{"paper_id":"a5e5b5a2-b111-48f9-8c41-b11da916ba72","body_text":"Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism, ovulation\ndisorder and polycystic ovaries (PCO) and the exclusion of other endocrinopaties\n( Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus\nWorkshop Group, 2004 ). PCOS affects 6-8% of women of reproductive age.\nAlthough PCOS was first described eighty years ago ( Stein & Leventhal, 1935 ), its aetiology is not yet fully elucidated,\nas it is a heterogeneous and complex disorder with metabolic and reproductive\nimplications. PCOS represents the major ovulatory cause of infertility, which leads\nsome PCOS patients to pursue  in vitro  fertilization (IVF)\ntreatments ( Dumesic  et al .,\n2015 ).\nThe follicular fluid (FF) that surrounds the cumulus-oocyte complex contains several\nfactors that originate from the blood transudate and are secreted by cumulus cells,\nsuch as proteins, steroids, polysaccharides and other metabolites; thus, FF provides\na unique microenvironment in which to study oocyte development and maturation ( Schweigert  et al. , 2006 ;  Appasamy  et al. , 2008 )\nIt is recognized that the FF from women with PCOS is characterized by deregulated\nexpression of several compounds, including anti-Müllerian hormone (AMH),\ninhibin-B, activin-A, amphiregulin, heparan sulfate proteoglycan 2; tumour necrosis\nfactor (TNF), α-induced protein 6 and plasminogen ( Ambekar  et al ., 2015 ). Although previous\nstudies have identified molecules in the FF of PCOS patients that are associated\nwith the deregulation of follicle maturation, this process is not completely\nunderstood. We aimed to identify putative differences in the FF profiles of PCOS\npatients and fertile women, represented by egg donors, using mass spectrometric\nanalysis to better understand the mechanisms that lead to deregulated oocyte\ndevelopment.\n\nThis prospective study evaluated the protein components of FF from oocyte donors\n(ODs) in comparison to those of FF from infertile women with PCOS who underwent\nIVF at Huntington Reproductive Medicine Centre and the Reproductive Unit of the\nFederal University of São Paulo (UNIFESP) from 2012 to 2015. This study\nprotocol was approved by the ethics committee of Federal University of\nSão Paulo (No. 1620/2011), and informed written consent was obtained from\neach patient.\nThirteen (13) patients were enrolled and divided into two groups: ODs (n=7) and\ninfertile PCOS patients (PCOS; n=6). The ODs were healthy female volunteers\nunder the age of 32 years with body mass indices between 18 and 30\nkg/m 2 , antral follicle counts ≥10, normal karyotypes, and\nthe absence of endometriosis who had been screened and tested for infectious\ndiseases. The PCOS patients were diagnosed with infertility according the\nRotterdam criteria ( Rotterdam\nESHRE/ASRM-Sponsored PCOS Consensus Workshop Group, 2004 ). All the\nPCOS patients presented body mass indices (BMI) below 25 kg/m 2 , basal\nfollicular stimulating hormone (FSH) levels below 15 IU/L, basal oestradiol\nlevels below 50 pg/mL, the presence of both ovaries, and no ongoing infectious\ndiseases or uterine abnormalities, and they had undergone intracytoplasmic sperm\ninjection (ICSI) cycles with ejaculated sperm. For both groups, patients who\npresented gynaecological bleeding, hydrosalpinx, allergy to gonadotropins or\nother medications used in the treatment, severe oligo- or azoospermia, abusive\nuse of any medications or ovarian hyperstimulation syndrome (OHSS) during the\ntreatment were excluded.\nFF was obtained from women who underwent the standard short protocol of IVF\n(using a GnRH antagonist - Cetrotide ® , Merck, Germany).\nControlled ovarian stimulation was performed using recombinant FSH (rFSH -\nGonal-F ® , Merck, Germany) and was monitored with\nultrasound. Ovulation was triggered with a GnRH agonist (aGnRH - Gonapeptyl,\nFerring, Germany) when at least two follicles reached 20 mm. The FF was\ncollected from the dominant follicles through aspiration between 34 and 36 h\nafter aGnRH administration, using transvaginal ultrasound guidance. Only clear\nFF samples, without blood or flushing medium contamination, were processed. The\nselected FF samples were centrifuged at 1200 rpm for 10 to 15 min to remove\ncellular debris. The supernatants were stored at −80ºC until\npurification.\nBefore analysis, albumin and immunoglobulins were removed from the FF samples (25\nµL) using the Albumin & IgG Depletion SpinTrap (GE Healthcare Life\nSciences™) according to the manufacturer’s protocol. The protein\nconcentration in each FF sample was measured in triplicate using a bicinchoninic\nacid assay (BCA assay) ( Smith  et\nal ., 1985 ). Twenty-five to thirty micrograms of\nalbumin/IgG-depleted FF protein was subjected to electrophoresis via 12.5%\nsodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under\nreducing conditions ( Laemmli, 1970 ) and\nstained with Coomassie brilliant blue R-250.\nAll gels were analysed, and ten bands were cut equally for each sample and\nprocessed separately for in-gel digestion according to the protocol described by\n Westermeier  et al .\n(2002) , with slight modifications.\nLiquid chromatography-mass spectrometry (LC-MS) analyses were performed using an\nElectrospray-Ion Trap-Time of Flight system (ESI-IT-TOF) (Shimadzu Co., Japan)\nequipped with a binary Ultra-Fast Liquid Chromatography system (UFLC) (20A\nProminence, Shimadzu) at the Laboratory of Biochemistry and Biophysics of the\nButantan Institute (São Paulo, Brazil). First, each band sample was\nlyophilized, resuspended in 50 µL of 0.1% acetic acid and loaded on a C18\ncolumn (Discovery C18, 5 µm; 50 × 2.1 mm) in a binary solvent\nsystem: (A2) water/acetic acid (999/1, v/v) and (B2) ACN/water/acetic acid\n(900/99/1, v/v/v). The column was eluted at a constant flow rate of 0.2 mL.min−1\nwith a 5 to 70% gradient of solvent B2 over 35 min. The eluates were monitored\nby a Shimadzu SPD-M20A PDA detector before introduction into the mass\nspectrometer. The interface voltage was adjusted to 4.5 kV, and the capillary\nvoltage was 1.76 kV at 200ºC. MS spectra were acquired in positive mode\nand collected in the 80-2000 mass charge (m/z) range. MS/MS spectra were\ncollected in the 50-1950 m/z range. Instrument control, data acquisition, and\ndata processing were performed with LabSolutions (LCMSsolution 3.60.361 version,\nShimadzu).\nProteomic analysis was performed using the Mascot Server (ion search) in house\nversion (2.4) and Peaks Studio V7 (Bioinformatics Solutions, Inc., Waterloo,\nCanada). The following parameters were adjusted for the search: parent mass and\nfragment mass error tolerance: 0.1 Da; enzyme: trypsin; fixed modification:\ncarbamidomethylation; variable modification: methionine oxidation; precursor\nmass search type: monoisotopic; max missed cleavages: 3; non-specific cleavages:\none; database: SwissProt, taxon:  Homo sapiens ; peptide - 10\nlgP: ≥15; and protein - 10 lgP: ≥20. The false discovery rate\n(FDR) for peptide-spectrum matches was ≤1%.\nAlthough each band was analysed separately in LC-MS, we performed a protein\nsearch combining all ten bands obtained from each patient. A protein was\nconsidered exclusive when it was detected in the FF of patients in either the OD\nor PCOS group and was totally absent in all of the samples from the other group;\na protein was considered overexpressed when it was detected in both groups, but\none group had a mean detected peptide level greater than that of the other group\nby 50% (greater than 1.5-fold in one group and less than 0.5-fold in the other\ngroup).\nThe identified proteins were classified according to their classes, locations,\nbiological functions and processes using the PANTHER Classification System (Gene\nOntology Phylogenetic Annotation Project, Los Angeles, USA) ( Mi  et al ., 2016 ). System\nbiology analysis was carried out using Ingenuity™ Pathway Analysis\nsoftware (IPA™, QIAGEN, Redwood, USA). The overexpressed proteins were\nselected for the analysis of canonical pathways and biological interaction\nnetworks. The biological processes were staggered according to the IPA™\nKnowledge Base. The associations between the identified proteins and canonical\npathways in the database were assessed with Ingenuity™ software using\nFisher's exact test (significance of  p <0.01).\nClinical proteomic studies is a multistage biomarker pipeline that begin with the\nidentification of a large number of proteins in a small set of sample. This\nscreening step, as this is our study, the number of samples included was based\non the principle that a minimum number of samples considering biological and\ntechnical variation inherent in the experiment. Thus, we included a small number\nof samples and non-parametric statistic was applied.\nThe patients’ demographic data were evaluated using descriptive statistics.\nNormality was evaluated with the Kolmogorov-Smirnov test. Non-paired continuous\ndata were compared using the Mann-Whitney test for means comparisons and paired\ndata were compared using Wilcoxon’s signed-rank test. Data analyses were\nperformed using SPSS 22 (IBM SPSS Software, USA), and significance was accepted\nfor  p -values ≤0.05.\n\nThe patients’ demographics and clinical outcomes are described in  Table 1 . The ovarian reserves of the patients\nin both groups had similar profiles in terms of basal FSH dosages and antral\nfollicle counts. As expected, the OD patients were younger, and the PCOS patients\nhad longer menstrual cycle intervals. The parameters related to ovarian induction\n(length, serum hormone levels, and mature (metaphase II-MII) oocytes collected) were\nsimilar between the groups, except for the amount of gonadotropin administered,\nwhich was higher in the OD group. The OD group had a higher number of top-quality\nembryos (3 rd  day) than the PCOS group.\nDemographic and clinical data for the patients in the PCOS and OD groups\nMeasured prior to oocyte collection.\nThe proteomic analysis of the proteins from the FF samples matched 229 proteins in\nthe SwissProt database. Forty-five (45) proteins were detected in both groups. Three\nof these shared proteins were excluded from analyses, as they were contaminants\n(trypsins and keratins), resulting in 42 proteins shared between the two groups.\nThere were 61 proteins that were exclusive to the PCOS group, and 123 proteins that\nwere exclusive to the OD group ( Supplemental Tables\nI  and  II ). To refine the SwissProt\nresults, only proteins that were expressed in at least two patients from each group\nwere considered. Five proteins were selected from those exclusively expressed in the\nPCOS group, and three proteins were selected from those exclusively expressed in the\nOD group ( Table 2 ).\nProteins that were exclusively detected in at least two FF samples from\neither the PCOS or OD group\nThe differentially expressed proteins were rated and selected. Six proteins were the\nhighest occurring peptides in the PCOS group, and ten proteins were the highest\noccurring peptides in the OD group ( Table 3 ).\nThe most significant proteins, which were expressed in the FF of at least two\npatients, were the complement C3 protein, which was overexpressed in the PCOS group,\nand titin, serum albumin, complement C4-A, complement C4-B, alpha-1-acid\nglycoprotein 1 and alpha-2-macroglobulin, which were overexpressed in the OD\ngroup.\nProteins that were differentially expressed in the FF of PCOS and OD patients\nand for which one group had at least 50% more peptides than the other\nThe proteins that were identified as exclusive or overexpressed were classified\naccording to the Gene Ontology database and analysed with respect to biological\npathways with the Ingenuity™ software. Six molecular functions were\nidentified, and four of them were very similar between the two groups (GO:0005488,\nGO:0004872, GO:0005198 and GO:0003824). The PCOS patients had fewer proteins related\nto transporter activity (GO:0005215) (7.10% OD  vs.  2.30% PCOS).\nAdditionally, translation regulation activity (GO:0045182) was detected only in the\nOD patients (1.80%) but was represented by only one protein.\nThe evaluation of protein classes resulted in nineteen different classes ( Figure 1 ). The most representative classes for\nthe OD group were cell junction, cell adhesion and transmembrane receptor\nregulatory/adaptor, which were exclusive to this group. The PCOS group presented\nmore proteins related to the oxireductase, membrane traffic protein and ligase\nclasses. The distribution of the protein classes in terms of cellular components\ndiffered between the groups: the PCOS group had more extracellular proteins, and the\nOD group had more membrane and membrane-related proteins ( Figure 2 ).\nFigure 1 Chart indicating the percentages of exclusive and upregulated FF proteins\nfrom the PCOS (dark grey) and OD (light grey) groups classified\naccording to protein classes based on the Gene Ontology database\nChart indicating the percentages of exclusive and upregulated FF proteins\nfrom the PCOS (dark grey) and OD (light grey) groups classified\naccording to protein classes based on the Gene Ontology database\nFigure 2 Chart indicating the percentages of exclusive and upregulated FF proteins\nfrom the PCOS and OD groups, classified according to cellular components\nbased on the Gene Ontology database\nChart indicating the percentages of exclusive and upregulated FF proteins\nfrom the PCOS and OD groups, classified according to cellular components\nbased on the Gene Ontology database\nThe biological processes associated with the detected proteins differed remarkably\nbetween the groups ( Figure 3 ). The PCOS group\nhad more proteins associated with immune process, cell localization and biological\nadhesion molecules. The OD group had more proteins associated with metabolic\nprocesses and cell component organization, suggesting that the OD group was more\nmetabolically active.\nFigure 3 Chart indicating the percentages of exclusive and upregulated FF proteins\nfrom the PCOS (dark grey) and OD (light grey) groups classified\naccording to biological processes based on the Gene Ontology\ndatabase\nChart indicating the percentages of exclusive and upregulated FF proteins\nfrom the PCOS (dark grey) and OD (light grey) groups classified\naccording to biological processes based on the Gene Ontology\ndatabase\nThese results were corroborated by the biological pathway analysis ( Table 4 ), as the proteins identified in the FF\nof the OD patients were related to cellular assembly and organization and cellular\nfunction and maintenance. The PCOS group had fewer proteins matched to cellular\nassembly and organization. As expected, the proteins of the OD group matched\nbiological functions related to embryo and general organism development; only two of\nthese proteins were detected in the FF of the patients in the PCOS group. The main\ncanonical pathways ( Supplemental Table III )\nfound only for the proteins in the FF from the PCOS patients were LXR/RXR activation\n( p =9.04 E -11 , overlap 7%) and FXR/RXR activation\n( p =1.67 E -10 , overlap 6.6%), which are key for the\nmetabolism of lipids, lipoproteins and glucose, reflecting the disrupted metabolism\nexhibited by PCOS patients. In addition, proteins associated with the intrinsic\n( p =1.70E -07 , overlap 22.2%) and extrinsic\n( p =1.48E -06 , overlap 13.3%) prothrombin activation\npathways were identified in the FF from the PCOS group.\nMolecular and cellular function and physiological system development and\nfunction matched to the proteins detected in the FF from PCOS and OD\npatients\n\nOur findings showed significantly diminished expression of proteins involved in key\nprocesses associated with oocyte competence and embryo development in PCOS patients.\nIn addition, overexpression of proteins related to oxidative stress, the immune\nresponse and lipid, lipoprotein and carbohydrate metabolism was observed in these\npatients. Although many proteomics analyses of FF have been published recently, the\nfunctional correlations among these proteins are still poorly recognized. We\nattempted to correlate the differentially expressed proteins in the FF from PCOS\npatients with physiological pathways. We believe that the observed differences may\nreflect the PCOS patients’ diminished embryo quality, as this factor is directly\nreliant on oocyte characteristics.\nIn our study, the inflammatory pathway represented by complement C3 protein and\nvitronectin was overexpressed in the FF from the PCOS group. The augmented levels of\nthese proteins in the FF seems to be related to poor oocyte quality, potentially\nexplaining IVF failure ( Estes  et\nal ., 2009 ). Additionally, excess complement cascade activation\nleads to deficiencies in vascular endothelial growth factor (VEGF) activity, which\nis essential for proper oocyte maturation ( Jarkovska\n et al ., 2010 ). Another marker for the disruption of\nthe inflammatory pathway in PCOS patients is the overexpression of\nalpha-2-HS-glycoprotein (fetuin-A). This protein is an acute-phase inflammatory\nregulator that is usually upregulated in OHSS ( Jarkovska  et al ., 2011 ). As we excluded OHSS patients\nand applied a GnRH agonist analogue to trigger ovulation, the presence of fetuin-A\nwas not expected and may contribute to the decreased oocyte quality in those\npatients.\nMoreover, the poor oocyte quality and deregulated inflammatory status of PCOS\npatients may be related to the overexpression of vitamin D-binding protein (VDBP) in\ntheir FF. VDBP was another protein found exclusively in the FF of the PCOS group,\nand according to the literature, this protein may be related to decreased\nimplantation, pregnancy ( Estes  et\nal ., 2009 ), and live birth rates ( Benkhalifa  et al ., 2015 ); VDBP is even more strongly\nassociated with a higher risk of miscarriages ( Kushnir  et al ., 2012 ) and foetal growth restriction\n( Wookey  et al .,\n2017 ).\nThe overexpressed coagulation pathway found in PCOS-FF, characterized by intrinsic\nand extrinsic prothrombin activation, is also linked to an inflammatory response;\nthis pathway has important roles in follicle physiology ( de Agostini, 2006 ) and may be associated with poor IVF outcomes\n( Bianchi  et al .,\n2016 ).\nThe exclusive and overexpressed proteins in the OD group, such as 26S protease,\nalpha-1-acid glycoprotein 1 and alpha-2-macroglobulin, are correlated with a better\novarian stimulation response. The 26S protease is a highly specialized, conserved\nribonucleoprotein that facilitates assembly of proteasome complexes; this protein is\ndirectly and indirectly involved in the regulation of gene expression ( Mittenberg, 2014 ). Alpha-2-macroglobulin is\nlinked to intrinsic and extrinsic coagulation cascades and is correlated with the\ncomplement pathway ( Hanrieder  et\nal. , 2009 ). The adequate regulation of coagulation and\nimmune response pathways is essential for the extracellular matrix (ECM) modelling\nthat facilitates follicular growth, ovulation and corpus luteum formation ( Kamat  et al ., 1995 ;  Curry & Smith, 2006 ), which may be more\neffective in fertile women.\nFurthermore, PCOS is frequently associated with disrupted lipid and carbohydrate\nmetabolism ( Dumesic  et al .,\n2015 ). We found some proteins in the PCOS-FF that were absent in the OD\ngroup; these proteins represented metabolic pathways, and their presence\ncorroborated previous findings ( Dai & Lu,\n2012 ;  Ambekar  et al .,\n2015 ). Our findings at the FF level suggest that the impairment of lipid\nand lipoprotein metabolism also occurs within a specific microenvironment, such as\nthat of infertile women with PCOS and a normal BMI. The increased inflammatory\nstatus and metabolic disruption observed through the protein composition of the FF\nfrom our PCOS patients seem to lead to a worse prognosis for oocyte viability and\nmay affect IVF outcomes. Previous studies of PCOS patients undergoing IVF treatment\nobtained a great number of oocytes but reported poor fertilization and embryo\ndevelopment rates, an outcome that may be linked to deregulated oocyte activation\nthrough a damaged microenvironment ( Jungheim\n et al ., 2009 ).\nTo find potential markers of oocyte quality, our inclusion and exclusion criteria\nwere very strict to allow us to identify markers that are exclusive to PCOS-FF\nwithout overlapping with other pathologies and conditions, such as obesity or OHSS.\nWe hypothesize that the evaluation of fetuin-A, VDBP, complement C3 and 26S protease\nexpression in the FF of PCOS patients undergoing IVF could be associated with oocyte\nquality. The limitations of these findings include the absence of experimental\nvalidation of the candidate markers through other techniques, such as Western\nblotting; additionally, the differentially expressed proteins must be correlated\nwith the final IVF outcomes to endorse their use in clinical practice.","source_license":"CC-BY-4.0","license_restricted":false}