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RBMOnline - Vol 19. No 5. 2009 737–744 Reproductive BioMedicine Online; on web 22 September 2009 Article The effect of hysteroscopic polypectomy on the concentrations of endometrial implantation factors in uterine flushings Dr Ben-Nagi has carried out research under the supervision of Mr Jurkovic in early pregnancy complications and endometrial implantation factors in women with endometrial polyps, submucous fibroids and with history of previous Caesarean sections
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   ArticleThe effect of hysteroscopic polypectomy on theconcentrations of endometrial implantationfactors in uterine flushings Dr Ben-Nagi  Dr Ben-Nagi has carried out research under the supervision of Mr Jurkovic in earlypregnancy complications and endometrial implantation factors in women with endometrialpolyps, submucous fibroids and with history of previous Caesarean sections. This researchhas been presented in international conferences and published in peer-reviewed journals.J Ben-Nagi 1,3 , J Miell 2 , J Yazbek 1 , T Holland 1 , D Jurkovic 11 Early Pregnancy and Gynaecology Assessment Unit, King’s College Hospital, Denmark Hill, London SE5 8RX, UK; 2 Department of Endocrinology, University Hospital Lewisham, London SE13 6LH, UK; 3 Correspondence: e-mail-address:  Abstract Endometrial polyps have been associated with infertility and early pregnancy loss. The aim of this study was to inves-tigate the effect of hysteroscopic polypectomy on the concentrations of endometrial implantation factors in uterineflushings. Pre-menopausal women with a certain diagnosis of endometrial polyp on contrast-enhanced transvaginalultrasound scan were recruited into this prospective study. In all women, paired samples of uterine flushings wereobtained on the same day of the menstrual cycle prior to and post hysteroscopic polypectomy. Enzyme-linked immu-noassays were performed to analyse glycodelin, interleukin-6 (IL-6), interleukin-10 (IL-10), tumour necrosis factor  a (TNF a ) and osteopontin, whilst immunoradiometric assay was used to analyse insulin-like growth factor bindingprotein-1 (IGFBP-1). Concentrations of IGFBP-1, TNF a  and osteopontin in uterine flushings were significantlylower in the mid-secretory phase prior to polypectomy in comparison to the measurements obtained after completesurgical removal of the polyp ( P   < 0.05). There were no differences in the concentrations of glycodelin, IL-6 andIL-10 in paired samples prior to and post-polypectomy. The presence of endometrial polyps is associated withdecreased mid-secretory concentrations of IGFBP-1, TNF a  and osteopontin, which are reversed following surgicalpolypectomy. Keywords : cytokines, endometrial polyps, glycodelin, IGFBP-1, osteopontin Introduction Polyps are one of the most common endometrial abnormal-ities with a reported prevalence of 9–25% in the generalpopulation of women (Anastasiadis  et al. , 2000; Sherman et al. , 2002). They are often asymptomatic but they cansometimes cause menstrual irregularities such as intermen-strual bleeding. Some studies have suggested that endome-trial polyps may be associated with infertility and earlypregnancy loss (Sanders, 2006; Tur-Kaspa  et al. , 2006).However, the pathophysiological processes by which polypsmay affect women’s fertility are not clear. It has been pos-tulated that polyps affect the endometrial environment bycausing abnormal bleeding or by presenting an abnormalsite for implantation. Assessment of the uterine cavityand removal of endometrial polyps is routinely performedin women undergoing fertility treatment. There is some evi-dence to suggest that pregnancy rates in infertile women areimproved following hysteroscopic polypectomy (Varasteh et al. , 1999).Glycodelin is one of the most abundantly secreted endome-trial glycoproteins. Its secretion is limited to the mid- andlate luteal phase of the cycle, which suggests that it plays RBMOnline  - Vol 19. No 5. 2009 737–744 Reproductive BioMedicine Online; on web 22 September 2009   2009 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK 737  a role in implantation and early pregnancy development (Li et al. , 1993; Seppala  et al. , 2001). Studies by Richlin  et al. (2002) have shown that concentrations of glycodelin inuterine flushings are raised in women with polyps and fib-roids compared with the control group in the proliferativephase of the cycle.Insulin growth factor binding protein-1 (IGFBP-1) isthought to have major effects on decidualization, implanta-tion and trophoblast invasion in the female reproductivetract. The expression of IGFBP-1 is tightly controlled andit is predominant in the secretory phase endometrium anddecidualized stromal endometrial cells (Fowler  et al. , 2000).Cytokines are produced by the human endometrium andare important mediators between the embryo and deciduaduring implantation. Piccinni  et al.  (1998) have shown thatthere are significantly higher concentrations of CD4 + T-helper 2 cells producing interleukin-4 (IL-4), interleu-kin-6 (IL-6) and interleukin-10 (IL-10) in decidua fromwomen with normal reproductive histories compared withwomen with recurrent miscarriages. Lim  et al.  (1998)reported a predominant type-2 cytokine expression profile(IL-4, IL-6 and IL-10) and lack of type-1 cytokine expres-sion (interferon- c , IL-2 and tumour necrosis factor- b ) innormal fertile women.Tumour necrosis factor- a  (TNF a ) is known to be one of themost versatile cytokines. It is speculated that TNF a  pro-motes DNA synthesis in the early proliferative phase andparticipates in cell differentiation and tissue remodelling,which is required to support embryonic attachment(Terranova  et al. , 1995). TNF a  also facilitates apoptosisand therefore initiates menstrual shedding (Tabibzadeh et al. , 1995).Osteopontin is a progesterone-regulated glycoprotein com-ponent of the extracellular matrix (Apparao  et al. , 2003). Itis secreted by the glandular epithelium of mammalian uteriand later by the decidualized stroma. Osteopontin is recog-nized by the integrin family to facilitate cell–cell attachmentand adhesion (Apparao  et al. , 2001).Although endometrial polyps have been identified as a pos-sible cause of infertility, their effect on endometrial implan-tation factors has been little studied so far. This prospectivestudy examined changes in the concentrations of endome-trial markers in paired samples of uterine flushings priorto and post hysteroscopic polypectomy. It was postulatedthat removal of polyps will have a significant impact onthe concentrations of endometrial markers, which shouldhelp us to understand better the pathophysiological mecha-nisms of impaired endometrial receptivity in the presence of endometrial polyps. Materials and methods Non-pregnant,pre-menopausalwomenattendingthegynae-cology out-patient clinic at King’s College Hospital, Lon-don, UK with a history of abnormal vaginal bleeding whowere found to have an endometrial polyp on transvaginalultrasound scan were invited to participate in this prospec-tive interventional study. The inclusion criteria were: (i)age between 18 and 45 years; (ii) regular menstrual cycles;(iii)notusing anyhormonal contraceptionor treatmentwithan effect on endometrium; (iv) presence of an endometrialpolyp on two-dimensional ultrasound scan, which was alsoconfirmed on saline infusion sonohysterosonography; (v)absence of any other endometrial pathology on transvaginalscan; (vi) ability to give written consent; and (vii) acceptanceof a second examination at follow-up, which was performedon the same cycle day of the first hysteroscopy. The studywasapprovedbyKing’sCollegeHospitalethicsandresearchand development committee.All women had a two-dimensional transvaginal ultrasoundscan performed by gynaecologists with expertise intransvaginal scanning using high-frequency transducers of 5–7.5 MHz (Aloka SSD-5000; Aloka, Tokyo, Japan). Thiswas followed by saline infusion sonohysterosonography toconfirm the presence of the endometrial polyp (Lee  et al. ,2006).Subsequently, all women included in the study had uterineflushings, performed prior to the commencement of the hys-teroscopy and polypectomy. Uterine flushings were per-formed in the dorsal lithotomy position. A Cusco’sbivalve speculum was inserted in the vagina in order toexpose the cervix. The cervix was cleaned with sterile salineto remove any visible potential contaminants. An 8 F pae-diatric Foley balloon catheter (Schering AG, Berlin) wasthen passed into the uterine cavity through the cervix andthe balloon was then inflated with 2 ml of air. Under simul-taneous transvaginal ultrasound scan guidance, the balloonwas withdrawn to lie above the level of the internal cervicalos. This was identified as the reflection of the urinary blad-der. Five aliquots of 2 ml sterile 0.154 mol/l sodium chlo-ride solution were then sequentially injected and aspiratedfrom the uterine cavity over approximately 10 s. Ultra-sound scan guidance was used to confirm that the fluiddid not enter the Fallopian tubes or cervical canal, withre-aspiration being done when the fluid was noted to be dis-tending the upper aspect of the uterine cavity. The five 2-mlaliquots were collected in five separate universal specimenpots, immediately frozen and stored at   20  C.Following hysteroscopic polypectomy women were askedto attend for a follow-up appointment, which included atransvaginal scan and repeat uterine flushings. Women wereadvised to wait until they had at least two menstrual bleeds(at least one complete normal physiological cycle) betweenthe hysteroscopy and the follow-up visit. The follow-upappointments were timed to coincide with the same dayof the cycle when the hysteroscopy and pre-polypectomyuterine flushings were performed.Prior to the commencement of any assay, the five 2-ml ali-quots obtained per patient were thawed at room tempera-ture and then pooled. The total pooled volume of uterineflushing was thawed for analysis on a single occasion only.The total volume of fluid retrieved from the uterine cavityper patient was recorded. The protein content was  Article - Hysteroscopic polypectomy and endometrial implantation factors -  J Ben-Nagi et al. RBMOnline  738  calculated in the total volume of uterine fluid recovered.The amount of total protein (mg/l) measured was adjustedper ml of fluid obtained in all the samples. Glycodelin,IGFBP-1 and osteopontin were expressed as ng per mg/ml of protein and IL-6, IL-10 and TNF a  was expressedas pg per mg/ml of protein. Laboratory assays Fluid protein The Bayer Advia method for the measurement of fluid pro-tein is a non-enzymatic assay (Siemens Medical SolutionsDiagnostics Limited, UK). Under acid conditions and inthe presence of molybdate ions, proteins form a blue-col-oured complex with pyrogallol red. The absorbance of thiscomplex is measured at 596 nm. The sensitivity of the assayis the lowest concentration that can be distinguished fromzero is 10 mg/l. Glycodelin Glycodelin was measured using a solid-phase enzyme-linked immunoassay (ELISA) based on the sandwich prin-ciple (DRG Instruments, Germany). When a qualitycontrol value differed by greater than 10% from the previ-ous assay means, the assay was repeated. The assay wastested for specificity, by the manufacturer against HCG(2000 IU/l), prolactin (200  l g/l), human placental lactogen(20  l g/l) and alpha feto protein (300 mIU/ml) withundetectable glycodelin concentrations in all cases. Thesensitivity of this assay is 6 ng/ml. Both the intra-assayand inter-assay coefficients of variation were 9.4% at ananalyte concentration of 118.39 ng/ml and 3.9% at ananalyte concentration of 99.6 ng/ml, respectively. IGFBP-1 IGFBP-1 was measured by immunoradiometric assay(IRMA). This is a non competitive assay where the analyteis‘sandwiched’betweentwoantibodies(DiagnosticsSystemsLaboratories,Webster,USA).Thesensitivitywas0.33 ng/ml.The intra- and inter-assay coefficients of variation were 5.2%atananalyteconcentrationof5.23 ng/mland3.5%atanana-lyte concentration of 5.16 ng/ml, respectively. IL-6, IL-10 and TNF a IL-6, IL-10 and TNF a  were analysed using commerciallyavailable solid-phase sandwich ELISA (Diaclone,Besancon, France). The sensitivity of the IL-6 assay was2 pg/ml with intra- and inter-assay coefficients of variationof 4.8% at an analyte concentration of 52.2 pg/ml and 5.5%at an analyte concentration of 53 pg/ml, respectively. ForIL-10, the sensitivity was 5 pg/ml with intra- and inter-assay coefficients of variation of 5% at an analyte concen-tration 55.1 pg/ml and 8.6% at a concentration of 58.3 pg/ml, respectively.The sensitivity of the TNF a  assay was 8 pg/ml. The intra-and inter-assay coefficients of variation were 5.3% at ananalyte concentration of 171.7 pg/ml and 12% at a concen-tration of 163 pg/ml, respectively. Osteopontin The osteopontin assay employed the quantitative sandwichenzyme immunoassay technique (R and D Systems, Minne-apolis, USA). Three samples were tested 20 times on oneplate to assess intra-assay sensitivity. The intra-assay coef-ficients of variation for samples 1, 2 and 3 were 4% at ananalyte concentration of 2.3 ng/ml, 2.6% at a concentrationof 4.9 ng/ml and 2.9% at a concentration of 9.3 ng/ml,respectively. A further three samples were tested in 40 sep-arate assays to assess inter-assay sensitivity. The inter-assaycoefficients of variation for samples 1, 2 and 3 were 6.6% atan analyte concentration of 2.36 ng/ml, 5.7% at an analyteconcentration of 4.81 ng/ml and 5.4% at a concentration of 9.17 ng/ml, respectively. Statistical analysis All statistical analyses were carried using Medcalc version9.2.0.2 (Medcalc Software, Mariakerke, Belgium). Pairedsample  t -test was usedto analyse the difference between totalvolumes retrieved in patients with and without polyps as thedata forvolume of uterine flushingretrieved (ml) is normallydistributed. Wilcoxon signed rank test was used to assess thedifference between glycodelin, IGFBP-1, IL-6, IL-10, TNF a and osteopontin in the pre- and post-polypectomy groups asthedatafortheseendometrialproteinsandcytokinesarenotnormally distributed. Spearman rank correlation was usedfor non-parametric correlation to examine the relationshipbetweenglycodelin,IGFBP-1,IL-6,IL-10,TNF a andosteo-pontin concentrations and protein content and volume of uterine flushing retrieved.  P   < 0.05 was considered statisti-cally significant. Results A total of 20 women were recruited into the study. Of these,8/20 (40%) failed to attend the post-operative transvaginalultrasound scan and uterine flushings and thus wereexcluded from the final data analysis. In the remaining 12(60%) women who attended for post-operative follow-up,11 (92%, 95% CI 65–99) women had a single polyp andone (8%, 95% CI 1–35) had two polyps on ultrasound scan.The median polyp volume was 4.7 cm 3 (range 1.32–16.99).Women’s median age was 36 years (range 29–45), gravidity2 (range 0–3) and parity 0 (range 0–2). The median lengthof the cycle was 28 days (range 26–28). Of the 12 patients,eight (67%, 95% CI 40–94) were in the luteal phase of thecycle (range 20–21) and were included in the analysis of the mid-secretory concentrations. The remaining four(33%, 95% CI 6–60) patients were in the proliferative phaseof the cycle (range 5–14).Uterine flushings were performed on the same day of thecycle prior and post-polypectomy (median 20; range5–21). The median time between the initial and follow-upvisit was 56 days (range 52–84). The median volume of fluid  Article - Hysteroscopic polypectomy and endometrial implantation factors -  J Ben-Nagi et al. RBMOnline  739  retrieved prior to polypectomy was 4.0 ml (range 1–10),which was not significantly different from the median vol-ume of 4.25 ml (range 3–8) following polypectomy. Theprocedure for uterine flushings was well-tolerated by allthe patients and in none of the cases was the procedureabandoned because of discomfort, pain or any othercomplications.The histologies of the hysteroscopically excised endometrialpolyps were all benign. The maturation status of the endo-metrium was described for five (42%, 95% CI 19–68) cases:four proliferative and one secretory endometrium.The protein content (mg) in the total volume (ml) of uterineflushing retrieved was analysed in the pre-polypectomygroup.Therewasnosignificantcorrelationdetectedbetweenthe total protein concentration and retrieved volume( r  =  0.02,). Glycodelin showed a stronger correlation withproteincontent( r  = 0.76,  P   = 0.01)thanwithrecoveredvol-umeofuterineflushings( r  =  0.07,notsignificant).Il-6alsoshowed a stronger correlation with total protein content( r  = 0.73,  P   = 0.03) than with the volume retrieved( r  = 0.13, not significant). However, the correlation did notreach statistical significance between the IGFBP-1, IL-10,TNF a  and osteopontin and total protein or total volume.Thus, all markers were expressed as per mg of total proteinas it appeared a more reliable indicator of the efficiency of the flushing process than the volume retrieved.There was no correlation between expression levels of theimplantation markers with age and parity. Glycodelin The overall median glycodelin prior to polypectomy was169.5 ng/mg/ml  10  –3 (range 64–902), which was not sig-nificantly different from 202 ng/mg/ml  10  –3 (range 16– 1886) post polypectomy. Similar results were obtainedwhen the analysis was limited only to the measurements,which were performed in the mid-secretory phase ( Table1 ,  Figure 1 ). IGFBP-1 The overall median IGFBP-1 prior to polypectomywas 1 ng/mg/ml  10  –3 (range 0–4), which increased to8 ng/mg/ml  10  –3 (range 4–29) post polypectomy( P   = 0.002). The changes in IGFBP-1 concentrationsremained significant when the analysis was limited to themid-secretory phase only ( P   = 0.03) ( Table 1 ,  Figure 2 ). IL-6, IL-10 and TNF a ThemedianIL-6andIL-10priortopolypectomygroupwere7.5 pg/mg/ml  10  –3 (range1–141)and3.5 pg/mg/ml  10  –3 (range 0–36), respectively. In the post-polypectomy group,the values were 13.5 pg/mg/ml  10  –3 (2–38) for IL-6 and11.5 pg/mg/ml  10  –3 (1–32) for IL-10. Similar results werealso obtained when the analysis was performed in the mid-secretoryphase( Table1 , Figures3 and 4 ).ThemedianTNF a increased after removal of polyps, being 5 pg/mg/ml  10  –3 (0–73) pre-polypectomy versus 69.5 pg/mg/ml  10  –3 (0–289) post-polypectomy ( P   = 0.01)). TNF a  changesremainedsignificantlydifferentwhencomparingitssecretionbetween the two groups in the mid-secretory phase only( P   = 0.03) ( Table 1 ,  Figure 5 ). Osteopontin Before polypectomy the median osteopontin was 1 ng/mg/ml  10  –3 (range 0–21) compared with 4 ng/mg/ml  10  –3 (range 0–69) after surgery but the difference was not Table 1 . Median concentrations of endometrial proteins and cytokines in the pre- and post-polypectomy groups in the mid-secretory phase. Pre-polypectomy Post-polypectomy  P -value Glycodelin (ng/mg/ml  10  3 ) 169.5 (64–785) 202.5 (60–1886) NSIGFBP-1 (ng/mg/ml  10  3 ) 1 (0–4) 7.5 (4–29) 0.03IL-6 (pg/mg/ml  10  3 ) 6 (1–141) 13.5 (2–38) NSIL-10 (pg/mg/ml  10  3 ) 3.5 (0–27) 13 (1–32) NSTNF a  (pg/mg/ml  10  3 ) 5 (0–16) 94 (0–289) 0.03Osteopontin (ng/mg/ml  10  3 ) 1 (0–21) 4 (0–69) 0.04 Values are median (range); IGFBP-1, insulin-like growth factor binding protein-1; IL, interleukin; NS, not statistically signifi-cant; TNF a , tumour necrosis factor- a . Figure 1.  Presence of glycodelin (ng/mg/ml  10  –3 )throughout the cycle in the pre- and post-polypectomygroups. Spearman’s rank correlations ( r ) for the pre- andpost-polypectomy groups are 0.21 and 0.04, respectively.  Article - Hysteroscopic polypectomy and endometrial implantation factors -  J Ben-Nagi et al. RBMOnline  740
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