{"paper_id":"59cf4b2f-99fc-436f-8144-b0e0c917a377","body_text":"1 \n \nTargeted follicular fluid proteomics using reverse phase protein arrays (RPPA); a \nfeasibility study \n \nMichael S. Bloom PhDa, Veronica G. Sanchez BSb, Victor Y. Fujimoto MDc, Makeda Tamratb, \nJenna R. Krall PhDd, Virginia Espina PhDb \n \naDepartment of Global and Community Health, College of Public Health, George Mason \nUniversity, Fairfax, Virginia, USA; bCenter for Applied Proteomics and Molecular Medicine, \nGeorge Mason University, Manassas, Virginia, USA; cDepartment of Obstetrics, Gynecology, \nand Reproductive Sciences, University of California at San Francisco, San Francisco, California, \nUSA \n \nCorresponding Author:  \nMichael S. Bloom, PhD \nDepartment of Global and Community Health \nGeorge Mason University \n4400 University Dr., MS 5B7 \nFairfax, Virginia, 22030 \n(703) 998-8588 \nmbloom22@gmu.edu \n \n \nAbstract \nThis small pilot feasibility study shows that reverse phase protein array (RPPA) technology is a \nuseful tool for targeted proteomics analysis in human ovarian follicular fluid. RPPA supplements \nmass spectrometry approaches that are currently used by providing functional signal \ntransduction data that drive cellular biology. Herein, we present the first report of using RPPA in \nfollicular fluid to elucidate protein signaling pathways. The results show potential associations \nbetween follicular fluid proteins measured with RPPA and reproductive outcomes from in vitro \nfertilization, including oocyte maturity, oocyte fertilization, embryo quality, and pregnancy. This \nstudy provides evidence that RPPA is a feasible approach to be used in clinical studies of \nreproductive endpoints. However, a larger study of RPPA to identify diagnostic and prognostic \nfollicular fluid protein biomarkers of infertility is needed.  \n \n  \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 4, 2026. ; https://doi.org/10.64898/2026.02.02.26345389doi: medRxiv preprint \nNOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.\n\n2 \n \nIntroduction \nMore than 20% of married and cohabiting women without a previous birth, approximately 5 \nmillion US women, experienced infertility between 2015 and 2019 [1]. Infertility, defined as 12 \nmonths of unprotected intercourse without a pregnancy, has driven increased use of in vitro \nfertilization (IVF) [2]. In 2022, US clinics initiated over 206,304 IVF cycles with intended embryo \ntransfers, resulting in 98,289 live births, yet more than 110,000 IVF cycles failed [3]. Many \nwomen require multiple IVF cycles, facing psychological distress [4,5], higher obstetrical risks \n[6,7], and potential long-term adverse health consequences [8–11], along with financial costs \nthat often exceed $19,000 per cycle [12]. Transferring multiple embryos to improve the chances \nof a live birth also raises the risks of multiple gestations and adverse birth outcome [13–15]. \nWith two-thirds of IVF cycles failing to produce a live birth, there is a critical need to identify \nbiological targets to improve outcomes [3,16,17]. \n \nProteomic profiling of follicular fluid (FF) collected and retained during IVF can offer direct \nmeasures of critical proteins that drive oocyte maturation and the subsequent developmental \nevents that eventually lead to live birth. While great advances have been made in describing the \nFF proteome, results have been inconsistent and few clinically-actionable data have emerged \n[18]. Mass spectrometry (MS) methods have identified highly abundant FF proteins and \npeptides, but these have limited sensitivity for post-translationally modified proteins, and require \nextensive expertise and data processing, large sample volumes, and complementary multiple \nreaction monitoring with heavy isotope labeled peptides for quantification.  \n \nAs a complementary approach to MS, reverse phase protein arrays (RPPA) offer a cost \neffective and multiplex strategy that has been extensively validated with biological fluids and \ntissues and is used in oncology clinical trials for developing precision tumor treatments [19–23]. \nHowever, RPPA has not been previously reported with FF. RPPA allows analysis of small \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 4, 2026. ; https://doi.org/10.64898/2026.02.02.26345389doi: medRxiv preprint \n\n3 \n \nsample volumes (e.g., nanograms), with the sensitivity (e.g., femtograms) and specificity of \nmonoclonal antibodies, while quantifying proteins and protein modifications  that govern their \nbiological activity, such as phosphorylation, acetylation, or cleavage [22]. To assess the \nfeasibility, we conducted an outcome-blinded pilot assessment of 21 FF proteins from critical \nbiological pathways selected a priori in 6 FF specimens from women undergoing IVF.  \n \nMethods \nStudy Protocol: \nUp to 4 independent FF samples were collected from 56 women undergoing IVF and enrolled in \nthe Study of Metals and Assisted Reproductive Technologies (SMART) in 2015-2017. The study \nprotocol was described in detail in a previous publication [24]. Briefly, women underwent \ngonadotropin-induced ovarian stimulation with serial ultrasounds and estrogen measures. \nHuman chorionic gonadotropin was administered approximately 2 weeks later, after follicles \ndeveloped to ≥17mm diameter, and oocytes were retrieved after 34-36 hours by transvaginal \nfine needle aspiration. In each ovary, the largest follicle was aspirated; after evacuation, the \nneedle was flushed with saline before sampling a second follicle, but the follicle itself was not \nflushed to preserve native analyte concentrations. After the oocyte was removed, each \nindividual 3.5-5mL FF sample was centrifuged to pellet residual cells and debris and aliquoted \nthe supernatant into 1.8mL cryovials, which were frozen at -80 °C. Any samples showing \nevidence of red blood were discarded [25]. \n \nMature oocytes collected from ovarian follicles in metaphase-2 arrest (MII arrest) were fertilized \nwith sperm using intracytoplasmic sperm injection or conventional insemination. Fertilization \nwas confirmed after 16–20 hours by the appearance of 2 pronuclei (2PN). Embryo and \nblastocyst quality were categorized as “high quality” or “low quality’ based on a day 2 or 3 \nembryo examination of blastomere fragmentation, cleavage rate, and blastomere symmetry \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 4, 2026. ; https://doi.org/10.64898/2026.02.02.26345389doi: medRxiv preprint \n\n4 \n \n[26–30], or Gardner’s scale for day 5 blastocysts [31]. Fresh embryos or blastocysts were \ntransferred and a positive serum hCG test 2 weeks later indicated pregnancy. All participants \ncompleted written informed consent and the study protocol was approved by the UCSF \nCommittee on Human Research. \n \nReverse Phase Protein Arrays (RPPA): \nRPPA analysis was used to quantify 21 FF proteins, normalized to total FF protein \nconcentration, in a single FF from 3 women with an IVF pregnancy and 3 without an IVF \npregnancy, randomly selected from SMART participants. FF was printed in two-fold serial \ndilutions on a nitrocellulose-coated slide (Grace BioLabs, Oncyte Avid, Bend, Oregon, USA) that \nincluded calibrators and controls for rigorous, clinical diagnostic level analysis. Twenty-one FF \nproteins were selected a priori based on validated antibodies available in our Center from the \nendocannabinoid system, oxidative stress and inflammatory response, epigenetic markers, \ntryptophan metabolism, cell division and migration, hormone synthesis and function, DNA \ndamage and repair, and vitamin D homeostasis pathways as described in Table 1.  \n \nEach array was probed with a validated, commercially available monoclonal or polyclonal \nantibody. Antibody validation is performed for each antibody and whenever a new lot number of \nantibody is received, following CAP/CLIA compliant standard operating procedures. There is a \ncompendium of more than 400 validated antibodies in our Center. Each sample, control, and \ncalibrator was printed in technical replicates on the arrays (required coefficient of variation < \n15%) [32] (Figure 1). Bovine serum albumin was used as the total protein calibrator. \nCommercial cell lysates served as process controls. Calibrators, created from cell lines, \nspanned the limits of quantification for the proteins of interest. The calibrators may also be used \nto interpolate the relative intensity values of each spot on the array. The pixel intensity of each \nspot was quantified using a calibrated flatbed scanner (UMAX PowerLook, UMAX  Technologies, \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 4, 2026. ; https://doi.org/10.64898/2026.02.02.26345389doi: medRxiv preprint \n\n5 \n \nDallas, Texas, USA) for chromogenic detection or a laser scanner (Tecan Group Ltd., Zurich, \nSwitzerland) for fluorescent detection.  \n \nSignal intensities greater than 3 standard deviations above background were considered to \nhave an adequate signal-to-noise ratio for the analysis [33]. RPPA spot intensity was \ndetermined using ImageQuant v.5.2 software (Cytiva, Marlborough, Massachusetts, USA). The \nlocal area background was subtracted from each spot and the data were normalized to total \nprotein using an in-house VBA Macro (RPPA Analysis Suite) [33].  \n \nData Analysis: \nWe estimated the associations between FF proteins, oocyte quality, and embryo quality \noutcomes using Spearman correlation coefficients and compared differences in mean FF \nprotein concentrations between women with and without an IVF pregnancy. We defined \nstatistical significance as P<0.10 for a 2-tailed test to accommodate the limited sample size and \ngenerate hypotheses for future confirmation. \n \nResults \nSeventeen of 21 FF proteins were quantified using RPPA in our CAP/CLIA-accredited \nproteomics laboratory, including TTP1, NFKB, p53BP1, Vitamin D Binding Protein, IL-1b, \nCleaved Caspase3, Prolactin Receptor, Prdx-1, Manganese Super Oxide Dismutase, IL-6, \nWnt5ab, Vitamin D Receptor, PPARgamma, IGF1R-beta, IDO, BLM, and DAG Lipase alpha \n(abbreviations are defined in Table 1). FF glutamine dehydrogenase, HDAC3, CuZnSOD, and \nERβ were not quantifiable.  \n \nAs shown in Figure 2, we found a mixed pattern of moderate to strong pairwise Spearman \ncorrelations between FF proteins and intermediate IVF outcomes. Oocyte maturity (MII arrest) \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 4, 2026. ; https://doi.org/10.64898/2026.02.02.26345389doi: medRxiv preprint \n\n6 \n \nand fertilization (2PN) correlated negatively to TPP1 and P53BP1, but positively correlated to \nNFKB, VitDBP, IL-1b, ClCasp3, ProR, Prdx-1, MnSOD, IL-6, Wnt5ab, VitDR, PPARgamma, \nIGF1Rbeta, and IDO. In contrast, greater TPP1 and P53BP1 correlated to high cleavage stage \nembryo quality, but greater FF VitDBP and IGF1Rbeta were associated with low cleavage stage \nembryo quality. There were no statistically significant correlations with blastocyst quality, which \nmay have been due to the limited number of high quality blastocysts in the sample (i.e., 6 high \nquality blastocysts of 42 total blastocysts). \n \nAs shown in Figure 3, we found greater mean levels of NFKB (37.3%), P53BP1 (25.5%), \nVitDBP (29.8%), IL-1b (42.8%), ClCasp3 (35.6%), ProR (18.0%), Prdx-1 (15.6%), MnSOD \n(70.7%), IL-6 (43.0%), Wnt5ab (70.8%), VitDR (81.3%), IGF1R-beta (535.9%), and \nDAGLipalpha (44.2%) among non-pregnant than pregnant women. However, the sample size \nwas too limited for formal hypothesis testing.  \n \nDiscussion \nIn this small feasibility study, we found different FF protein concentrations according to oocyte \nfertilization, embryo quality, and pregnancy outcomes using RPPA. Our results suggest that FF \nproteins in the endocannabinoid system (DAG-lipase α findings) [34–36], Wnt signaling pathway \n(Wnt5ab findings) [37,38], acute phase immune response (IL-6 and NFKB findings) [39,40], \noxidative stress response (MnSOD and p53BP1 findings) [41,42], innate immune response \n(IL-1β and NFKB findings) [40,43], and vitamin D signaling pathway (VD3R and VDBP findings) \n[44–46] may be important determinants of IVF outcomes. However, these results are \npreliminary and a larger and more comprehensive analysis will be necessary for definitive \nresults.  \n \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 4, 2026. ; https://doi.org/10.64898/2026.02.02.26345389doi: medRxiv preprint \n\n7 \n \nThe potential for an oocyte to complete meiosis, fertilize normally, and support early embryo \ndevelopment, collectively referred to as oocyte maturation, depends on nuclear, epigenetic, and \ncytoplasmic processes that influence IVF outcomes [47,48]. Throughout development, oocytes \nare immersed in FF, which mediates nutrient exchange and biochemical communication with \nmural granulosa cells, cumulus cells, and the vascular compartment [49,50]. FF contains a \nplasma ultrafiltrate restricted by the blood–follicle barrier to molecules less than ~300 kDa, \nalong with factors produced in situ by granulosa cells and the oocyte itself, creating a \nspecialized microenvironment [51–53]. For example, 22 FF proteins differed between women \nwith and without ovarian pathology in a recent study, while only 2 were detectable in matched \nserum [54]. Because FF is the fluid most proximate to the oocyte, it provides a unique window \ninto the microfollicular environment and can reveal protein markers predictive of IVF outcomes \nthat are diluted or undetectable in peripheral blood [49,55]. \n \nProteomic profiling of FF collected during IVF provides insight into the protein systems that drive \noocyte maturation and the downstream developmental events that lead to pregnancy and live \nbirth [49,56,57]. Unlike genomic or transcriptomic assays, which may correlate only weakly with \nprotein abundance or activation status, proteomic approaches directly characterize follicular \nphenotype in real time [58]. Over the past 15 years, FF proteome profiling has expanded \nsubstantially [17,56]. Investigators have increasingly identified and quantified proteins from \nmajor biological pathways in efforts to develop diagnostic and prognostic biomarkers. A \nsynthesis of 617 FF proteins reported through 2014 highlighted acute phase inflammatory \nresponse, wound response, complement, coagulation, lipid metabolism, and cytoskeletal \npathways as most frequently represented, with matrix metalloproteinases (MMPs), thrombin, \nand vitamin D/Retinoid X Receptor alpha emerging as central hubs [59]. Another study detected \n742 proteins in pooled FF from 3 ovum donors using MS, primarily related to growth factor and \nreceptor signaling, immunity, anti-apoptosis, MMP activity, and complement [60]. An MS-based \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 4, 2026. ; https://doi.org/10.64898/2026.02.02.26345389doi: medRxiv preprint \n\n8 \n \nbiological pathway analysis similarly confirmed the prominence of complement and coagulation \nsystems, with additional emphasis on innate immunity and angiogenesis [61]. \n \nMore recent studies have expanded the FF proteome further. One MS analysis identified 2461 \nFF proteins, including 1108 detected for the first time, enriched in metabolic processes and \nbiological regulation [62]. A SWATH (Sequential Window Acquisition of all Theoretical Mass \nSpectra)-MS study detected 2182 FF proteins, most lacking known metabolic functions, and \nothers involved in coagulation, integrin signaling, gonadotropin-releasing hormone signaling, \nplasminogen activation, and Wnt signaling [63]. Despite substantial progress, findings across \nstudies remain inconsistent and few clinically actionable biomarkers have emerged \n[18,49,56,59,64]. \n \nWhile MS-based approaches have identified highly abundant FF proteins and peptides, these \nmethods require complex, specialized workflows for measuring low-abundance proteins and \npeptides in FF and their post-translational modifications [65]. The results of this feasibility study \nsuggest that RPPA is a feasible complementary technology to quantify low-abundance ovarian \nFF proteins and their post-translationally modified forms that predict IVF outcomes, and may \nserve as diagnostic/prognostic indicators and targets for interventions. However, a larger \nvalidation study will be necessary to confirm that RPPA technology is a feasible approach to \ninvestigate FF proteins as potential clinical and prognostic indicators of IVF outcomes or targets \nfor clinical intervention. \n  \n . 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Using reverse-phase protein arrays as pharmacodynamic \nassays for functional proteomics, biomarker discovery, and drug development in cancer. \nPharmacodyn Cancer Drug Dev 2016;43:476–83. \nhttps://doi.org/10.1053/j.seminoncol.2016.06.005. \n \n  \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 4, 2026. ; https://doi.org/10.64898/2026.02.02.26345389doi: medRxiv preprint \n\n13 \n \nTable 1. Proteins analyzed in human ovarian follicular fluid using RPPA \nProtein Abbreviation Source \nTripeptidyl peptidase 1 TTP1 Cell Signaling Technology \nNuclear factor-κB  NFKB Cell Signaling Technology \nP53 binding protein P53BP1 Cell Signaling Technology \nVitamin D binding protein VitDBP Abcam \nInterleukin-1 β IL-1b Cell Signaling Technology \nEstrogen receptor β ERbeta DSHB \nCopper-Zinc superoxide dismutase CuZnSOD StressGen Biotechnologies \nCleaved caspase 3 (Asp175) ClCasp3 Cell Signaling Technology \nProlactin receptor ProR Epitomics \nPhosphorylated (Tyr 194) peroxiredoxin 1 Prdx-1 Cell Signaling Technology \nManganese superoxide dismutase MnSOD Assay Designs \nInterleukin-6 IL-6 BioVision \nWingless integrated 5a/5b Wnt5ab Cell Signaling Technology \nVitamin D3 receptor VitDR Cell Signaling Technology \nPeroxisome proliferator-activated receptor γ PPARgamma Cell Signaling Technology \nPhosphorylated insulin-like growth factor  \nreceptor β (Tyr 1135/36) \nIGF1R-beta Cell Signaling Technology \nIndoleamine-pyrrole 2,3-dioxygenase  IDO Cell Signaling Technology \nCleaved histone deacetylase 3 HDAC3 Cell Signaling Technology \nBloom syndrome protein BLM Cell Signaling Technology \nGlutamine dehydrogenase GDH Cell Signaling Technology \nDiacylglycerol lipase α  DAGLipalpha Cell Signaling Technology \nAbbreviations: RPPA, reverse phase protein arrays \n \n  \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 4, 2026. ; https://doi.org/10.64898/2026.02.02.26345389doi: medRxiv preprint \n\n14 \n \nFigure 1. Reverse Phase Protein Arrays (RPPA) provide quantitative data for cell \nsignaling kinases and their post-translational modified forms. Specimens, control, and \ncalibrators are printed on nitrocellulose coated slides in replicate dilution curves. Each array is \nprobed with a single, validated antibody and catalyzed signal amplification chemistries.  \n \n  \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 4, 2026. ; https://doi.org/10.64898/2026.02.02.26345389doi: medRxiv preprint \n\n15 \n \nFigure 2. Spearman correlation coefficients between follicular fluid proteins measured using \nRPPA and intermediate IVF outcomes among women using IVF  \n \nAbbreviations: BLM, Bloom syndrome protein; ClCasp3, cleaved caspase 3; DAGLipalpha, \ndiacylglycerol lipase α; HQ, high quality; IDO, indoleamine-pyrrole 2,3-dioxygenase; IGF1Rbeta, \nphosphorylated insulin-like growth factor receptor β; IL-1b, interleukin-1 β; IL-6, interleukin 6; \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 4, 2026. ; https://doi.org/10.64898/2026.02.02.26345389doi: medRxiv preprint \n\n16 \n \nIVF, in vitro fertilization; MnSOD, manganese superoxide dismutase; NFKB, nuclear factor \nkappa β; NS, not significant; P53BP1, P53 binding protein; PPARgamma, peroxisome \nproliferator-activated receptor γ; Prdx-1, peroxiredoxin 1; ProR, prolactin receptor; RPPA, \nreverse phase protein array; TPP1, Tripeptidyl peptidase 1; VitDBP, vitamin D binding protein; \nVitDR, vitamin D3 receptor; Wnt5ab, Wingless integrated 5a/5b \n \nNOTE: Colors in the boxes correspond to the Spearman correlation coefficients between \nindividual follicular fluid proteins and the intermediate IVF outcomes (n for each test listed in \neach column), Darker green indicates a more positive correlation and darker red indicates a \nmore negative correlation. Statistical significance was defined as P<0.10. \n \n \n  \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 4, 2026. ; https://doi.org/10.64898/2026.02.02.26345389doi: medRxiv preprint \n\n17 \n \nFigure 3. Mean (95% confidence interval) follicular fluid protein concentrations measured \nusing RPPA, between women with (n=3) and without (n=3) an IVF pregnancy. \n \nAbbreviations: BLM, Bloom syndrome protein; ClCasp3, cleaved caspase 3; DAGLipalpha, \ndiacylglycerol lipase α; HQ, high quality; IDO, indoleamine-pyrrole 2,3-dioxygenase; IGF1Rbeta, \nphosphorylated insulin-like growth factor receptor β; IL-1b, interleukin-1 β; IL-6, interleukin 6; \nIVF, in vitro fertilization; MnSOD, manganese superoxide dismutase; NFKB, nuclear factor \nkappa β; P53BP1, P53 binding protein; PPARgamma, peroxisome proliferator-activated \nreceptor γ; Prdx-1, peroxiredoxin 1; ProR, prolactin receptor; RPPA, reverse phase protein \narray; TPP1, Tripeptidyl peptidase 1; VitDBP, vitamin D binding protein; VitDR, vitamin D3 \nreceptor; Wnt5ab, Wingless integrated 5a/5b \n \nNOTE: Symbols correspond to mean follicular fluid protein concentrations (n=3 in each \npregnancy group) and whiskers represent 95% confidence intervals. Symbols without whiskers \nrepresent uniformly measured values for a pregnancy group (i.e., BLM, IDO, PPARgamma), or \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 4, 2026. ; https://doi.org/10.64898/2026.02.02.26345389doi: medRxiv preprint \n\n18 \n \nmeasurement of n=1 or n=2 for a pregnancy group (i.e., BLM, IDO, IGF1Rbeta, PPARgamma, \nVitDR, VitDBP). \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted February 4, 2026. ; https://doi.org/10.64898/2026.02.02.26345389doi: medRxiv preprint","source_license":"CC0","license_restricted":false}