Ovarian Tissue Oocyte-In Vitro Maturation for Fertility Preservation

The present study on OTO-IVM has been approved by the local Ethical Committee of UZ-Brussels (addendum of project 2008/068 and project 2022/303). All patients signed written informed consent. Each patient was individually assessed by a reproductive medicine specialist physician, navigator nurse, and the referring oncologist to compose the optimal FP treatment plan, taking into account the patient's preferences¹⁴. In short, patients eligible for OTC are in urgent need of FP and are less than 36 years of age¹⁴. To combine OTC with OTO-IVM, chemo- or radiotherapy cannot have been administered in the 6 months preceding OTC.

1. Laboratory environment and personnel

1. Perform OTC in a Class A laminar flow cabinet.
2. Perform OTC with two operators: one working aseptically in the laminal flow cabinet, and a second cleaning all non-sterile materials with a bactericide and sporicide decontaminant spray and handing over materials and supplies aseptically to the first operator. Process the ovarian cortex on a benchtop cooler lid (± 4 °C).
3. Perform the cumulus-oocyte complex (COC) search in a second laminar flow cabinet with a stereomicroscope in a MAR lab on a heated stage (37 °C).
4. Perform retrieval of the COC from the medullar remnants (sections 3 and 4) and initiate the IVM process (section 5). This is done by a third operator.

2. Media preparation

NOTE: Five types of media are used for this procedure (detailed below): OTC handling medium, OTC freezing medium, Search medium, LAG medium, and IVM medium. When preparing media, work aseptically in the flow cabinet, as detailed in section 1. Use new, unopened reagents for every procedure and maintain the sterility of all disposables used to ascertain the sterility of the produced media.

1. OTC handling medium
   1. Supplement Leibovitz's L15 medium with 4 mg/mL human serum albumin (HSA), 100 U/mL penicillin, and 100 µg/mL streptomycin (see Table of Materials).
   2. Keep the OTC handling medium refrigerated until use (for a maximum of 2 days).
      NOTE: Use OTC handling medium to rinse and process the ovarian tissue and use it cold (0-4 °C).
2. OTC freezing medium
   1. Supplement Leibovitz's L15 medium with 1.5 M dimethyl sulfoxide (DMSO) and 4 mg/mL HSA.
   2. Keep the OTC freezing medium refrigerated until use (for a maximum of 2 days).
3. Search medium
   NOTE: The Search medium (a HEPES-buffered medium for oocyte handling) is commercially available (see Table of Materials) and ready to use. Use the Search medium to rinse the filter and collect the COC while searching for COCs in between the medulla fragments (section 4).
   1. Fill six 14 mL round-bottom, sterile tubes with 6 mL of Search medium.
   2. Prepare a 4-well dish with 500 µL of Search medium covered with 350 µL of oil (see Table of Materials).
   3. Heat the tubes and the 4-well dish at 37 °C for at least 1 h prior to the arrival of the ovarian tissue. HEPES-buffered media do not require CO₂ incubation.
4. LAG medium
   NOTE: The commercially available IVM system (see Table of Materials) comprises two different media: LAG medium and IVM medium, which needs to be supplemented as detailed below. LAG medium is used to rinse the COCs, but can also be used for a 2-3 h incubation period preceeding IVM in IVM medium, as suggested by the insert of the IVM system.
   1. Use the commercially available, ready-to-use LAG medium (see Table of Materials).
5. IVM medium
   1. Supplement commercially available IVM medium with 10 mg/mL HSA, 75 mIU/mL follicle stimulating hormone (FSH), and 100 mIU/mL human chorionic gonadotropin (hCG).
   2. Prepare a 4-well culture dish with one well of LAG medium and three wells of supplemented IVM medium: 500 µL of medium covered with 350 µL of oil overlay.
   3. Equilibrate the dish overnight in an incubator at 37 °C in 6% CO₂ and 20% O₂, the optimal environment for IVM culture.

3. Ovarian cortex preparation

NOTE: Laparoscopic whole ovary removal was performed as described by Jadoul et al.¹⁵.

1. Upon arrival of the ovary in the laboratory, wash the ovary in the OTC handling medium (step 2.1) twice by transferring the ovary into a 100 mm Petri dish with the OTC handling medium.
2. With a scalpel, cut open the ovary in half longitudinally (Figure 1A).
3. Make several incisions in the medullar tissue both vertically and horizontally with a fresh scalpel. Avoid damaging the cortex. Pierce the firm antral follicles of various sizes within the medulla gently with the scalpel to release the follicular fluid in the OTC handling medium.
4. Pare down the soft medulla tissue with surgical scissors (Figure 1B). This procedure may take several minutes.
5. Put the ovarian shell in a new dish with fresh OTC handling medium during the trimming process.
6. Transfer the dish with medullar fragments and released follicular fluid (Figure 1C) to the second flow cabinet to start the search for COCs.
7. Repeat steps 3.4 to 3.6 until the desired thickness (1-2 mm) of the cortex tissue is obtained.
8. Slice the cortex into pieces of approximately 8 mm x 5 mm.
9. Incubate the pieces 3x consecutively for 10 min in approximately 25 mL of OTC freezing medium (step 2.2) in 100 mm Petri dishes.
10. Place one piece in 800 µL of OTC freezing medium in a cryovial.
11. Cryopreserve the ovarian tissue using a slow freezing protocol in a cryochamber controlled by a temperature controller (see Table of Materials): from 4 °C to -7 °C at -2 °C/min, manual seeding at -7 °C, from -7° C to -40 °C at -0.3 °C/min, from -40 °C to -100 °C at -10 °C/min, and at -100 °C plunge the cryovials in liquid nitrogen and store them.

4. Search for COCs

1. Prepare the laminar flow cabinet for the COC search.
   1. Warm 60 mm culture dishes on the heated stage under the stereomicroscope in the laminar flow cabinet.
   2. Put the 14 mL tubes with 6 mL of preheated Search medium (step 2.3.1) in a heated block, and the preheated 4-well dish with Search medium on the heated stage under the microscope in the laminar flow cabinet.
   3. Place a sterile filter (cell strainer, 70 µm mesh size; see Table of Materials) bottom-down in a culture dish.
   4. Take a 290-310 µm glass capillary for use: do not use smaller capillaries than 290-310 µm, in order to avoid damage to the oocyte-cumulus cell connectivity.
   5. Ensure 1 mL filter tips and a 1 mL pipet are available.
2. Take the first dish with medullar fragments from the OTC laminar flow cabinet (4 °C) and put the dish on the heated stage (37 °C) in the IVM laminar flow cabinet as soon as possible.
   NOTE: The first dish of medullar fragments and follicular fluid might be contaminated with blood from blood-filled ovulated follicles or the vasculature of the ovary itself. Red blood cells blur clear vision and complicate the search for COCs. The contaminated medium must be removed to wash away the red blood cells (see step 4.3).
3. Remove the blood cell contamination by following the steps below.
   1. Pipet 2 mL of Search medium over the filter membrane (70 µm cell strainer) to wet the membrane and put the dish, which captured the medium, aside on the heated stage.
   2. Flip the filter upside-down and place it with the handle against the rim of a new culture dish, creating a slope with the bottom of the filter.
   3. Collect blood-contaminated medium in between medullar fragments using a 1 mL tip and blow it gently over the filter membrane (Figure 1D) to capture the COCs present in the contaminated medium.
   4. Repeat step 4.3.3 until most of the blood-contaminated medium is removed.
   5. Pour fresh Search medium over the medullar fragments immediately to keep the COC suspended in the culture medium at all times.
   6. Rinse the sloped filter membrane gently with 2 mL of Search medium to rinse away red blood cells on the filter membrane.
   7. Place the filter bottom-down in a new dish and pour 3-4 mL of Search medium into the filter to expel the COC from the filter membrane into the medium that fills the dish. Remove the filter. To ensure that the filter membrane does not dry out (and any possible COCs that are not yet expelled from it), immerse the filter bottom-down in the first dish, which was used to wet the filter for the first time.
   8. Immediately examine the expelled medium for COCs by visual inspection under a stereo microscope with a magnification of 10x-50x. Search for clear translucent oocytes with a rim of dark, compact surrounding cumulus cells (Figure 1E). Swirl the medium in the dish and examine the medium again.
   9. Rinse the filter a second time in a new dish and examine the expelled medium for COCs.
   10. Examine the medium in the dish where the filter is immersed for the remaining COCs.
   11. Transfer COCs with a glass capillary to the 4-well dish containing the Search medium (Figure 1E). Keep the dishes on the heated stage at all times.
4. Search in between the medullar fragments for COCs after removing the contaminating blood cells from the dish with medullar fragments and replenishing the dish with clear Search medium. Use the glass capillary to move around the medullar fragments and swirl the medium in the dish in order to find COCs. Pull apart large medullar fragments with tuberculin needles on a 1 mL syringe if the presence of an oocyte is suspected.
5. Collect all intact COCs in the 4-well dish with Search medium.
   ​NOTE: In a typical OTC procedure, cortex tissue is pared down from the medulla using three consecutive 100 mm dishes, where the ovarian shell is moved to a new dish with fresh medium and where the remnants are transferred to the IVM flow for COC search. Usually, only the first dish requires the removal of blood cells; in the second and third dishes, fewer medullar fragments are present, and COC search is performed directly in between fragments and in the medium.

5. IVM of COCs

1. Rinse the COCs in a clean well with Search medium.
2. Move the pre-equilibrated IVM culture dish (the 4-well dish that contains one well of LAG medium and three wells of IVM medium) from the incubator to the heated stage in the IVM flow cabinet.
3. Label the IVM culture dish containing COCs with patient information.
4. Rinse the COCs in LAG medium (2-3 s) and place them in one of the three wells with supplemented IVM medium. Culture the COCs in groups of approximately 10 COCs per well. Omit naked oocytes-oocytes devoid of attached cumulus cells-from the culture, as they are known to have severely compromised developmental potential.
5. Place the dish in an incubator at 37 °C, 6% CO₂, and 20% O₂ for 30 h.

6. Handling mature oocytes

1. Strip oocytes of their surrounding cumulus cells by brief exposure to 80 IU of hyaluronidase (see Table of Materials) and gentle mechanical pipetting after 30 h of IVM.
2. Identify mature oocytes by the extrusion of the first polar body.
3. Vitrify or inseminate the mature oocytes, depending on the patient's preference.
   NOTE: If the maturation check after 30 h of IVM falls outside the acceptable working hours, the IVM duration can be shortened to 28 h or extended up to 42 h. If only a few mature oocytes are obtained after 30 h, immature oocytes can be cultured additionally overnight in the original IVM well, devoid of cumulus cells, and used the next morning for vitrification or intracytoplasmic sperm injection (ICSI) when mature.