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

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.