{"paper_id":"2690fa30-b23c-4d0e-9191-d1a2e21e2f39","body_text":"In vitro  fertilization (IVF) has gained attention since its\nintroduction in 1978 and, to date, more than 2 million babies have been born\nworldwide through assisted reproductive technologies (ART). Developments in\ntechnologies for IVF, embryo culture and therapies for ovarian stimulation make ART\nrelatively successful. However, it has its limitations with >50% of IVF embryos\nfailing to implant. Selecting viable embryos for transfer is a key factor for the\nsuccess of IVF treatment; and requires accurate pre-transfer assessment of embryo\nviability and morphology. However, the available methods to identify high\nimplantation potential embryos are still limited ( Gardner  et al ., 2015 ).\nThe field of human ART would therefore benefit from more quantitative methods of\ndetermining embryo viability and implantation potential to further improve the\npregnancy rates. It is known that soluble ligands and its receptors mediate human\npre-implantation embryo development and implantation ( Thouas  et al ., 2015 ). Non-invasive analyses of\nthe embryonic secretome, including proteins secreted by the embryo into the\nsurrounding media, provide an alternative method for assessing an embryo's\nviability. These results may promote an understanding of the biological mechanisms,\nand potentially allow for the success of IVF developmental biomarkers.\nA study evaluating conditioned media from non-manipulated human embryos cultured in\nvitro demonstrated that it contains extracellular vesicles and bearing the\ntraditional microvesicle and exosome marker proteins CD63, CD9 and ALIX, which may\nsuggested a way of communication at the maternal-fetal interface ( Giacomini  et al ., 2017 ). A\nnumber of researchers has used the proteomics approach related to human reproduction\nin several subareas as ectopic pregnancy ( Gerton\n et al ., 2004 ), miscarriage ( Liu  et al ., 2006 ), follicular fluid ( Lo Turco  et al ., 2010 ;  Kim  et al ., 2006 ;  Silberstein  et al ., 2009 ;\n Estes  et al ., 2009 ;\n Twigt  et al ., 2012 ;\n Liu  et al ., 2007 ),\noocytes ( Ferreira  et al .,\n2010 ), endometrium ( Matorras  et\nal ., 2018 ) and embryos ( Ferreira  et al ., 2010 ;  Katz-Jaffe & Gardner, 2007 ;  Katz-Jaffe  et al ., 2009 ;  Dominguez  et al ., 2009 ). Recently, various\nproteins have been investigated as an embryo biomarker in the spent culture media\n( Butler  et al ., 2013 ;\n Mains  et al ., 2011 ;\n Ziebe  et al ., 2013 ;\n Cortezzi  et al ., 2013 ;\n Dominguez  et al ., 2008 )\ngiven that the embryo culture medium secretome reflects the embryo development. We\nhypothesize that protein profiles are affected according with infertility factors,\nwhich can be responsible for detrimental embryonic developmental competence, ensuing\non lower IVF success rates. Hence, we studied samples from patients presenting deep\ninfiltrating endometriosis (ENDO) and polycystic ovarian syndrome (PCOS), which are\nthe most frequent female factors of infertility. Endometriosis is present in until\n50% ( Missmer & Cramer, 2003 ) of infertile\nwomen and PCOS is present in the main cause of anovulatory infertility ( Thessaloniki ESHRE/ASRM-Sponsored PCOS Consensus\nWorkshop Group, 2008 ). It is clear that the ovarian microenviroment is\naffected by the presence of endometriosis ( Regiani\n et al ., 2015 ; Singh  et al ., 2013;\n Karita  et al ., 2011 ) and\nPCOS ( Huang  et al ., 2013 ;\n Ambekar  et al ., 2015 ;\n Roth  et al ., 2014 )\nBased on the knowledge that the potential of embryo development is an extension of\noocyte quality, and the last is directly influenced by the microenviroment of ovary,\npossibly being affected by the presence of PCOS or ENDO, we hypothesized the embryo\nsecretoma during its pre-implantation development could also vary reflecting the\ninfertility factor condition. The aim of this study was to screen the protein\nprofile of conditioned embryo culture media in patients with PCOS and ENDO\nundergoing IVF by proteomics approaches.\n\nIt was included in the study conditioned embryo culture media samples obtained\nfrom patients submitted to IVF cycle at the Huntington - Reproductive Medicine,\nSao Paulo - Brazil. Institutional Ethical approval secured for the use of\nsamples for the purpose of this research and the samples were donated by\nconsenting patients whose signed the Informed Consenting Form as established by\nethics for assisted reproduction treatment and research ( CFM, 2013 ). Clinical characteristics and outcomes were\nobtained from patients' charts.\nAll women presented with the following inclusion criteria: infertile patients\nundergoing ICSI cycles with ejaculated sperm, presence of both ovaries, regular\nmenstrual cycle, body mass index (BMI) lower than 35 Kg/m 2 , no\nongoing infectious disease, no uterus pathology, basal follicular stimulating\nhormone (FSH) measurement <14IU/L, and basal estradiol measurement\n<70pg/mL. The exclusion criteria were presence of gynecological bleeding,\nhydrosalpinx, allergy to gonadotropins or other medications used in the\ntreatment, abusive use of any other medications during treatment, and male\npartners presenting with severe oligozoospermia.\nAmong patients included, three groups were analyzed according to infertility\netiology: tubal factor patients who were considered the control group for this\nstudy (CONTROL; n=6), infertile patients presenting PCOS (PCOS; n=7) and\ninfertile patients presenting endometriosis grades III and IV (ENDO; n=14). The\ndiagnosis of infertility was carried out according to international\npatterns.\nPatients were submitted to pituitary blockage and controlled ovarian stimulation\nas routine. The pituitary blockage was obtained with a GnRH agonist (Lupron\nkit™, Abbot SA Societé Française des Laboratories, France),\nand the ovarian stimulation was performed using recombinant FSH (rFSH, Gonal-F\n ®  Serono, Switzerland). When at least two follicles\nreached a diameter of 16 mm, the final follicular maturation was triggered with\n250µg of recombinant hCG (rhCG, Ovidrel ® , Serono,\nSwitzerland). Oocyte retrieval was performed after 35 to 36 hours by\ntransvaginal ultrasound-guided aspiration, and the luteal phase was supported by\n90mg of daily progesterone (Crinone ® , Serono, Switzerland) via\nvaginal approach.\nAfter oocyte recovery and denudation, all of the mature oocytes were fertilised\nby ICSI ( Palermo  et al .,\n1992 ) as per the routine of the clinic. The normally fertilized\noocytes were identified and cultured in groups until day 3 (D3) in 1 mL of cell\nculture medium (G-1 Plus, Vitrolife) under a layer of paraffin oil (OVOIL,\nVitrolife), in incubators with 5% O 2  and 5% CO 2 . From D3\nuntil the blastocyst stage (D5), the embryos were cultured in 1 mL of medium\ncontaining 10% human albumin (CSCM, Irvine Scientific) under a layer of paraffin\noil in triple gas incubators (90% N2, 5% O 2  and 5% CO 2 ).\nThe blastocysts were morphologically classified and the highest grades ones were\nselected for transfer to the women's uterus using a catheter guided by\nultrasound. The culture media was collected on day 3 of development and stored\nat -80ºC until use. For the proteomics analysis, two pools of samples\nwere prepared for groups CONTROL and PCOS, and four pools for group ENDO.\nAll analyzes in this step of study were carried out at Proteomics and Mass\nSpectrometry Facility, Center for Drug Discovery and Innovation (CDDI),\nUniversity of South Florida (USF), Tampa - FL, USA. Arbitrary samples were used\nfor methods standardization, and clinical samples were pooled into 3 to 4\nsamples pools according to pre-established groups (Control, PCOS and ENDO).\nSamples were prepared by using two steps of purification in order to eliminate\nthe excess of albumin. First, samples were submitted to microfiltration using a\n30KDa pore filter (Amicon ®  Ultra-0.5, Centrifugal Filter\nDevices - 30 kDa, Millipore, USA) and the filtrated material was collected. The\nmaterial retained in the filter (higher than 30 KDa) was albumin depleted using\naffinity chromatography columns (Albumin & IgG Depletion SpinTrap, GE\nHealthcare Life Sciences, USA) according to manufacturer's protocol. The two\naliquots of each sample were joined and protein concentrations were measured by\nBradford method (Pierce 660nm Protein Assay Reagent, Thermo Scientific), using\npre-diluted albumin standard curve (Pre-Diluted Protein Assay Standards: Bovine\nSerum Albumin, Thermo Scientific) and the absorbance's were acquired at 660 nm.\nTwo hundred and fifty micrograms (250µg) of protein for each sample were\ndiluted in 8M urea buffer and digested using FASP kits (Expedeon, Inc, USA) as\nper vendor specifications. Tryptic peptides were acidified by adding\ntrifluoroacetic acid (TFA 1%) to get the 0.1% of TFA concentration, and then\ndesalted on solid phase columns (DSC-18, Solid Phase Extraction - SPE,\nDiscovery ®  DSC-18 SPE Products, Sigma Aldrich). Samples\nwere vacuum dried ( Wiśniewski  et\nal ., 2009 ).\nTryptic peptides resulting from the preparation were fractionated by liquid\nchromatography mass spectrometry (HPLC-MS/MS). Five microliters injection of\neach sample loaded with aqueous solvent (0.1% formic acid in water) were\nseparated by nano-flow reversed phase HPLC using a Nano-LC Ultra 2D+ (Eksigent,\nDublin, CA) equipped with a Proteopep 2 Integra Fit trapping column (100\nµm i.d. x 2.5 cm; C18, 5 µm, 300Ǻ) and a Proteopep 2\nIntegra Fit analytical column (75 µm i.d. x 10 cm; C18, 5 µm,\n300Ǻ, New Objective, Woburn, MA). Samples (0.5 to 3 µg in 5\nµL) were loaded onto the trap column at 2 µL/min (Solvent A) for\n12 minutes, after which a valve was switched to include the analytical column.\nPeptides were then eluted with a gradient (300 nL/min) of 2% B to 35% B over 240\nminutes (Solvent A: 100% H 2 O, 0.1% formic acid, Solvent B: 98%\nacetonitrile, 0.5% formic acid). Eluates were delivered by electrospray\nionization (ESI) at 2 kV and analyzed by data-dependent MS/MS on a LTQ XL\n(Thermo Scientific, San Jose, CA, USA) mass spectrometer equipped with XCalibur\n(version 2.0.7) data acquisition software. Full MS scans were set for centroid\nmode at normal resolution. MS/MS scans were performed on the top ten most\nintense ions from each full scan to acquire spectral data for peptide\nidentification. Dynamic exclusion durations were set to 180s with one repeat and\na list size of 500.\nRaw data acquired were searched against the European Bioinformatics Institute's\n(EBI) universal protein resource database (UniProt, November 2014) using Mascot\n(version 2.2). Peptide and protein validations were performed using the Scaffold\nplataform (version 3.00.08). Search parameters used were as follows: precursor\nmass error tolerance of 20 ppm; fragment mass error tolerance of 0.1 Da; trypsin\nas a protease with one missed cleavage allowed; and carbamidomethylation of\ncysteine as fixed modification and oxidation of methionine as a variable\nmodification. Identified peptides and proteins were validated and visualized\nwith Scaffold 3.6 (Proteome Software, Portland, OR).\nThe proteins identified in the previous analysis were analysed using\nIngenuity™ Pathway Analysis software (IPA™, QIAGEN, Redwood, USA).\nWe considered peptide threshold of 20% and the biological processes were\nstaggered according to the IPA™ Knowledge Base. The association between\nthe identified proteins and canonical pathways of the database was also accessed\nwith IPA™ software using Fisher's exact test (significance of\n p <0.01). From 2880 proteins identified, decoy proteins\nthat are considered false identification were excluded. Two sets of analysis\nwere carried out with remaining proteins using the IPA™. First of all we\nidentified proteins exclusive in each study group (PCOS or ENDO) compared with\nCONTROL. We considered exclusively expressed those protein expressed in at least\ntwo sample pools of each group and absent on the other comparison groups. Then,\nwe evaluated proteins differentially expressed in the study groups (PCOS or\nENDO) compared with CONTROL and considered fold change higher than two.\n\nTable 1  describes the demographic data regards\nthe patients included in this study. In the ENDO group, 17 proteins were exclusively\nexpressed (present in at least two of four samples pools), and two were over\nexpressed compared to CONTROL ( Table 2 ). The\ncanonical pathways identified which were related to proteins exclusively expressed\nin ENDO group were associated with calcium metabolism [ calcium signaling\n( p =0.0057) and transport ( p =0.0058) and\ncalcium induced T -lymphocyte apoptosis ( p =0.041) ] and EGF\nsignaling ( p =0.035) ( Figure\n1 ).\nDemographic characteristics of women included in this study\nANOVA\nProteins exclusively expressed and over expressed in the ENDO compared to\nCONTROL group\nFC=Fold Change. KDa=kilodaltons\nFigure 1 Comparative analysis of canonical pathways based on proteins\nover-expressed in ENDO group compared to CONTROL.  ■  CONTROL\n ■ \nENDO.\nComparative analysis of canonical pathways based on proteins\nover-expressed in ENDO group compared to CONTROL.  ■  CONTROL\n ■ \nENDO.\nOn the other hand, in the PCOS group presented 284 proteins exclusively expressed and\none overexpressed with fold change higher than 2 compared to CONTROL, which were\nassociated with the following pathways: Protein Kinase A signaling and calcium\nsignaling were downregulated, and GADD45 signaling, hydrocarbon receptor signaling\nand GDP-L fucose biosynthesis II were upregulated ( Figure 2 ). The PCOS group had two pools of samples analysed and from 284\nproteins exclusively expressed in the PCOS group, six were present in both pools of\nsamples ( Table 3 ).\nProteins exclusively expressed and over expressed in the PCOS compared to\nCONTROL group\nFC=Fold Change. KDa=kilodaltons\nBased on proteins identified, the CONTROL group had the following cellular and\nmolecular function highlighted: cellular development, cellular movement, amino acid\nmetabolism, small molecule biochemistry, cellular assembly and organization, which\nwere function associated to general cellular development. Also, the embryonic organ\nand tissue development were physiological functions activated based on proteins\nidentified in the 3 study groups of samples.\nFigure 2 Comparative analysis of canonical pathways based on proteins\nover-expressed in PCOS group compared to CONTROL.  ■  CONTROL\n ■ \nPCOS.\nComparative analysis of canonical pathways based on proteins\nover-expressed in PCOS group compared to CONTROL.  ■  CONTROL\n ■ \nPCOS.\n\nIn the last decade, the application of proteomics high throughput methodologies to\nhuman reproductive fluids and cells have delineated novel biochemical functional\nprofiles and molecular processes that characterize and may affect folliculogenesis,\noocyte maturation and quality, and as consequence the embryo development potential\nand clinical outcomes. Embryos are programmed to produce soluble ligands and\nreceptors, which elicit changes in embryo developmental phenotype and also\nmodulating local responses in the receptive endometrium determining an embryo\nmaternal cross talk during peri-implantational period. These signaling pathways are\nhighly complex and it understanding has been gradually improved ( Thouas  et al ., 2015 ). The\nembryos from  in vitro  fertilization also produce soluble factors\nthat are secreted in the spent culture media. Proteomics in spent culture media\ninvolved the measurement of amino acids ( Brison\n et al ., 2004 ;  Sturmey  et al ., 2008 ), proteins ( Katz-Jaffe  et al. , 2006a ; b ;  2009 ;  Nyalwidhe  et al ., 2013 ) and\nmetabolomics evaluates how the embryo alters its microenvironment ( Scott  et al ., 2008 ;  Leese  et al ., 2008 ). Authors\nhave been trying to find if changes in the levels of some molecules are associated\nwith implantation potential of embryos and clinical outcomes of IVF cycles.\nDifferent approaches were necessary based on the nature of the specimens and the\ntypes of analyses being done. The standardization of proteomics approaches embryo\nculture medium was developed for this study, there we could obtain data available to\na general protein profile in the samples. Depletion of abundant proteins combined\nwith multidimensional protein fractionation was instrumental in allowing the study\nof middle- and lower-abundance proteins. Our study demonstrates that the technology\ncan provide a consistent result given the restriction of starting material and time\nto analysis. These proteins with their regulatory pathways may play a vital role in\nreproductive process.\nIn embryos derived from ENDO group, we observed over expression of EGF signaling\npathway compared to CONTROL. EGF is a growth factor family which has been already\nidentified to be expressed by the human preimplantation embryo ( Chia  et al ., 1995 ) and\n in vitro  studies also showed the EGF treatment increases early\nhuman embryo development and blastocyst formation ( Yu  et al ., 2012 ). Animal studies have shown that EGF\nenhanced developmental competence of cat embryos by stimulating cell proliferation\nand modulating the EGFR expression at various developmental stages ( Thongkittidilok  et al ., 2015 ).\nOn the other hand, an  in vitro  study cultured mice embryos with\nhuman tubal fluid from endometriosis patients and observed that the levels of\nembryonic EGF, IGF-I, and their receptors were increased, and it attenuated embryo\ndevelopment by impairing embryonic growth factor/receptor/signal transduction ( Ding  et al ., 2010 ).\nEndometriosis has been associated with impaired IVF outcomes ( Harb  et al ., 2013 ; Somigliana &\nGarcia-Velasco, 2015). Based on that, we can speculate the higher expression of EGF\nin endometriosis group may have an ovarian origin and it can be associated with the\nmechanism of embryo development of those patients.\nOther pathways highlighted were associated with calcium signaling. Studies have shown\nthat mitochondria has an enormous capacity to regulate Ca 2+  ( Giacomello  et al ., 2007 ). The\nembryo development is strongly correlated with the activity of mitochondria, and the\nabnormal distribution of mitochondria exert negative effects on the embryogenesis\ndue to the abnormal ATP distribution ( Nagai\n et al ., 2006 ), because high energy supply around\nnucleus is very important during embryonic development ( Wang  et al ., 2009 ), and may perform an\nimportant function in embryonic cell-cycle transition and embryonic axis\nestablishment ( Whitaker, 2008 ). A study as\nshown that conditioned medium from human embryos also trigger calcium oscillations\nin human endometrial epithelial cells, and importantly, the endometrial responses\nare affected by developmentally competency of embryos as low-quality human embryos\ntrigger prolonged and disorganized calcium oscillations, leading to a uterine stress\nresponse ( Brosens  et al .,\n2014 ). In our study, while samples coming from endometriosis patients\npresented calcium-signaling upregulated, the PCOS samples showed it downregulated.\nIn spite of most of publication find similar clinical outcomes after IVF in PCOS\npatients, it is related to alterations in oocyte quality and consequently in embryo\nquality, may be due to endocrine and intra-ovarian environment ( Sermondade  et al ., 2013 ). The\ncalcium metabolism downregulated in the PCOS embryos might reflect the poorer oocyte\nquality observed in those patients.\nProtein kinase A signaling is also down regulated in PCOS samples. A study in\nnon-human primate showed that mechanisms underlying adrenal hyperandrogenism seen in\nthe human condition of PCOS are further considered in terms of the effects of\naltered relative expression of CYP17, HSD3B2 and CYB5, as well as the altered\nsignaling responses of various kinases including protein kinase A ( Abbott & Bird, 2009 ). GADD45, another\nprotein over expressed in PCOS samples, is a protein that is often induced by DNA\ndamage and other stress signals associated with growth arrest and apoptosis ( Salvador  et al ., 2013 ).\nThe data on secretory activity can vary considerably suggest that embryo can\nintrinsically modulate the microenvironment and may reflect developmental plasticity\nrather than quality.  In vitro  studies provided evidences for a\nputative mechanism by which the decidualized stromal cells sense developmental\nembryos through the molecules secreted in the culture media ( Brosens  et al ., 2014 ). Also, signals emanating\nfrom competent human embryos triggered a very specific transcriptional response in\nthe mouse uterus, characterized by the induction of multiple metabolic genes ( Teklenburg et al, 2010 ). Hence, focusing on\nexposures that infertility factors per se influence differential proteome profiles in\nthe embryo culture media, can help understand its effects in the embryo\ndevelopmental plasticity and competence, and consequently in IVF outcomes.\nThis study relies on the proteomic profile of conditioned embryo culture media in\ndifferent infertility conditions. We did not correlated the proteins identified with\nthe embryo characteristics as samples come from embryos cultured in groups and they\nwere group cultured and we joined them into pools according to infertility factors\nto be analyzed. Also, the sample size is reduced and outcomes should be confirmed\nusing immunodetection technique and in a higher number of samples. Due to high\nconcentration of contaminants in the culture media, as albumin, samples were\nsubmitted to a number of process which might depleted other less abundant proteins.\nTo date, the proteomic approach has proven to be a challenging task due to the\ncomplexity and diversity of the human embryo and heterogeneity across patients and\nwithin embryo cohorts.\nIn summary, the embryonic, organ and tissue development were physiological functions\nactivated based on proteins identified in the three study groups of samples. The\nembryos coming from endometriosis patients present a high calcium activity and on\nthe other hand, embryos coming from PCOS patients showed a decreased calcium action,\nwhich may be related to embryo developmental competence or plasticity. Other\npathways as grow factors through the EGF signaling pathway overexpressed in\nendometriosis embryos and protein kinase A in PCOS were also observed.\nCharacterizing the proteomic embryonic secretome will advance our knowledge of early\nembryogenesis and the embryo's role during the initial stages of implantation.\nAdditionally, the activity in embryo culture medium could lead to improved selection\nof embryos for transfer warrants further investigation.\n\nThe authors gratefully acknowledge the assistance in sample collection of the IVF\nlaboratory team in the Huntington Reproductive Medicine, São Paulo, Brazil,\nand the researchers at the Proteomics and Mass Spectrometry Facility, Center for\nDrug Discovery and Innovation (CDDI), University of South Florida (USF), Tampa - FL,\nUSA, for their assistance with proteomics analysis.\nThey also thank Gabriela Venturini, PhD, University of Sao Paulo Brazil, for helping\nwith bioinformatics analysis and Adriana Invitti, PhD, Federal University of\nSão Paulo, for reading the manuscript and stimulating discussions.","source_license":"public-domain-us","license_restricted":false}