DNA, translocate to the nucleus and activate the transcription of several target genes [18,19]. Although NF-kB pathway was indicated to be active in the normal endometrium of healthy women [8,10,14], its expression in endometrium of women with endometrial polyp and the effect of hysteroscopic polypectomy on the endometrial NF-kB activity is unknown. In the present study, we attempted to investigate whether hysteroscopic polypectomy alters the NF-kB expression in the endometrium of women with endometrial polyp. We evaluated the NF-kB1 level and NF-kB p65 expression in the endometrium before and after hysteroscopic removal of the endometrial polyp during the mid-secretory phase. In addition, to assess the specificity of the results, patients with healthy endometrium included as controls. Materials and methods This prospective interventional study was carried out in Kafkas University Medical Faculty. The study protocol was approved by the Institutional Ethical Committee for Research on Human Subjects. Informed written consent form was obtained from all the women. We evaluated the NF-kB1 subunit level and NF-kB p65 subunit expression in the endometrial tissues obtained prior to hysteroscopy and respective endometrial tissues after the hysteroscopic polypectomy during mid-luteal phase in 15 infertile women with endometrial polyp and 5 control cases with unexplained infertility with normal endometrium. Five fertile women enrolled as the control groups had at least two children, and had no history of infertility or habitual abortion. All the women were recruited to the study had regular menstrual cycles, ranged between 21 and 35 days. All participants were required to meet the following inclusion criteria: (1) age ranging from 18 to 35 years; (2) regular menstrual cycles; (3) presence of endometrial polyp on Transvaginal ultrasonography, which was also confirmed on saline infusion sonohysterogram; 4) not taking any hormonal medications or treatment with an effect on endometrium at least 3 months before surgery. The exclusion criteria were: (1) having any confounding medical conditions known to affect endometrial receptivity such as endometriosis/endometrioma, submucosal fibroids, polycystic ovarian syndrome (PCOS) or hydrosalpinges; (2) presence of any other endometrial pathology including ashermann syndrome, endometrial cancer or endometrial hyperplasia; (3) diagnosis of pelvic inflammatory disease at the time of the study; (4) previous history of endometrial surgery; (5) absence of any systemic and/or rheumatologic disease which may lead to an inflammation. All women had two-dimensional transvaginal ultrasound scan (Voluson 730 Pro; GE Healthcare Kretztechnik, Zipf, Austria) using high frequency transducers of 7.5 MHz. This was followed by saline infusion hysterosonography for the confirmation of endometrial polyps. Subsequently, all women recruited for the study had endometrial sampling through pipelle curettage (Type IIA Endometrial Suction Curette, Jiangsu Suyun Med. Co., Ltd) performed prior to the commencement of polypectomy in the secretory phase of menstrual cycle. The endometrial specimen was divided for pathological evaluation and laboratory investigation. When endometrial polyp was present, it is excised under hysteroscopic guidance. The blood samples were obtained from all participants at the time of endometrial biopsy. The ones with endometrial polyp or unexplained infertility underwent a second procedure 4 months after the surgical removal of the polyp. The secretory phase was calculated as the 7–9 days after the ultrasonographic confirmation of ovulation and was confirmed by endometrial
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histological dating. Endometrial dating was performed by an independent pathologist experienced in gynecological pathology. Endometrial samplings were evaluated according to the histopathological criteria of Noyes et al. [20]. An out of date biopsy was defined as a lag of 3 days between the chronological and the histological day [21]. NF-kB1 determination in the endometrial tissues In all women with endometrial polyp and control subjects, endometrial NF-kB1 (p105) subunit concentrations were measured by Enzyme-Linked ImmunoSorbent Assay (ELISA). This immunoassay kit (NF-kB ELISA kit, USCN Life Sci Inc) allows for in vitro quantitative determination of human NF-kB1 (p105) concentrations in serum, plasma, urine, tissue homogenates, cell culture supernatants and other biological fluids. For this assay, each endometrial sample was rinsed with 1X PBS to remove excess blood, and subsequently, weighted as a wet weight. Then, the clean endometrial samples were homogenized in 1 mL of 1X PBS and stored overnight at a temperature 20 8C. After two freeze-thaw cycles were performed to break the cell membranes, the homogenates were centrifuged for 5 min at 4000 rpm. The supernatant was stored at 80 8C to avoid loss of bioactivity and contamination. The detection range of kit was 0.312–20 ng/ mL. The intra-(within day) and inter-assay (between days) coefficients of variation for endometrial NF-kB1 (p105) were <10% and <12%, respectively. The results of NF-kB1 (p105) concentrations detected by immunoassay kit were divided by the weights of endometrial tissue. The results were presented as ng/ mg tissue. Immunohistochemical method for NF-kappaB/p65 Four micrometer paraffin sections on poly-L-lysine coated slides were used after drying in an oven for 1 h at 60 8C. The sections were de-waxed in xylene, rehydrated in ethanol and then, incubated for 10 min in 3% hydrogen peroxide to block endogenous peroxidase. After washing in phosphate buffer saline, the sections incubated 8 min at ultra V block. The immunoreaction was performed for 60 min with ready to use NF Kappa B/p65 (Rel A) Ab-1 antibody (NeoMarkers, Labvision corp. CA, USA). After washing in PBS, slides were incubated with horseradish peroxidase kit. For negative control, the endometrial tissue was incubated with non-immune rabbit serum in place of the primary antibody. Finally, the preparations were developed in AEC chromogen counterstained with hematoxylin and mounted with aqueous-mount. Third trimester human placenta served as the positive control. To evaluate immunhistochemical NF-kB p65 (Rel A) expression in the endometrium, the H-Score method was used [22]. This immunohistochemical semi-quantitative method consists of the percentages of positively stained cells multiplied by a weighted intensity of staining: H-Score = SPi (i + 1), where Pi is the percentage of stained cells in each intensity category (0–100%), and i is the intensity indicating weak (i = 1), moderate (i = 2) or strong staining (i = 3) [23]. Statistical analysis The normality distribution of data was performed by using Kolmogorov–Smirnov test and all variables were skewed normally. The continuous variables were analyzed by ANOVA test with post hoc Tukey’s procedure and Mann–Whitney U test. A P value <0.05 was considered significant. The results are expressed as mean and standard deviation (SD). The data was analyzed by using the Statistical Package for Social Sciences software 21.0 for Windows package software (SPSS IBM, New York, USA).
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Table 1 Demographic features and of each group. Baseline variable
Endometrial polyp group (n = 15)
Unexplained infertile group (n = 5)
Fertile group (n = 5)
P
Age, years BMI Infertility duration, years Size of polyp, mm Number of polyp
27 21 4 17 2
29 (24–34) 20 (18–23) 4 (4.0–3.6) – –
28 (23–31) 21 (18–24) – – –
NS
(20–33) (19–23) (5–4.0) (9–40) (1–4)
NS – –
Values are presented as mean and SD unless otherwise stated; NS = non-significant.
Results The demographic characteristics of each group were presented in Table 1. Each group of patients had normal body mass index. All endometrial tissue samples and hysteroscopically excised endometrial polyps underwent histological evaluation; endometrial polyps were identified, and biopsy samples revealed normal secretory endometrium. The mean endometrial NF-kB1 (p105) level was significantly higher in women with polyp before the removal of polyp compared to unexplained infertile cases and fertile control cases (P < 0.001 and P < 0.001, respectively). The mean endometrial NF-kB1 (p105) levels were similar in the unexplained infertile cases with fertile women (P = 0.66). Surgical removal of the polyp decreased the mean endometrial NF-kB1 (p105) levels to the level of those with unexplained infertile and fertile cases (Table 2). Immunohistochemical analysis confirmed the ELISA findings in each group. The increased NF-kB p65 (Rel A) immunoreactivity was predominantly localized to the cytoplasm of luminal and glandular epithelial cells in the polyp group (Fig. 1). After surgical removal of polyp, the mean H-Score of endometrial NF-kB p65 (Rel A) expression significantly decreased to a level, which is similar to that of the unexplained infertile and fertile groups. Further, there was no statistically significant difference in the mean H-Score of endometrial NF-kB p65 (Rel A) expression between the unexplained infertile and fertile groups (P = 0.74). Comments Different underlying molecular defects might be involved in the mechanisms of endometrial polyp-associated sub-fertility. Several studies have shown a significant improvement in pregnancy rate in subfertile or infertile women who underwent hysteroscopic polypectomy [24–26]. The result of one randomized controlled trial concerning effect of endometrial polypectomy on sub-fertility
has demonstrated that pregnancy rate considerably increased in women who underwent endometrial polypectomy [27]. Another prospective study comparing concentrations of endometrial markers in consecutive endometrial flushing fluid prior to with post hysteroscopic polypectomy has shown a significant increase in IGFBP-1, TNF alpha and osteopontin concentrations after hysteroscopic polypectomy [28]. Further, a case-control study has reported a significant lower mRNA expression of HOXA10 and HOXA11 in endometrium from women with polyps than controls [6]. Although the synthesis and secretion of NF-kB was studied for human proliferative, secretory, menstruel and eutopic endometrium [7,8,29] there are no comprehensive data concerning the concentration of NF-KB in the endometrium of women with polyp. It was postulated that the presence of endometrial polyp in uterine cavity may alter endometrial signaling pathways lead to impaired endometrial receptivity; therefore, removal of polyps will have beneficial impact on endometrial receptivity [6]. In the present study NF-kB1 expression and NF-kB p65 immunoreactivity in the endometrium was significantly higher in women with polyp compared to unexplained infertile and fertile controls. Hysterescopic polypectomy resulted in a significant decrease in endometrium NF-kB1 and NF-kB p65 activity. The reduced endometrial NF-kB1 after hysterescopic polypectomy suggests that high NF-kB1 activity may disturb transcription of mediators involved in physiological immunological and cell survival processes as well to modulate transcription of proteins involved in implantation. Therefore endometrial NF-kB expression may contribute to polypassociated sub-fertility. A limitation of the current study is that we did not evaluate the DNA binding or IkB phosphorylation of NF-kB in the endometrium of cases with endometrial polyp and controls Therefore, increased NF-kB expression does not mean increased NF-kB activity and we are not able to comment on whether the elevated NF-kB level and expression reflected to altered gene transcription. A separate limitation of this study is about the HSCORE method. SPi(i + 1) formula has 2 major sources of bias,
Table 2 Comparisons of NF-kB1 (p50/105) and H-Score of immunohistochemical NF kappa B/65 (RelA) expression in the groups. NF-kB (p50/105) concentrations (ng/ mg tissue)
I – Endometrial polyp (n = 15) II – Unexplained infertile (n = 5) III – Fertile group (n = 5)
I vs. II I vs. III II vs. III
Preoperative
Postoperative
0.24 0.12 0.07 0.23 0.08 0.03
0.13 0.13 – –
P1
0.001* NA NA
H-Score of NF kappa B/65 (RelA) expression Preoperative
Postoperative
4.12 0.56 2.81 0.77 2.87 0.40
2.33 0.76 – –
P2
0.001* NA NA
NF-kB (p50/105) concentrations (ng/mg tissue)
H-Score of NF kappa B/65 (RelA) expression
P value
P value
Preoperative
Preoperative
0.001* 0.001* 0.66
0.001* 0.001* 0.74
Data are presented as mean and SD. Comparisons between each outcome group with fertile group (ANOVA test with post hoc Tukey’s procedure for continuous variables). P1 represent for comparison of NF-kB1 (p50/105) between the groups; P2 represent for comparison of H-Score of immunohistochemical NF kappa B/65 (RelA) expression between the groups. * P < 0.05; NA = not applicable.
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Fig. 1. Immunohistochemical staining of NF kappa B/65 (Rel A) in endometrium from women with endometrial ployp before (A) and after (B) hysterscopic polypectomy; unexplained infertile (C) and fertile (D) controls. Inlet (A) shows intense glandular NF kappa B/65 staining in the endometrium from women with polyp before surgery significantly decreased after surgery (B; circle). The intensity of glandular NF kappa B/65 staining in the endometrium from women with unexplained infertile (C; arrow) was similar to fertile control (D; arrow). (E) Positive control (third trimester placenta showing circumferential cytoplasmic positivity (arrow) in the trophoblastic cells).
including the percentage of the area examined and the perceived intensity of the staining. Therefore, the skill and experience of the observer could influence the results obtained. To avoid this handicap, HSCORE performed by an expert in the field with 20+ years of experience in reading immunohistochemical analyses. Embryo implantation in human endometrium is associated with inflammatory-like responses [30]. The endometrial epithelium expresses several mediators which may be regulated by the NF-kB [31,32]. The NF-kB signal transduction pathway is involved in the up-regulation of inflammatory genes, including cyclooxygenase-2 and chemokines may have a role in the control of the inflammatory events associated with implantation [33,34]. A previous study showed that Rel A, a component of the NF-kB, is present in the glandular epithelium and provided evidence that the activation of NF-kB in these cells may be responsible for the increase in the expression of interleukin 6 and leukemia inhibitory factor during the implantation window [14]. Increased endometrial NF-kB expression in women with polyp may be one of the possible underlying mechanisms of sub-fertility. Activation of inflammatory transcription factors could compete with some genes such as FOXO1 or HOXA10 [35] and disrupt the interaction between the embryo and endometrium. Moreover, inflammation is associated with free radical production, which in turn will alter the post-translational code of the progesterone receptors [36] which may interact with the NF-kB pathway [13] and disturb embryo implantation. It has been reported that the expression of genes responsible for endometrial receptivity fail to rise during the implantation window in cases with hydrosalphinx in which is a typical inflammatory condition associated with a 50% reduction in
implantation rates [37–39]. Likewise, increased amount of NF-kB1 and NF-kB p65 expression in endometrium of women with polyp may suggest a mechanism to increase proinflammatory signaling to NF-kB at a time when local immunosuppression is necessary. Overactivation of NF-kB pathway may prevent inflammationinduced expression of implantation genes and possibly blastocysts binding to the epithelium. The present findings suggest that endometrial polyps increase the endometrial NF-kB activity and may influence implantation dysfunctions and thus polyp-associated sub-fertility. Histerescopic removal of polyps decreases endometrium NF-kB1 activity and may improve endometrial receptivity. Contributions Murat Bozkurt: Design of study, collection data, statistical analysis, writing the paper and intellectual contributions. Levent S¸ahin: Collection data, English language editing. Mustafa Ulas¸: Collection data. Funding The authors declare no relevant financial support requiring to disclosure. Disclosure The authors declare no relevant financial or religious interests requiring to disclosure.
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Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Hysteroscopic polypectomy decreases NF-kB1 expression in the mid-secretory endometrium of women with endometrial polyp Murat Bozkurt a,*, Levent S¸ahin a, Mustafa Ulas¸ b a b
Kafkas University School of Medicine, Department of Obstetrics and Gynecology, Kars, Turkey Fırat University School of Medicine, Department of Physiology, Elazıg˘, Turkey
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 July 2014 Received in revised form 8 March 2015 Accepted 31 March 2015
Objectives: Endometrial polyps have been identified as a possible factor for sub-fertility, their implication on endometrial NF-kB1 activity; the central regulator of the immune system gene expression involved in implantation has yet been little studied. We evaluated the NF-kB1 level and NF-kB p65 expression in the endometrium before and after hysteroscopic removal of the endometrial polyp during the mid-secretory phase. Study design: Infertile women with polyp (n = 15) were enrolled. Unexplained infertile patients with normal endometrium (n = 5) and healthy fertile women (n = 5) were recruited as controls. Endometrial samples were obtained before and 4 months after the hysteroscopic polypectomy. NF-kB1 levels were analyzed by ELISA in the endometrium of all groups before and after polypectomy. The H-Score method was used to evaluate immunohistochemical NF-kB p65 expression in the endometrium. Results: NF-kB1 expression and NF-kB p65 immunoreactivity in the endometrium were significantly higher in women with polyp compared to unexplained infertile and fertile controls. Hysteroscopic polypectomy resulted in a significant decrease in endometrium NF-kB1 and NF-kB p65 activity. Conclusions: Endometrium of women with endometrial polyp has abnormalities in expression of NFkB1 and NF-kB p65 which may contribute to endometrial receptivity and so polyp related sub-fertility. Hysteroscopic polypectomy may help to the normalization of endometrial NF-kB concentrations. ß 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: NF-kB1 Endometrial polyps Hysteroscopic polypectomy
Introduction Endometrial polyps, which are benign, hyperplastic-localized overgrowths of glands and stroma have been reported in 15–25% of infertile women and in about 1.4% of women undergoing in vitro fertilization (IVF) [1–3]. The mechanism by which endometrial polyp influences fertility is not clearly understood yet; however, it has been suggested the endometrial polyps may be associated with mechanical interference with sperm transport, embryo implantation or aberrant expression of endometrial markers consisting of HOXA, insulin growth factor binding protein 1 (IGFBP-1), tumor necrotizing factor alpha (TNF alpha) [4–6]. Nuclear factor-kappa B (NF-kB) is a transcription factor involved in endometrial pathologies and known to be a proinflammatory, mitogenic and antiapoptotic factor in many cell types [7–9]. Its function was recognized in TNF and IL1B receptors signal transduction pathways [10,11].
¨ niversitesi Kampu¨su¨ Sag˘lık Aras¸tırma ve * Corresponding author at: Kafkas U Uygulama Hastanesi, Kars, Turkey. Tel.: +90 5322279072/5056330044; fax: +90 0474 225 14 30. E-mail address: [email protected] (M. Bozkurt). http://dx.doi.org/10.1016/j.ejogrb.2015.03.032 0301-2115/ß 2015 Elsevier Ireland Ltd. All rights reserved.
Little is known about endometrial NF-kB activity in women with endometrial polyp. Basal NF-kB activity has been demonstrated in normal human endometrium and endometrial cells [8,10]. Recent clinical study has demonstrated that physiologic endometrial NF-kB activation in the three different phases of the menstrual cycle [7]. During menstruation, NF-kB is activated in the glandular epithelium and endothelium of the endometrium [12]. Estrogen and progesterone receptors may interact with the NF-kB protein in a repressive way [13]. Alterations of the NF-kB expression were shown in eutopic endometrium of endometriosis patients [8,14]. The NF-kB peptides construct homodimers or heterodimers from five subunits: p50/p105 (NF-kB1), p52/p100 (NF-kB2), p65 (RelA), c-Rel, and RelB. Rel-A/NF-kB1 heterodimer is the more commonly found in vivo and Rel-A is the subunit that most frequently participates in the active NF-kB complex [15,16] NFkB1 (p105) and NF-kB2 (p100) are converted to p50 and p52, respectively, which can pass to the nucleus and bind to DNA for activation of gene transcription. However, p65 (Rel A) can readily translocate to the nucleus without any conversion. NF-kB dimers are located in the cell cytoplasm bound to its inhibitory protein IkBa, which blocks the nuclear translocation of NF-kB. In response
M. Bozkurt et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 189 (2015) 96–100
to an extracellular signal, NF-kB cascade is activated and IkBa is phosphorylated allowing the release of NF-kB [16,17] dimers capable of binding to DNA, translocate to the nucleus and activate the transcription of several target genes [18,19]. Although NF-kB pathway was indicated to be active in the normal endometrium of healthy women [8,10,14], its expression in endometrium of women with endometrial polyp and the effect of hysteroscopic polypectomy on the endometrial NF-kB activity is unknown. In the present study, we attempted to investigate whether hysteroscopic polypectomy alters the NF-kB expression in the endometrium of women with endometrial polyp. We evaluated the NF-kB1 level and NF-kB p65 expression in the endometrium before and after hysteroscopic removal of the endometrial polyp during the mid-secretory phase. In addition, to assess the specificity of the results, patients with healthy endometrium included as controls. Materials and methods This prospective interventional study was carried out in Kafkas University Medical Faculty. The study protocol was approved by the Institutional Ethical Committee for Research on Human Subjects. Informed written consent form was obtained from all the women. We evaluated the NF-kB1 subunit level and NF-kB p65 subunit expression in the endometrial tissues obtained prior to hysteroscopy and respective endometrial tissues after the hysteroscopic polypectomy during mid-luteal phase in 15 infertile women with endometrial polyp and 5 control cases with unexplained infertility with normal endometrium. Five fertile women enrolled as the control groups had at least two children, and had no history of infertility or habitual abortion. All the women were recruited to the study had regular menstrual cycles, ranged between 21 and 35 days. All participants were required to meet the following inclusion criteria: (1) age ranging from 18 to 35 years; (2) regular menstrual cycles; (3) presence of endometrial polyp on Transvaginal ultrasonography, which was also confirmed on saline infusion sonohysterogram; 4) not taking any hormonal medications or treatment with an effect on endometrium at least 3 months before surgery. The exclusion criteria were: (1) having any confounding medical conditions known to affect endometrial receptivity such as endometriosis/endometrioma, submucosal fibroids, polycystic ovarian syndrome (PCOS) or hydrosalpinges; (2) presence of any other endometrial pathology including ashermann syndrome, endometrial cancer or endometrial hyperplasia; (3) diagnosis of pelvic inflammatory disease at the time of the study; (4) previous history of endometrial surgery; (5) absence of any systemic and/or rheumatologic disease which may lead to an inflammation. All women had two-dimensional transvaginal ultrasound scan (Voluson 730 Pro; GE Healthcare Kretztechnik, Zipf, Austria) using high frequency transducers of 7.5 MHz. This was followed by saline infusion hysterosonography for the confirmation of endometrial polyps. Subsequently, all women recruited for the study had endometrial sampling through pipelle curettage (Type IIA Endometrial Suction Curette, Jiangsu Suyun Med. Co., Ltd) performed prior to the commencement of polypectomy in the secretory phase of menstrual cycle. The endometrial specimen was divided for pathological evaluation and laboratory investigation. When endometrial polyp was present, it is excised under hysteroscopic guidance. The blood samples were obtained from all participants at the time of endometrial biopsy. The ones with endometrial polyp or unexplained infertility underwent a second procedure 4 months after the surgical removal of the polyp. The secretory phase was calculated as the 7–9 days after the ultrasonographic confirmation of ovulation and was confirmed by endometrial
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histological dating. Endometrial dating was performed by an independent pathologist experienced in gynecological pathology. Endometrial samplings were evaluated according to the histopathological criteria of Noyes et al. [20]. An out of date biopsy was defined as a lag of 3 days between the chronological and the histological day [21]. NF-kB1 determination in the endometrial tissues In all women with endometrial polyp and control subjects, endometrial NF-kB1 (p105) subunit concentrations were measured by Enzyme-Linked ImmunoSorbent Assay (ELISA). This immunoassay kit (NF-kB ELISA kit, USCN Life Sci Inc) allows for in vitro quantitative determination of human NF-kB1 (p105) concentrations in serum, plasma, urine, tissue homogenates, cell culture supernatants and other biological fluids. For this assay, each endometrial sample was rinsed with 1X PBS to remove excess blood, and subsequently, weighted as a wet weight. Then, the clean endometrial samples were homogenized in 1 mL of 1X PBS and stored overnight at a temperature 20 8C. After two freeze-thaw cycles were performed to break the cell membranes, the homogenates were centrifuged for 5 min at 4000 rpm. The supernatant was stored at 80 8C to avoid loss of bioactivity and contamination. The detection range of kit was 0.312–20 ng/ mL. The intra-(within day) and inter-assay (between days) coefficients of variation for endometrial NF-kB1 (p105) were <10% and <12%, respectively. The results of NF-kB1 (p105) concentrations detected by immunoassay kit were divided by the weights of endometrial tissue. The results were presented as ng/ mg tissue. Immunohistochemical method for NF-kappaB/p65 Four micrometer paraffin sections on poly-L-lysine coated slides were used after drying in an oven for 1 h at 60 8C. The sections were de-waxed in xylene, rehydrated in ethanol and then, incubated for 10 min in 3% hydrogen peroxide to block endogenous peroxidase. After washing in phosphate buffer saline, the sections incubated 8 min at ultra V block. The immunoreaction was performed for 60 min with ready to use NF Kappa B/p65 (Rel A) Ab-1 antibody (NeoMarkers, Labvision corp. CA, USA). After washing in PBS, slides were incubated with horseradish peroxidase kit. For negative control, the endometrial tissue was incubated with non-immune rabbit serum in place of the primary antibody. Finally, the preparations were developed in AEC chromogen counterstained with hematoxylin and mounted with aqueous-mount. Third trimester human placenta served as the positive control. To evaluate immunhistochemical NF-kB p65 (Rel A) expression in the endometrium, the H-Score method was used [22]. This immunohistochemical semi-quantitative method consists of the percentages of positively stained cells multiplied by a weighted intensity of staining: H-Score = SPi (i + 1), where Pi is the percentage of stained cells in each intensity category (0–100%), and i is the intensity indicating weak (i = 1), moderate (i = 2) or strong staining (i = 3) [23]. Statistical analysis The normality distribution of data was performed by using Kolmogorov–Smirnov test and all variables were skewed normally. The continuous variables were analyzed by ANOVA test with post hoc Tukey’s procedure and Mann–Whitney U test. A P value <0.05 was considered significant. The results are expressed as mean and standard deviation (SD). The data was analyzed by using the Statistical Package for Social Sciences software 21.0 for Windows package software (SPSS IBM, New York, USA).
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Table 1 Demographic features and of each group. Baseline variable
Endometrial polyp group (n = 15)
Unexplained infertile group (n = 5)
Fertile group (n = 5)
P
Age, years BMI Infertility duration, years Size of polyp, mm Number of polyp
27 21 4 17 2
29 (24–34) 20 (18–23) 4 (4.0–3.6) – –
28 (23–31) 21 (18–24) – – –
NS
(20–33) (19–23) (5–4.0) (9–40) (1–4)
NS – –
Values are presented as mean and SD unless otherwise stated; NS = non-significant.
Results The demographic characteristics of each group were presented in Table 1. Each group of patients had normal body mass index. All endometrial tissue samples and hysteroscopically excised endometrial polyps underwent histological evaluation; endometrial polyps were identified, and biopsy samples revealed normal secretory endometrium. The mean endometrial NF-kB1 (p105) level was significantly higher in women with polyp before the removal of polyp compared to unexplained infertile cases and fertile control cases (P < 0.001 and P < 0.001, respectively). The mean endometrial NF-kB1 (p105) levels were similar in the unexplained infertile cases with fertile women (P = 0.66). Surgical removal of the polyp decreased the mean endometrial NF-kB1 (p105) levels to the level of those with unexplained infertile and fertile cases (Table 2). Immunohistochemical analysis confirmed the ELISA findings in each group. The increased NF-kB p65 (Rel A) immunoreactivity was predominantly localized to the cytoplasm of luminal and glandular epithelial cells in the polyp group (Fig. 1). After surgical removal of polyp, the mean H-Score of endometrial NF-kB p65 (Rel A) expression significantly decreased to a level, which is similar to that of the unexplained infertile and fertile groups. Further, there was no statistically significant difference in the mean H-Score of endometrial NF-kB p65 (Rel A) expression between the unexplained infertile and fertile groups (P = 0.74). Comments Different underlying molecular defects might be involved in the mechanisms of endometrial polyp-associated sub-fertility. Several studies have shown a significant improvement in pregnancy rate in subfertile or infertile women who underwent hysteroscopic polypectomy [24–26]. The result of one randomized controlled trial concerning effect of endometrial polypectomy on sub-fertility
has demonstrated that pregnancy rate considerably increased in women who underwent endometrial polypectomy [27]. Another prospective study comparing concentrations of endometrial markers in consecutive endometrial flushing fluid prior to with post hysteroscopic polypectomy has shown a significant increase in IGFBP-1, TNF alpha and osteopontin concentrations after hysteroscopic polypectomy [28]. Further, a case-control study has reported a significant lower mRNA expression of HOXA10 and HOXA11 in endometrium from women with polyps than controls [6]. Although the synthesis and secretion of NF-kB was studied for human proliferative, secretory, menstruel and eutopic endometrium [7,8,29] there are no comprehensive data concerning the concentration of NF-KB in the endometrium of women with polyp. It was postulated that the presence of endometrial polyp in uterine cavity may alter endometrial signaling pathways lead to impaired endometrial receptivity; therefore, removal of polyps will have beneficial impact on endometrial receptivity [6]. In the present study NF-kB1 expression and NF-kB p65 immunoreactivity in the endometrium was significantly higher in women with polyp compared to unexplained infertile and fertile controls. Hysterescopic polypectomy resulted in a significant decrease in endometrium NF-kB1 and NF-kB p65 activity. The reduced endometrial NF-kB1 after hysterescopic polypectomy suggests that high NF-kB1 activity may disturb transcription of mediators involved in physiological immunological and cell survival processes as well to modulate transcription of proteins involved in implantation. Therefore endometrial NF-kB expression may contribute to polypassociated sub-fertility. A limitation of the current study is that we did not evaluate the DNA binding or IkB phosphorylation of NF-kB in the endometrium of cases with endometrial polyp and controls Therefore, increased NF-kB expression does not mean increased NF-kB activity and we are not able to comment on whether the elevated NF-kB level and expression reflected to altered gene transcription. A separate limitation of this study is about the HSCORE method. SPi(i + 1) formula has 2 major sources of bias,
Table 2 Comparisons of NF-kB1 (p50/105) and H-Score of immunohistochemical NF kappa B/65 (RelA) expression in the groups. NF-kB (p50/105) concentrations (ng/ mg tissue)
I – Endometrial polyp (n = 15) II – Unexplained infertile (n = 5) III – Fertile group (n = 5)
I vs. II I vs. III II vs. III
Preoperative
Postoperative
0.24 0.12 0.07 0.23 0.08 0.03
0.13 0.13 – –
P1
0.001* NA NA
H-Score of NF kappa B/65 (RelA) expression Preoperative
Postoperative
4.12 0.56 2.81 0.77 2.87 0.40
2.33 0.76 – –
P2
0.001* NA NA
NF-kB (p50/105) concentrations (ng/mg tissue)
H-Score of NF kappa B/65 (RelA) expression
P value
P value
Preoperative
Preoperative
0.001* 0.001* 0.66
0.001* 0.001* 0.74
Data are presented as mean and SD. Comparisons between each outcome group with fertile group (ANOVA test with post hoc Tukey’s procedure for continuous variables). P1 represent for comparison of NF-kB1 (p50/105) between the groups; P2 represent for comparison of H-Score of immunohistochemical NF kappa B/65 (RelA) expression between the groups. * P < 0.05; NA = not applicable.
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Fig. 1. Immunohistochemical staining of NF kappa B/65 (Rel A) in endometrium from women with endometrial ployp before (A) and after (B) hysterscopic polypectomy; unexplained infertile (C) and fertile (D) controls. Inlet (A) shows intense glandular NF kappa B/65 staining in the endometrium from women with polyp before surgery significantly decreased after surgery (B; circle). The intensity of glandular NF kappa B/65 staining in the endometrium from women with unexplained infertile (C; arrow) was similar to fertile control (D; arrow). (E) Positive control (third trimester placenta showing circumferential cytoplasmic positivity (arrow) in the trophoblastic cells).
including the percentage of the area examined and the perceived intensity of the staining. Therefore, the skill and experience of the observer could influence the results obtained. To avoid this handicap, HSCORE performed by an expert in the field with 20+ years of experience in reading immunohistochemical analyses. Embryo implantation in human endometrium is associated with inflammatory-like responses [30]. The endometrial epithelium expresses several mediators which may be regulated by the NF-kB [31,32]. The NF-kB signal transduction pathway is involved in the up-regulation of inflammatory genes, including cyclooxygenase-2 and chemokines may have a role in the control of the inflammatory events associated with implantation [33,34]. A previous study showed that Rel A, a component of the NF-kB, is present in the glandular epithelium and provided evidence that the activation of NF-kB in these cells may be responsible for the increase in the expression of interleukin 6 and leukemia inhibitory factor during the implantation window [14]. Increased endometrial NF-kB expression in women with polyp may be one of the possible underlying mechanisms of sub-fertility. Activation of inflammatory transcription factors could compete with some genes such as FOXO1 or HOXA10 [35] and disrupt the interaction between the embryo and endometrium. Moreover, inflammation is associated with free radical production, which in turn will alter the post-translational code of the progesterone receptors [36] which may interact with the NF-kB pathway [13] and disturb embryo implantation. It has been reported that the expression of genes responsible for endometrial receptivity fail to rise during the implantation window in cases with hydrosalphinx in which is a typical inflammatory condition associated with a 50% reduction in
implantation rates [37–39]. Likewise, increased amount of NF-kB1 and NF-kB p65 expression in endometrium of women with polyp may suggest a mechanism to increase proinflammatory signaling to NF-kB at a time when local immunosuppression is necessary. Overactivation of NF-kB pathway may prevent inflammationinduced expression of implantation genes and possibly blastocysts binding to the epithelium. The present findings suggest that endometrial polyps increase the endometrial NF-kB activity and may influence implantation dysfunctions and thus polyp-associated sub-fertility. Histerescopic removal of polyps decreases endometrium NF-kB1 activity and may improve endometrial receptivity. Contributions Murat Bozkurt: Design of study, collection data, statistical analysis, writing the paper and intellectual contributions. Levent S¸ahin: Collection data, English language editing. Mustafa Ulas¸: Collection data. Funding The authors declare no relevant financial support requiring to disclosure. Disclosure The authors declare no relevant financial or religious interests requiring to disclosure.
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