NICHD Ovarian Nomenclature Workshop 2021

Investigation into the ovary spurred by OTC, has demonstrated that our traditional description of the ovarian anatomy is inadequate as research in the field progresses. One obstacle to the advancement of knowledge in female fertility and reproduction is that there is a paucity of distinction between the different cellular and anatomical compartments of the ovary. In May-June 2021, Dr. Gomez-Lobo convened scientists from around the U.S. for several Division of Intramural Research (DIR) supported Ovarian Tissue Nomenclature Workshop meetings. Experts in reproductive endocrinology, gynecologic oncology, radiology, pathology, bioinformatics, and ovarian tissue research, met and created workgroups to further delineate ovarian follicle description, tissue ontology, and the 3-D description of the ovary. Resulting recommendations will establish the new national and international classification standards for ovary research which will allow for clearer evaluation of the function of the ovary at a gross and sub-anatomic level.

Workshop recommendations:

Workshop Sub-Groups

Gross Anatomy Workgroup Recommendations

The Ovarian Gross Anatomy Working Group recommended use of a three-dimensional (3D) regional model of the ovary based on the organ’s macroscopically visible structures and major points of fixation.

Refer to caption.
Figure. Proposed regional model for gross anatomy of the ovary
Sub-anatomy Workgroup Recommendations

The Subanatomy Working Group reviewed the current ontology of the ovary and proposed updates to the Uberon classification by referencing histologic features with a revised list of subanatomic structures, including cell types and extracellular features.

Uberon Icon

Follicle Workgroup Recommendations

The Follicle Classification Working Group recommended the expansion of the current classification system to include six stages of preantral follicles, five stages of antral follicles, three categories of abnormal preantral follicles, abnormal antral follicles, multi-oocytic follicles, and follicle variations of unknown significance (FVUS).

Table 1. Classification System of Human Ovarian Follicle Morphology

Follicle ClassesMorphologic Characteristics
Preantral FolliclePreantral Follicles with Normal MorphologyPrimordial ovarian follicleOocyte surrounded by a continuous or an incomplete single layer of squamous granulosa cells. Follicle diameter ~ 40-44 μm; oocyte diameter ~ 29-35 μm*1-5
Transitional primordial ovarian follicleOocyte surrounded by a continuous or incomplete single layer consisting of both squamous and cuboidal granulosa cells.6, 7 Follicle diameter ~ 44 μm; oocyte diameter ~ 36 μm*1
Primary ovarian follicleOocyte surrounded by a complete single layer of cuboidal granulosa cells. Follicle diameter ~ 41-100 μm; oocyte diameter ~ 29-40 μm*1-4,8
Transitional primary ovarian follicleOocyte surrounded by reorganizing cuboidal granulosa cells that exist in a partial single and double layer; zona pellucida and theca layer may be visible.6,7,9
Secondary ovarian follicleOocyte surrounded by 2-3 layers of cuboidal granulosa cells surrounded by the theca interna layer. Zona pellucida present. Follicle diameter ~ 56-200 μm; oocyte diameter ~ 35-55 μm*1-3,8
Multilayer ovarian follicleOocyte surrounded by >3 layers of cuboidal granulosa cells with outer layer of theca cells. Zona pellucida present. Follicle diameter ~250-300 μm; oocyte diameter 100-120 μm.4,10
Preantral Follicle with Abnormal Morphological FeaturesPreantral Follicles with abnormal morphological features of oocyte [AMF-o]Preantral follicles with vacuoles in oocyte and/or containing degenerating oocytes.
Preantral Follicles with abnormal morphological features of granulosa cells [AMF-g]Preantral follicles with vacuoles in granulosa cells and/or containing degenerating granulosa cells.
Preantral Follicles with abnormal morphological features in both oocytes and granulosa cells [AMF-og]Preantral follicles with vacuoles in both oocytes and granulosa cells and/or containing both degenerating oocytes and granulosa cells.
Antral FollicleAntral Follicles with Normal Morphology Cumulus-enclosed oocyte in a fluid-filled space (antrum) surrounded by multiple layers of mural granulosa cells separated by a basement membrane from the adjacent layer of theca interna cells and the outermost layer of theca externa cells.10  Measured either histologically or on ultrasound. Human follicle has the ability to resume meiosis, with a follicle diameter 2-5mm and oocyte diameter ~ 100-120 μm.11,12 Minimal follicle diameter for developmental competence ~5-7mm.13
Early antralAntral follicle with antrum diameter < 2 mm on histology.5 Follicle diameter ~250-400 μm; oocyte diameter 100-120 μm2-4,11
Pre-selectionAntral follicle with antrum diameter 2-5 mm on imaging (possibly histology**)5
SelectionAntral follicle with antrum diameter 6-9 mm on imaging (possibly histology**)14
DominanceAntral follicle with antrum diameter 10-15 mm on imaging (possibly histology**)5-17
Pre-ovulatoryAntral follicle with antrum diameter 16-28 mm on imaging (possibly histology**). Human oocyte diameter ~ 100-140 μm.2,11,18
Atretic Antral Follicle with Normal Morphology Antral follicle of any stage wherein granulosa cells proximal to the antrum detach from the mural layers that appear as single or multiple cells within the antrum; these granulosa cells typically have pyknotic nuclei; follicle may contain an oocyte with abnormal morphology.
Antral Follicle with Abnormal Morphological Features Antral follicles containing a subset of atretic-appearing granulosa cells enclosing an oocyte with normal or abnormal morphology.
Corpus Luteum (CL) and derivativesCL of the Menstrual Cycle Grows to ~1.5cm diameter.  It contains 1) luteal tissue granulosa-lutein layer plus the theca-lutein area; 2) inner boundary of connective tissue; 3) central cavity that was filled with follicular fluid or blood.19 There is a combination of small and large luteal cells, each with a round, centrally located nucleus and prominent nucleolus.20 The luteal cells are surrounded by a connective tissue capsule and is adjacent to a highly vascularized capillary network. Fibroblast cells, collagen fibers, endothelial cells and macrophages are present. Variations of cell composition, vascularity, and quantity of collagen occur based on timing within the luteal phase. As the CL undergoes luteolysis and loses its ability to produce progesterone, there are degenerative changes, cytoplasmic vacuolization, macrophage marker expression, leading to apoptosis.19  Limited data in humans, different stages noted in the Buffalo.20
CL of Pregnancy Same histologic appearance as CL of the menstrual cycle. (Above).  Limited data.20
Corpora Albicans Small white scars typically present in the medulla that consist of collagen Type I from fibroblasts. Generally separated from the cortex by a thin band of medullary stromal cells.
Hemorrhagic Anovulatory Follicle  An inner lining of fibrous tissue (2–8 layers thick) surrounded by luteinized cells (up to 50 layers thick) Arterioles and venules, extended into the center of the follicle with a central cavity filled with red blood cells. Seen in humans21 and mammals22.
Luteinized Unruptured Follicle  Oocytes trapped within CLs and expanded cumulus oocyte complexes within unruptured antral follicles. Found in reproductive humans3-25 and mammals26-28
Multi-oocytic follicles  ≥2 oocytes surrounded by squamous or cuboidal granulosa cells; represent germ cell cysts that have failed to breakdown.
Follicle Variations of Unknown Significance (FVUS)  Follicles that do not fit any above definition; i.e. large primordial follicles as observed in prepubertal ovaries.
Tangential  Cluster of granulosa cells without an oocyte indicating a follicle edge, but unable to distinguish stage of follicle.

*Size of follicle and oocyte may vary depending on fresh or frozen tissue as well as processing techniques that potentially can cause shrinkage. 
**Antral follicles can be seen histologically. The accuracy of the size may vary depending on whether the tissue is fresh or frozen. Follicle may rupture or become distorted during tissue biopsy, fixation, and sectioning, therefore altering the diameter of the antrum. Histology measurements may not match ultrasound measurements. Oocytes may not be seen since some are aspirated or extruded into the media during processing for ovarian tissue cryopreservation.

Table 2. Abnormal morphological features sometimes observed in follicles29,30

Vacuoles or spaces inside the oocyte, although the granulosa cells look healthy
Large spaces between the granulosa cells, but the oocyte appears normal
Dissociated granulosa cells and abnormal oocyte
Large vacuoles in the oocyte
Misshapen oocyte with normal granulosa cell layers
Degenerate oocyte and pyknotic granulosa cells

Figure 1: Follicle Nomenclature H&E Stained Examples

Refer to caption.

Figure 1: A. Primordial: Oocyte surrounded by a continuous or an incomplete single layer of squamous granulosa cells. B. Transitional Primordial: Oocyte surrounded by a continuous or incomplete single layer consisting of both squamous and cuboidal granulosa cells. C. Primary: Oocyte surrounded by a complete single layer of cuboidal granulosa cells. D. Transitional Primary: Oocyte surrounded by reorganizing cuboidal granulosa cells in a partial single and double layer; zona pellucida and theca layer may be visible. E. Secondary: Oocyte surrounded by 2-3 layers of cuboidal granulosa cells with outer layer of theca cells. F. Multilayer: Oocyte surrounded by >3 layers of cuboidal granulosa cells with outer layer of theca cells.G. Abnormal Morphological features of the oocyte (AMF-o): Vacuoles in oocyte and/or containing degenerating oocytes. H. Abnormal Morphological features of granulosa cells (AMF-g): Vacuoles in granulosa cells and/or containing degenerating granulosa cells. I. Abnormal morphological features in both oocytes and gcs (AMF-og): Vacuoles in both oocytes and granulosa cells and/or containing both degenerating oocytes and granulosa cells. J. Multi-oocytic: ≥2 oocytes surrounded by squamous or cuboidal granulosa cells; represent germ cell cysts that have failed to breakdown. K: Early Antral Follicle (<2mm diameter). L: Pre-selection Antral Follicle (2-5mm diameter). M: Selection Antral Follicle (6-9 mm diameter). N: Atretic Antral with Normal Morphology. O: Atretic Antral with Abnormal Morphology. P: Corpus Luteum. Q: Corpus Albicans. R: Tangential cut. S: Tangential antral follicle cut. T: Capillary. U: Cross section of ovary. Example of calculating follicle density by using 1mm depth of the cortex to calculate area. V: Zoomed in view of cross section of ovary.

Figure 2: Follicle morphology comparison with different fixatives.

Refer to caption.

Figure 2A and 2B are from the same human ovary (Patient 1), comparing 10% Neutral Buffered Formalin (NBF) vs. 4% paraformaldehyde (PFA). Figure 2C and 2D are from the same human ovary (Patient 2), comparing NBF vs. modified Davidson’s Fixative (mDF). 

Box 1: Suggested Follicle Density Guidelines:

SECTIONS AND AREA
Section using 5μm
Count every 5th section
Area for Follicle density
Use area that is 1mm in depth from the edge of the cortex
Calculate follicles/mm2
Average follicles/mm<2 based on the number of sections
FOLLICLE COUNTING QUALIFICATIONS
Primordial/ Transitional Primordial: Count all*
Primary/ Transitional Primary: Count all*
Secondary/Multilayer: Count only ones with oocyte nucleus present
All Antral Stages: Count only ones with oocyte nucleus present
Count abnormal follicles separately - AMF-o, AMF-g, AMF-og, multioocytic, and FVUS  

*If there is only one section to analyze, follicle counting guidelines include counting all Primordial, Transitional Primordial, Primary, and Transitional Primary regardless of whether the oocyte nucleus is present or absent. If there is more than 1 section available, only count if the oocyte nucleus is present to avoid double counting of follicles because the average diameter of an oocyte in a primordial follicle is ~ 29-35 μm.*1-5

Workshop Participants

NameAffiliation
Christina Annunziata, MD, PhDNational Cancer Institute
G Thomas Brown, MD, PhDArtificial Intelligence Resource; National Cancer Institute
Alan H DeCherney, MDReproductive Endocrinology and Infertility; Eunice Kennedy Shriver National Institute of Child Health and Human Development
Tazim Dowlut-McElroy, MDPediatric and Adolescent Gynecology Program; Eunice Kennedy Shriver National Institute of Child Health and Human Development
Francesca E Duncan, PhDDepartment of Obstetrics and Gynecology; Feinberg School of Medicine, Northwestern University
Veronica Gomez-Lobo, MDPediatric and Adolescent Gynecology Program; Eunice Kennedy Shriver National Institute of Child Health and Human Development
Clarisa R Gracia, MDDivision of Reproductive Endocrinology and Infertility/Fertility Preservation;
University of Pennsylvania Perelman School of Medicine
Stefan Gysler, MDDivision of Gynecologic Oncology; University of Pennsylvania Perelman School of Medicine
Lisa M Halvorson, MDUSMA-Bayer Pharmaceuticals (Current Affiliation)
Affiliation at time of workshop:
Gynecologic Health and Disease Branch; Eunice Kennedy Shriver National Institute of Child Health and Human Development
Tracy Harrison, MDReproductive Sciences; University of California San Diego
Junhyong Kim, PhDUniversity of Pennsylvania Perelman School of Medicine
Monica M Laronda, PhDStanley Manne Children's Research Institute, Ann & Robert H Lurie Children's Hospital of Chicago, Feinberg School of Medicine, Northwestern University
Richard D LeDuc, PhDChildren's Hospital Research Institute of Manitoba
Hong Lou, MDPediatric and Adolescent Gynecology Program; Eunice Kennedy Shriver National Institute of Child Health and Human Development
Marla Lujan, PhDDivision of Nutritional Sciences; Cornell University
Jacqueline Y Maher, MDPediatric and Adolescent Gynecology Program; Eunice Kennedy Shriver National Institute of Child Health and Human Development
Kathleen O’Neill, MD, MSTRDivision of Reproductive Endocrinology and Infertility; University of Pennsylvania Perelman School of Medicine
Kutluk H Oktay, MD, PhDDepartment of Obstetrics, Gynecology, and Reproductive Sciences; Yale University School of Medicine
Stephanie A Pangas, PhDDepartment of Pathology and Immunology; Baylor College of Medicine
Alison Pouch, PhDDepartments of Radiology & Bioengineering: University of Pennsylvania Perelman School of Medicine
Rebecca Rakow-Penner, MD, PhDDepartment of Radiology; University of California San Diego
Julie Rios, MDReproductive Endocrinology and Infertility; University of Pittsburgh (Present affiliation)
Erin Rowell, MDAssociate Professor of Surgery; Northwestern University Feinberg School of Medicine
Lauren E Schwartz, MDDepartment of Pathology; University of Pennsylvania Perelman School of Medicine
James Segars, MDReproductive Science and Women’s Health Research; Johns Hopkins University
Ariella Shikanov, PhDDepartment of Biomedical Engineering, Department of Obstetrics and Gynecology; University of Michigan
Susan Taymans, PhDEunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health
Candace Tingen, PhDGynecologic Health and Disease Branch;  Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health
Elizabeth L Tsui, BSStanley Manne Children's Research Institute, Ann & Robert H Lurie Children's Hospital of Chicago, Feinberg School of Medicine, Northwestern University
Mary B Zelinski, PhDOregon National Primate Research Center; Oregon Health & Science University

Publications

  • Tsui EL, O'Neill KE, LeDuc RD, Shikanov A, Gomez-Lobo V, Laronda MM. Creating a common language for the subanatomy of the ovary. Biol Reprod. 2022 Oct 29:ioac199. doi: 10.1093/biolre/ioac199. Epub ahead of print. PMID: 36308436.
  • O'Neill KE, Maher JY, Laronda MM, Duncan FE, Leduc RD, Lujan ME, Oktay KH, Pouch AM, Segars JH, Tsui EL, Zelinski MB, Halvorson LM, Gomez-Lobo V. Anatomic Nomenclature and 3D Regional Model of the Human Ovary: Call for a New Paradigm. Am J Obstet Gynecol. 2022 Sep 30:S0002-9378(22)00795-5. doi: 10.1016/j.ajog.2022.09.040. Epub ahead of print. PMID: 36191605.

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