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

Summary

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.

Abstract

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 >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.

Introduction

Infertility affects millions of individuals and couples worldwide, with estimates that one in six people and one in eight couples face difficulties conceiving naturally^1,2,3. 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 medicine

Protocol

Before detailing the protocols, emphasis should be placed on the critical importance of collaboration among reproductive endocrinologists, clinical staff, embryologists, research coordinators, and basic science researchers. Successful collection and processing of valuable reproductive tissues for research depends on a coordinated, multidisciplinary effort. Research coordinators and clinical teams play a key role in obtaining Institutional Review Board (IRB) approval and ensuring ongoing regulatory compliance. Designated personnel, typically research coordinators, graduate students, or postdoctoral fellows, serve as liaisons between the research a....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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