Targeted follicular fluid proteomics using reverse phase protein arrays (RPPA); a feasibility study

This small pilot feasibility study shows that reverse phase protein array (RPPA) technology is a useful tool for targeted proteomics analysis in human ovarian follicular fluid. RPPA supplements mass spectrometry approaches that are currently used by providing functional signal transduction data that drive cellular biology. Herein, we present the first report of using RPPA in follicular fluid to elucidate protein signaling pathways. The results show potential associations between follicular fluid proteins measured with RPPA and reproductive outcomes from in vitro fertilization, including oocyte maturity, oocyte fertilization, embryo quality, and pregnancy. This study provides evidence that RPPA is a feasible approach to be used in clinical studies of reproductive endpoints. However, a larger study of RPPA to identify diagnostic and prognostic follicular fluid protein biomarkers of infertility is needed. . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 4, 2026. ; https://doi.org/10.64898/2026.02.02.26345389doi: medRxiv preprint NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. 2

More than 20% of married and cohabiting women without a previous birth, approximately 5 million US women, experienced infertility between 2015 and 2019 [1]. Infertility, defined as 12 months of unprotected intercourse without a pregnancy, has driven increased use of in vitro fertilization (IVF) [2]. In 2022, US clinics initiated over 206,304 IVF cycles with intended embryo transfers, resulting in 98,289 live births, yet more than 110,000 IVF cycles failed [3]. Many women require multiple IVF cycles, facing psychological distress [4,5], higher obstetrical risks [6,7], and potential long-term adverse health consequences [8–11], along with financial costs that often exceed $19,000 per cycle [12]. Transferring multiple embryos to improve the chances of a live birth also raises the risks of multiple gestations and adverse birth outcome [13–15]. With two-thirds of IVF cycles failing to produce a live birth, there is a critical need to identify biological targets to improve outcomes [3,16,17]. Proteomic profiling of follicular fluid (FF) collected and retained during IVF can offer direct measures of critical proteins that drive oocyte maturation and the subsequent developmental events that eventually lead to live birth. While great advances have been made in describing the FF proteome, results have been inconsistent and few clinically-actionable data have emerged [18]. Mass spectrometry (MS) methods have identified highly abundant FF proteins and peptides, but these have limited sensitivity for post-translationally modified proteins, and require extensive expertise and data processing, large sample volumes, and complementary multiple reaction monitoring with heavy isotope labeled peptides for quantification. As a complementary approach to MS, reverse phase protein arrays (RPPA) offer a cost effective and multiplex strategy that has been extensively validated with biological fluids and tissues and is used in oncology clinical trials for developing precision tumor treatments [19–23]. However, RPPA has not been previously reported with FF. RPPA allows analysis of small . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 4, 2026. ; https://doi.org/10.64898/2026.02.02.26345389doi: medRxiv preprint 3 sample volumes (e.g., nanograms), with the sensitivity (e.g., femtograms) and specificity of monoclonal antibodies, while quantifying proteins and protein modifications that govern their biological activity, such as phosphorylation, acetylation, or cleavage [22]. To assess the feasibility, we conducted an outcome-blinded pilot assessment of 21 FF proteins from critical biological pathways selected a priori in 6 FF specimens from women undergoing IVF.

Study Protocol: Up to 4 independent FF samples were collected from 56 women undergoing IVF and enrolled in the Study of Metals and Assisted Reproductive Technologies (SMART) in 2015-2017. The study protocol was described in detail in a previous publication [24]. Briefly, women underwent gonadotropin-induced ovarian stimulation with serial ultrasounds and estrogen measures. Human chorionic gonadotropin was administered approximately 2 weeks later, after follicles developed to ≥17mm diameter, and oocytes were retrieved after 34-36 hours by transvaginal fine needle aspiration. In each ovary, the largest follicle was aspirated; after evacuation, the needle was flushed with saline before sampling a second follicle, but the follicle itself was not flushed to preserve native analyte concentrations. After the oocyte was removed, each individual 3.5-5mL FF sample was centrifuged to pellet residual cells and debris and aliquoted the supernatant into 1.8mL cryovials, which were frozen at -80 °C. Any samples showing evidence of red blood were discarded [25]. Mature oocytes collected from ovarian follicles in metaphase-2 arrest (MII arrest) were fertilized with sperm using intracytoplasmic sperm injection or conventional insemination. Fertilization was confirmed after 16–20 hours by the appearance of 2 pronuclei (2PN). Embryo and blastocyst quality were categorized as “high quality” or “low quality’ based on a day 2 or 3 embryo examination of blastomere fragmentation, cleavage rate, and blastomere symmetry . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 4, 2026. ; https://doi.org/10.64898/2026.02.02.26345389doi: medRxiv preprint 4 [26–30], or Gardner’s scale for day 5 blastocysts [31]. Fresh embryos or blastocysts were transferred and a positive serum hCG test 2 weeks later indicated pregnancy. All participants completed written informed consent and the study protocol was approved by the UCSF Committee on Human Research. Reverse Phase Protein Arrays (RPPA): RPPA analysis was used to quantify 21 FF proteins, normalized to total FF protein concentration, in a single FF from 3 women with an IVF pregnancy and 3 without an IVF pregnancy, randomly selected from SMART participants. FF was printed in two-fold serial dilutions on a nitrocellulose-coated slide (Grace BioLabs, Oncyte Avid, Bend, Oregon, USA) that included calibrators and controls for rigorous, clinical diagnostic level analysis. Twenty-one FF proteins were selected a priori based on validated antibodies available in our Center from the endocannabinoid system, oxidative stress and inflammatory response, epigenetic markers, tryptophan metabolism, cell division and migration, hormone synthesis and function, DNA damage and repair, and vitamin D homeostasis pathways as described in Table 1. Each array was probed with a validated, commercially available monoclonal or polyclonal antibody. Antibody validation is performed for each antibody and whenever a new lot number of antibody is received, following CAP/CLIA compliant standard operating procedures. There is a compendium of more than 400 validated antibodies in our Center. Each sample, control, and calibrator was printed in technical replicates on the arrays (required coefficient of variation < 15%) [32] (Figure 1). Bovine serum albumin was used as the total protein calibrator. Commercial cell lysates served as process controls. Calibrators, created from cell lines, spanned the limits of quantification for the proteins of interest. The calibrators may also be used to interpolate the relative intensity values of each spot on the array. The pixel intensity of each spot was quantified using a calibrated flatbed scanner (UMAX PowerLook, UMAX Technologies, . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 4, 2026. ; https://doi.org/10.64898/2026.02.02.26345389doi: medRxiv preprint 5 Dallas, Texas, USA) for chromogenic detection or a laser scanner (Tecan Group Ltd., Zurich, Switzerland) for fluorescent detection. Signal intensities greater than 3 standard deviations above background were considered to have an adequate signal-to-noise ratio for the analysis [33]. RPPA spot intensity was determined using ImageQuant v.5.2 software (Cytiva, Marlborough, Massachusetts, USA). The local area background was subtracted from each spot and the data were normalized to total protein using an in-house VBA Macro (RPPA Analysis Suite) [33]. Data Analysis: We estimated the associations between FF proteins, oocyte quality, and embryo quality outcomes using Spearman correlation coefficients and compared differences in mean FF protein concentrations between women with and without an IVF pregnancy. We defined statistical significance as P<0.10 for a 2-tailed test to accommodate the limited sample size and generate hypotheses for future confirmation.

Seventeen of 21 FF proteins were quantified using RPPA in our CAP/CLIA-accredited proteomics laboratory, including TTP1, NFKB, p53BP1, Vitamin D Binding Protein, IL-1b, Cleaved Caspase3, Prolactin Receptor, Prdx-1, Manganese Super Oxide Dismutase, IL-6, Wnt5ab, Vitamin D Receptor, PPARgamma, IGF1R-beta, IDO, BLM, and DAG Lipase alpha (abbreviations are defined in Table 1). FF glutamine dehydrogenase, HDAC3, CuZnSOD, and ERβ were not quantifiable. As shown in Figure 2, we found a mixed pattern of moderate to strong pairwise Spearman correlations between FF proteins and intermediate IVF outcomes. Oocyte maturity (MII arrest) . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 4, 2026. ; https://doi.org/10.64898/2026.02.02.26345389doi: medRxiv preprint 6 and fertilization (2PN) correlated negatively to TPP1 and P53BP1, but positively correlated to NFKB, VitDBP, IL-1b, ClCasp3, ProR, Prdx-1, MnSOD, IL-6, Wnt5ab, VitDR, PPARgamma, IGF1Rbeta, and IDO. In contrast, greater TPP1 and P53BP1 correlated to high cleavage stage embryo quality, but greater FF VitDBP and IGF1Rbeta were associated with low cleavage stage embryo quality. There were no statistically significant correlations with blastocyst quality, which may have been due to the limited number of high quality blastocysts in the sample (i.e., 6 high quality blastocysts of 42 total blastocysts). As shown in Figure 3, we found greater mean levels of NFKB (37.3%), P53BP1 (25.5%), VitDBP (29.8%), IL-1b (42.8%), ClCasp3 (35.6%), ProR (18.0%), Prdx-1 (15.6%), MnSOD (70.7%), IL-6 (43.0%), Wnt5ab (70.8%), VitDR (81.3%), IGF1R-beta (535.9%), and DAGLipalpha (44.2%) among non-pregnant than pregnant women. However, the sample size was too limited for formal hypothesis testing.

In this small feasibility study, we found different FF protein concentrations according to oocyte fertilization, embryo quality, and pregnancy outcomes using RPPA. Our results suggest that FF proteins in the endocannabinoid system (DAG-lipase α findings) [34–36], Wnt signaling pathway (Wnt5ab findings) [37,38], acute phase immune response (IL-6 and NFKB findings) [39,40], oxidative stress response (MnSOD and p53BP1 findings) [41,42], innate immune response (IL-1β and NFKB findings) [40,43], and vitamin D signaling pathway (VD3R and VDBP findings) [44–46] may be important determinants of IVF outcomes. However, these results are preliminary and a larger and more comprehensive analysis will be necessary for definitive results. . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 4, 2026. ; https://doi.org/10.64898/2026.02.02.26345389doi: medRxiv preprint 7 The potential for an oocyte to complete meiosis, fertilize normally, and support early embryo development, collectively referred to as oocyte maturation, depends on nuclear, epigenetic, and cytoplasmic processes that influence IVF outcomes [47,48]. Throughout development, oocytes are immersed in FF, which mediates nutrient exchange and biochemical communication with mural granulosa cells, cumulus cells, and the vascular compartment [49,50]. FF contains a plasma ultrafiltrate restricted by the blood–follicle barrier to molecules less than ~300 kDa, along with factors produced in situ by granulosa cells and the oocyte itself, creating a specialized microenvironment [51–53]. For example, 22 FF proteins differed between women with and without ovarian pathology in a recent study, while only 2 were detectable in matched serum [54]. Because FF is the fluid most proximate to the oocyte, it provides a unique window into the microfollicular environment and can reveal protein markers predictive of IVF outcomes that are diluted or undetectable in peripheral blood [49,55]. Proteomic profiling of FF collected during IVF provides insight into the protein systems that drive oocyte maturation and the downstream developmental events that lead to pregnancy and live birth [49,56,57]. Unlike genomic or transcriptomic assays, which may correlate only weakly with protein abundance or activation status, proteomic approaches directly characterize follicular phenotype in real time [58]. Over the past 15 years, FF proteome profiling has expanded substantially [17,56]. Investigators have increasingly identified and quantified proteins from major biological pathways in efforts to develop diagnostic and prognostic biomarkers. A synthesis of 617 FF proteins reported through 2014 highlighted acute phase inflammatory response, wound response, complement, coagulation, lipid metabolism, and cytoskeletal pathways as most frequently represented, with matrix metalloproteinases (MMPs), thrombin, and vitamin D/Retinoid X Receptor alpha emerging as central hubs [59]. Another study detected 742 proteins in pooled FF from 3 ovum donors using MS, primarily related to growth factor and receptor signaling, immunity, anti-apoptosis, MMP activity, and complement [60]. An MS-based . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 4, 2026. ; https://doi.org/10.64898/2026.02.02.26345389doi: medRxiv preprint 8 biological pathway analysis similarly confirmed the prominence of complement and coagulation systems, with additional emphasis on innate immunity and angiogenesis [61]. More recent studies have expanded the FF proteome further. One MS analysis identified 2461 FF proteins, including 1108 detected for the first time, enriched in metabolic processes and biological regulation [62]. A SWATH (Sequential Window Acquisition of all Theoretical Mass Spectra)-MS study detected 2182 FF proteins, most lacking known metabolic functions, and others involved in coagulation, integrin signaling, gonadotropin-releasing hormone signaling, plasminogen activation, and Wnt signaling [63]. Despite substantial progress, findings across studies remain inconsistent and few clinically actionable biomarkers have emerged [18,49,56,59,64]. While MS-based approaches have identified highly abundant FF proteins and peptides, these

require complex, specialized workflows for measuring low-abundance proteins and peptides in FF and their post-translational modifications [65]. The results of this feasibility study suggest that RPPA is a feasible complementary technology to quantify low-abundance ovarian FF proteins and their post-translationally modified forms that predict IVF outcomes, and may serve as diagnostic/prognostic indicators and targets for interventions. However, a larger validation study will be necessary to confirm that RPPA technology is a feasible approach to investigate FF proteins as potential clinical and prognostic indicators of IVF outcomes or targets for clinical intervention. . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 4, 2026. ; https://doi.org/10.64898/2026.02.02.26345389doi: medRxiv preprint 9

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Proteins analyzed in human ovarian follicular fluid using RPPA Protein Abbreviation Source Tripeptidyl peptidase 1 TTP1 Cell Signaling Technology Nuclear factor-κB NFKB Cell Signaling Technology P53 binding protein P53BP1 Cell Signaling Technology Vitamin D binding protein VitDBP Abcam Interleukin-1 β IL-1b Cell Signaling Technology Estrogen receptor β ERbeta DSHB Copper-Zinc superoxide dismutase CuZnSOD StressGen Biotechnologies Cleaved caspase 3 (Asp175) ClCasp3 Cell Signaling Technology Prolactin receptor ProR Epitomics Phosphorylated (Tyr 194) peroxiredoxin 1 Prdx-1 Cell Signaling Technology Manganese superoxide dismutase MnSOD Assay Designs Interleukin-6 IL-6 BioVision Wingless integrated 5a/5b Wnt5ab Cell Signaling Technology Vitamin D3 receptor VitDR Cell Signaling Technology Peroxisome proliferator-activated receptor γ PPARgamma Cell Signaling Technology Phosphorylated insulin-like growth factor receptor β (Tyr 1135/36) IGF1R-beta Cell Signaling Technology Indoleamine-pyrrole 2,3-dioxygenase IDO Cell Signaling Technology Cleaved histone deacetylase 3 HDAC3 Cell Signaling Technology Bloom syndrome protein BLM Cell Signaling Technology Glutamine dehydrogenase GDH Cell Signaling Technology Diacylglycerol lipase α DAGLipalpha Cell Signaling Technology Abbreviations: RPPA, reverse phase protein arrays . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 4, 2026. ; https://doi.org/10.64898/2026.02.02.26345389doi: medRxiv preprint 14 Figure 1. Reverse Phase Protein Arrays (RPPA) provide quantitative data for cell signaling kinases and their post-translational modified forms. Specimens, control, and calibrators are printed on nitrocellulose coated slides in replicate dilution curves. Each array is probed with a single, validated antibody and catalyzed signal amplification chemistries. . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 4, 2026. ; https://doi.org/10.64898/2026.02.02.26345389doi: medRxiv preprint 15 Figure 2. Spearman correlation coefficients between follicular fluid proteins measured using RPPA and intermediate IVF outcomes among women using IVF Abbreviations: BLM, Bloom syndrome protein; ClCasp3, cleaved caspase 3; DAGLipalpha, diacylglycerol lipase α; HQ, high quality; IDO, indoleamine-pyrrole 2,3-dioxygenase; IGF1Rbeta, phosphorylated insulin-like growth factor receptor β; IL-1b, interleukin-1 β; IL-6, interleukin 6; . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 4, 2026. ; https://doi.org/10.64898/2026.02.02.26345389doi: medRxiv preprint 16 IVF, in vitro fertilization; MnSOD, manganese superoxide dismutase; NFKB, nuclear factor kappa β; NS, not significant; P53BP1, P53 binding protein; PPARgamma, peroxisome proliferator-activated receptor γ; Prdx-1, peroxiredoxin 1; ProR, prolactin receptor; RPPA, reverse phase protein array; TPP1, Tripeptidyl peptidase 1; VitDBP, vitamin D binding protein; VitDR, vitamin D3 receptor; Wnt5ab, Wingless integrated 5a/5b NOTE: Colors in the boxes correspond to the Spearman correlation coefficients between individual follicular fluid proteins and the intermediate IVF outcomes (n for each test listed in each column), Darker green indicates a more positive correlation and darker red indicates a more negative correlation. Statistical significance was defined as P<0.10. . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 4, 2026. ; https://doi.org/10.64898/2026.02.02.26345389doi: medRxiv preprint 17 Figure 3. Mean (95% confidence interval) follicular fluid protein concentrations measured using RPPA, between women with (n=3) and without (n=3) an IVF pregnancy. Abbreviations: BLM, Bloom syndrome protein; ClCasp3, cleaved caspase 3; DAGLipalpha, diacylglycerol lipase α; HQ, high quality; IDO, indoleamine-pyrrole 2,3-dioxygenase; IGF1Rbeta, phosphorylated insulin-like growth factor receptor β; IL-1b, interleukin-1 β; IL-6, interleukin 6; IVF, in vitro fertilization; MnSOD, manganese superoxide dismutase; NFKB, nuclear factor kappa β; P53BP1, P53 binding protein; PPARgamma, peroxisome proliferator-activated receptor γ; Prdx-1, peroxiredoxin 1; ProR, prolactin receptor; RPPA, reverse phase protein array; TPP1, Tripeptidyl peptidase 1; VitDBP, vitamin D binding protein; VitDR, vitamin D3 receptor; Wnt5ab, Wingless integrated 5a/5b NOTE: Symbols correspond to mean follicular fluid protein concentrations (n=3 in each pregnancy group) and whiskers represent 95% confidence intervals. Symbols without whiskers represent uniformly measured values for a pregnancy group (i.e., BLM, IDO, PPARgamma), or . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 4, 2026. ; https://doi.org/10.64898/2026.02.02.26345389doi: medRxiv preprint 18 measurement of n=1 or n=2 for a pregnancy group (i.e., BLM, IDO, IGF1Rbeta, PPARgamma, VitDR, VitDBP). . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 4, 2026. ; https://doi.org/10.64898/2026.02.02.26345389doi: medRxiv preprint