{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T22:22:33Z","timestamp":1792794153770,"version":"4.5.0"},"reference-count":46,"publisher":"Oxford University Press (OUP)","issue":"5","license":[{"start":{"date-parts":[[2025,2,4]],"date-time":"2025-02-04T00:00:00Z","timestamp":1738627200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/academic.oup.com\/pages\/standard-publication-reuse-rights"}],"funder":[{"name":"NIH\/NCATS Colorado CTSA","award":["T32TR004367"],"award-info":[{"award-number":["T32TR004367"]}],"id":[{"id":"https:\/\/ror.org\/03xsh7c04","id-type":"ROR","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R00HD103778"],"award-info":[{"award-number":["R00HD103778"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["K99HD103778"],"award-info":[{"award-number":["K99HD103778"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Department of Pediatrics at CU Anschutz Medical Campus","id":[{"id":"https:\/\/ror.org\/03wmf1y16","id-type":"ROR","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2025,11,14]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>The number and distribution of follicles in each growth stage provide a reliable readout of ovarian health and function. Leveraging techniques for three-dimensional imaging of ovaries in toto has the potential to uncover total, accurate ovarian follicle counts. Due to the size and holistic nature of these images, counting oocytes is time consuming and difficult. The advent of machine-learning algorithms has allowed for the development of ultra-fast, automated methods to analyze microscopy images. In recent years, these pipelines have become more accessible to non-specialists. We used these tools to create OoCount, a high-throughput, open-source method for automatic oocyte segmentation and classification from fluorescent three-dimensional microscopy images of whole mouse ovaries using a deep-learning convolutional neural network\u2013based approach. We developed a fast tissue-clearing and imaging protocol to obtain three-dimensional images of whole-mount mouse ovaries. Fluorescently labeled oocytes from three-dimensional images were manually annotated in Napari to develop a training dataset. This dataset was used to retrain StarDist using a convolutional neural network within DL4MicEverywhere to automatically label all oocytes in the ovary. In a second phase, we utilize Accelerated Pixel and Object Classification, a Napari plugin, to sort oocytes into growth stages. Here, we provide an end-to-end pipeline for producing high-quality three-dimensional images of mouse ovaries and obtaining follicle counts and staging. We demonstrate how to customize OoCount to fit images produced in any lab. Using OoCount, we obtain oocyte counts from each growth stage in the perinatal and adult ovary, improving our ability to study ovarian function and fertility.<\/jats:p>","DOI":"10.1093\/biolre\/ioaf023","type":"journal-article","created":{"date-parts":[[2025,2,2]],"date-time":"2025-02-02T07:12:20Z","timestamp":1738480340000},"page":"1083-1101","source":"Crossref","is-referenced-by-count":9,"title":["OoCount: a machine-learning-based approach to mouse ovarian follicle counting and classification"],"prefix":"10.1093","volume":"113","author":[{"given":"Lillian","family":"Folts","sequence":"first","affiliation":[{"name":"Section of Developmental Biology, Department of Pediatrics, University of Colorado Anschutz Medical Campus , Aurora, CO ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anthony S","family":"Martinez","sequence":"additional","affiliation":[{"name":"Section of Developmental Biology, Department of Pediatrics, University of Colorado Anschutz Medical Campus , Aurora, CO ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jaelyn A","family":"Williams","sequence":"additional","affiliation":[{"name":"Section of Developmental Biology, Department of Pediatrics, University of Colorado Anschutz Medical Campus , Aurora, CO ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Corey","family":"Bunce","sequence":"additional","affiliation":[{"name":"Konrad Lorenz Institute for Evolution and Cognition Research , Klosterneuburg ,","place":["Austria"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Blanche","family":"Capel","sequence":"additional","affiliation":[{"name":"Department of Cell Biology, Duke University Medical Center , Durham, NC ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jennifer","family":"McKey","sequence":"additional","affiliation":[{"name":"Section of Developmental Biology, Department of Pediatrics, University of Colorado Anschutz Medical Campus , Aurora, CO ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2025,2,4]]},"reference":[{"key":"2025111708574951700_ref1","first-page":"200","article-title":"Initial and cyclic recruitment of ovarian follicles","volume":"21","author":"McGee","year":"2000","journal-title":"Endocr Rev"},{"key":"2025111708574951700_ref2","doi-asserted-by":"crossref","first-page":"197","DOI":"10.1016\/j.ydbio.2008.06.017","article-title":"Foxo3 is a PI3K-dependent molecular switch controlling the initiation of oocyte growth","volume":"321","author":"John","year":"2008","journal-title":"Dev Biol"},{"key":"2025111708574951700_ref3","doi-asserted-by":"crossref","first-page":"575706","DOI":"10.3389\/fcell.2020.575706","article-title":"The factors and pathways regulating the activation of mammalian primordial follicles in vivo","volume":"8","author":"Chen","year":"2020","journal-title":"Front Cell Dev Biol"},{"key":"2025111708574951700_ref4","doi-asserted-by":"crossref","first-page":"111","DOI":"10.1095\/biolreprod.115.133652","article-title":"How is the number of primordial follicles in the ovarian reserve established?","volume":"93","author":"Findlay","year":"2015","journal-title":"Biol Reprod"},{"key":"2025111708574951700_ref5","doi-asserted-by":"crossref","first-page":"569","DOI":"10.1530\/rep.1.00095","article-title":"Methods for quantifying follicular numbers within the mouse ovary","volume":"127","author":"Myers","year":"2004","journal-title":"Reproduction"},{"key":"2025111708574951700_ref6","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1186\/1477-7827-1-11","article-title":"Ovarian follicle counts \u2013 not as simple as 1, 2, 3","volume":"1","author":"Tilly","year":"2003","journal-title":"Reprod Biol Endocrinol"},{"key":"2025111708574951700_ref7","doi-asserted-by":"crossref","first-page":"e81088","DOI":"10.7554\/eLife.81088","article-title":"Integration of mouse ovary morphogenesis with developmental dynamics of the oviduct, ovarian ligaments, and rete ovarii","volume":"11","author":"McKey","year":"2022","journal-title":"elife"},{"key":"2025111708574951700_ref8","doi-asserted-by":"crossref","first-page":"1033","DOI":"10.1002\/dvdy.21925","article-title":"Three-dimensional visualization of testis cord morphogenesis, a novel tubulogenic mechanism in development","volume":"238","author":"Combes","year":"2009","journal-title":"Dev Dyn"},{"key":"2025111708574951700_ref9","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1095\/biolreprod.115.132118","article-title":"A whole-mount approach for accurate quantitative and spatial assessment of Fetal oocyte dynamics in Mice1","volume":"93","author":"Malki","year":"2015","journal-title":"Biol Reprod"},{"key":"2025111708574951700_ref10","doi-asserted-by":"crossref","first-page":"9","DOI":"10.3389\/fcell.2021.698315","article-title":"Ovary development: insights from a three-dimensional imaging revolution","volume":"9","author":"Soygur","year":"2021","journal-title":"Front Cell Dev Biol"},{"key":"2025111708574951700_ref11","doi-asserted-by":"crossref","first-page":"203","DOI":"10.1007\/978-1-0716-3259-8_12","volume-title":"Germline Stem Cells: Methods and Protocols","author":"Soygur","year":"2023"},{"issue":"175","key":"2025111708574951700_ref12","doi-asserted-by":"crossref","DOI":"10.3791\/62972","article-title":"Whole ovary immunofluorescence, clearing, and multiphoton microscopy for quantitative 3D analysis of the developing ovarian Reserve in Mouse","author":"Boateng","year":"2021","journal-title":"J Vis Exp"},{"key":"2025111708574951700_ref13","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1016\/j.ydbio.2015.04.006","article-title":"Follicle dynamics and global organization in the intact mouse ovary","volume":"403","author":"Faire","year":"2015","journal-title":"Dev Biol"},{"key":"2025111708574951700_ref14","doi-asserted-by":"crossref","first-page":"44810","DOI":"10.1038\/srep44810","article-title":"CLARITY reveals dynamics of ovarian follicular architecture and vasculature in three-dimensions","volume":"7","author":"Feng","year":"2017","journal-title":"Sci Rep"},{"key":"2025111708574951700_ref15","doi-asserted-by":"crossref","first-page":"1080","DOI":"10.1093\/biolre\/ioaa012","article-title":"Combined iDISCO and CUBIC tissue clearing and lightsheet microscopy for in toto analysis of the adult mouse ovary","volume":"102","author":"McKey","year":"2020","journal-title":"Biol Reprod"},{"key":"2025111708574951700_ref16","doi-asserted-by":"crossref","first-page":"dev201185","DOI":"10.1242\/dev.201185","article-title":"An end-to-end pipeline based on open source deep learning tools for reliable analysis of complex 3D images of ovaries","volume":"150","author":"Lesage","year":"2023","journal-title":"Development"},{"key":"2025111708574951700_ref17","doi-asserted-by":"crossref","first-page":"1041","DOI":"10.1093\/biolre\/ioad182","article-title":"Tissue clearing and imaging approaches for in toto analysis of the reproductive system","volume":"110","author":"Folts","year":"2023","journal-title":"Biol Reprod"},{"key":"2025111708574951700_ref18","doi-asserted-by":"crossref","first-page":"223","DOI":"10.1146\/annurev-biodatasci-072018-021305","article-title":"Imaging, visualization, and computation in developmental biology","volume":"2","author":"Cutrale","year":"2019","journal-title":"Annu Rev Biomed Data Sci"},{"key":"2025111708574951700_ref19","doi-asserted-by":"crossref","first-page":"dev199616","DOI":"10.1242\/dev.199616","article-title":"Deep learning for bioimage analysis in developmental biology","volume":"148","author":"Hallou","year":"2021","journal-title":"Development"},{"key":"2025111708574951700_ref20","doi-asserted-by":"crossref","first-page":"1233","DOI":"10.1038\/s41592-019-0403-1","article-title":"Deep learning for cellular image analysis","volume":"16","author":"Moen","year":"2019","journal-title":"Nat Methods"},{"key":"2025111708574951700_ref21","doi-asserted-by":"crossref","DOI":"10.1111\/jmi.13282","article-title":"The rise of data-driven microscopy powered by machine learning","volume-title":"J Microsc","author":"Morgado","year":"2024"},{"key":"2025111708574951700_ref22","article-title":"DL4MicEverywhere: deep learning for microscopy made flexible, shareable and reproducible","year":"2024"},{"key":"2025111708574951700_ref23","doi-asserted-by":"crossref","first-page":"2276","DOI":"10.1038\/s41467-021-22518-0","article-title":"Democratising deep learning for microscopy with ZeroCostDL4Mic","volume":"12","author":"Chamier","year":"2021","journal-title":"Nat Commun"},{"key":"2025111708574951700_ref24","doi-asserted-by":"crossref","first-page":"100","DOI":"10.1038\/s41592-020-01018-x","article-title":"Cellpose: a generalist algorithm for cellular segmentation","volume":"18","author":"Stringer","year":"2021","journal-title":"Nat Methods"},{"key":"2025111708574951700_ref25","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1007\/978-3-030-00934-2_30","volume-title":"Medical Image Computing and Computer Assisted Intervention \u2013 MICCAI 2018","author":"Schmidt","year":"2018"},{"key":"2025111708574951700_ref26","doi-asserted-by":"crossref","first-page":"2472","DOI":"10.1002\/1873-3468.14451","article-title":"A Hitchhiker\u2019s guide through the bio-image analysis software universe","volume":"596","author":"Haase","year":"2022","journal-title":"FEBS Lett"},{"key":"2025111708574951700_ref27","doi-asserted-by":"crossref","first-page":"3655","DOI":"10.1109\/WACV45572.2020.9093435","article-title":"Star-convex Polyhedra for 3D object detection and segmentation in microscopy","volume":"2020","author":"Weigert","year":"2020","journal-title":"IEEE Winter Conference on Applications of Computer Vision (WACV)"},{"key":"2025111708574951700_ref28","article-title":"Napari\/napari","author":"Sofroniew","year":"2022"},{"key":"2025111708574951700_ref29","article-title":"Haesleinhuepf\/napari-accelerated-pixel-and-object-classification: 0.8.2","author":"Haase"},{"key":"2025111708574951700_ref30","first-page":"52458","article-title":"Culture and Co-culture of mouse ovaries and ovarian follicles","volume":"97","author":"Morgan","year":"2015","journal-title":"J Vis Exp"},{"key":"2025111708574951700_ref31","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1093\/biolre\/ioac176","article-title":"Recapitulating folliculogenesis and oogenesis outside the body: encapsulated in vitro follicle growth\u2020","volume":"108","author":"Converse","year":"2023","journal-title":"Biol Reprod"},{"key":"2025111708574951700_ref32","doi-asserted-by":"crossref","first-page":"e2100920118","DOI":"10.1073\/pnas.2100920118","article-title":"Single-cell sequencing reveals suppressive transcriptional programs regulated by MIS\/AMH in neonatal ovaries","volume":"118","author":"Meinsohn","year":"2021","journal-title":"Proc Natl Acad Sci USA"},{"key":"2025111708574951700_ref33","doi-asserted-by":"crossref","first-page":"24","DOI":"10.1210\/js.2017-00329","article-title":"The orphan nuclear receptor liver homolog Receptor-1 (Nr5a2) regulates ovarian granulosa cell proliferation","volume":"2","author":"Meinsohn","year":"2018","journal-title":"J Endocr Soc"},{"key":"2025111708574951700_ref34","doi-asserted-by":"crossref","first-page":"3398","DOI":"10.2741\/2321","article-title":"Orphan nuclear receptor function in the ovary","volume":"12","author":"Zhao","year":"2007","journal-title":"Front Biosci"},{"key":"2025111708574951700_ref35","doi-asserted-by":"crossref","first-page":"5570","DOI":"10.1073\/pnas.1814930116","article-title":"Neural crest-derived neurons invade the ovary but not the testis during mouse gonad development","volume":"116","author":"McKey","year":"2019","journal-title":"Proc Natl Acad Sci USA"},{"key":"2025111708574951700_ref36","doi-asserted-by":"crossref","first-page":"676","DOI":"10.1038\/nmeth.2019","article-title":"Fiji: an open-source platform for biological-image analysis","volume":"9","author":"Schindelin","year":"2012","journal-title":"Nat Methods"},{"key":"2025111708574951700_ref37","article-title":"Napari: a multi-dimensional image viewer for python","author":"Sofroniew","year":"2022"},{"key":"2025111708574951700_ref38","doi-asserted-by":"crossref","first-page":"1371","DOI":"10.1038\/s41592-024-02310-w","article-title":"Omega \u2013 harnessing the power of large language models for bioimage analysis","volume":"21","author":"Royer","year":"2024","journal-title":"Nat Methods"},{"key":"2025111708574951700_ref39","doi-asserted-by":"crossref","unstructured":"Folts L, Martinez A, Bunce C, Capel B, McKey J. Data from: OoCount: a machine-learning based approach to mouse ovarian follicle counting and classification [dataset]. Dryad \u00a02024. 10.5061\/dryad.nk98sf81r.","DOI":"10.1101\/2024.05.13.593993"},{"key":"2025111708574951700_ref40","first-page":"2","article-title":"Docker: lightweight Linux containers for consistent development and deployment","volume":"2014","author":"Merkel","year":"2014","journal-title":"Linux J"},{"key":"2025111708574951700_ref41","article-title":"Svetlana a supervised segmentation classifier for Napari","year":"2024"},{"key":"2025111708574951700_ref42","doi-asserted-by":"crossref","first-page":"1423","DOI":"10.1038\/s41556-020-00603-8","article-title":"Apoptosis in the foetal testis eliminates developmentally defective germ cell clones","volume":"22","author":"Nguyen","year":"2020","journal-title":"Nat Cell Biol"},{"key":"2025111708574951700_ref43","doi-asserted-by":"crossref","first-page":"6125","DOI":"10.1002\/jcp.27367","article-title":"Autophagy participates in cyst breakdown and primordial folliculogenesis by reducing reactive oxygen species levels in perinatal mouse ovaries","volume":"234","author":"Zhihan","year":"2019","journal-title":"J Cell Physiol"},{"key":"2025111708574951700_ref44","doi-asserted-by":"crossref","first-page":"e1589","DOI":"10.1038\/cddis.2014.559","article-title":"Germ cell-specific Atg7 knockout results in primary ovarian insufficiency in female mice","volume":"6","author":"Song","year":"2015","journal-title":"Cell Death Dis"},{"key":"2025111708574951700_ref45","doi-asserted-by":"crossref","first-page":"16","DOI":"10.5653\/cerm.2021.04917","article-title":"Lifetime changes of the oocyte pool: contributing factors with a focus on ovulatory inflammation","volume":"49","author":"Park","year":"2022","journal-title":"Clin Exp Reprod Med"},{"key":"2025111708574951700_ref46","doi-asserted-by":"crossref","first-page":"1086","DOI":"10.1093\/biolre\/ioae048","article-title":"Quantification of follicles in human ovarian tissue using image processing software and trained artificial intelligence","volume":"110","author":"Blevins","year":"2024","journal-title":"Biol Reprod"}],"container-title":["Biology of Reproduction"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/academic.oup.com\/biolreprod\/advance-article-pdf\/doi\/10.1093\/biolre\/ioaf023\/61752796\/ioaf023.pdf","content-type":"application\/pdf","content-version":"am","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/biolreprod\/article-pdf\/113\/5\/1083\/61752796\/ioaf023.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/biolreprod\/article-pdf\/113\/5\/1083\/61752796\/ioaf023.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,11,17]],"date-time":"2025-11-17T13:58:12Z","timestamp":1763387892000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/biolreprod\/article\/113\/5\/1083\/7998806"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,2,4]]},"references-count":46,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2025,2,4]]},"published-print":{"date-parts":[[2025,11,14]]}},"URL":"https:\/\/doi.org\/10.1093\/biolre\/ioaf023","relation":{"has-preprint":[{"id-type":"doi","id":"10.1101\/2024.05.13.593993","asserted-by":"object"}]},"ISSN":["0006-3363","1529-7268"],"issn-type":[{"value":"0006-3363","type":"print"},{"value":"1529-7268","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2025,11]]},"published":{"date-parts":[[2025,2,4]]}}}