Name: Collection of Human Follicular Fluid, Follicle Somatic Cells, and Immature Oocytes from Individuals Undergoing In Vitro Fertilization (Video) | JoVE Journal
Uploaded: 2025-10-24T14:10:00.000+00:00
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Description: Learn standardized methods to isolate follicular fluid, somatic cells, and immature oocytes from IVF procedures for reproductive biology research. This protocol enables high-quality molecular analyses of human fertility and ovarian function.

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The data presented in this manuscript were generated with funding from the Friends of Prentice Foundation. E.B. is supported by the Women&#39;s Reproductive Health Research (WRHR) Career Development Program (K12 HD050121). Additional support was provided by the Northwestern University NUSeq Core Facility.

The authors have no conflict of interest to disclose.

ART generates a wealth of biological material that is discarded during routine clinical care. However, these tissues represent a powerful and largely untapped resource for advancing our understanding of human ovarian physiology, oocyte competence, and the microenvironment that supports reproduction. In this manuscript, we outline a set of standardized, scalable protocols for the systematic collection and processing of these materials from women undergoing oocyte retrieval. These methods support a wide range of downstream...

Infertility affects millions of individuals and couples worldwide, with estimates that one in six people and one in eight couples face difficulties conceiving naturally1,2,3. Advances in assisted reproductive technologies (ART) have transformed the management of infertility, offering hope to those affected by age-related fertility decline, ovulatory dysfunction, male factor or unexplained infertility. Since the birth of the first baby via in vitro fertilization (IVF) in 1978, the use of ART has grown substantially, becoming an integral part of reproductive medicine4. In the United States alone, ART cycle numbers have steadily increased over the past decade, with over 400,000 cycles in 20225. This rising demand highlights both the social and clinical importance of refining ART techniques and understanding the biological factors that contribute to reproductive success.
In parallel, fertility preservation has emerged as a critical tool to safeguard future reproductive potential in a diverse range of individuals. Over 40,000 fertility preservation IVF cycles were performed in 20225. For cancer patients, cryopreservation of oocytes or embryos before initiating gonadotoxic chemotherapy has become standard of care6,7,8,9. Similarly, transgender individuals undergoing cross-sex hormone therapy may choose to preserve gametes to maintain their reproductive autonomy10,11. Even for individuals without immediate medical concerns, elective oocyte freezing is increasingly utilized to mitigate the effects of reproductive aging and extend fertility options12. These varied clinical indications have expanded the scope and relevance of fertility preservation, providing new opportunities to study reproductive biology in unique patient populations.
ART procedures yield mature oocytes that are either cryopreserved or fertilized as part of clinical care. In addition, a substantial amount of biological material, including follicular fluid, somatic cells from follicles, and immature oocytes, is typically discarded during the IVF cycle. Yet these cells and tissues represent valuable sources of insight into the ovarian microenvironment, oocyte quality, and broader aspects of reproductive physiology. The cumulus-oocyte complex (COC), for example, provides a valuable window into the cellular and molecular interactions that support oocyte maturation. During follicular aspiration, the follicular fluid collected from the antral follicle often contains both soluble factors and cells that are dislodged or aspirated along with the oocyte. This fluid harbors a diverse array of metabolites, hormones, growth factors, cytokines, lipids, nucleic acids, and environmental signals that reflect both systemic and local aspects of reproductive health13,14,15,16,17,18,19,20,21. In addition to these soluble components, follicular fluid contains a heterogeneous mix of cells: cumulus cells (CCs), which surround the oocyte, provide metabolic support and facilitate its maturation; mural granulosa cells, which line the follicle wall and produce steroid hormones such as estradiol; immune cells, including macrophages and T cells, which play roles in tissue remodeling and ovulation; and theca cells, stromal cells, or blood cells-often introduced during follicle aspiration16. Because these tissues are obtained as part of routine clinical practice and would otherwise be discarded, they represent an ethical, readily available, and biologically rich resource for translational research that cannot be matched by animal models or other human sample types.
Our team at the Northwestern Fertility and Reproductive Medicine Center and Northwestern University has developed and refined protocols for collecting and studying human follicular fluid, cumulus, granulosa, and other follicle somatic cells, and immature oocytes from patients undergoing IVF, under approved research protocols (Figure 1and Figure 2).&#160;We also collect matched biofluids such as serum/plasma and urine from these patients. While typically discarded during routine clinical care, these materials offer a unique and underutilized opportunity to explore the cellular, molecular, and endocrine landscape of the human ovarian follicle. Importantly, the applicability of this protocol depends on timely sample handling and preservation, as RNA integrity and cell viability can rapidly decline if processing is delayed, making early processing a critical factor for success. The analysis of these cells and tissues can yield critical insights into reproductive physiology and pathophysiology. Studying these components can enhance our understanding of conditions such as diminished ovarian reserve, polycystic ovary syndrome, environmental toxin exposure, and age-associated reproductive decline, while also supporting the development of non-invasive biomarkers for ovarian function, oocyte quality, and overall reproductive potential17,22,23,24,25,26,27,28. Moreover, they serve as powerful platforms for advancing experimental approaches in contraception, environmental reproductive toxicology, and infertility treatment23,29,30,31,32,33,34. Compared to traditional enzymatic or bulk isolation techniques, the approach here emphasizes gentle microdissection and fresh single-cell preparation, which preserve transcriptomic integrity and cellular heterogeneity more effectively for downstream analyses such as single-cell RNA-Sequencing. The objective of this study is to describe and validate standardized protocols for the systematic collection, processing, and analysis of discarded human follicular components, with the hypothesis that these materials can yield high-quality molecular and cellular data that advance research in reproductive biology and fertility.

<table><tbody><tr><td>4-well dish</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>144444</td><td></td></tr><tr><td>ACK RBC Lysis Buffer</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>A1049201-100mL</td><td></td></tr><tr><td>Acridine Orange / Propidium Iodide Stain (2 x 0.5 mL)</td><td>Logos Biosystems INC, Anyang, South Korea</td><td>F23001</td><td></td></tr><tr><td>Agilent 2100 Bioanalyzer RNA 6000 Pico Chip</td><td>Agilent Technologies, Santa Clara, CA, USA</td><td>5067-1513</td><td></td></tr><tr><td>Anti-AMHR2 antibody</td><td>Abcam, Cambridge, UK</td><td>ab197148</td><td></td></tr><tr><td>Anti-FOXL2 antibody</td><td>Abcam, Cambridge, UK</td><td>ab246511</td><td></td></tr><tr><td>Bovine serum albumin (BSA) 10%</td><td>Sigma, St. Louis, MO, USA</td><td>A1595-50mL</td><td></td></tr><tr><td>Bovine Serum Albumin, chromatographically purified, New Zealand origin, low endotoxin, suitable for cell culture, pH 7, &amp;ge;98%</td><td>Sigma -Aldrich, St. Louis, MO, USA</td><td>A1933-25G</td><td></td></tr><tr><td>Center well dishes</td><td>Falcon, Corning, NY, USA</td><td>353037</td><td></td></tr><tr><td>Corning Dispase</td><td>Fisher Scientific, Waltham, MA, USA</td><td>CB-40235</td><td></td></tr><tr><td>Cover Glass (15mm diameter, for 24-Well Plate, 100/cs)</td><td>CELLTREAT Scientific Products, Pepperell, MA, USA</td><td>229172</td><td></td></tr><tr><td>Cryogenic vials</td><td>Fisher Scientific, Waltham, MA, USA</td><td>430488</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO) &amp;ge;99%, liquid</td><td>MP Biomedicals, Irvine, CA, USA</td><td>219018690</td><td></td></tr><tr><td>Disposable vacuum line</td><td>Cook Medical, Bloomington, IN, USA</td><td>G51609</td><td></td></tr><tr><td>DMEM</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>10566024</td><td></td></tr><tr><td>DNase 1 ( &gt;2000 units/mg protein)</td><td>Sigma, St. Louis, MO, USA</td><td>D5025-150KU</td><td></td></tr><tr><td>Dulbecco&amp;prime;s Phosphate Buffered Saline (DPBS), no calcium, no magnesium</td><td>Fisher Scientific, Waltham, MA, USA</td><td>20012027</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco, Waltham, MA, USA</td><td>10082-147</td><td></td></tr><tr><td>Gibco DMEM/F12</td><td>Fisher Scientific, Waltham, MA, USA</td><td>11-320-033</td><td></td></tr><tr><td>Gibco DPBS, no calcium, no magnesium</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>14-190-250</td><td></td></tr><tr><td>Gibco HEPES</td><td>Fisher Scientific, Waltham, MA, USA</td><td>15630-080</td><td></td></tr><tr><td>Gibco PBS, pH 7.4</td><td>Fisher Scientific, Waltham, MA, USA</td><td>10010023-500mL</td><td></td></tr><tr><td>Gibco Trypsin-EDTA (0.25%)</td><td>Fisher Scientific, Waltham, MA, USA</td><td>25-200-056</td><td></td></tr><tr><td>Goat Anti-Rabbit IgG Antibody (H+L), Unconjugated</td><td>Vector Laboratories, Newark, CA, USA</td><td>AI-1000-1.5</td><td></td></tr><tr><td>Goat Anti-Rabbit IgG H&amp;amp;L (Alexa Fluor 647)</td><td>Abcam, Cambridge, UK</td><td>ab150079</td><td></td></tr><tr><td>Halt Phosphatase Inhibitor Single-Use Cocktail (100x)</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>78420</td><td></td></tr><tr><td>Halt Protease Inhibitor Cocktail (100x)</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>87786</td><td></td></tr><tr><td>HBSS</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>14175095-500mL</td><td></td></tr><tr><td>L-Glutamine</td><td>Gibco, Waltham, MA, USA</td><td>A2916801</td><td></td></tr><tr><td>Light mineral oil for embryo culture</td><td>Fujifilm Irvine Scientific, Santa Ana, CA, USA</td><td>9305</td><td></td></tr><tr><td>Mr. Frosty Freezing container</td><td>Fisher Scientific, Waltham, MA, USA</td><td>5100-0050</td><td></td></tr><tr><td>Omega FBS</td><td>Fisher Scientific, Waltham, MA, USA</td><td>NC0570754 -500mL</td><td></td></tr><tr><td>Ovum aspiration needle</td><td>Cook Medical, Bloomington, IN, USA</td><td>G55477</td><td></td></tr><tr><td>Paraformaldehyde 4% Aqueous Solution, EM Grade</td><td>Electron Microscopy Sciences, Hatfield, PA, USA</td><td>157-4-SP</td><td></td></tr><tr><td>Penicillin Streptomycin (10,000 U/mL) (Pen Strep)</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>15140122</td><td></td></tr><tr><td>Percoll</td><td>Sigma, St. Louis, MO, USA</td><td>P1644-100ML</td><td></td></tr><tr><td>Qubit fluorometer</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>Q33238</td><td></td></tr><tr><td>Quinn&amp;rsquo;s Advantage Fertilization Medium supplemented with 5% human serum albumin</td><td>CooperSurgical Fertility Companies, Trumbull, CT, USA</td><td>ART-1021 and ART-3001</td><td></td></tr><tr><td>Recovery Cell Culture Freezing Medium</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>12648010-50mL</td><td></td></tr><tr><td>RNAeasy Micro Kit</td><td>Qiagen, Hilden, Germany</td><td>74004</td><td></td></tr><tr><td>RNAlater</td><td>Fisher Scientific, Waltham, MA, USA</td><td>AM7024</td><td>Storage reagent</td></tr><tr><td>SAGE In-Vitro Maturation Media</td><td>CooperSurgical Fertility Companies, Trumbull, CT, USA</td><td>ART-1600</td><td>Oocyte maturation medium&amp;nbsp;</td></tr><tr><td>Triton X-100</td><td>Sigma -Aldrich, St. Louis, MO, USA</td><td>9036-19-5</td><td></td></tr><tr><td>Trypan Blue Solution</td><td>Fisher Scientific, Waltham, MA, USA</td><td>T8154-100ML</td><td></td></tr><tr><td>VECTASHIELD PLUS Antifade Mounting Medium with DAPI</td><td>Vector Laboratories, Newark, CA, USA</td><td>H-2000-10</td><td>&amp;nbsp;Antifade Mounting Medium</td></tr></tbody></table>

collection of human follicular fluid, follicle somatic cells, and immature oocytes from individuals undergoing in vitro fertilization

Collection of Human Follicular Fluid, Follicle Somatic Cells, and Immature Oocytes from Individuals Undergoing In Vitro Fertilization

Human ovary follicular contents, including follicular fluid (FF), oocytes, cumulus cells, and somatic cells, offer a powerful yet underutilized opportunity to study ovarian physiology. Assisted reproductive technologies (ART) routinely generate these biological byproducts, which are often discarded after mature oocytes are used for clinical care. However, they provide valuable insights into fertility, reproductive aging, and ovarian dysfunction. Here, we present a standardized protocol for the systematic collection and downstream processing of follicular components from women undergoing oocyte retrieval. Fully expanded cumulus-oocyte complexes (COCs) are microdissected to isolate cumulus cells without enzymatic denudation, preserving transcriptomic integrity. FF is centrifuged, with the supernatant analyzed for cytokines, hormones, metabolites, and environmental toxicants, while the pellet is cryopreserved and processed via density gradient centrifugation to isolate viable granulosa cells. A stepwise protocol enables the preparation of single-cell suspensions from fresh FF for single-cell RNA sequencing (scRNA-Seq), including enzymatic digestion, serial filtration, and red blood cell lysis, and yields &#62;65% viable cells representing a diverse composition, including granulosa cells, macrophages, T cells, smooth muscle-like cells, and antigen-presenting cells. In addition, immature germinal vesicle (GV) oocytes can be matured in vitro, with 56% reaching the MII stage within 24 h. Together, these protocols yield high-quality RNA, enriched granulosa cell populations, and complex single-cell datasets that capture the heterogeneity of the ovarian microenvironment. They provide a framework to leverage discarded reproductive tissues for translational research in human fertility and reproductive biology.

Distinct compartments of the human antral follicle, including the oocyte, cumulus cells, granulosa cells, theca cells, and follicular fluid (Figure 1), offer unique insights into ovarian physiology, reproductive aging, and oocyte competence. A schematic flowchart summarizing the collection and processing of these compartments is shown in Figure 2. Techniques such as IVM, single-cell and bulk RNA sequencing, cytokine profiling, me...

This protocol outlines standardized methods to isolate and process follicular fluid, somatic cells, and immature oocytes from IVF procedures, enabling high-quality molecular and cellular analyses. These approaches support translational research into fertility, reproductive aging, and ovarian dysfunction using materials typically discarded in clinical care.

Human ovary follicular contents, including follicular fluid (FF), oocytes, cumulus cells, and somatic cells, offer a powerful yet underutilized ...

We developed reproducible methods to collect and analyze IVF byproducts to study human fertility, infertility, and reproductive aging. Maintaining RNA integrity and cell viability is a challenge that requires rapid, coordinated sample handling between clinical and research teams. To begin, obtain 50 milliliter conical tubes containing follicular fluid.Centrifuge the tubes at 400 x g for 10 minutes at four degrees Celsius. Discard the supernatant from each tube, leaving behind the cell pellet. Add four to five milliliters of DMEM/F-12 to each cell pellet and resuspend the pellet by gentle pipetting.Now add 100 microliters of dimethyl sulfoxide and 100 microliters of fetal bovine serum into each tube. Aliquot 800 microliters of the resuspended cell suspension into cryogenic vials. Then gently pipette up and down to mix the contents before sealing the cryovials.Transfer the sealed vials into a freezing container filled with 100%isopropyl alcohol. Place it in a minus 80 degrees Celsius freezer for 24 hours to allow controlled cooling at approximately 1 degree Celsius per minute. The next day, move the cryovials to a liquid nitrogen tank for long-term storage.Thaw fetal bovine serum, L-glutamine, DMEM/F-12, and penicillin-streptomycin in a 37 degrees Celsius bead or water bath. In a 15 milliliter conical tube, prepare four milliliters of thaw media and eight milliliters of resuspension media. In a 50 milliliter conical tube, prepare up to 50 milliliters of culture media and place it in a 37 degrees Celsius bead bath.Next, thaw the frozen cryovials in a 37 degrees Celsius bead bath for approximately one minute or until the pellet begins to loosen. Using a P-1000 pipette, transfer the entire contents of each cryovial into thaw media and gently resuspend. Centrifuge the thawed cell suspension in thaw media at 400 x g for 10 minutes at four degrees Celsius.Then aspirate the supernatant, leaving only the cell pellet in the 15 milliliter conical tube. Now add four milliliters of resuspension media to the pellet and vigorously pipette to ensure complete resuspension without clumps. Next, gently pipette one part density gradient medium at the bottom of a tube containing the cell suspension, releasing slowly to form a clear layer.Then add DPBS gently to maintain layering. Centrifuge the tube at 850 x g for 10 minutes at four degrees Celsius to activate the density gradient medium. After centrifugation, identify the top white cotton-like granulosa cell layer and the bottom layer as primarily red blood cells.Using a P-1000 pipette, gently remove the top layer of cells and resuspend them in the remaining four milliliters of resuspension media. Now combine 10 microliters of the resuspended cells with 10 microliters of trypan blue solution in a 0.5 microliter microcentrifuge tube. Pipette 10 microliters of this mixture onto a hemocytometer for cell counting.Then centrifuge the cell suspension at 380 x g for five minutes at room temperature. Aspirate the supernatant from the centrifuge cell suspension, leaving behind the cell pellet. Then add the required volume of warmed culture media and resuspend the pellet thoroughly.Plate the cells at the desired seeding density. View the cells under a microscope and then incubate the cells. Rinse the cells with warmed DPBS and replace the media every two days.To process human follicular fluid, first centrifuge the samples at 380 x g for 10 minutes at four degrees Celsius. Then transfer the supernatant from each tube into fresh 15 milliliter conical tubes. Add protease inhibitor cocktail and phosphatase inhibitor in a 1 to 100 ratio to the follicular fluid and mix thoroughly by pipetting.Distribute one milliliter of the prepared follicular fluid into sterile 1.5 milliliter microcentrifuge tubes. Store the tubes at 80 degrees Celsius until further analysis. Immunocytochemistry performed on day four of culture showed that approximately 90%of cells stained positive for AMHR2, confirming strong expression of this granulosa cell marker.Quantification of AMHR2 staining confirmed that approximately 90%of the cells were AMHR2 positive. Further immunocytochemistry confirmed granulosa cell identity with approximately 80%of cells staining positive for FOXL2. Quantification of FOXL2 staining showed approximately 80%FOXL2 positive cells.Our protocol yields high-quality RNA, viable granulosa cells, and single cell dataset, revealing molecular underpinnings of human reproductive function. We aim to identify ovarian cell types with age-related gene expression changes to uncover the mechanisms of reproductive aging and fertility decline. Future research will focus on integrating multiomics and longitudinal studies that will map dynamic follicular changes across age, environment, and disease states.

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Learn standardized methods to isolate follicular fluid, somatic cells, and immature oocytes from IVF procedures for reproductive biology research. This protocol enables high-quality molecular analyses of human fertility and ovarian function.

Collection of Human Follicular Fluid, Follicle Somatic Cells, and Immature Oocytes from Individuals Undergoing In Vitro Fertilization (Video) | JoVE Journal

Collection of Human Follicular Fluid, Follicle Somatic Cells, and Immature Oocytes from Individuals Undergoing In Vitro Fertilization

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