Ovarian Tissue Oocyte-In Vitro Maturation for Fertility Preservation

Abstract

Mature oocyte vitrification is the standard of care to preserve fertility in women at risk of infertility. However, ovarian tissue cryopreservation (OTC) is still the only option to preserve fertility in women who need to start gonadotoxic treatment urgently or in prepubertal children. During ovarian cortex preparation for cryopreservation, medullar tissue is removed. Growing antral follicles reside at the border of the cortex-medullar interface of the ovary and are broken during this process, releasing their cumulus-oocyte complex (COC). By thoroughly inspecting the medium and fragmented medullar tissue, these immature cumulus-oocyte complexes can be identified without interfering with the OTC procedure. The ovarian tissue-derived immature oocytes can be successfully matured in vitro, creating an additional source of gametes for fertility preservation. If OTC is performed within or near a medical assisted reproduction laboratory, all necessary in vitro maturation (IVM) and oocyte vitrification tools can be at hand. Furthermore, upon remission and child wish, the patient has multiple options for fertility restoration: ovarian tissue transplantation or embryo transfer after the insemination of vitrified/warmed oocytes. Hence, ovarian tissue oocyte-in vitro maturation (OTO-IVM) can be a valuable adjunct fertility preservation technique.

Introduction

Fertility preservation (FP) options for women planned for gonadotoxic treatment, sex-reassignment therapy, or women who have a genetic predisposition for premature ovarian failure, depend on the health and age of the patient, available timeframe, type of treatment, patient's preference, and FP procedures available at the fertility center of choice. Vitrification of mature oocytes obtained after ovarian stimulation with gonadotropins and oocyte retrieval in a medical assisted reproduction (MAR) laboratory cycle is considered the preferred option for FP^1,2. However, for prepubertal girls, women in whom the urgent start of gonadotoxic treatment or gonadectomy is required, or women with a high risk of permanent amenorrhea due to gonadotoxic treatment, a cycle of ovarian stimulation with gonadotropins is not possible, and ovarian tissue cryopreservation (OTC), which is an accepted and valid technique for FP^1,2,3, is the only option. The goal of OTC is to cryopreserve thousands of dormant primordial follicles in the ovarian cortex tissue, which can resume growth after the transplantation of frozen/thawed tissue onto the remaining ovary or in a peritoneal pocket after the careful screening of minimal residual disease in representative tissue fragments.

In order to obtain cortical fragments of 1-2 mm thickness suitable for cryopreservation, the soft medullar tissue needs to be removed. This medullar tissue typically entails growing follicles in various stages of development that escape the stiff ovarian cortex to allow for their growth and expansion⁴. For many years, several labs have been investigating the potential of these oocytes recovered from follicles residing in the remnant medullar tissue after ovarian cortical fragment preparation using in vitro maturation (IVM)^5,6,7, referred to as ovarian tissue oocyte IVM (OTO-IVM). Antral follicles, even those less than 6 mm in diameter, contain immature oocytes surrounded by cumulus cells that can mature, fertilize, and develop into healthy babies using an IVM system^8,9. IVM is considered the standard of care for women at risk for ovarian hyperstimulation syndrome (OHSS), such as polycystic ovary syndrome (PCOS) patients. However, in the field of FP, there are limited data available for IVM in cases with a contraindication for ovarian stimulation; IVM of oocytes collected transvaginally is still considered innovative, and OTO-IVM is considered experimental^2,10. That said, the reports of the first live births after OTO-IVM^11,12,13 highlight the potential of using OTO-IVM as an add-on technique when OTC is required for FP in patients¹⁴.

This study provides technical details to adopt OTO-IVM in the MAR laboratory and illustrates the results obtained in a single center.

Protocol

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.

Representative Results

Over the past decade, 98 patients undergoing oophorectomy or ovarian biopsy for OTC were also offered OTO-IVM. The results presented here are an update of the clinical program as published before^7,13. Immature oocytes obtained during ovarian tissue processing were matured in vitro predominantly for 30 h. However, a more flexible maturation time was allowed for practical reasons or because of low maturation, ranging from 28-42 h. Patients opted predominantly for oocyte vitrification (85/98) or-upon specific request of the patient-embryos were created by inseminating mature oocytes and embryo vitrification (13/98). A total of 1,417 oocytes were collected in 98 patients aged between 1 and 38 years old who were offered OTC with OTO-IVM. The mean oocyte maturation rate was 40% ± 22% (mean ± SD). The mean maturation rate was lower in prepubertal girls compared to postpubertal women (26% vs. 42%, respectively; Table 1). For 94/98 patients, immature oocytes were found, and at least one metaphase II (MII) was obtained for 86/94 patients after OTO-IVM, indicating that OTO-IVM provided an additional benefit of mature oocytes for cryopreservation in 88% of the OTC population.

Overall, an average of 6.0 ± 6 mature oocytes was obtained in the OTC population (intention to treat, ITT). However, some patients were very young (<5 years old; n = 4) or had a limited amount of ovarian tissue available for OTO-IVM (ovarian biopsy [n = 4] or tumor involvement in the ovary [n = 4]). If only patients with successful OTO-IVM were considered (at least one MII; n = 86), an average of 6.5 ± 6 MII was obtained.

In order to preserve the fertility chances of women, oocyte vitrification was advised for the patients. However, for 13 patients, the couple insisted on creating embryos for FP. Fertilization success was assessed by the presence of two pronuclei (2PN) at approximately 18 h after ICSI. OTO-IVM oocytes showed a 63% fertilization rate (40 2PN/64 MII); 68% of the zygotes developed into cleavage stage embryos of sufficient quality for cryopreservation (27 good quality embryos [GQE]/40 2PN), generating on average 2.1 ± 2 embryos per patient being vitrified. In brief, GQEs sufficient for cryopreservation are defined as six cells or more and a maximum of 20% fragmentation⁷.

Data on the warming of OTO-IVM oocytes or embryos are scarce. Of 86 patients with cryopreserved gametes/embryos, 17 patients returned to the MAR clinic with a desire for pregnancy. Ten decided to use their cryopreserved material obtained after OTO-IVM; seven patients showed sufficient ovarian activity to start an in vitro fertilization (IVF)/ICSI stimulation cycle with fresh oocytes. Five patients had OTO-IVM oocytes warmed, with a survival rate of 75% (58/77). These oocytes showed a similar potential for fertilization (48%; 28/58) compared to fresh OTO-IVM oocytes, but a lower embryo development rate, generating only 29% (8/28) GQEs for transfer or cryopreservation, an average of 1.3 ± 1 per patient. Out of eight created embryos, seven were warmed, of which six embryos did not lead to pregnancy, and one embryo led to a healthy live birth.

For five patients, OTO-IVM embryos were warmed. A total of nine embryos were warmed, of which one did not survive, two were genetically tested for breast cancer (BRCA) genes and considered unsuitable for transfer, four did not lead to a pregnancy, and two embryos led to two healthy babies born in two patients.

Overall, three of the 10 patients who returned to the MAR clinic with a desire for pregnancy had a healthy baby with their cryopreserved OTO-IVM material (30%), without the need for additional laparoscopic surgery for tissue transplantation and the risk of reintroducing malignancy.

Figure 1: Ovary incision and isolation of ovarian cortex and COCs. (A) Longitudinal incision of the ovary through the cauterization lesion to cut the ovary in half. (B) Removal of the inner medullar tissue to isolate the cortical ovarian tissue with scissors. (C) Medullar fragments and follicular fluids remaining after ovarian cortex isolation. (D) Filtration of the medium through a 70 µm mesh filter to isolate COCs and remove contaminating red blood cells. (E) Representative image of a COC before IVM; scale bar corresponds to 100 µm. Please click here to view a larger version of this figure.

ITT	Total	Prepubertal	Adult
n	98	11	87
average # Cortex fragments/patient	18.5 ± 10	11.5 ± 7	19.4 ± 10
average # COC/patient	14.5 ± 12	16.2 ± 16	14.2 ± 12
average # MII/patient	6.0 ± 6	4.3 ± 5	6.2 ± 6
total # COC	1417	178	1239
total # MII	563	47	516
MII rate	40%	26%	42%

Table 1: The outcome of OTO-IVM in the total population and stratified according to menarche. Abbreviations: ITT = intention to treat; n = amount of subjects; COC = cumulus oocyte complex; MII = metaphase II oocyte.

Discussion

The priority of the FP procedure is always to manipulate and freeze the ovarian cortex according to the standard operating protocol that has been validated in the clinic. A drawback in FP is the absence of a standard protocol available in the published literature regarding OTC and OTO-IVM. It is difficult to assess the efficiency and validity of the techniques and adaptations since there is a large time gap between freezing/vitrification and thawing/warming in a clinical setting. If changes to the OTC protocol are made to augment the efficiency of OTO-IVM, it is vital to validate the adapted OTC protocol for tissue health upon cryopreservation and thawing. In this report, the protocol for OTC, routinely used at Brussels IVF at the University Hospital of VUB, Brussels, prescribes that the medulla is pared down from the ovarian cortex using scissors. As such, medullar tissue is systematically cut into small fragments, and COCs are set free. Other labs prepare the ovarian cortex using scalpels, where the bulk of the medullar tissue is scooped from the cortex shell. Here, additional cutting of the large medullar piece is needed to release the COC. Puncturing visible, large antral follicles with a needle upon receiving the ovary in the laboratory is contraindicated, as these punctures damage the ovarian cortex. Secondly, the yield of COC aspiration from large antral follicles in an ovary ex vivo is low.

If ovarian tissue is transported from a local hospital to a center of expertise for tissue cryopreservation, this transport is performed under cold conditions (0-4 °C) for up to 24 h with minimal tissue damage¹⁶. During transport, the ovary is suspended in saline solution, phosphate-buffered saline, or more complex media like Leibowitz's L15, alpha modification of Eagle's medium, or Custodiol; however, data about tissue viability (and OTO-IVM success) related to different transport media are scarce¹⁶. The OTC protocol states that the ovarian cortex should be processed at 0-4 °C to slow metabolism and reduce ischemic damage. However, chilling oocytes influences their cytoskeletal conformation¹⁷ and membrane integrity¹⁸, and impairs the maturation potential of ovarian tissue-derived oocytes¹⁹ (immediate processing at 37 °C results in 42% MII, whereas transport for 2-5 h on ice reduces the MII rate to 27%¹⁹). Other centers perform ovarian tissue processing at room temperature or 37 °C, but the presented results reflect tissue processing in cold conditions with intermittent transport of medullar fragments and medium to a 37 °C heated stage for COC search.

For IVM the standard protocol, used for infertility patients with a PCOS background, was used⁹. A commercial IVM medium is used to culture immature oocytes, where the manufacturer recommends supplementing the IVM medium with maternal serum. In order to avoid bias of unknown substances delivered by maternal serum, the currently presented IVM results are obtained using commercial HSA as the protein source in the IVM medium. Novel biphasic IVM culture systems have been developed, which augment the yield in mature oocytes in an infertile PCOS population²⁰ as well as in OTO-IVM²¹. The continued efforts in optimizing IVM protocols will likely enhance, also the efficiency of OTO-IVM in fertility preservation patients. Immature oocytes are incubated at 20% O₂ during the maturation phase, unlike incubation under 6% O₂ for embryonic preimplantation development. IVM in reduced oxygen conditions impairs blastocyst development²², hence it is critical to perform the 30 h incubation period in IVM medium in 20% O₂. Maturation is assessed after 30 h by the enzymatic and mechanical removal of surrounding cumulus cells. Sometimes, an additional overnight culture in IVM medium of cumulus-free oocytes was applied to increase the yield of mature oocytes; However, only a limited amount of additional mature oocytes were obtained after this prolonged maturation time. Mature oocytes were cryopreserved using the standard protocol for oocyte vitrification at Brussels IVF²³.

As shown in the results, OTO-IVM can be offered to most patients undergoing OTC; however, for patients under 5 years of age, the likelihood of obtaining oocytes is low⁷ due to the limited differentiation between cortex and medulla tissue and the absence of antral follicles. In general, maturation rates of OTO-IVM are lower for children as compared to adults, most likely due to the intrinsic differences between adult and prepubertal folliculogenesis and hence the compromised developmental capacity due to, for example, increased aneuploidy^24,25. Similarly, advanced age (>30 years) has been described to affect the OTO-IVM maturation rate²⁵. Further, when ovaries have cysts or malignant involvement, the yield after OTO-IVM can be low²⁶, as is also seen when an ovarian biopsy is taken instead of a whole ovary. The selection of patients for OTO-IVM is important in determining successful outcomes.

A subset of FP patients have a contraindication for ovarian tissue transplantation (high risk of reintroducing malignant cells in, for example, ovarian cancers) or host-hostility in autoimmune diseases, and need to wait until human in vitro folliculogenesis is clinically applicable to have their genetically-own child. OTO-IVM is the only clinically applicable FP option for these patients.

Although the clinical relevance of OTO-IVM is evident, some issues need to be solved to validate this technique before the experimental label of the technique can be lifted. The impact of the temperature of transport and processing requires investigation in more detail. The standard IVM protocol used for infertile patients may not be efficacious for immature oocytes harvested from ovarian tissue, and IVM culture media and methods require finetuning to enhance oocyte quality after maturation. The lower oocyte quality of OTO-IVM oocytes is illustrated by a lower survival rate after vitrification/warming of OTO-IVM oocytes (75%), as compared to mature oocytes harvested after ovarian stimulation in an oocyte donation program (93.7%)²³. More data on oocyte vitrification efficiency and the developmental capacity of OTO-IVM oocytes are necessary to estimate the true potential of OTO-IVM in FP. The population of patients who are suitable for oophorectomy/ovarian biopsy for FP is shrinking, as a result of the advances in therapeutical agents rendering cancer therapies more efficient and less gonadotoxic.

In conclusion, performing OTO-IVM creates an additional option for FP and offers a realistic additional chance of achieving pregnancy, as shown by the three healthy children born in three individual patients in this small cohort of 10 patients who warmed their cryopreserved OTO-IVM oocytes or embryos. Therefore, OTO-IVM is considered a valuable add-on tool that can be used when cryopreservation of ovarian tissue is the best approach for preserving a patient's fertility.

Materials

Name	Company	Catalog Number	Comments
1000 µL filter tips	Eppendorf/VWR International	613-6780	COC search
Benchtop Cooler	Fisher Scientific	15-350-54	Benchtop Cooler lid is used to prepare the tissue, Benchtop Cooler tube holder to keep cryovials with freezing medium cooled
Corning Cell culture dish, non-treated, 100 mm	Corning/VWR International	430591	Dish for ovarian tissue preparation
CryoSure-DMSO	WAK Chemicals	0482	Cryoprotectant for ovarian tissue cryopreservation
Cumulase	Origio/CooperSurgical	16125000A	recombinant human hyaluronidase enzyme for cumulus cell removal after IVM
Decontamination spray: Suprox	Medipure LTD	MP016	Desinfectant solution for aseptic handling with bactericide and sporicide action
Disposable scalpels	Swann-Morton	0511	Ovarian tissue preparation
Falcon 14 mL Round Bottom Polystyrene Test Tube, with Snap Cap, Sterile	Falcon/VWR International	BDAA352057	Medium container
Falcon Cell strainer 70 µm	Falcon/VWR International	352350	Filter for elimination of red blood cell contamination and COC search
Freeze control Ampoule Cryochamber and Temperature Controller	Cryologic	CL-8800i   CC60AS	Slow freezing machine
FSH: Menopur 75 IU	Ferring	BE197504	Follicle Stimulating Hormone : Supplement for IVM medium
Handling pipette 290-310 µm	Vitrolife	15538	COC search: gentle transfer of COC without damaging oocyte-cumulus cell connectivity
hCG: Brevactid 5000 IE	Ferring	5008001036	Human Chorionic Gonadotropin : Supplement for IVM medium
High security tube	CryoBioSystem	022252	cryovial, heat-sealed for safe cryostorage
HSA-solution	Vitrolife	10064	Human serum Albumin: supplement for IVM medium
Leibovitz's L-15 medium	Life Technologies Europe	31415-029	Handling medium for ovarian tissue preparation
MediCult IVM system	Origio/CooperSurgical	82214010	medium for IVM containing both LAG and IVM medium. IVM medium needs to be supplemented as detailed in the protocol
METZENBAUM fino scissors 140 mm	Chirurgical Maintenance	VIZ08280314	Medium size scissors for initial medulla removal
Nunc 4-well dishes for IVF	Nunc/VWR International	144444	COC collection during COC search and IVM culture
Nunc Invitro fertilization Petri Dish with Vented Lid, 60 mm, Non-Pyrogenic, Sterile	Thermo Scientific/VWR	NUNC150270	Dish for COC search
Oocyte handling medium : Flushing Medium with heparin	Origio/CooperSurgical	10765060	Search medium for COC search
Ovoil	Vitrolife	10029	oil for IVM culture
Penicillin/Streptomicin mix	Life Technologies Europe	15140-148	Supplement for OTC handling medium
Scissors, curved, 150 mm long, 20 mm blade	Chirurgical Maintenance	VIREBST999-SC	Small size scissors for residual medulla removal