{"paper_id":"61e1af7d-bfa4-4f4c-a207-3d90a0673af4","body_text":"Endometriosis is defined as the presence and development of endometrial cells outside\nthe uterine cavity ( Kennedy  et al .,\n2005 ). For women with infertility, the prevalence of endometriosis is\n25-50% and, for women with endometriosis diagnosis, approximately 30-50% them have\nreduced fertility ( Missmer  et al .,\n2004 ).\nThe American Society of Reproductive Medicine (ASRM) classifies endometriosis as:\nminimal (I), mild (II), moderate (III) and severe (IV) ( ASRM, 1997 ). Most women with endometriosis III/IV have pelvic\nanatomical alterations that impede ovulation and tubal transport of the embryo\n( de Ziegler  et al .,\n2010 ). However, women with endometriosis I/II, in which there are no\nanatomical alterations of the pelvic cavity, also have decreased fertility rates\n( Bérubé  et al .,\n1998 ;  Parazzini, 1999 ).\nThe decrease of oocyte quality in women with endometriosis contributes to infertility\n( Mansour  et al ., 2010 ;\n Da Broi  et al ., 2014 ;\n Barcelos  et al ., 2009 )\nbut the mechanisms responsible for this effect are unknown ( Simón  et al ., 1994 ;  Díaz  et al ., 2000 ).\nAddition of follicular fluid (FF) from infertile women with endometriosis stages\nI/II into an  in vitro  maturation (IVM) medium of bovine oocytes\nreduces the quality of oocytes ( Giorgi  et\nal ., 2016 ) and embryos in vitro produced from these oocytes\n( Giorgi  et al ., 2021 ).\nThe antioxidants L-carnitine (LC) and N-acetyl-cysteine (NAC) protect against\nmeiotic damages to these oocytes, suggesting that oxidative stress (OS) has a role\nin the etiopathogenesis of infertility and endometriosis ( Giorgi  et al. , 2016 ;  2021 ).\nLC also functions as part of transport of long chain fatty acids from the cytosol\ninto the mitochondria for β-oxidation ( Evans\n& Fornasini, 2003 ). β-oxidation is essential for the\nresumption of oocyte meiosis and nuclear maturation in mice ( Downs  et al ., 2009 ;  Paczkowski  et al ., 2013 ;  Valsangkar & Downs, 2013 ), swine and cattle ( Paczkowski  et al ., 2013 ).\nPrevious studies evaluated the addition of omega-3 fatty acids into the IVM medium of\nbovine oocytes ( Oseikria  et al .,\n2016 ;  Nikoloff  et al .,\n2017 ).  Oseikria  et al .\n(2016 ) showed that addition of low concentrations of docosahexaenoic acid\n(DHA) increased oocyte competence as indicated by increased cleavage rates and\nblastocyst formation after parthenogenetic activation ( Oseikria  et al ., 2016 ).  Nikoloff  et al . (2017 ) showed that the\naddition of eicosapentaenoic acid (EPA) improved the oocyte quality, as indicated by\nincreased  cumulus  cell expansion.\nNo studies have yet evaluated the impact of the endometriosis stage on oocyte\nquality, and the possible prevention of oocyte damage by the combined administration\nof LC, DHA and EPA. Therefore, we used confocal microscopy to compare the impact of\nFF from different groups of women (controls, with endometriosis I/II, III/IV without\nendometrioma and III/IV with endometrioma) to the IVM medium of bovine oocytes on\nnuclear maturation and organization of the meiotic spindle and chromosomes. We then\nexamined the impact of LC and DHA/EPA(n3) on the amelioration of FF-induced oocyte\ndamage.\n\nThis study was approved by the Research Ethics Committee of the Clinic Hospital of\nthe Medical School of Ribeirão Preto (HC-FMRP), University of São\nPaulo (USP) (Process HCRP nº 12201/2008) and by the Ethics Committee in Animal\nExperimentation of FMRP - USP (nº 169/2008).\nFF samples acquired from March to October of 2013 and from May 2016 to September\n2017 were from infertile women who underwent ovarian hyperstimulation for\nintracytoplasmic sperm injection (ICSI) in the Sector of Human Reproduction,\nDepartment of Gynecology and Obstetrics, Faculty of Medicine of Ribeirão\nPreto, University of São Paulo (FMRP-USP) (eligible patients). All\npatients provided written informed consent prior to participation.\nAll women with endometriosis had the following characteristics: younger than 40\nyears; body mass index (BMI) of 30kg/m 2  or less; serum concentration\nof follicle stimulating hormone (FSH) of 12 mIU/mL or less; free of chronic\nanovulation, hydrosalpinx, chronic diseases, endocrinopathy, cardiovascular\nconditions, and infection; non-smoking; not use of anti-inflammatory agents,\nhormonal medications, or vitamin complexes in the 6 months before treatment of\nassisted reproduction techniques (ART); and previous diagnosis of endometriosis\nbased on videolaparoscopy. Women in the control group had the same\ncharacteristics and were infertile due to tubal and/or male factors.\nWomen with endometriosis were subdivided into 3 groups [early stage endometriosis\n(EI/II) and advanced endometriosis without (EIII/IV) or with an active\nendometrioma in the cycle visualized by transvaginal ultrasonography\n(Endometrioma)].\nThe Endometrioma group represented women with active lesion of endometriosis\nsince all of endometriosis women underwent to treatment during\nvideolaparoscopy.\nSamples of FF obtained during 2013 were previously tested in experiments of\nbovine IVM for evaluate their potentiality to cause meiotic abnormalities.\nResults are presented in supplementary material.\nPreviously to controlled ovarian stimulation (COS), all patients used combined\noral contraceptive and in accordance with the characteristics of each patient,\none of two COS protocols was chosen:\n- Flexible antagonist protocol: gonadotrophins (150 to 300IU/day) administered\ndaily on the first 6 days, with the dose adjusted daily according to follicular\ngrowth.\n- Minimal stimulation protocol (clomiphene citrate plus gonadotrophins and a GnRH\nantagonist), in which clomiphene citrate (100 mg/day) was administered daily on\nthe first 5 days and gonadotrophins (150 IU/day) was administered on days 2 and\n4, and daily from day 6.\nAdministration of GnRH antagonist (ganirelix or cetrorelix 0.25mg/day) began when\nthe mean diameter of the largest follicle was 14 mm or more. Recombinant hCG\n(250 µg, Ovidrel ® , Serono, Brazil) or urinary hCG\n(10,000 IU, Choriomon ® , Meizler, Brazil) was administered when\nat least one follicle had a diameter of 18 mm. Oocytes were collected 34 to 36 h\nafter administration of recombinant hCG, and the luteal phase was maintained by\nmicronized progesterone (600 mg/day).\nFF samples were obtained during oocyte recovery for ICSI. To prevent repetitive\npunctures, FF was only acquired from the first follicle (diameter ≥15mm)\nof the first punctured ovary.\nIn Endometrioma group, samples of FF were from ovary with (2/8) or without\nendometrioma (6/8).\nSamples were immediately taken to the embryology laboratory, where embryologists\nchecked for the presence of oocytes and/or granulosa cells. Oocytes were\nseparated from the FF for use during ART. The FF was centrifuged at 300\n g  for 10 min, aliquoted, and stored at -80 o C.\nAll FF samples without oocyte and/or granulosa cells, and samples contaminated\nwith blood were discarded.\nThe ovaries of cows were collected after slaughter and transferred into\nphysiological saline at 35 to 38.5 o C. In the laboratory, follicles\nwith diameters of 2 to 8 mm were aspirated, and  cumulus -oocyte\ncomplexes (COCs) with uniform cytoplasm and three or more layers of cumulus\noophorus cells were selected ( Adona & Lima\nVerde Leal, 2004 ;  Ferreira  et\nal ., 2009 ).\nCOCs (about 20 per drop) were cultivated without mineral oil in TCM-199\ncontaining Earle’s salts and bicarbonate (Invitrogen, Gibco Laboratories Life\nTechnologies, Inc., Grand Island, NY, USA) supplemented with 0.4mM sodium\npyruvate, 0.5µg/mL gentamicin, 5µg/mL FSH, 2.5 UI/mL hCG\n(Chorulon ® ), 1 µg/mL estradiol and 10% foetal calf\nserum (FCS; Gibco); at 38.5°C, 95% humidity and 5% CO 2  ( Hashimoto  et al.  2002 ;\n Adona & Lima Verde Leal, 2004 ;\n Ferreira  et al .,\n2009 ). The duration of IVM was 22-24 h.\nThe concentration of FF added to IVM medium was 1% based on previous study of\n Da Broi  et al . (2014 )\nthat tested different concentrations of FF from infertile women with and without\nmild endometriosis on medium of IVM of bovine oocytes. The concentrations of FF\ntested were 1%, 5%, 10% and 15%, and no dose-response was observed ( Da Broi  et al ., 2014 ). So,\nwe used the lowest tested concentration (1%).\nThe concentration of LC (Sigma Aldrich C0283) in the IVM medium was 0.6 mg/mL\n( Mansour  et al .,\n2009 ;  Giorgi  et al. ,\n2016 ), and LC was stored in a 100 x stock solution that was prepared\nwith water, filtered (0.22 µm), aliquoted, and stored at -20°C prior to\nuse.\nThe concentration of omega-3 fatty acids in the IVM medium was 1 nM [0.4nM was\nDHA (Sigma Aldrich D2534) and 0.6 nM EPA (Sigma Aldrich E2011)] ( Nikoloff  et al ., 2017 ).\nThe ratio of 2:3 DHA:EPA was chosen based on previous randomized clinical trials\n( Nadjarzadeh  et al .,\n2015 ;  Haghiac  et\nal ., 2015 ;  Rahbar  et\nal. , 2012 ). A stock solution (100x) was prepared using\nDMSO, filtered (0.22µm), aliquoted, and stored at -20°C.\nAfter 22-24h of IVM,  cumulus  cells were removed by pipetting,\nand the oocytes were fixed in a buffer for microtubule stabilization ( Liu  et al ., 1998 ;  Ferreira  et al ., 2009 ).\nThe oocytes were then washed and blocked in washing medium [phosphate buffer\nsaline (PBS) with 0.02% NaN 3 , 0.01% Triton X-100, 0.2% defatted dry\nmilk, 2% goat serum, 2% bovine serum albumin, and 0.1 M glycine] for 2 h at\n37ºC. Incubation with an anti-β-tubulin murine monoclonal antibody\n(1:1000) was performed overnight at 4 o C. After washing, a secondary\nfluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG antibody (1:500;\nZymed Laboratories, Invitrogen, Carlsbad, CA, USA) was added at\n38.5 o C for 2 h. The oocytes were washed again and labelled with\nHoechst 33342 (10 mg/mL) in Vectashield mounting medium (H-1000, Vector,\nBurlingame, CA, USA), placed on a glass slide, and covered with a coverslip.\nA confocal microscope (Confocal Leica TCS SP5, Leica Microsystems, Mannheim,\nGermany) with 405 nm diode UV and a 543 nm HeNe laser was used to visualize the\noocytes at 40 x.\nFirst, oocytes were classified according to nuclear maturation, as being in\nmetaphase I (MI), telophase I (TI), metaphase II (MII), or undergoing\nparthenogenetic activation (PA). MII oocytes were categorized based on\nmetaphasic plate visualization, as analyzable (meiotic spindle in lateral or\nsagittal position) or non-analyzable (meiotic spindle in polar position) ( Ju  et al. , 2005 ).\nMII oocytes observed in lateral/sagittal position were considered: “normal” when\nmeiotic spindle had typical barrel shape and chromosomes arranged in line at the\nequator of the spindle ( Figure 1D ); and as\n“abnormal” when meiotic spindle had reduced size and was disarranged or\ndispersed from the plane of the metaphasic plate ( Figure 1A-C ). PA was defined as the presence of two polar bodies or\nthe presence of telophase II.\nFigure 1 Representative confocal microscopy images (40x) of bovine oocytes\nmatured  in vitro  during metaphase II, based on\norganization of the meiotic spindle and chromosomal alignment.\nNormal MII: A; Abnormal MII: B, C, D.  Note: Scale bar: 10\nµm; White arrows: misaligned chromosomes .\nRepresentative confocal microscopy images (40x) of bovine oocytes\nmatured  in vitro  during metaphase II, based on\norganization of the meiotic spindle and chromosomal alignment.\nNormal MII: A; Abnormal MII: B, C, D.  Note: Scale bar: 10\nµm; White arrows: misaligned chromosomes .\nData were analyzed using R Studio software version 1.0.153 ( https://www.R-project.org ).\nClinical variables, response to COS, and ICSI results of women donors of FF were\ncompared between the four groups (control, EI/II, EIII/IV and Endometrioma)\nusing Kruskall Wallis test with Dunn post test.\nThe categorical variables (rates of MI, TI, PA, MII, MII analyzable, normal MII)\nwere compared between the 9 groups using the Chi-square test.\nFor all comparisons a  p  value below 0.05 was considered.\n\nEight IVM experiments were performed, and one FF sample of each group was used\nindividually in each experiment.\nFigure 2  represents Flow-chart of selection\nof FF donors.\nFigure 2 Flow-chart of recruitment of donors of follicular fluid.\nFlow-chart of recruitment of donors of follicular fluid.\nA total of 39 FF samples were processed and stored at -196°C until use. The\nchoice of samples in each experiment was chosen based on age, BMI and COS\nprotocol. In Control and EIII/IV groups, only FF samples from women with BMI\nbetween 19 and 29 were included.  Table 1 \nshows characteristics of FF donors used in each group.\nClinical variables, response to controlled ovarian stimulation, and ICSI\nresults of infertile women with no endometriosis (control), women with\nendometriosis I/II (EI/II), women with endometriosis III/IV without\nendometrioma (EIII/IV) and women with endometriosis III/IV with\nendometrioma (Endometrioma).\nDuring the 8 IVM experiments (performed between November 2017 and January 2018),\n1686 immature COCs were submitted to IVM, 1561 oocytes were fixed for\nimmunofluorescence, and 1401 oocytes were visualized by confocal microscopy. A\ntotal of 167 oocytes were in MI, 25 were in TI, 1188 in were in MII, and 21 were\nPA. Among the 1188 oocytes in MII, 735 were analyzable.\nThe 9 experimental groups had no differences in TI ( p =0.05467),\nPA ( p =0.8854), and analyzable MII ( p =0.5651)\n( Table 2 ).\nStages of nuclear maturation, and percentages of normal MII oocytes\nmatured in vitro in medium without follicular fluid (No-FF), with\naddition of 1% FF from infertile patients without endometriosis (FFC),\nwith early endometriosis (FFEI/II), with advanced endometriosis without\n(FFEIII/IV) or with endometrioma (FFEndometrioma), supplemented with 0.6\nmg/mL L-carnitine and 1 nM omega-3 (LC+n3) visualized by confocal\nmicroscopy.\nThe rate of MI, in the No-FF group (6.0%) was similar to that of the FFC group\n(6.1%,  p =1), the FFC+LC+n3 group (11.7%,\n p =0.1277), the FFEI/II group (12.7%,\n p =0.07406), the FFEI/II+LC+n3 group (9.8%,\n p =0.3085), the FFEIII/IV+LC+n3 group (7.7%,\n p =0.3085), and the FFEndometrioma+LC+n3 group (11.1%,\n p =0.166). In the other hand, the rate of MI was lower in\nthe No-FF and FFC groups when compared to the FFEIII/IV (16.9%;\n vs . No-FF:  p =0.00504;  vs. \nFFC:  p =0.00537) and FFEndometrioma (24.7%,  vs. \nNo-FF:  p <0.0001,  vs.  FFC:\n p <0.0001). The addition of LC+n3 had no effect on the MI\nrate in the FFC group (6.1%  vs.  11.7%,\n p =0.192) and FFEI/II group (12.7%  vs.  9.8%,\n p =0.5288). However, the FFEIII/IV+LC+n3 group had a lower\nMI rate than the FFEIII/IV group (7.7%  vs.  16.9%,\n p =0.0259), and the FFEndometrioma+LC+n3 group also had\nlower MI rate than FFEndometrioma group (11.1%  vs.  24.7%,\n p =0.0028).\nThe total MII rate was 91.9% in the No-FF group, similar to the FFC group (89.2%,\n p =0.5389), the FFC+LC+n3 group (84.7%,\n p =0.06992), the FFEI/II group (85.4%,\n p =0.1069), the FFEI/II+LC+n3 group (85.3%,\n p =0.09598), the FFEIII/IV+LC+n3 group (90.8%,\n p =0.1069) and the FFEndometrioma+LC+n3 group (86.4%,\n p =0.1681). The lowest total MII rate was in the\nFFEndometrioma group (69.3%), and this was significantly lower than all other\ngroups ( vs.  No-FF:  p <0.0001;\n vs.  FFC:  p <0.0001;  vs. \nFFC+LC+n3:  p =0.00194,  vs.  FFEI/II:\n p =0.00114,  vs.  FFEI/II+LC+n3:\n p =0.00117,  vs.  FFEIII/IV:\n p =0.02681,  vs.  FFEIII/IV+LC+n3:\n p <0.0001;  vs.  FFEndometrioma+LC+n3:\n p =0.00044). The No-FF group had a higher rate of total MII\nthan the FFEIII/IV group (80.7%,  p =0.00681). The total MII rate\nwas similar in the FFC group (89.2%) and the FFC+LC+n3 group (89.2%,\n p =0.3122), the total MII rate was also similar in the\nFFEI/II+LC+n3 group (85.3%) and the FFEI/II group (85.4%,\n p =1.0). However, the addition of LC+n3 increased the rate of\ntotal MII in the FFEIII/IV group and the FFEndometrioma group [(FFEIII/IV\n vs . FFEIII/IV+LC+n3:  p =0.0190) and\n(FFEndometrioma  vs.  FFEndometrioma+LC+n3:\n p =0.0004)].\nThe percentage of normal MII was 87.2% in the No-FF group, similar to the FFC\ngroup (87.2%,  p =1.0), the FFC+LC+n3 group (82.5%,\n p =0.54), the FFEI/II+LC+n3 group (84.5%,\n p =0.7615), the FFEIII/IV+LC+n3 group (84.1%,\n p =0.7122) and the FFEndometrioma+LC+n3 group (75.3%,\n p =0.0623). The percentage of normal MII was significantly\ngreater in the No-FF group (87.2%) than in the FFEI/II group (62.2%,\n p= 0.00023), the FFEIII/IV group (70.2%,\n p =0.0092) and the FFEndometrioma group (72.7%,\n p =0.03497). The FFC group also had a significantly higher\npercentage of normal MII than the FFEI/II group ( p =0.00059),\nthe FFEIII/IV group ( p =0.01523) and the FFEndometrioma group\n( p =0.0486). The addition of LC+n3 during IVM did not alter\nthe rate of normal MII in the FFC group ( p =0.5792) nor in the\nFFEndometrioma group ( p =0.865). However, LC+n3 increased the\nrate of normal MII in the FFEI/II group ( p =0.00205) and the\nFFEIII/IV group ( p =0.04995). Although the FFEndometrioma group\n(72.7%) and the FFEndometrioma+LC+n3 group (75.3%) had similar percentages of\nnormal MII ( p =0.865), the FFEndometrioma+LC+n3 group had a\nsimilar percentage of normal MII as the No-FF group ( p =0.062)\nand the FFC group ( p =0.083).\n\nThis study demonstrated that the FF from infertile women with any stage of\nendometriosis decreased the quality of bovine oocytes that were cultured in IVM\nmedium. The FF of women with endometrioma had an even more detrimental impact on\noocyte quality, in that this FF affected nuclear maturation and promoted meiotic\nabnormalities. The addition of LC+n3 prevented the oocyte damages induced by FF from\nwomen with endometriosis. These results suggest that OS and alterations in\nβ-oxidation decrease oocyte quality during the early and advanced stages of\nendometriosis.\nThe results presented here confirm previous data of our group, which showed that\naddition of FF from infertile women with mild endometriosis to IVM medium of bovine\noocytes led to damage of the meiotic spindle ( Da Broi\n et al ., 2014 ;  Giorgi\n et al ., 2016 ). A novel finding of the present study\nis that the FF of infertile women with advanced endometriosis also led to meiotic\ndamage of bovine oocytes.\nWe observed an impairment of maturation rate of bovine oocytes in the presence of FF\nfrom women with endometriosis in stage III/IV with or without endometrioma.  Hamdan  et al . (2016)  also\nobserved that the FF from women with severe endometriosis impaired  in\nvitro  oocyte maturation. These authors suggested that the decreased\npolar body extrusion rate is a consequence of DNA damage caused by OS ( Hamdan  et al ., 2016 ).\nWe demonstrated that the FF from infertile women with endometriosis III/IV with\nendometrioma had the greatest impact on nuclear maturation. A recent retrospective\ncohort study showed a lower number of quality embryos in the group of women with\nendometrioma compared to women without endometriosis, although the cumulative live\nbirth rate did not differ between groups ( Zeng\n et al ., 2022 ). An endometrioma is characterized by\nan accumulation of iron and its derivatives, making the environment toxic and\nhostile to folliculogenesis ( Sanchez  et\nal ., 2014 ). Analysis of FF by mass spectrometry indicated\nthe presence of 535 expressed proteins, and that 139 of these proteins occurred in\nthe FF of both ovaries of women with unilateral endometrioma and control women\n( Regiani  et al ., 2015 ),\ndemonstrating the impact of endometriosis (or presence of an active endometrioma in\nthe cycle, regardless of the follicular proximity) on the composition of FF ( Regiani  et al. , 2015 ).\nHowever, it would be interesting future studies assessing FF of both ovaries from\nwomen with unilateral endometrioma on oocyte quality and OS markers.\nWe found that LC+n3 prevented the oocyte damage induced by FF from women with\nendometriosis. Our previous study reported that LC protected against injuries to the\nmeiotic spindle of  in vitro  bovine oocytes induced by FF from women\nwith mild endometriosis ( Giorgi  et\nal ., 2016 ). Thus, a novel finding of the present study is\nthat LC+n3 together prevent oocyte damage.\nA review highlighted the important role of carnitine in female fertility by analysis\nof  in vivo  and  in vitro  studies with humans and\nanimal models, and described the possible mechanisms by which LC improves female\nfertility ( Agarwal  et al. ,\n2018 ). Experimental studies showed that supplementation with n3 improves\noocyte quality ( Nehra  et al .,\n2012 ), regulates the endometrium ( Waters\n et al. , 2014 ) and increases the pregnancy rate\n( Wathes  et al ., 2007 ).\nAlso recent studies showed the importance of EPA/DHA rich dietary in composition of\nFF and in improve cleavage rate in patients of ART ( Kermack  et al. , 2020 ;  2021 ).\nLC is an antioxidant that reduces OS and lipotoxicity by eliminating free radicals,\nand thereby decreases apoptosis and promotes oocyte growth and development ( Agarwal  et al ., 2018 ). LC and\nfatty acids have roles in β-oxidation, an important energy production pathway\nduring oocyte maturation ( Downs  et\nal ., 2009 ;  Paczkowski\n et al ., 2013 ;  Valsangkar & Downs, 2013 ;  Dunning\n et al. , 2014 ). Oocytes from infertile women with\nendometriosis I/II have mitochondrial alterations, based on analysis by transmission\nelectron microscopy and RT-PCR ( Xu  et\nal ., 2015 ). Although we did not directly investigate oocyte\nmitochondria in this study, we suggest that the FF of women with endometriosis\nalters mitochondrial function, and thereby reduces oocyte quality.\nOur evaluation of the clinical data of the different FF donor groups (FFC, FFEI/II,\nFFEIII/IV and FFEndometrioma) indicated a difference in the duration of infertility\nfor the control group and the group with endometriosis III/IV with endometrioma. We\nbelieve this difference is not relevant to our outcome because these two groups had\na similar median age (a factor strongly related to worsening oocyte quality).\nAnother important point is that the FF samples were not paired by type of COS\nprotocol, and in the literature the role of stimulation in oocyte quality is still\ncontroversial ( Thaker  et al .,\n2020 ;  Montoya-Botero  et\nal ., 2021 ;  Jirge  et\nal ., 2022 ); however, the number of days of stimulation and\nthe amount of FSH used by women were similar comparing the groups.The present study\nhelped to elucidate the etiopathogenesis of infertility due to endometriosis.\nHowever, there were some limitations. First, the sample size was small, limiting the\ngeneralizability of the results; however, we used rigorous criteria for selection of\nall FF donors. Second, we used FF of women who were submitted to COS, so\nextrapolation of our findings to women undergoing natural cycles is questionable;\nhowever, women in the control group were scheduled for COS, making our comparisons\nvalid. Third, we used an  in vitro  bovine oocyte maturation assay,\nso direct extrapolation to humans is not possible. Studies with human oocytes\nmatured  in vivo  are needed to corroborate our findings. And fourth,\nthe technique used to analyze the oocytes (confocal microscopy) was limited to\nassessed oocytes fixed in polar position, resulting in 58%-68% of MII\nanalyzable.\nOur major findings were that FF from infertile women with endometriosis damages\nmeiotic spindle assembly and alters chromosome alignment of MII bovine oocytes. The\nFF from women with endometriosis III/IV besides damage the meiotic spindle, also\nimpairs nuclear maturation; and FF from women with endometrioma leads to additional\nimpairment of nuclear maturation. Supplementation with LC+n3 prevented all these\ndamaging effects.\nThe currently available treatments for infertility due to endometriosis are surgery\nand/or ART ( Kennedy  et al .,\n2005 ). These treatments are invasive and/or costly, and therefore\nunavailable to many people. New therapeutic approaches are needed to help many women\nwhose infertility is due to endometriosis. Our findings suggest that clinical\nstudies should investigate the impact of a combination of surgical treatment with\nLC+n3 supplementation for preventing the recurrence and/or progression of\nendometriosis and improving natural fertility.","source_license":"public-domain-us","license_restricted":false}