{"paper_id":"3b9886c8-eb86-4d4a-9cb1-74abd38142af","body_text":"Endometriosis is one of the most common gynecologic diseases, and its clinical\nmanifestations include dyspareunia, dysmenorrhea, chronic pelvic pain, and\ninfertility ( Howard, 2009 ;  ASRM, 2006 ). It is present in approximately 10%\nof women in fertile age ( Härkki  et\nal ., 2010 ), but among infertile patients, endometriosis\nprevalence can be as high as 25-50% ( Dong  et\nal ., 2013 ). Although it is known for decades that\nendometriosis is associated with infertility, the pathogenesis of this association\nis still not completely understood ( Härkki\net al., 2010 ). Nevertheless, several studies have shown that\nendometriosis is associated with abnormal folliculogenesis, luteal insufficiency\n( Cunha-Filho  et al .,\n2003 ;  2001 ), and abnormal embryo\nfertilization and implantation ( Fadhlaoui  et\nal ., 2014 ), which contribute to infertility. Regarding lower\nimplantation rates, it has been shown that some endometrial receptivity markers may\nbe impaired in the endometrium of these patients ( Lu\n et al ., 2013 ;  Dimitriadis  et al ., 2006 ;  Dong et al., 2013 ).\nLeukemia inhibitory factor (LIF) and integrin αvβ3 are well-known\nendometrial receptivity markers. LIF is a polyfunctional pleiotropic cytokine, which\nbelongs to the IL-6 family ( Tawfeek  et\nal ., 2012 ).  Stewart\n et al . (1992)  have shown for the first time that\nmice lacking a functional LIF gene fail to implant blastocysts in their endometrium.\nSince then, many authors have demonstrated that proper LIF expression by the\nendometrium is also important for female fertility ( Lalitkumar  et al ., 2013 ;  Mikolajczyk  et al ., 2007 ). LIF is expressed\nmainly in the glandular and luminal epithelium of the endometrium during the window\nof implantation ( Vogiagis  et al .,\n1996 ). Impaired LIF expression has been shown in patients with\nunexplained infertility, uterine anatomical abnormalities, and endometriosis ( Mikolajczyk et al., 2007 ;  Hasegawa  et al ., 2012 ;  Dimitriadis  et al ., 2006 ).\nIntegrin αvβ3 is a transmembrane glycoprotein that has been extensively\nstudied in the human endometrium. It can be detected in the epithelial layer of mice\nand the human endometrium during the window of implantation ( Apparao  et al ., 2001 ;  Liu  et al ., 2013 ;  Franchi  et al ., 2011 ). Several studies have\ndemonstrated that decreased expression of this protein can impair embryo\nimplantation in vitro ( Zhang  et\nal ., 2011 ;  Kaneko  et\nal ., 2011 ;  Kang  et\nal ., 2014 ;  Schmitz\n et al ., 2014 ). Moreover, patients with hydrosalpinx,\nunexplained infertility and recurrent pregnancy loss have presented impaired\nintegrin αvβ3 expression ( Daftary\n et al ., 2007 ;  Tei\n et al ., 2003 ;  Germeyer  et al ., 2014 ). Nevertheless, the relationship\nbetween this integrin and endometriosis has been controversial in the literature\n( Lessey  et al ., 1994 ;\n Ordi  et al ., 2003 ;  Casals  et al ., 2012 ).\nMilk fat globule epidermal growth factor 8 (MFG-E8) is a novel protein recently\nassociated to the implantation process ( Mirkin\n et al ., 2005 ).  Franchi  et al.  (2011)  demonstrated for the first time\nthat MFG-E8 is expressed in the human endometrial epithelium and that it is\nup-regulated during the window of implantation. Besides, MFG-E8 histological\nsequence in epithelial cell location suggests luminal secretion of the protein\n( Franchi et al., 2011 ). Moreover, we\nhave demonstrated that blocking this protein in an in vitro trophoblast/endometrial\nepithelium model can impair the implantation process ( Schmitz  et al ., 2014 ). Nevertheless, this\nprotein has never been studied in the endometrium of infertile patients.\nUltimately, what constitutes adequate expression of endometrial receptivity markers\nintegrin αvβ3 and LIF is still controversial in endometriosis\npatients, while MFG-E8 has not yet been studied in these patients. Considering that\nendometriosis patients may have an impaired implantation process, this study aimed\nto compare the endometrial expression of MFG-E8, integrin αvβ3, and\nLIF between patients with infertility and endometriosis and healthy fertile patients\n(controls) during the window of implantation.\n\nThis prospective case-control study was carried out in the Department of\nGynecology of the Hospital de Clínicas de Porto Alegre and in the Jones\nInstitute for Reproductive Medicine. The STROBE guideline was used ( von Elm  et al ., 2007 ).\nFive patients with peritoneal endometriosis and infertility (case group) and four\nhealthy fertile patients (control group) were recruited between January 2014 and\nNovember 2014 to take part in the study. Diagnosis of infertility was considered\nwhen the couple had not conceived after 12 months of contraceptive-free\nintercourse ( ASRM, 2008 ). The case group\nincluded consecutive patients diagnosed with peritoneal endometriosis during\nlaparoscopic surgery meeting the enrollment criteria described below. The degree\nof endometriosis was categorized based on to the revised classification of\nendometriosis of the American Society for Reproductive Medicine ( ASRM, 1997 ). Patients submitted to elective\nlaparoscopic tubal ligation were invited to join the control group. These\nindividuals were ruled out for endometriosis (by laparoscopy), had a history of\nnormal fertility, and were non-smokers.\nThe individuals in the case and control groups had to meet the following\nenrollment criteria: (i) age between 25 and 38 years (ii) regular menstrual\ncycles, (iii) presence of both ovaries, (iv) no endocrine disorder and (v) no\nfamily history of genetic disease. The case group also had normal sperm\nanalysis. Patients with abnormal ovarian reserve (antral follicle count under\n10), obesity (BMI ≥30), history of miscarriage, and smokers were\nexcluded.\nThe local ethics committee approved this study and a written informed consent was\nprovided to all subjects prior to sample collection (IRB equivalent).\nEndometrial biopsies were performed during the natural cycle during the putative\nwindow of implantation (LH+8 to LH+10). LH + 1 was considered the day of\novulation ( Kao  et al .,\n2003 ). Ovulation was detected by serial ultrasound exams, and it was\ndefined as the 24-h period that separated the identification of a mature,\npre-ovulatory follicle on one scan and either of the following on the next scan:\n(i) follicle rupture; (ii) presence of an early corpus luteum; (iii) presence of\nfree fluid in the cul-de-sac ( Ecochard\n et al ., 2013 ).\nThe biopsies were performed with a Pipelle ®  catheter (CCD,\nParis, France). Each endometrial biopsy specimen was fixed in formalin and\nembedded in paraffin in preparation for histological examination and detection\nof MFG-E8, integrin αvβ3, and LIF by immunostaining.\nParaffin-embedded tissue blocks of the endometrial biopsy specimens were cut into\n5-µm sections. Immunohistochemistry was performed as previously described\n( Chandra  et al .,\n2013 ). Briefly, the slides were deparaffinized, dehydrated, and\nrehydrated followed by immersion in retrieval solution 1:10 (Dako). Endogenous\nperoxidases were quenched with 3% hydrogen peroxide for 10 min and non-specific\nbinding sites were blocked with 1.5% normal goat or horse serum (Vector\nLaboratories) for 30 min at room temperature. The sections were then covered by\nappropriate dilutions of primary antibody, MFG-E8 (Abcam) 1:100, integrin\nαvβ3 (Santa Cruz Biotechnologies) 1:10 or LIF (Sigma) 1:750, and\nplaced in a refrigerator overnight. After primary antibody incubation, the\nsections were washed with PBS and incubated with anti-mouse or anti-rabbit\nsecondary antibody (Vector Lab) at a dilution of 1:200 for 30 min at room\ntemperature. After incubation with secondary antibodies, the tissues were\nincubated with ABC reagent (Vector Laboratories) for 30 min, followed by PBS\nwash. The antigens were localized by incubation with AEC chromogen-substrate\n(skyTek Labs) and finally mounted with Accergyl mounting media (Accurate\nChemicals) with a cover slip. Negative controls included sections treated with a\nsimilar dilution of a non-immune IgG1 (isotype control, eBioscience, San Diego,\nCA, USA). Representative images were photographed with an Olympus BX50\nmicroscope using an Olympus DP70 Q-color 3 camera ( Franchi  et al ., 2011 ).\nThe assessment of staining intensity and distribution for integrin and LIF was\nmade using the semi-quantitative histologic score (HSCORE) system. The HSCORE\nwas calculated using the following equation: HSCORE: Σ Pi (i + 1), where\ni represents the intensity of staining on a scale from 1 to 3 (1 for weak, 2 for\nmoderate, and 3 for strong staining) and Pi the percentage of stained\nendometrial stromal and epithelial cells for each intensity, varying from\n0-100%, as previously described ( Lessey\n et al ., 2006 ).\nThe assessment for staining intensity and distribution for MFG-E8 was made using\ncomputerized image analysis using a modification of  Fuhrich  et al . (2013) , with the aid of\nMetamorph TM  (Molecular Devices) instead of software program Image\nJ. The original method was found to be highly correlated with HSCORE values\nobtained by expert evaluators. Color images collected using an Olympus 20x\nobjective were automatically thresholded rather than manually circumscribed, and\nthe thresholded areas from three different 20x fields were averaged and\nsubtracted from total white (255) values on an eight byte scale, as published\nearlier ( Fuhrich et al., 2013 ). A size\nfilter setting was used to exclude stray pixels so that only cell structures\nwere analyzed.\nThe statistical analysis was carried out using software package SPSS 18.0. The\nmeasure of central tendency used was the mean and the measure of variability was\nthe standard deviation ( Lambalk  et\nal ., 2004 ). Categorical variables in the two groups were\ncompared using the 2-sided Pearson Chi-squared test. Continuous variables were\ncompared using Student's t-test. Differences with a  p -value\n<5% were considered significant.\n\nFive patients with endometriosis and infertility were included in the case group and\nfour healthy fertile patients were enrolled in the control group.  Table 1  shows patient demographic\ncharacteristics. In the case group, four patients had stage I endometriosis and one\nhad stage II endometriosis.\nDemographic characteristics of women with infertility and endometriosis (case\ngroup) and healthy fertile women (control group).\nAll values are means±SD;\nBMI = body mass index;\nAFC = antral follicle count;\nStudent’s t-test;\nChi-squared test.\nImmunohistochemistry confirmed previous findings and revealed that MFG-E8 was\npredominantly located in glandular epithelium. MFG-E8´s receptor, integrin\nαvβ3, was localized in the epithelial, as well as in the stromal\nlayer. Immunolocalization showed that LIF was preferentially observed in the luminal\nepithelium ( Figure 1 ).\nFigure 1 Immunohistochemical localization of MFG-E8, integrin αvβ3,\nand LIF in the human endometrium. Representative images of: (A) negative\ncontrol from the control group (endometriosis and infertility), (B) and\nfrom the case group (healthy fertile patients); (C) MFG-E8 staining from\nthe control group, (D) and from the case group; (E) Integrin\nαvβ3 staining from the control group, (F) and from the\ncase group; and (G) LIF staining from the control group and from the\ncase group (H).\nImmunohistochemical localization of MFG-E8, integrin αvβ3,\nand LIF in the human endometrium. Representative images of: (A) negative\ncontrol from the control group (endometriosis and infertility), (B) and\nfrom the case group (healthy fertile patients); (C) MFG-E8 staining from\nthe control group, (D) and from the case group; (E) Integrin\nαvβ3 staining from the control group, (F) and from the\ncase group; and (G) LIF staining from the control group and from the\ncase group (H).\nFigure 2  shows the mean HSCORE for MFG-E8,\nintegrin αvβ3 and LIF. Statistically significant differences were\nfound for MFG-E8 ( p <0.001) and LIF ( p =0.033)\nbetween the control and case groups. No significant difference was found for\nintegrin αvβ3 ( p =0.084).\nFigure 2 MFG-E8 (A), integrin αvβ3 (B) and LIF (C) HSCORE in healthy\nfertile patients (control group) and in patients with endometriosis and\ninfertility. ** p <0.001 compared with controls.\n* p <0.05 compared with controls.\nMFG-E8 (A), integrin αvβ3 (B) and LIF (C) HSCORE in healthy\nfertile patients (control group) and in patients with endometriosis and\ninfertility. ** p <0.001 compared with controls.\n* p <0.05 compared with controls.\n\nThis study showed for the first time that patients with endometriosis and infertility\nhave increased MFG-E8 expression in the endometrium during the putative window of\nimplantation. Patients also had decreased LIF expression during the same period.\nNevertheless, no differences were found in the expression of integrin\nαvβ3 between the groups.\nMFG-E8 is a glycoprotein identified for the first time in 1990 ( Stubbs  et al ., 1990 ). Since then, it has been\nextensively studied in many physiological and pathological processes, especially in\nthe immune system ( Matsuda  et al .,\n2011 ;  Oba  et al .,\n2011 ). A possible role for MFG-E8 in the implantation process was pointed\nout for the first time in 2005, when it was found up-regulated during the window of\nimplantation ( Mirkin  et al .,\n2005 ). After that, other studies suggested its participation in the\nimplantation process ( Franchi  et\nal ., 2011 ;  Schmitz  et\nal ., 2014 ); however, to the best of our knowledge, it had\nnever been studied in the endometrium of infertile patients.\nMFG-E8 is known to be involved in inflammatory processes ( Komura  et al ., 2009 ), and it is regulated by\nTNF-α in the human endometrium ( Yu  et\nal ., 2014 ). Moreover, endometriosis patients are known to\npresent chronic inflammation ( Zhao  et\nal ., 2015a ;  Berkes  et\nal ., 2014 ). Therefore, our hypothesis that\nendometriotic/infertile patients have increased MFG-E8 expression during the window\nof implantation was confirmed in the current study. We had previously shown in an\nin-vitro model that blocking MFG-E8 impairs the implantation process ( Schmitz  et al ., 2014 ).\nNevertheless, it seems that either the down regulation ( Sinningen  et al ., 2015 ) or the up-regulation\nof MFG-E8 may impair physiological processes ( Zhao\net al., 2015b ;  Yamamoto  et\nal ., 2014 ).\nMFG-E8 receptor integrin αvβ3 is a well-established endometrial\nreceptivity marker. Nevertheless, its expression in the endometrium of individuals\nwith endometriosis has been a topic of controversy in the literature. Our study\nfound no differences in the expression of integrin αvβ3 between the\ngroups, although levels tended to be lower in the case group\n( p =0.084). In 1994, a large study with 241 individuals with\nendometriosis showed that subjects with stage I/II endometriosis had decreased\nintegrin expression ( Lessey  et al .,\n1994 ). However, the authors included biopsies after day 19 of the cycle,\nand not only during the window of implantation. Moreover, the study did not mention\nif all patients were infertile. On the other hand, two smaller studies failed to\nfind such difference ( Ordi  et al .,\n2003 ;  Casals  et al .,\n2012 ) after analyzing integrin αvβ3 expression during the\nwindow of implantation.\nA possible cause for the decrease in integrin αvβ3 expression is the\nfact that these patients had impaired HOXA10 production, which is responsible for\nthe expression of subunit β3 ( Taylor\n et al ., 1999 ;  Lu\n et al ., 2013 ;  Zhu\n et al ., 2013 ). The overexpression of MFG-E8 may also\ndown regulate the production of its receptor.\nAnother important endometrial receptivity marker that seems to be impaired in the\nendometrium of individuals with endometriosis is LIF. As also seen in our results,\n Dimitriadis  et al. \n(2006)  previously described diminished expression of LIF in patients with\nstage I/II endometriosis during the window of implantation. In addition,  Alizadeh  et al.  (2011)  also\nreported impaired LIF expression in a similar group of patients. Nevertheless, a\nstudy with 14 endometriosis patients showed no difference in LIF levels in uterine\nflushings when compared to fertile controls (21 patients) ( Mikolajczyk  et al ., 2007 ).\nThe method used in this study is a modification of a previously published technique\n( Fuhrich  et al ., 2013 )\nthat allows for faster and more accurate results; the thresholding function in\nMetamorph TM  removes holes in background objects, thus allowing the\nquantification of thresholded areas only. Manually circumscribing objects as in\n Fuhrich et al., 2013  is much slower and\nmay also include holes, which reduces the averaged staining intensity in a variable\nfashion depending on the individual object's hole area. Size filter settings in\nMetamorph TM  were also used to exclude stray pixels and small debris,\nenabling a more accurate measurement of cell structures. Unfortunately, not all\ntypes of staining can be adequately analyzed by this method, as integrin and LIF.\nThis is why the traditional H-score was used in this study.\nAlthough it may be argued that the study sample was relatively small, statistical\nsignificant differences were found. Moreover, the results agreed with what we were\nexpecting, based on biological plausibility. Although we did not measure serum or\nurinary LH levels to further define the ovulation day, previous studies have defined\novulation only with ultrasound ( Ecochard  et\nal ., 2013 ).\nIn conclusion, our study showed for the first time that patients with endometriosis\nand infertility have altered MFG-E8 expression in the endometrium during the\nputative window of implantation. Moreover, we also demonstrated that these patients\nhave diminished LIF, as shown before. Nevertheless, there was no difference in the\nexpression of integrin αvβ3, although in the case group levels tended\nto be lower. The endometrium of patients with endometriosis must be more thoroughly\ncharacterized to improve the understanding of the association between this condition\nand infertility.","source_license":"public-domain-us","license_restricted":false}