Fertility Preservation Through Oocyte Vitrification: Clinical and Laboratory Perspectives

Summary

Oocyte cryopreservation is recognized by several international scientific societies as the gold standard for fertility preservation in postpubertal women. Appropriate clinical and laboratory strategies ensure maximum efficacy, efficiency, and safety of fertility preservation treatments.

Abstract

Preserving female fertility is crucial in a multifunctional healthcare system that takes care of patients' future quality of life. Oocyte cryopreservation is recognized by several international scientific societies as the gold standard for fertility preservation in postpubertal women, for both medical and non-medical indications. The main medical indications are oncologic diseases, gynecologic diseases such as severe endometriosis, systemic diseases compromising the ovarian reserve, and genetic conditions involving premature menopause. This paper describes the whole clinical and laboratory work-up of a fertility preservation treatment by outlining recommendations for objective and evidence-based counseling. Furthermore, it focuses on the effectiveness of the procedure and describes the most appropriate strategies to fully exploit the ovarian reserve and maximize the number of oocytes retrieved in the shortest possible time. The evaluation of the ovarian reserve, the definition of an ideal stimulation protocol, as well as oocyte retrieval, denudation, and vitrification procedures have been detailed along with approaches to maximize their efficacy, efficiency, and safety.

Introduction

The development and implementation of an efficient cryopreservation program for human oocytes has been a significant breakthrough in reproductive medicine. According to recent evidence, vitrification is the most effective strategy to cryopreserve metaphase II (MII) oocytes, as it results in statistically higher survival rates compared to slow freezing, independently of the patient population (infertile patients or oocyte donation program)^1,2,3. The remarkable achievements of oocyte vitrification led the Practice Committees of the American Society for Reproductive Medicine (ASRM) and the Society for Assisted Reproductive Technology (SART) to pronounce this technique to be the most effective for elective fertility preservation in postpubertal women, for both medical and non-medical indications^4,5,6. Medical indications for fertility preservation include (i) cancer and autoimmune diseases that require therapies⁷ such as radiotherapy, cytotoxic chemotherapy, and endocrine therapy (whose detrimental effect on the ovarian reserve is associated with maternal age as well as type and dose of the treatment); (ii) ovarian diseases requiring repeated or radical surgery (such as endometriosis)⁸; and (iii) genetic conditions (e.g., X-fragile) or premature ovarian failure. In addition, fertility preservation has become a valuable option for all women who have not accomplished their parental objective for non-medical reasons (also known as social freezing).

Regardless of the indication for fertility preservation and according to the major international guidelines on fertility preservation, all patients willing to vitrify their oocytes should receive appropriate counseling to be informed about their realistic chance of success, the costs, risks, and limitations of the procedure^{9,10,11,12,13}. Most importantly, it should be clear that vitrifying a cohort of MII oocytes does not ensure a pregnancy, but that it offers a higher chance of success for future in vitro fertilization (IVF) treatment, if necessary¹⁴. In this regard, the woman's age at the time of oocyte vitrification is certainly the most important limiting factor¹⁵ as advanced maternal age (AMA; >35 years) is the main cause of female infertility¹⁶. Besides a progressive reduction in the ovarian reserve, AMA is associated with an impairment of oocyte competence due to defective physiological pathways such as metabolism, epigenetic regulation, cell cycle checkpoints, and meiotic segregation¹⁷. Therefore, the reasonable number of eggs to vitrify mainly depends on maternal age. In women younger than 36 years, at least 8-10 MII oocytes¹⁸ are required to maximize the chance of success. In general, the higher the number of vitrified oocytes, the higher is the likelihood of success. Therefore, tailoring ovarian stimulation according to ovarian reserve markers such as anti-Müllerian hormone (AMH) levels or antral follicle count (AFC) is crucial to fully exploit the ovarian reserve in the shortest possible time.

The safety of the whole procedure is the other key issue when enrolling patients for fertility preservation. Clinicians should employ the best strategies to minimize the risks and prevent (i)ovarian hyperstimulation syndrome (OHSS) by using safe approaches such as the gonadotrophin-releasing hormone (GnRH) antagonist protocol followed by a GnRH agonist trigger¹⁹ and (ii) the remote, yet possible, risks of peritoneal bleeding, injury to the pelvic structures (ureter, bowel, appendix, nerves), or pelvic infection during oocyte retrieval. Lastly, (iii) traditional regimens for stimulation are associated with supraphysiologic serum estradiol and therefore, are not recommended in estrogen-sensitive diseases such as breast cancer. Protocols involving aromatase inhibitors (such as letrozole or tamoxifen) are more suitable in these cases^20,21. In the laboratory setting, the most widespread protocol for oocyte vitrification is still the one first described by Kuwayama and colleagues^2,23, which consists of a stepwise procedure involving the gradual addition of cryoprotectants (CPAs). In the first phase (equilibrium/dehydration), oocytes are exposed in a CPA solution containing 7.5% v/v ethylene glycol and 7.5% v/v dimethyl sulfoxide (DMSO), while in the second phase, oocytes are moved to a vitrification solution with 15% v/v ethylene glycol and 15% v/v DMSO, plus 0.5 mol/L sucrose. After a short incubation in the medium of vitrification, the oocytes can be placed in specifically designed, open cryodevices and finally plunged in liquid nitrogen at -196 °C to be stored until use.

Here, the whole clinical and laboratory work-up of a fertility preservation treatment has been described by (i) outlining recommendations for objective and evidence-based counseling, (ii) focusing on the cost-effectiveness of the procedure, and (iii) describing the most appropriate strategies to fully exploit the ovarian reserve and maximize the number of oocytes retrieved in the shortest possible time. The evaluation of the ovarian reserve, the definition of an ideal stimulation protocol, as well as oocyte retrieval, denudation, and vitrification procedures will be detailed along with approaches to maximize their efficacy, efficiency, and safety. As other protocols or adaptations of this protocol exist in the literature, the representative results and the discussion sections of this manuscript only apply to this procedure.

Protocol

1. Work-up and clinical counseling

NOTE: In case of patients requiring fertility preservation for oncologic reasons, ensure that there is no waiting list for scheduling consultation, and the appointment is provided as soon as possible.

1. Examine the medical history and previous documentation, and assess the patient's general health status.
2. Record all information (including the oncologist's approval to undergo ovarian stimulation in case of cancer patients) in a relational database.
3. Provide the patient with specific counseling about the feasibility of the procedure. Explain the steps of the procedure (ovarian stimulation, oocyte retrieval, oocyte vitrification), and inform her about the realistic chances of success (mainly dependent on maternal age and expected number of MII oocytes at the time of oocyte retrieval), as well as the cost and limitations of the procedure.
4. Perform transvaginal ultrasound to obtain information on the ovarian reserve (i.e., AFC) and to assess the accessibility of the ovaries for egg collection.
5. Request blood tests to assess blood group and Rhesus factor, coagulation screening (blood count, prothrombin, thromboplastin, fibrinogen, Protein C, Protein S, anti-thrombin III, homocysteine), and infectious diseases (Hepatitis B, Hepatitis C, HIV, Venereal Disease Research Laboratory/Treponema pallidum Hemagglutination Assay).
   NOTE: In case of patients accessing a fertility preservation program for non-urgent medical reasons, a more comprehensive assessment may include basal follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol, AMH, breast examination, Papanicolaou test, genetic screening of coagulation (factor V of Leiden and prothrombin), and TORCH screening (toxoplasmosis, rubella, cytomegalovirus).
6. Request a cardiological evaluation (electrocardiogram).
7. Recommend psychological counseling.

2. Controlled ovarian stimulation protocols for fertility preservation

NOTE: When the time available before starting the cancer treatment is limited, the random-start protocol (i.e., starting ovarian stimulation at any time during the menstrual cycle) is recommended for the ovarian stimulation in oncologic patients who are candidates for fertility preservation. In a fertility preservation program for non-urgent medical reasons or social reasons, conventional stimulation starting in the early follicular phase is preferable, and ovarian stimulation is started based on the menstrual cycle. Controlled ovarian stimulation (COS) approaches should be performed according to the recent European Society of Human Reproduction and Embryology (ESHRE) guidelines²⁴.

1. Tailor COS according to the patient's characteristics and ovarian reserve markers, mainly maternal age, FSH, AMH, and AFC.
2. Start COS on day 5 of the menstrual cycle using recombinant or urinary FSH with a fixed dose of 150-300 IU/day (antagonist protocol).
   NOTE: In a specific patient population with LH deficiency or poor-suboptimal response, additional LH 75/150 IU/day may be administered according to the ovarian response, LH levels, and the gynecologist's judgement.
3. In patients with estrogen-sensitive diseases, include gonadotropins associated with aromatase inhibitors (letrozole) from day 1 of stimulation until day 7 after oocyte retrieval.
4. Administer a fixed dose of gonadotropins for 4 days.
5. Monitor follicular growth on day 5 and then every 2-3 days; eventually, adjust the gonadotropin dosage.
6. Once at least 3 follicles reach 17-18 mm in diameter, administer the trigger for final oocyte maturation with a single subcutaneous bolus of GnRH agonist at the dose of 0.5 mL.

3. Oocyte retrieval

1. Preparing for oocyte retrieval
   1. For materials required, refer to the Table of Materials, and keep ready laboratory footwear and outfit, surgical facemask, hair cover, surgical gloves, a permanent non-toxic marker, tweezers, sterile small gauzes, disposable or reusable speculum, vaginal surgery equipment and surface disinfectant. Ensure the availability of resuscitation equipment, anesthetic drugs for reversal, a kit prepared for the treatment of anaphylactic shock, and oxygen in the operating room.
   2. Perform the oocyte retrieval procedure according to the recommendations of the ESHRE Working Group on Ultrasound in assisted reproduction technologies (ART)²⁵.
      1. Administer sedation or general anesthesia, and antibiotics for prophylaxis.
         ​NOTE: In this protocol, deep sedation was achieved by administrating propofol (whose dosage is adjusted according to the patient's weight) and 50-100 µg of fentanyl, 1000 mg of paracetamol, and assisted mask ventilation with oxygen.
      2. Perform oocyte retrieval using an aspiration unit composed of a vacuum pump, a collection tube connected to a 17 G single-lumen needle, and an oocyte-collecting tube. During the collection, do not exceed a pressure of ~120 mmHg to avoid the risk of damage to the oocytes such as stripping off the cumulus cells or fracturing the zona pellucida.
      3. Calibrate working surface temperature to ensure 37 °C in the culture media. During the whole procedure, minimize oocyte exposure to even transient temperature that may affect their developmental competence.
      4. At the end of oocyte retrieval, observe the patient for 3-4 h before discharge.
2. Operation theater
   1. Before entering the operating room, identify the patient and confirm the time of ovulation trigger.
   2. Have the patient lie down on the operating table in a gynecological position.
   3. Cleanse the vagina/cervix prior to oocyte retrieval to minimize bacterial contamination.
   4. Perform a preliminary transvaginal ultrasound to assess the position of the ovaries and the anatomical relationships with the various organs and blood vessels.
   5. Under ultrasound guidance, insert a single-lumen needle through the vaginal wall and into an ovarian follicle, taking care not to injure the organs or blood vessels located between the vaginal wall and the ovary.
   6. Start aspiration from the closest follicle and moving on to the most distal ones.
   7. Puncture all follicles of a diameter larger than 11-12 mm, performing "twisting movements" of the needle to aspirate the whole follicular fluid, which is then released into a sterile tube (round bottom 14 mL) preheated in the block heater of the operating room.
   8. Immediately after retrieval, seal and label the tube with details of the patient's identity.
   9. Ensure that the nurse brings the tube to the laboratory, where it is immediately screened for the presence of cumulus-oocyte complexes (COCs).
   10. Instruct the embryologists to inform the clinician of the total number of COCs retrieved.
   11. Once the procedure is complete for the first ovary, flush the needle with clean medium, and proceed with the second ovary using the same procedure.
   12. After oocyte retrieval, evaluate any bleeding from the ovaries or blood vessels of the parametrium and free fluid in the pouch of Douglas.
       ​NOTE: To automate and improve the effectiveness of gamete and embryo traceability at the clinical level, an electronic witnessing system (EWS) was implemented in the center²⁶. Nevertheless, this protocol does not mention the EWS, to ensure reproducibility of the protocol in any IVF laboratory. Still, consider that all steps of the procedure require a second operator (i.e. a witness) to ensure gamete and embryo traceability.

4. IVF laboratory

1. The day before the oocyte retrieval procedure
   1. Prepare oocyte collecting tubes (refer to the Table of Materials).
      1. Dispense 1 mL of IVF medium (refer to the Table of Materials) in each oocyte collection tube (round bottom, 5 mL), and cover with 0.2 mL of mineral oil (refer to the Table of Materials) for embryo culture.
         ​NOTE: The number of tubes will be defined according to the number of follicles expected to be retrieved. Each tube might contain up to 4 COCs.
   2. Seal the tubes with the cap (first snap). Label the tubes with details of the type of medium and date of preparation. Incubate the tubes overnight at 37 °C in a controlled atmosphere (6% CO₂, 5% 0₂).
2. On the day of the oocyte retrieval
   1. Prewarm the plastic supplies at 37 °C (sterile culture dishes and Pasteur pipettes).
   2. Ask the patients to confirm their identity (full name and date of birth) and the time of ovulation trigger. Annotate on the laboratory sheet that the identification (ID) procedure has been accomplished, and that the time of ovulation trigger has been confirmed.
   3. Take the oocyte collection tubes off the incubator (right before the procedure begins), and push down the cap to ensure tight closure. Label the oocyte collection tubes with the patient's information. Place the oocyte collection tubes in the block heater at 37 °C.
   4. Examine the follicular fluid in the prewarmed sterile culture dishes, and identify COCs. Once one or more COCs are identified, rinse them twice in two drops of medium to remove the follicular fluid and blood contamination.
   5. Transfer the COCs to the oocyte collection tubes and annotate the number of COCs on the tube. Loosen the caps of the medium tubes, and promptly incubate them at 37 °C in an a atmosphere of 6% CO₂, 5% O₂.
   6. Repeat steps 7 to 9 according to the number of oocytes retrieved. Update the laboratory sheet.
      ​NOTE: Ensure that a witness checks that all the tube-warming blocks (including the ones in the operating theater) are empty and signs the closing of the procedure on the laboratory sheet.
   7. Wipe down the working stage after completion of the procedure.

5. Oocyte denudation

1. Prewarm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered medium and hyaluronidase (refer to the Table of Materials) at 37 °C at least 1 h before starting.
2. Prepare a 4-well IVF plate (refer to the Table of Materials) with 0.6 mL/well of prewarmed HEPES-buffered medium (supplemented with 5% human serum albumin [HSA]) covered with 0.3 mL of mineral oil for embryo culture, and warm at 37 °C for at least 30 min.
3. Label the oocyte denudation plate with details of the patient's identity.
4. Immediately before starting the procedure, add hyaluronidase to the first well to obtain a final concentration of about 20 IU/mL.
5. Place a limited number of COCs (up to 6) in the first well containing the enzyme to disperse the cumulus cells.
6. To enhance enzymatic removal of the cumulus and corona cells, perform the stripping of cumulus cells by pipetting the oocytes repeatedly through a Pasteur pipette with an inner diameter of approximately 250 μm for up to 30 s. After initial cell dissociation is observed, transfer the oocytes to the second well containing only HEPES-buffered medium, taking care to carry over a minimum amount of the enzyme.
7. Perform further denudation to remove the corona cells by using denuding pipettes with decreasing inner diameters (170-145 µm). Use lower diameters (135µm) only if strictly necessary.
8. Carefully wash the denuded oocytes to wash out the enzyme.
9. After denudation, examine the oocytes under an inverted microscope to assess their integrity and stage of maturation. Separate MII oocytes from the immature ones (germinal vesicle and metaphase-I).
10. Move the MII oocytes to the vitrification area to immediately perform cryopreservation. Update the laboratory sheet.

6. Oocyte vitrification

NOTE: Perform oocyte vitrification preferably within 38 h of oocyte retrieval and immediately after denudation. The vitrification procedure described here has to be accomplished at room temperature (RT) and by using a stripper pipette with an inner diameter of 170 µm so as not to damage oocytes during manipulation.

1. Approximately 30 min before the procedure, bring the equilibration solution (ES) and vitrification solution (VS) to RT (25-27 °C).
2. Properly label the cryodevices with the patient's name and ID, treatment ID, date of freezing, number of oocytes, and cryobarcode.
3. Fill a disposable cooling rack up to the top with fresh liquid nitrogen, and start the sterilization process.
4. Label the vitrification plate (refer to the Table of Materials) with the patient's name and ID. Ask a witness to check the correct ID of the cryodevices.
5. Carefully invert each vial twice to mix its contents before use, and prepare the lid of a 6 mm Petri dish with one drop of 30 µL of HEPES-buffered medium (with 5% HSA) and one adjacent drop of 30 µL of ES.
   NOTE: Place drops just before use to limit medium evaporation.
6. Using a 170 µm diameter stripper pipette, place the oocytes (up to 9) in the first drop with the smallest possible volume of medium. Using the stripper pipette, create a bridge of medium between the drop n.1 and n.2 to obtain a gradual increase in concentration of the CPAs (Figure 1A).
7. Incubate the oocytes in the first drop for 3 minutes. Add a third drop of 30 µL of ES (n.3). After 3 min, transfer the oocytes into the second drop of ES, and create a medium bridge between drops n.3 and n.2 (Figure 1B). Incubate the oocytes in drop n.3 for 3 min.
8. During the incubation, add one 30 µL drop of ES for each cryodevice that will be used (if 9 oocytes are to be cryopreserved, place 3 drops of ES with 3 oocytes in each drop of ES). Move the oocytes into pure ES solution, and leave them for 6-9 min (until they recover their initial size after shrinkage) (Figure 1C).
9. Prepare a central well dish (60 x 15 mm) with 1 mL of VS. At the end of the first 6 min, transfer the oocytes to be cryopreserved into the VS solution, releasing as little medium as possible. Leave the oocytes in VS for 1 min, and wash them carefully by moving them from the bottom to the top of the dish to remove the excess of ES.
10. Approximately 10 s before the end of the minute of incubation, place the cryodevice under the microscope, and adjust the focus on the black mark (i.e., the tip of the cryodevice). Place the oocytes on the cryodevice beside the black mark with the minimum amount of VS (Figure 2A).
11. Move the stripper pipette away from the oocytes, and remove the excess of VS medium (Figure 2B) so that the oocytes remain covered by a thin layer of medium (Figure 2C).
12. Quickly plunge the cryodevice in liquid nitrogen, rapidly shaking it to remove air bubbles from its surface. Hold the protective cap of the cryodevice with tweezers, and fill it with liquid nitrogen; then insert the cryodevice into it while keeping the propylene strips in liquid nitrogen.
13. Store the cryodevice in a visiotube previously labeled with the patient's information. Update the laboratory sheet.

7. Oocyte warming

1. Approximately 30 min before the procedure, warm the thawing solution (TS), dilution solution (DS), and washing solution (WS) to RT (25-27 °C). Carefully invert each vial twice to mix its contents. Place 1 mL of TS in a central well Petri dish, and warm it at 37 °C for at least 1 h before starting the procedure.
2. Label all plastic supplies with the patient's name and ID and the type of solution. Ask a witness to confirm the patient's information on the cryodevice.
3. For each cryodevice to be warmed, prepare a 6-well plate with 200 µL of DS in the first well and an equal amount of WS in the second and third ones (named WS1 and WS2, respectively). Add phosphate buffered saline (PBS) or sterile water in the area outside the wells to prevent evaporation.
4. Take the TS dish out from the incubator, and place it under the microscope. Adjust the focus of the microscope to the center of the Petri dish.
5. Carefully twist and remove the protective cap of the cryodevice, while keeping the propylene strips in liquid nitrogen. Transfer the cryodevice from liquid nitrogen into the TS as quickly as possible to avoid the risk of devitrification and initiate the countdown (1 min).
6. Localize the oocytes by focusing on the tip of the cryodevice (i.e., the black mark). Using a stripper pipette, release the oocytes from the cryodevice.
   NOTE: Try not to aspirate the oocytes from the cryodevice; gently release some media on them until they move into the TS.
7. Using a 170 µm diameter stripper pipette, transfer the oocytes to DS with a little amount of TS (to create a gradient), and leave them in the DS for 3 min. Move the oocytes to WS1 likewise, and leave them for 5 min. Finally, transfer the oocytes to WS2 for 1 min.
8. Transfer the oocytes into an appropriate preequilibrated IVF culture medium, and incubate them for 1 h before proceeding with intracytoplasmic sperm injection (ICSI). Update the laboratory sheet.

Results

Overview of the fertility preservation program at the center

Over a 12-year period (2008-2020), 285 women underwent at least one oocyte retrieval entailing the vitrification of the whole cohort of mature eggs collected. Most of these women (n=250) underwent a single retrieval, and 35 underwent multiple retrievals. The reasons for undergoing oocyte retrieval for egg vitrification are summarized into 4 categories: medical (except for cancer), cancer, non-medical, and others. Amo...

Discussion

Clinical considerations

Although emerging strategies, such as ovarian tissue cryopreservation and in vitro maturation, have been explored, oocyte vitrification after COS is the gold standard technique for fertility preservation. In this scenario, the number of oocytes retrieved and cryopreserved should be maximized in the shortest possible time as most cancer patients might benefit solely from one ovarian cycle before they have to commence their cancer treatment(s). Thus, a p...

Materials

Name	Company	Catalog Number	Comments
Collection
Equipment
Hot plate	IVF TECH
Lab Markers	Sigma Aldrich
Laminar Flow Hood	IVF TECH		Grade A air flow
Stereomicroscope	Leica	Leica M80
Thermometer
Test tube Warmer
Tri-gas incubator	Panasonic	MCO-5M-PE	02/CO2
Vacuum Pump	Cook	K-MAR-5200
Consumables
CSCM (Continuos single culture complete) medium	Fujifilm Irvine Scientific	90165	IVF culture medium supplemented with HSA
Mineral Oil for embryo culture	Fujifilm Irvine Scientific	9305
Ovum Aspiration Needle (Single lumen)	Cook	K-OSN-1730-B-60
Primaria Dish	Corning	353803	Corning Primaria Dish 100x20 mm style standard cell culture dish
Round- bottom tubes	Falcon	352001	Falcon 14ml Round Bottom Polystyrene Test tube with snap cap
Round- bottom tubes	Falcon	352003	Oocyte collection tubes/ Falcon 5ml 12x75 Round Bottom Polipropilene Test tube with snap cap
Rubber Bulb	Sigma Aldrich	Z111589-12EA
Sterile glass Pasteur pipettes	Hunter Scientific	PPB150-100PL	Pipette Pasteur Cotonate, 150mm, MEA e CE
Denudation
Equipment
CO2 incubator	Eppendorf	Galaxy 14S
Flexipet adjustable handle set	Cook	G18674	Stripper  holder
Gilson Pipetman	Gilson	66003	p20
k-System Incubator	Coopersurgical	G210Invicell
Lab Markers	Sigma Aldrich
Laminar Flow Hood	IVF TECH		Grade A air flow
Stereomicroscope	Leica	Leica M80
Consumables
Biopur epTIPS Rack	Eppendorf	30075331	Micropipettes epTIPS Biopur 2-200 µl
Human Serum Albumin	thermoFisher Scietific	9988
Hyaluronidase	Fujifilm Irvine Scientific	90101	80 IU/mL of hyaluronidase enzyme in HEPES-buffered HTF
IVF culture dish (60 x 15mm)	Corning	353802	Corning Primaria Falcon Dish 60X15mm TC Primaria standard cell culture dish
IVF dish 4-well plate with sliding lid	ThermoFisher Scietific	176740	Multidishes 4 wells (Nunc)
IVF One well dish	Falcon	353653	Falcon 60 x 15 mm TC treated center-well IVF
Mineral Oil for embryo culture	Fujifilm Irvine Scientific	9305
Modified HTF Medium	Fujifilm Irvine Scientific	90126	HEPES-Buffered medium
Rubber Bulb	Sigma Aldrich	Z111589-12EA	1 mL for pasteur pipettes
Sterile glass Pasteur pipettes	Hunter Scientific	PPB150-100PL	Pipette Pasteur Cotonate, 150 mm, MEA e CE
stripping pipette  tips (140 µm)	Cook	K-FPIP-1140-10BS-6	PIPETTE FLEXIPETS PER DENUDING
stripping pipette tips (130 µm )	Cook	K-FPIP-1130-10BS-7	PIPETTE FLEXIPETS PER DENUDING
stripping pipette tips (170 µm)	Cook	K-FPIP-1170-10BS-5	PIPETTE FLEXIPETS PER DENUDING
Vitrification
Equipment
Electronic Timer
Flexipet adjustable handle set	Cook	G18674	Stripper  holder
Gilson Pipetman	Gilson	F123601	p200
Lab Markers	Sigma Aldrich
Laminar Flow Hood	IVF TECH		Grade A air flow
Stainless Container for Cooling Rack	Kitazato		Liquid nitrogen container for vitrification
Stereomicroscope	Leica	Leica M80
Consumables
Biopur epTIPS Rack	Eppendorf	30075331	Micropipettes epTIPS Biopur 2-200 µL
Human Serum Albumin	Fujifilm Irvine Scientific	9988
IVF culture dish (60 x 15 mm)	Corning	353802	Corning Primaria Falcon Dish 60 x 15 mm TC Primaria standard cell culture dish
IVF dish 6-well	Oosafe	OOPW-SW02	OOSAFE 6 WELL DISH WITH STRAW HOLDER
Modified HTF Medium	Fujifilm Irvine Scientific	90126	HEPES-Buffered medium
stripping pipette tips (170 µm)	Cook	K-FPIP-1170-10BS-5	PIPETTE FLEXIPETS PER DENUDING
Vitrification Freeze kit	Fujifilm Irvine Scientific	90133-so	2 Vials of ES (Equilibration Solution, 2 x 1 mL) and 2 Vials of VS (Vitrification Solution, 2 x 1 mL)
Vitrifit	Coopersurgical Origio	42782001A	VitriFit  Box
Warming
Equipment
Electronic Timer
Flexipet adjustable handle set	Cook	G18674	Stripper  holder
Gilson Pipetman	Gilson	F123601	p200
k-System Incubator	Coopersurgical	G210Invicell
Lab Markers	Sigma Aldrich
Laminar Flow Hood	IVF TECH		Grade A air flow
Stainless Container for Cooling Rack	Kitazato		Liquid nitrogen container for vitrification
Stereomicroscope	Leica	Leica M80
Consumables
Biopur epTIPS Rack	Eppendorf	30075331	Micropipettes epTIPS Biopur 2-200 µL
CSCM (Continuos single culture complete) medium	Fujifilm Irvine Scientific	90165	IVF culture medium supplemented with HSA
IVF culture dish (60 x 15 mm)	Corning	353802	Corning Primaria Falcon Dish 60X15mm TC Primaria standard cell culture dish
IVF dish 4-well plate with sliding lid	ThermoFisher Scietific	176740	Multidishes 4 wells (Nunc)
IVF dish 6-well	Oosafe	OOPW-SW02	OOSAFE® 6 WELL DISH WITH STRAW HOLDER
Mineral Oil for embryo culture	Fujifilm Irvine Scientific	9305
SAtripping pipette tips (300µm)	Cook	K-FPIP-1300-10BS-5	PIPETTE FLEXIPETS PER DENUDING
Vitrification Thaw kit	Fujifilm Irvine Scientific	90137-so	4 Vials of TS (Thawing Solution, 4 x 2 mL) + 1 Vial of DS (Dilution Solution, 1 x 2 mL) +1 Vial of WS (Washing Solution, 1 x 2 mL)