{"paper_id":"bafbb16c-a895-4f66-ae4a-d5d82cadc989","body_text":"R E S E A R C H Open Access\nAge-related trends in anti-Mullerian\nhormone serum level in women with\nunilateral and bilateral ovarian\nendometriomas prior to surgery\nDorota Nieweglowska 1, Iwona Hajdyla-Banas 2, Kazimierz Pitynski 1*, Tomasz Banas 1, Oliwia Grabowska 3,\nGrzegorz Juszczyk 4, Artur Ludwin 1 and Robert Jach 5\nAbstract\nBackground: Endometriosis is a well-known cause of infertility, and the anti-Mullerian hormone (AMH) is an\naccepted biomarker of ovarian reserve and response to artificial reproductive technology procedures. The present\nstudy was a prospective analysis of age-dependent AMH serum concentration in women with bilateral and\nunilateral ovarian endometriomas before therapy onset compared with healthy controls.\nMethods: This prospective cross-sectional study included 384 women aged 18 –48 years. AMH serum concentration\nwas assessed between days 3 and 6 of the menstrual cycle in 78 patients with bilateral and 157 patients with\nunilateral ovarian endometriomas and compared with 149 healthy controls. Ovarian endometriosis was confirmed\nhistopathologically, and data were presented as medians with interquartile range (IQR).\nResults: Stage III endometriosis was diagnosed in 53.2 %, stage IV in 18.3 %, stage V in 23.4 % and stage VI in 5.4 %\nof the patients. Patients with bilateral ovarian endometriomas showed the lowest median AMH levels compared\nwith patients suffering from unilateral ovarian endometriosis (0.55; IQR: 0.59 vs. 2.00; IQR: 2.80; p < 0.001) and the\ncontrol group (0.55; IQR: 0.59 vs. 2.84; IQR: 3.2; p < 0.001). Median AMH concentration values were not significantly\ndifferent between patients with unilateral ovarian endometriosis and the healthy controls (2.00; IQR: 2.80 vs. 2.84;\nIQR: 3.2; p = 0.182). A strongly negative correlation between AMH levels and age was confirmed in healthy\nindividuals (R = −0.834; p < 0.001) and women with unilateral ovarian endometriomas (R = −0.774; p < 0.001).\nPatients with bilateral ovarian endometriosis showed a significantly negative but only moderate correlation\nbetween AMH levels and age (R = −0.633; p < 0.001), which was significantly lower than in the healthy controls\n(R = −0.633 vs. R= −0.834; p = 0.006) but not in the patients with unilateral ovarian endometriosis (R = −0.663 vs.\nR-0.774; p = 0.093). Based on a multivariate regression analysis, only bilateral localization of ovarian endometrial\ncysts ( p = 0.003) and patient age ( p < 0.001), but not left/right localization of unilateral cyst or cyst volume, were\nnegatively associated with AMH serum concentration.\nConclusion: According to our data, unilateral ovarian endometriosis had a moderately negative and nonsignificant\neffect on AMH-based ovarian reserve evaluated prior to surgery, irrespective of age. In contrast, the ovarian reserve\nwas significantly reduced in women with bilateral ovarian endometriomas.\nKeywords: Anti-Mullerian hormone, AMH, Ovarian endometrioma, Endometriosis\n* Correspondence: pitynski@wp.eu\n1Department of Gynecology and Oncology, Jagiellonian University, Chair of\nGynecology and Obstetrics, Krakow, 21 Kopernika Str, 30-501 Krakow, Poland\nFull list of author information is available at the end of the article\n© 2015 Nieweglowska et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0\nInternational License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and\nreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to\nthe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver\n(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.\nNieweglowska et al. Reproductive Biology and Endocrinology  (2015) 13:128 \nDOI 10.1186/s12958-015-0125-x\n\nBackground\nInfertility is an increasing medical and socioeconomic\nproblem affecting up to 15 % of couples [1]. Fortunately,\nmany of these patients have an opportunity for parent-\ning, due to rapidly developing diagnostics and artificial\nreproduction techniques (ARTs). Among many factors\nresponsible for fertility problems, endometriosis is often\nrecognized in women diagnosed with infertility [2].\nEndometriosis affects up to 10 % of women of repro-\nductive age, and its pathogenesis, despite numerous\nstudies, has not been elucidated [3]. As many as 40 % of\npatients with endometriosis will face fertility problems\n[4], which can be caused by direct ovarian destruction,\nimpotence of fallopian tubes, due to intraperitoneal ad-\nhesions or impaired follicle growth, and ovulation disor-\nders resulting from local pelvic inflammation [5]. Women\ndiagnosed with endometriosis suffer from endometrial\npolyps and have a high rate of implantation failure [6].\nMoreover, endometriomas may damage otherwise healthy\novarian tissue. Hughesdon described the ovarian cortex\nnear an endometrioma as st retched and disorganized\nwith evidence of smooth muscle metaplasia [7]. Re-\ncent studies have identified several toxic agents, such\nas pro-inflammatory cytokines, reactive oxygen species\n(ROS) and iron deposits in endometriotic fluid [8, 9].\nBecause the barrier separating cyst fluid from normal\novarian tissue is 1 mm thick and composed of fibror-\neactive tissue and the ovarian cortex, endometriotic\nfluid is thought to be highly harmful to the surround-\ning cells and the healthy ovarian cortex tissue near\nthe endometrioma. ROS and cytokines cause fibrosis\nof the ovarian tissue and a reduction in cortex-specific\nstromal cells [9]. Subsequently, the fibrosis together with\nthe ROS-triggered decrease in angiogenesis and capillary\nloss in the ovarian cortex impair follicle nutrition and may\nbe responsible for lower follicular density and functional\nfollicle loss in the ovaries with endometriosis [10]. These\nfindings support the theory that endometriomas cause\novarian damage before surgical treatment.\nMany women with pelvic endometriosis and concomi-\ntant infertility require ART procedures. However, patients\nsuffering from endometriosis have also been reported to\nshow a decreased ovarian reserve compared to healthy in-\ndividuals of the same age [11, 12]. The evidence of endo-\nmetriosis was also proven to be a risk factor for a reduced\nresponse to controlled ovarian stimulation in women\nundergoing ART [13]. The anti-Mullerian hormone level\nis a useful marker of the ovarian reserve and response\nused in fertility therapy [14] and allows individualized\ntreatment, reducing clinical risk of ART-related complica-\ntions and improving the pregnancy rate [15].\nAMH was identified as a glycoprotein dimer com-\nposed of two monomers of 72 kDa, each connected with\ndisulfide bridges and belongs to the transforming growth\nfactor β (TNFβ) family [16]. AMH is produced exclu-\nsively by granulosa cells of ovarian follicles and may have\na regulatory effect on the axial folliculogenesis [17]. Ex-\nperiments in mice suggest that AMH inhibits the growth\nof primary follicles and is involved in the growth regula-\ntion of antral, preantral and small follicles by inhibiting\ntheir sensitivity to follicle-stimulating hormone (FSH)\n[18]. AMH secretion is strongly associated with age, with\nproduction starting in the 36 th week of fetal life and\nreaching a peak in puberty before continuously decreas-\ning until menopause when often reaches undetectable\nvalues [19, 20]. Many studies confirmed that AMH levels\ndecline with age in peripartum and in patients with se-\nvere pelvic edometriosis but remains stable during the\nmenstrual cycle, which allows for its random determin-\nation irrespective of the cycle day [17, 21 – 24]. However,\nHadlow et al. reported variations in AMH serum con-\ncentration during the mens trual cycle with a gradual\ndecrease from the early-fol licular to the late-luteal\nphase of the menstrual cycle with peak values in the\nmid-follicular phase [25].\nAMH is an accepted biomarker of the ovarian reserve\nand response to ART procedures; however, a lack of ref-\nerence values established separately for female cohorts\nwith different ovarian pathology can be a major draw-\nback in its clinical utility. Based on our literature review,\nwe hypothesize that endometriomas themselves can im-\npair ovarian reserve irrespective of surgical treatment.\nTo clear this hypothesis, we set up a prospective study\nevaluating AMH levels in women with ovarian endomet-\nriosis. The aim of the study was a preoperative evalu-\nation of serum AMH concentration in women with\nunilateral and bilateral endometriomas, depending of the\nages of the patients.\nMethods\nAll women referred to the Gynecology and Oncology\nDepartment and Endocrine Gynecology Department of\nthe Jagiellonian University Medical College, Krakow,\nfrom January 2009 to December 2014, were construct-\nively and prospectively evaluated for this study. Inclusion\ncriteria were age (18 – 48 years) and bilateral or unilateral\novarian endometriosis diagnosed during laparoscopy or\nlaparotomy and confirmed histopathologically. We ex-\ncluded the following subjects: (1) pregnant women; (2)\npatients with previous excision of ovarian cysts; (3) pa-\ntients diagnosed with infertility (unless solely related to\nendometriosis or the male factor infertility); (4) patients\nwho had received hormonal treatment during the prior\n36 months; (5) patients diagnosed with endocrine disor-\nders; (6) patients suffering from chronic disease (defined\nas illness lasting 3 months or more, according to the\nU.S. National Center for Health Statistics); and (7) pa-\ntients with a history of malignancy. The control group\nNieweglowska et al. Reproductive Biology and Endocrinology  (2015) 13:128 Page 2 of 9\n\nconsisted of healthy volunteers who participated in the\nNational Screening Program against Cervical Cancer.\nControl group exclusion criteria included a history of (1)\ninfertility, (2) endometriosis, (3) early pregnancy loss, (4)\nhormonal disorders, (5) malignancy, and (6) pelvic/\nabdominal cavity surgery. All the participating women\nsigned an informed consent document, and the research\nwas approved by the Jagiellonian University Ethics Board\n(KBET/21/B/2009).\nDiagnostic procedures\nEvery woman meeting the inclusion criteria had her\nmedical history recorded and underwent a bimanual\npelvic examination by an experienced gynecology or\ngynecologic endocrinology consultant. Subsequently, a\ntransvaginal ultrasonography was performed using a\nVoluson 730 Pro equipped with a 6.5 MHz trans-vaginal\nprobe (General Electric Medical Systems, Kretztechnik,\nZipf, Austria) to evaluate the uterus and ovaries and look\nfor the presence of endometrial cysts. The volume of\neach endometrial cyst was evaluated in every case and\nexpressed in cm 3.\nSurgical treatment\nThe majority of the patients (98.3 %) with an initial diag-\nnosis of endometrial ovarian cysts underwent routine\nunilateral/bilateral laparoscopic cyst enucleation and ex-\ncision of peritoneal endometriosis. In three cases, a con-\nversion to laparotomy was performed due to severe\nintraoperative bleeding ( n = 1) and the need for partial\nlarge bowel resection and anastomosis ( n = 3). None of\nthe patients required hysterectomy or adnexectomy. The\nbleeding was controlled by bipolar coagulation (85.5 %)\nand ovarian suturing (14.5 %).\nHistopathological examination: Although histopatho-\nlogical evaluation of endometriosis visualized during sur-\ngery was not necessary to make the final diagnosis, all of\nthe excised specimens were examined postoperatively by\nexperienced pathologists using hematoxylin-eosin stain-\ning and immunohistochemistry, according to our routine\nprocedures. The postoperative diagnosis was based on\nthe final histopathological report confirming ovarian\nendometriosis.\nBlood analysis\nVenous blood samples were analyzed between days 3\nand 6 of the menstrual cycle to determine the AMH\nlevels. In patients with ovarian endometriomas, the aver-\nage time between blood collection and surgery was 33\ndays (range 3 – 47 days) as the vast majority of women\nwere scheduled for operation during the next menstrual\ncycle after blood collection. Immediately after collection,\nthe samples were centrifuged for 15 min at 1400 rpm.\nThe serum was aspirated and transferred to 1.5 mL\nEppendorf tubes and stored for up to 3 months at −80 °\nC. Baseline blood samples were collected between days 3\nand 7 of the menstrual cycle. The biochemical analysis\nwas performed using the enzyme-linked immunosorbent\nassay (ELISA) method. The 96-well plates (Biokom) were\nincubated with the blood serum (50 μL per well) for 12\nh at 4 °C. Each plate was washed with 3 x 200 μL PBS\n(pH 7.0) using an automatic scrubber. The plates were\nre-incubated with the primary antibody at a dilution of\n1:10,000 (Thermo Scientific) for 12 h at +4 °C. Next, the\nplates were incubated for 2 h with secondary antibody\nlabeled with horseradish peroxidase (HRP) and a 15 min\nincubation with 3,3 ′,5,5′-tetramethylbenzidine (TMB,\nThermo Scientific). Immediately after putting on the\nstop solution (1 nM HCl, Thermo Scientific), the plates\nwere read using an ELISA reader with KC Junior\nprogram. Measurements were performed in duplicate.\nStandardization curves included 8 steps at the stage\nr = 0.95. The serum AMH concentration was expressed\nin ng/mL, and the functional detection limits ranged from\n0.05 to 15.00 ng/mL.\nStatistical analyses\nThe Kolmogorov-Smirnov test was used to evaluate the\ndistribution of variables. To compare the variables with\nnormal distribution, an analysis of variance (ANOVA)\ntest was performed, and the data were presented as the\nmean values with standard deviation (SD). To analyze\ncontinuous data with a different distribution than\nnormal and quantitive variables, a chi square test or\nKruskal-Wallis ANOVA was used, and baseline charac-\nteristics were presented as median values with interquar-\ntile range (IQR). Post hoc exploration (Fisher ’ s least\nsignificant difference test) was performed if significant\ndifferences were found during the ANOVA analysis.\nMultiple regression was used to analyze the impact of\ndifferent clinical variables on the serum AMH level.\nBecause serum AMH concentration showed a skewed\ndistribution, logarithmic transformation was applied for\nfurther testing. Spearman ’ s correlation test was used to\nanalyze the relationship between AMH serum level and\nage, and the results were presented using Spearman ’ s\ncorrelation index (R). A p-value of 0.05 was considered\nto indicate statistical significance. All calculations were\nperformed using STATISTICA data analysis software,\nversion 10.0 (StatSoft, Inc. 2011, Tulsa, OK, USA).\nResults\nA total of 498 women were eligible for the study, and\n235 fulfilled the inclusion criteria (78 women with bilat-\neral and 157 with unilateral and ovarian endometriomas;\nFig. 1). Among the patients with endometriosis, 68 were\npart of a couple experiencing fertility problems, 86 had\npelvic pain syndrome (PPS), and 91 were diagnosed with\nNieweglowska et al. Reproductive Biology and Endocrinology  (2015) 13:128 Page 3 of 9\n\novarian endometriosis during a routine gynecological\nexamination and referred for further management. Male\nfactor infertility was diagnosed in 17 of the 68 infertile\npairs. In 51 couples, a comprehensive infertility examin-\nation revealed no potential causes of infertility apart\nfrom the presence of endometriomas. These women\nwere diagnosed with endometriosis-related infertility\nand included in the study. The control group consisted\nof 149 women with no gynecological disorders, a history\nof pelvic/abdominal surgery, pregnancy or chronic diseases\nand who attended cervical cancer screening programs.\nMore than half of the patients (53.2 %) suffering from\nendometriosis were diagnosed with stage III disease ac-\ncording to the European Society of Human Reproduction\nand Embryology (ESHRE) criteria [26]. Stage IV endomet-\nriosis was recognized in 18.3 % of women, and stages V\nand VI were confirmed in 23.4 % and 51 %, respectively.\nThere were no cases of stage I-II endometriosis because\nsuch patients do not present with an ovarian mass. In pa-\ntients with unilaterality, ovarian endometrial cysts were\npredominantly localized in the right ovary (T able 1). There\nwere no differences in ovarian endometrioma volume be-\ntween patients with bilateral and unilateral ovarian mass\n(T able 1). Women with bilateral and unilateral ovarian\nendometriosis suffered significantly more frequently with\npainful and irregular menstruation compared to healthy\nwomen (T able 1). Patients suffering from bilateral but not\nunilateral ovarian endometriomas presented significantly\nlower gravidity ( p < 0.001) and parity ( p = 0.003) com-\npared to the control group. No significant differences in\nmedian age, median age of first period, body mass index\n(BMI) or amount of menstrual discharge were noticed be-\ntween the groups analyzed (T able 1).\nPatients with bilateral ovarian endometriomas pre-\nsented the lowest median AMH levels, compared to\nwomen suffering from unilateral ovarian endometri-\nosis (0.55; IQR: 0.59 vs. 2.00; IQR: 2.80; p < 0,001)\nand the control group (0.55; IQR: 0.59 vs. 2.84; IQR:\n3.2; p < 0.001). Patients with ovarian unilateral endo-\nmetriosis also showed lower but insignificant median\nAMH levels compared to the control group (2.00; IQR:\n2.80 vs. 2.84; IQR: 3.2; p = 0.182). Based on multivariate\nregression, only bilateral localization of ovarian endo-\nmetrial cyst ( p = 0.003) and patient age ( p <0 . 0 0 1 )b u t\nnot localization or cyst volume were negatively associ-\nated with the AMH serum concentration.\nEligible patients n=498\nExcluded patients n=263\n- pregnancy  (n=9)\n- history of ovarian surgery (n=45)\n- infertility (not related to \nendometriosis) (n=23)\n- treated with hormones during the \npast 36 months (n=31)\n- with endocrine disorders  (n=66)\n- with chronic diseases (n=51) \n- with history of malignancy (n=5)\n- lack of AMH evaluation (n=19)\n- age over 48 years (n=14)\nRecruited patients: total n = 235\nBilateral ovarian \nendometriosis n=78\nHealthy individuals\nn=149\nUnilateral ovarian \nendometriosis n=157\nTotal number of included women: total n =384\nFig. 1 Flow diagram demonstrating the patient recruitment procedure\nNieweglowska et al. Reproductive Biology and Endocrinology  (2015) 13:128 Page 4 of 9\n\nAn age-related decrease in AMH levels was confirmed\nin the control group and in patients with unilateral and\nbilateral ovarian endometriomas (Fig. 2).\nA strong negative correlation between AMH levels and\nage was confirmed in the healthy controls (R = −0.834;\np < 0.001) and women with unilateral ovarian endomet-\nriosis (R = −0.774; p < 0.001). In women with bilateral\novarian endometriosis, a moderately negative correlation\nbetween AMH levels and age was observed (R = −0.663;\np < 0.001). Correlation indices for AMH levels and age\nTable 1 The clinical characteristics of patients with bilateral and unilateral ovarian endometrioas and the healthy women\nPatients with bilateral ovarian\nendometrimas (n = 78)\nPatients with unilateral ovarian\nendometrimas (n = 157)\nHealthy controls\n(n = 149)\np\nMedian age [yers] (IQR b) 35,50 (17,00) 32,00 (15,00) 32,00 (18,00) NS c\nMean BMI [kg/m 2] (±SDa) 22,72 (±1,61) 23,19 (±2,13) 23,41 (±2,51) NS c\nEndometrial cyst localization\nRight ovary 78 (100,00 %) 93 (59,23 %) NA e NAe\nLeft ovary 78 (100,00 %) 64 (40,77 %)\nMean volume of ovarian endometriomas [cm 3] 6,23 (±1,12) 6,41 (±1,18) NA e NSc\nMean age of first menstrual period [years] (±SD a) 11,52 (±1,52) 11,38 (±1,64) 11,45 (±1,81) NS c\nMean duration of menstrual cycle [days] (±SD a) 28,50 (±2,5) 29,50 (±3,0) 28,00 (±2,0) NS c\nMenstrual cycles 42 (53,85 %) / 116 (73,89 %) / 124 (83,22) / <0,001 d\nRegular/Irregular 36 (46,15 %) 41 (26,11 %) 25 (16,78 %)\nMenstrual cycles 38 (48,72) / 42 (26,75 %) / 32 (21,48) / <0,001 d\nPainful/painless 40 (51,28 %) 115 (73,25 %) 117 (78,52 %)\nMean duration of menstruation [days] 4,25 (±1,52) 4,12 (±1,31) 4,32 (±0,97) NS c\nType of menstrual bleeding NSc\nScant 7 (11,54 %) 12 (7,64 %) 8 (10,69 %)\nNormal 52 (66,67 %) 115 (73,25 %) 117 (75,52 %)\nHeavy 17 (21,79 %) 30 (19,11 %) 24 (13,79 %)\nMedian number of gestations (IQR b) 1,0 (1,0) 2 (2) 3 (2) 0,028 d\nMedian number of deliveries (IQR b) 0,5 (1) 2 (1) 3 (1) 0,016 d\naSD – standard deviation; bIQR – interquarlite range; cNS – statistically not significant; dstatistically significant value; eNA – data not available\nFig. 2 Age-related anti-Mullerian hormone (AMH) distribution in patients with bilateral ovarian endometriomas, unilateral ovarian endometriomas\nand the controls\nNieweglowska et al. Reproductive Biology and Endocrinology  (2015) 13:128 Page 5 of 9\n\ndid not differ significantly between women with unilateral\nendometriosis and the controls (R = −0.774 vs. R= −0.834;\np = 0.140). However, patients with bilateral ovarian endo-\nmetriomas showed a significantly weaker correlation\nbetween AMH levels and age compared to the con-\ntrols (R = −0.633 vs. R= −0.834; p = 0.006) but\nnot the patients with unilateral ovarian endometri-\nosis (R = −0.663 vs. R= −0.774; p = 0.093).\nFor further analysis of AMH serum level, participants\nwere divided according to age into the following 6\ngroups: 18 – 22, 23 – 27, 28 – 32, 33 – 37, 38 – 42 and 43 – 48\nyears. Serum AMH levels were then evaluated in\npatients with bilateral and unilateral ovarian endome-\ntriomas and compared to healthy controls in the same\nage-related groups. In all age groups, serum AMH con-\ncentration was the highest in healthy controls. However,\nin women aged 43 years and above, no significant differ-\nences in AMH levels were observed between patients\nwith bilateral ovarian endometriomas, unilateral ovarian\nendometriomas and controls. In young women aged\n18– 22 years, we observed significantly lower median\nAMH levels in patients with bilateral ovarian endo-\nmetriomas compared to the controls (0.82; IQR: 1.12\nvs. 4.63; IQR: 1.09; p < 0.001) and between patients\nwith bilateral ovarian endometriosis and unilateral\novarian endometriosis (0.82; IQR: 1.12 vs. 4.24; IQR:\n1.24; p = 0.036), while differences were not observed\nbetween unilateral ovarian endometriosis patients and\nthe controls (4.24; IQR: 1.12 vs. 4.63; IQR: 1.09; p <0 . 0 0 1 ;\nFig. 3). Additionally, women aged 23 – 27 years with bilat-\neral ovarian endometriomas showed significantly lower\nmedian serum AMH concentrations compared to patients\nwith unilateral ovarian endometriomas (0.55; IQR: 1.07 vs.\n4.10; IQR: 1.17; p < 0.001) and to controls (0.55; IQR: 1.07\nvs. 4.24; IQR: 0.44; p < 0.001), while no significant differ-\nences in median AMH levels were found between unilat-\neral ovarian endometrioma patients and the healthy\ncontrols (Fig. 3). Similar to previous groups in women\naged 28– 32 years, patients with bilateral ovarian endome-\ntriomas showed the lowest median AMH serum levels\ncompared to women with unilateral ovarian involvement\n(0.95; IQR: 0.37 vs. 2.25; IQR: 1.02; p = 0.001) and healthy\ncontrols (0.95; IQR: 0.37 vs. 3.00; IQR: 1.86; p <0 . 0 0 1 ) ,\nwith no significant differences observed between unilateral\novarian endometriomas and the healthy controls (Fig. 3).\nIn women aged 33 – 37 years, the lowest median AMH\nconcentration was observed in patients with bilateral ovar-\nian endometriomas compared to patients with unilateral\novarian endometriomas (0.49; IQR: 0.45 vs. 1.75; IQR:\n0.38; p < 0.001) and controls (0.49; IQR: 0.45 vs. 1.98; IQR:\n0.84; p < 0.001; Fig. 3). Additionally, in women aged\n38– 42 years, the significantly lowest median serum\nAMH concentration was in patients with bilateral\novarian endometriomas compared to patients with\nunilateral ovarian endometriomas (0.35; IQR: 0.30 vs.\n1.05; IQR: 0.70; p < 0.001) and the controls (0.35;\nIQR: 0.30 vs. 1.35; IQR: 0.90; p <0 . 0 0 1 ;F i g .3 ) .I n\ncontrast to younger patients, women aged 43 – 48\nyears showed no statistically significant differences in\nthe median AMH concentration between women with\nbilateral ovarian endometriomas (0.18; IQR: 0.19),\nunilateral ovarian endom etriomas (0.14; IQR: 0.78)\nand the controls (0.49; IQR: 0.8; Fig. 3).\nDiscussion\nIn our study, we confirmed a significant age-related\nAMH decrease in healthy women and patients with\novarian endometriomas, both bilateral and unilateral,\n18-22 23-27 28-32 33-37 38-42 43-48\nAge groups [years]\n0\n1\n2\n3\n4\n5\n6\n7Anty-Mullerian Hormon (AMH) level [ng/ml]\n 1. Bilateral ovarian endometriomas\n 2 Unilateral ovarian endometriomas\n 3. Controlsp= 0 , 0 3 6\np< 0 , 0 0 1 p *<0,001\np*<0,001\np*<0,001\np*=0,001\np*<0,001\np*<0,001\np*<0,001\np< 0 , 0 0 1*\n*\n*\nFig. 3 Differences in serum anti-Mullerian hormone (AMH) concentration levels between patients with bilateral and unilateral ovarian endometrio-\nmas compared to healthy individuals in different age groups. *statistical significance at p < 0.05\nNieweglowska et al. Reproductive Biology and Endocrinology  (2015) 13:128 Page 6 of 9\n\nbefore the onset of surgical therapy [27 – 30]. Patients\nwith unilateral ovarian endometriosis showed a signifi-\ncantly negative correlation between serum AMH level\nand age. Correlation indices were not significantly differ-\nent between these patients and the healthy controls. On\nthe contrary, patients with bilateral endometriomas\nshowed a significantly weaker correlation between AMH\nlevels and age, which differed significantly from pa-\ntients with unilateral endometriomas. Moreover, pa-\ntients under 43 years of age with bilateral ovarian\nendometriosis showed significantly lower serum AMH\nconcentration compared to patients with unilateral\novarian endometriosis and healthy controls. The vast\nmajority of our participants were diagnosed with\nmoderate endometriosis, and only a few with a severe\ndisease. Interestingly Shebl et al. reported significant\ndifferences in AMH levels between patients with severe\nendometriosis and healthy individuals, while patients with\nmild endometriosis showed AMH serum concentration\ncomparable to healthy controls [27]. Similar to our find-\nings, Somigliana et al., showed that only patients with bi-\nlateral ovarian benign tumors (of which 72 % were\nendometrial cysts) had significantly lower AMH serum\nlevels when compared to patients with unilateral ovarian\ncysts and healthy women [31]. These results were again\nconfirmed in other studies showing no differences in\nserum AMH levels between patients with mild endometri-\nosis and healthy women [32 – 34]. Thus, unilateral endo-\nmetriosis negatively affects ovarian reserve although not\nto statistical significance, while bilateral ovarian endome-\ntriomas result in significantly decreased AMH levels from\nan early age. In women with unilateral ovarian endomet-\nrial cysts, procreative ovarian function is sustained and\ncomparable to that of healthy individuals.\nLind et al. investigated AMH levels in women who\nunderwent surgery for benign ovarian tumors and found\nthat the reduction in AMH levels depended on the\nhistological type of the ovarian cyst and preoperative\nAMH levels [35]. Somigalina et al. reported that presur-\ngical AMH serum concentrations were higher in patients\nwith dermoid cysts than in women with endometriomas;\nhowever, the difference was not statistically significant\n[31]. These clinical findings are consistent with the path-\nology of endometriotic and non-endometriotic ovarian\ncysts. Neither dermoid cysts nor simple ovarian cysts\ncause local inflammation. Thus, healthy ovarian cortical\ntissue surrounding a non-endometriotic cyst is not ex-\nposed to pro-inflammatory agents or ROS, which are\npresent in endometriotic fluid. Furthermore, in contrast\nto endometriomas, non-endometriotic ovarian cysts do\nnot contain iron deposits, which cause the hemosiderin-\nladen macrophages that trigger follicular destruction in\nthe surrounding cortical tissue. Thus, it appears that\ndermoid and simple ovarian cysts do not directly impair\novarian function. Nevertheless, it must be emphasized\nthat surgical intervention and ovarian cystectomy for\nbenign ovarian tumors are independent risk factors for\nreduced ovarian reserves.\nOvarian endometrial cysts may be directly responsible\nfor a decrease in AMH levels. However, surgical treat-\nment of endometriomas may be an additional and inde-\npendent risk factor for impaired ovarian function.\nSeveral studies have investigated the impact of surgery\non ovarian function in women with endometriosis. Inter-\nestingly, Streuli et al. reported that decreased serum\nAMH levels in women with endometriosis were limited\nonly to those with previous endometrioma surgery [36].\nMoreover, in patients undergoing laparoscopic cystec-\ntomy due to ovarian endometriosis, a significant reduc-\ntion in AMH secretion was reported [33]. Women with\nhigher preoperative AMH levels showed a more rapid\ndecrease in AMH levels. Similarly, in patients with uni-\nlateral ovarian endometrioma, the decrease in AMH\nlevels was more significant and lasted longer compared\nto women who underwent surgery for dermoid cysts\n[35]. In contrast, Vignali et al. reported that the postop-\nerative decrease in AMH levels in women who had\nundergone laparoscopic excision of ovarian endometrial\ncysts was temporary; AMH levels returned to preopera-\ntive values 12 months after surgery [37]. Angioni et al.\ninvestigated the feasibility of single-port access laparos-\ncopy (SPAL) compared with multiport laparoscopy\n(MPL) for cystectomy of ovarian endometriomas [38].\nOver a 3-month follow-up period, they observed a sig-\nnificant decrease in AMH serum concentrations and an-\ntral follicle count after SPAL compared with MPL and\nconcluded that SPAL cystectomy should not be recom-\nmended for women with endometriomas who desire\npregnancy [38]. However, in addition to surgical access,\nthe cyst extirpation method and bleeding control can in-\nfluence ovarian reserve. Although excisional surgery is\nthe gold standard treatment for endometriotic cysts, it\nmay result in unintended removal of healthy ovarian tis-\nsue. Moreover, the use of bipolar coagulation to control\nbleeding may damage healthy ovarian tissue, causing a\ndecrease in ovarian reserve. Nappi et al. found that laser\nhemostasis using a dual-wavelength system did not\nsignificantly reduce ovarian reserve and prevented fol-\nlicular loss after endometrioma surgery [39]. Thus, laser\nhemostasis may be a better choice than bipolar coagula-\ntion for women with endometriomas who wish to pre-\nserve fertility. Many studies were in accordance with our\nfindings that unilateral ovarian endometriosis, which is\nconsidered to be a moderate endometriosis according to\nESHRE criteria, does not impair ovarian procreation\nfunction at any age.\nAs the procreative function of ovaries declines rapidly\nafter 35 years of age, we analyzed the association between\nNieweglowska et al. Reproductive Biology and Endocrinology  (2015) 13:128 Page 7 of 9\n\nage and AMH levels after stratification of patients accord-\ning to age. Serum AMH levels were significantly lower in\nwomen with bilateral ovarian endometriosis who were\nunder 43 years of age than in age-matched patients with\nunilateral endometriomas and healthy individuals. How-\never, in women over the age of 42 years, no differences in\nmedian serum AMH concentration was observed between\npatients with bilateral or unilateral ovarian endometriosis\nand the controls. This result can be explained because\nafter the age of 40 years, ovarian follicle function has de-\ncreased to the extent that endometriosis does not further\nreduce ovarian reserve.\nTo our knowledge, this is the first comprehensive\nstudy evaluating the relationship between serum AMH\nconcentration levels and age in women with bilateral\nand unilateral ovarian endometriomas prior to surgery.\nAll laboratory evaluations were performed in one setting\nby staff highly experienced in blood collection for serum\nAMH level assessment, performed at a narrow point in\ntime to reduce research bias. However, this study had\nseveral limitations. The primary limitation was the low\nnumber of patients with bilateral ovarian endometrio-\nmas. Second, women with incidental endometriomas\nand PPS who experienced irregular periods were not\nevaluated to identify the cause of menstruation irregular-\nity. Thus, it is possible that undiagnosed disorders were\nthe cause of impaired ovarian function in these women.\nHowever, this potential bias was significantly mitigated\nby an extensive review of available medical records and\nhistories to detect risk factors for impaired ovarian func-\ntion apart from endometriosis, although the presence of\nan undiagnosed disorder could not be completely ruled\nout. The results obtained should be externally validated\nin a larger cohort to gain an epidemiological impact.\nOnly a prospective follow-up of AMH secretion in\nwomen with ovarian endometriomas conducted in a\nlarge population and with age-matched controls over a\nlonger period of time will allow us to fully elucidate its\nclinical utility.\nConclusions\nIn the present study, we showed a significantly negative\ncorrelation between serum AMH concentration and age\nin women with bilateral and unilateral ovarian endometri-\nosis. However, median serum AMH levels were signifi-\ncantly lower only in patients with bilateral ovarian\nendometriomas compared to controls, while in patients\nwith unilateral ovarian endometriomas, median serum\nAMH concentration was insignificantly lower compared\nto the healthy individuals. According to our data, unilat-\neral ovarian endometriosis moderately impaired AMH-\nbased ovarian reserve prior to surgery, irrespective of age.\nIn contrast, in women with bilateral ovarian endometrio-\nmas, a significant reduction in ovarian reserve was shown.\nAbbreviations\nANOVA: analysis of variance; AMH: anti-mullerian hormone; ART: artificial\nreproductive technology; ELISA: enzyme-linked immunosorbent assay;\nFSH: follicle-stimulating hormone; IQR: interquartile range; NS: statistically not\nsignificant; NA: data not available; TNF: tumor necrosis factor; NK: natural\nkiller cells; R: correlation index; SD: standard deviation.\nCompeting interests\nThe authors declare no competing interests.\nAuthors’ contributions\nDN and IHB were the chief investigators, responsible for the study concept\nand design; they collected data and wrote the manuscript. KP and TB shared\nin the initial conception of the study, referred the patients for hormonal\nanalysis, performed vaginal ultrasounds, qualified the patients for surgery\nand drafted and critically reviewed the manuscript. OG performed all the\nlaboratory tests and reviewed and agreed with the final version of the\nmanuscript. GJ, AL and RJ participated in the study design and data analysis\nand reviewed the final draft. All authors read and approved the final\nmanuscript.\nAcknowledgments\nThe publication of this paper was supported by the Faculty of Medicine,\nJagiellonian University Medical College, Leading National Research Centre\n(KNOW) 2012 –2017.\nWe would like to thank the American Journal Experts (AJE) (http://www.aje.com)\nfor a professional language editing of the manuscript.\nAuthor details\n1Department of Gynecology and Oncology, Jagiellonian University, Chair of\nGynecology and Obstetrics, Krakow, 21 Kopernika Str, 30-501 Krakow, Poland.\n2Center of Rheumatology, Immunology and Rehabilitation, Dietl Specialistic\nHospital, Krakow, Poland. 3Nuffield Division of Clinical Laboratory Science,\nUniversity of Oxford, Level 4, John Radcliffe Hospital, Headington, OX3 9DU\nOxford, UK. 4Department of Public Health, Medical University of Warsaw,\nWarsaw, Poland. 5Department of Gynecological Endocrinology, Jagiellonian\nUniversity Medical College, Krakow, Poland.\nReceived: 16 September 2015 Accepted: 17 November 2015\nReferences\n1. 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Nappi L, Angioni S, Sorrentino F, Cinnella G, Lombardi M, Greco P.\nAnti-Mullerian hormone trend evaluation after laparoscopic surgery of\nmonolateral endometrioma using a new dual wavelengths laser system\n(DWLS) for hemostasis. Gynecol Endocrinol. 2015. doi:10.3109/09513590.\n2015.1068754.\n•  We accept pre-submission inquiries \n  Our selector tool helps you to find the most relevant journal\n  We provide round the clock customer support \n  Convenient online submission\n  Thorough peer review\n  Inclusion in PubMed and all major indexing services \n  Maximum visibility for your research\nSubmit your manuscript at\nwww.biomedcentral.com/submit\nSubmit your next manuscript to BioMed Central \nand we will help you at every step:\nNieweglowska et al. Reproductive Biology and Endocrinology  (2015) 13:128 Page 9 of 9","source_license":"CC0","license_restricted":false}