Method Article

Fertility Preservation Through Oocyte Vitrification: Clinical and Laboratory Perspectives

DOI:

10.3791/61963

⸱

September 16th, 2021

 ,  ,  ,  ,  ,  ,  , 
1Clinica Valle Giulia, GeneraLife IVF

In This Article

Summary

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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 indications4,5,6. 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 procedure9,10,11,12,13. 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 cases20,21. In the laboratory setting, the most widespread protocol for oocyte vitrification is still the one first described by Kuwayama and colleagues2,23, 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.

Access restricted. Please log in or start a trial to view this content.

Protocol

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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.

Access restricted. Please log in or start a trial to view this content.

Results

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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...

Access restricted. Please log in or start a trial to view this content.

Discussion

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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...

Access restricted. Please log in or start a trial to view this content.

Disclosures

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

The authors have nothing to disclose.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Collection
Equipment
Hot plateIVF TECH
Lab MarkersSigma Aldrich
Laminar Flow HoodIVF TECHGrade A air flow
StereomicroscopeLeicaLeica M80
Thermometer
Test tube Warmer
Tri-gas incubatorPanasonicMCO-5M-PE02/CO2
Vacuum PumpCookK-MAR-5200
Consumables
CSCM (Continuos single culture complete) mediumFujifilm Irvine Scientific90165IVF culture medium supplemented with HSA
Mineral Oil for embryo cultureFujifilm Irvine Scientific9305
Ovum Aspiration Needle (Single lumen)CookK-OSN-1730-B-60
Primaria DishCorning353803Corning Primaria Dish 100x20 mm style standard cell culture dish
Round- bottom tubesFalcon352001Falcon 14ml Round Bottom Polystyrene Test tube with snap cap
Round- bottom tubesFalcon352003Oocyte collection tubes/ Falcon 5ml 12x75 Round Bottom Polipropilene Test tube with snap cap
Rubber BulbSigma AldrichZ111589-12EA
Sterile glass Pasteur pipettesHunter ScientificPPB150-100PLPipette Pasteur Cotonate, 150mm, MEA e CE
Denudation
Equipment
CO2 incubatorEppendorfGalaxy 14S
Flexipet adjustable handle setCookG18674Stripper  holder
Gilson PipetmanGilson66003p20
k-System IncubatorCoopersurgicalG210Invicell
Lab MarkersSigma Aldrich
Laminar Flow HoodIVF TECHGrade A air flow
StereomicroscopeLeicaLeica M80
Consumables
Biopur epTIPS RackEppendorf30075331Micropipettes epTIPS Biopur 2-200 µl
Human Serum AlbuminthermoFisher Scietific9988
HyaluronidaseFujifilm Irvine Scientific9010180 IU/mL of hyaluronidase enzyme in HEPES-buffered HTF
IVF culture dish (60 x 15mm)Corning353802Corning Primaria Falcon Dish 60X15mm TC Primaria standard cell culture dish
IVF dish 4-well plate with sliding lidThermoFisher Scietific176740Multidishes 4 wells (Nunc)
IVF One well dishFalcon353653Falcon 60 x 15 mm TC treated center-well IVF
Mineral Oil for embryo cultureFujifilm Irvine Scientific9305
Modified HTF MediumFujifilm Irvine Scientific90126HEPES-Buffered medium
Rubber BulbSigma AldrichZ111589-12EA1 mL for pasteur pipettes
Sterile glass Pasteur pipettesHunter ScientificPPB150-100PLPipette Pasteur Cotonate, 150 mm, MEA e CE
stripping pipette  tips (140 µm)CookK-FPIP-1140-10BS-6PIPETTE FLEXIPETS PER DENUDING
stripping pipette tips (130 µm )CookK-FPIP-1130-10BS-7PIPETTE FLEXIPETS PER DENUDING
stripping pipette tips (170 µm)CookK-FPIP-1170-10BS-5PIPETTE FLEXIPETS PER DENUDING
Vitrification
Equipment
Electronic Timer
Flexipet adjustable handle setCookG18674Stripper  holder
Gilson PipetmanGilsonF123601p200
Lab MarkersSigma Aldrich
Laminar Flow HoodIVF TECHGrade A air flow
Stainless Container for Cooling RackKitazatoLiquid nitrogen container for vitrification
StereomicroscopeLeicaLeica M80
Consumables
Biopur epTIPS RackEppendorf30075331Micropipettes epTIPS Biopur 2-200 µL
Human Serum AlbuminFujifilm Irvine Scientific9988
IVF culture dish (60 x 15 mm)Corning353802Corning Primaria Falcon Dish 60 x 15 mm TC Primaria standard cell culture dish
IVF dish 6-wellOosafeOOPW-SW02OOSAFE 6 WELL DISH WITH STRAW HOLDER
Modified HTF MediumFujifilm Irvine Scientific90126HEPES-Buffered medium
stripping pipette tips (170 µm)CookK-FPIP-1170-10BS-5PIPETTE FLEXIPETS PER DENUDING
Vitrification Freeze kitFujifilm Irvine Scientific90133-so2 Vials of ES (Equilibration Solution, 2 x 1 mL) and 2 Vials of VS (Vitrification Solution, 2 x 1 mL)
VitrifitCoopersurgical Origio42782001AVitriFit  Box
Warming
Equipment
Electronic Timer
Flexipet adjustable handle setCookG18674Stripper  holder
Gilson PipetmanGilsonF123601p200
k-System IncubatorCoopersurgicalG210Invicell
Lab MarkersSigma Aldrich
Laminar Flow HoodIVF TECHGrade A air flow
Stainless Container for Cooling RackKitazatoLiquid nitrogen container for vitrification
StereomicroscopeLeicaLeica M80
Consumables
Biopur epTIPS RackEppendorf30075331Micropipettes epTIPS Biopur 2-200 µL
CSCM (Continuos single culture complete) mediumFujifilm Irvine Scientific90165IVF culture medium supplemented with HSA
IVF culture dish (60 x 15 mm)Corning353802Corning Primaria Falcon Dish 60X15mm TC Primaria standard cell culture dish
IVF dish 4-well plate with sliding lidThermoFisher Scietific176740Multidishes 4 wells (Nunc)
IVF dish 6-wellOosafeOOPW-SW02OOSAFE® 6 WELL DISH WITH STRAW HOLDER
Mineral Oil for embryo cultureFujifilm Irvine Scientific9305
SAtripping pipette tips (300µm)CookK-FPIP-1300-10BS-5PIPETTE FLEXIPETS PER DENUDING
Vitrification Thaw kitFujifilm Irvine Scientific90137-so4 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)

References

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,
  1. Cobo, A., Diaz, C. Clinical application of oocyte vitrification: a systematic review and meta-analysis of randomized controlled trials. Fertility and Sterility. 96 (2), 277-285 (2011).
  2. Rienzi, L., et al. Oocyte, embryo and blastocyst cryopreservation in ART: systematic review and meta-analysis comparing slow-freezing versus vitrification to produce evidence for the development of global guidance. Human Reproduction Update. 23 (2), 139-155 (2017).
  3. Nagy, Z. P., Anderson, R. E., Feinberg, E. C., Hayward, B., Mahony, M. C. The Human Oocyte Preservation Experience (HOPE) Registry: evaluation of cryopreservation techniques and oocyte source on outcomes. Reproductive Biology and Endocrinology. 15 (1), (2017).
  4. Practice Committees of American Society for Reproductive, M., & Society for Assisted Reproductive, T. Mature oocyte cryopreservation: a guideline. Fertility and Sterility. 99 (1), 37-43 (2013).
  5. Lee, S. J., et al. American Society of Clinical Oncology recommendations on fertility preservation in cancer patients. Journal of Clinical Oncology. 24 (18), 2917-2931 (2006).
  6. Nakayama, K., Ueno, N. T. American Society of Clinical Oncology recommendations on fertility preservation should be implemented regardless of disease status or previous treatments. Journal of Clinical Oncology. 24 (33), 5334-5335 (2006).
  7. Martinez, F. Update on fertility preservation from the Barcelona International Society for Fertility Preservation-ESHRE-ASRM 2015 expert meeting: indications, results and future perspectives. Human Reproduction. 32 (9), 1802-1811 (2017).
  8. Lantsberg, D., Fernando, S., Cohen, Y., Rombauts, L. The role of fertility preservation in women with endometriosis: a systematic review. Journal of Minimally Invasive Gynecology. 27 (2), 362-372 (2020).
  9. Anderson, R. A., et al. Cancer treatment and gonadal function: experimental and established strategies for fertility preservation in children and young adults. Lancet Diabetes & Endocrinology. 3 (7), 556-567 (2015).
  10. Lambertini, M., et al. Cancer and fertility preservation: international recommendations from an expert meeting. BMC Medicine. 14, 1(2016).
  11. Kim, S. J., Kim, S. K., Lee, J. R., Suh, C. S., Kim, S. H. Oocyte cryopreservation for fertility preservation in women with ovarian endometriosis. Reproductive Biomedicine Online. 40 (6), 827-834 (2020).
  12. Loren, A. W., et al. Fertility preservation for patients with cancer: American Society of Clinical Oncology clinical practice guideline update. Journal of Clinical Oncology. 31 (19), 2500-2510 (2013).
  13. Peccatori, F. A., et al. Cancer, pregnancy and fertility: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Annals of Oncology. 24, Suppl 6 160-170 (2013).
  14. Dondorp, W. J., De Wert, G. M. Fertility preservation for healthy women: ethical aspects. Human Reproduction. 24 (8), 1779-1785 (2009).
  15. Cil, A. P., Bang, H., Oktay, K. Age-specific probability of live birth with oocyte cryopreservation: an individual patient data meta-analysis. Fertilility and Steriityl. 100 (2), 492-499 (2013).
  16. Ubaldi, F. M., et al. Advanced maternal age in IVF: still a challenge? The present and the future of its treatment. Frontiers in Endocrinology. 10, 94(2019).
  17. Cimadomo, D., et al. Impact of maternal age on oocyte and embryo competence. Frontiers in Endocrinology. 9, 327(2018).
  18. Cobo, A., et al. Oocyte vitrification as an efficient option for elective fertility preservation. Fertility and Sterility. 105 (3), 755-764 (2016).
  19. Devroey, P., Polyzos, N. P., Blockeel, C. An OHSS-Free Clinic by segmentation of IVF treatment. Human Reproduction. 26 (10), 2593-2597 (2011).
  20. Sonigo, C., et al. Impact of letrozole supplementation during ovarian stimulation for fertility preservation in breast cancer patients. European Journal of Obstetrics & Gynecology and Reproductive Biology X. 4, 100049(2019).
  21. Marklund, A., et al. Efficacy and safety of controlled ovarian stimulation using GnRH antagonist protocols for emergency fertility preservation in young women with breast cancer-a prospective nationwide Swedish multicenter study. Human Reproduction. 35 (4), 929-938 (2020).
  22. Kuwayama, M. Highly efficient vitrification for cryopreservation of human oocytes and embryos: the Cryotop method. Theriogenology. 67 (1), 73-80 (2007).
  23. Kuwayama, M., Vajta, G., Kato, O., Leibo, S. P. Highly efficient vitrification method for cryopreservation of human oocytes. Reproductive Biomedicine Online. 11 (3), 300-308 (2005).
  24. Bosch, E., et al. ESHRE guideline: ovarian stimulation for IVF/ICSI(dagger). Human Reproduction Open. 2020 (2), (2020).
  25. ESHRE Working Group on Ultrasound in ART. Recommendations for good practice in ultrasound: oocyte pick up(dagger). Human Reproduction Open. 2019 (4), 025(2019).
  26. Rienzi, L., et al. Failure mode and effects analysis of witnessing protocols for ensuring traceability during IVF. Reproductive Biomedicine Online. 31 (4), 516-522 (2015).
  27. Mazzilli, R., et al. Effect of the male factor on the clinical outcome of intracytoplasmic sperm injection combined with preimplantation aneuploidy testing: observational longitudinal cohort study of 1,219 consecutive cycles. Fertility and Sterility. 108 (6), 961-972 (2017).
  28. Polyzos, N. P., et al. Cumulative live birth rates according to the number of oocytes retrieved after the first ovarian stimulation for in vitro fertilization/intracytoplasmic sperm injection: a multicenter multinational analysis including approximately 15,000 women. Fertility and Steriityl. 110 (4), 661-670 (2018).
  29. Alteri, A., Pisaturo, V., Nogueira, D., D'Angelo, A. Elective egg freezing without medical indications. Acta Obstetricia et Gynecologica Scandinavica. 98 (5), 647-652 (2019).
  30. Mesen, T. B., Mersereau, J. E., Kane, J. B., Steiner, A. Z. Optimal timing for elective egg freezing. Fertility and Steriityl. 103 (6), 1551-1556 (2015).
  31. Doyle, J. O., et al. Successful elective and medically indicated oocyte vitrification and warming for autologous in vitro fertilization, with predicted birth probabilities for fertility preservation according to number of cryopreserved oocytes and age at retrieval. Fertility and Sterility. 105 (2), 459-466 (2016).
  32. Rienzi, L., et al. Significance of metaphase II human oocyte morphology on ICSI outcome. Fertility and Sterility. 90 (5), 1692-1700 (2008).
  33. Ubaldi, F. M., et al. Reduction of multiple pregnancies in the advanced maternal age population after implementation of an elective single embryo transfer policy coupled with enhanced embryo selection: pre- and post-intervention study. Human Reproduction. 30 (9), 2097-2106 (2015).
  34. Cakmak, H., Katz, A., Cedars, M. I., Rosen, M. P. Effective method for emergency fertility preservation: random-start controlled ovarian stimulation. Fertility and Sterility. 100 (6), 1673-1680 (2013).
  35. Moravek, M. B., et al. Long-term outcomes in cancer patients who did or did not pursue fertility preservation. Fertility and Sterility. 109 (2), 349-355 (2018).
  36. Vaiarelli, A., Venturella, R., Vizziello, D., Bulletti, F., Ubaldi, F. M. Dual ovarian stimulation and random start in assisted reproductive technologies: from ovarian biology to clinical application. Current Opinion in Obstetrics and Gynecology. 29 (3), 153-159 (2017).
  37. Vaiarelli, A., Cimadomo, D., Ubaldi, N., Rienzi, L., Ubaldi, F. M. What is new in the management of poor ovarian response in IVF. Current Opinion in Obstetrics and Gynecology. 30 (3), 155-162 (2018).
  38. Venturella, R., et al. State of the art and emerging drug therapies for female infertility. Gynecological Endocrinology. 35 (10), 835-841 (2019).
  39. Venturella, R., et al. A modern approach to the management of candidates for assisted reproductive technology procedures. Minerva Ginecologica. 70 (1), 69-83 (2018).
  40. Ferreiro, E., de Uralde, B. L., Abreu, R., Garcia-Velasco, J. A., Munoz, E. Aromatase inhibitors for ovarian stimulation in patients with breast cancer. Current Drug Targets. 21 (9), 910-921 (2020).
  41. Oktay, K., et al. Letrozole reduces estrogen and gonadotropin exposure in women with breast cancer undergoing ovarian stimulation before chemotherapy. Journal of Clinical Endocrinology and Metabolism. 91 (10), 3885-3890 (2006).
  42. Meirow, D., et al. Tamoxifen co-administration during controlled ovarian hyperstimulation for in vitro fertilization in breast cancer patients increases the safety of fertility-preservation treatment strategies. Fertility and Steriity. 102 (2), 488-495 (2014).
  43. Friedler, S., Koc, O., Gidoni, Y., Raziel, A., Ron-El, R. Ovarian response to stimulation for fertility preservation in women with malignant disease: a systematic review and meta-analysis. Fertility and Steriity. 97 (1), 125-133 (2012).
  44. Tsampras, N., Gould, D., Fitzgerald, C. T. Double ovarian stimulation (DuoStim) protocol for fertility preservation in female oncology patients. Human Fertility. 20 (4), 248-253 (2017).
  45. Vaiarelli, A., et al. Double stimulation in the same ovarian cycle (DuoStim) to maximize the number of oocytes retrieved from poor prognosis patients: a multicenter experience and SWOT analysis. Frontiers in Endocrinology. 9, 317(2018).
  46. Cimadomo, D., et al. Luteal phase anovulatory follicles result in the production of competent oocytes: intra-patient paired case-control study comparing follicular versus luteal phase stimulations in the same ovarian cycle. Human Reproduction. 33 (8), 1442-1448 (2018).
  47. Rienzi, L., Ubaldi, F. M. Oocyte versus embryo cryopreservation for fertility preservation in cancer patients: guaranteeing a women's autonomy. Journal of Assisted Reproduction and Genetics. 32 (8), 1195-1196 (2015).
  48. Oktay, K., Harvey, B. E., Loren, A. W. Fertility preservation in patients with cancer: ASCO clinical practice guideline update summary. JCO Oncology Practice. 14 (6), 381-385 (2018).
  49. Iussig, B., et al. A brief history of oocyte cryopreservation: Arguments and facts. Acta Obstetricia et Gynecologica Scandinavica. 98 (5), 550-558 (2019).
  50. Best, B. P. Cryoprotectant toxicity: facts, issues, and questions. Rejuvenation Research. 18 (5), 422-436 (2015).
  51. Fuller, B., Paynter, S. Fundamentals of cryobiology in reproductive medicine. Reproductive Biomedicine Online. 9 (6), 680-691 (2004).
  52. Konc, J., Kanyo, K., Kriston, R., Somoskoi, B., Cseh, S. Cryopreservation of embryos and oocytes in human assisted reproduction. BioMed Research International. 2014, 307268(2014).
  53. Erstad, B. L. Osmolality and osmolarity: narrowing the terminology gap. Pharmacotherapy. 23 (9), 1085-1086 (2003).
  54. Sunde, A., et al. Time to take human embryo culture seriously. Human Reproduction. 31 (10), 2174-2182 (2016).
  55. Swain, J. E., Cabrera, L., Xu, X., Smith, G. D. Microdrop preparation factors influence culture-media osmolality, which can impair mouse embryo preimplantation development. Reproductive Biomedicine Online. 24 (2), 142-147 (2012).
  56. Wale, P. L., Gardner, D. K. The effects of chemical and physical factors on mammalian embryo culture and their importance for the practice of assisted human reproduction. Human Reproduction Update. 22 (1), 2-22 (2016).
  57. Seki, S., Mazur, P. The dominance of warming rate over cooling rate in the survival of mouse oocytes subjected to a vitrification procedure. Cryobiology. 59 (1), 75-82 (2009).
  58. Karlsson, J. O. A theoretical model of intracellular devitrification. Cryobiology. 42 (3), 154-169 (2001).
  59. Jin, B., et al. Equilibrium vitrification of mouse embryos. Biololgy of Reproduction. 82 (2), 444-450 (2010).
  60. Mazur, P. Equilibrium, quasi-equilibrium, and nonequilibrium freezing of mammalian embryos. Cell Biophysics. 17 (1), 53-92 (1990).
  61. Parmegiani, L., et al. "Universal Warming" protocol for vitrified oocytes to streamline cell exchange for transnational donation programs: a multi-center study. Journal of Assisted Reproduction and Genetics. 37 (6), 1379-1385 (2020).
  62. Vajta, G., Rienzi, L., Ubaldi, F. M. Open versus closed systems for vitrification of human oocytes and embryos. Reproductive Biomedicine Online. 30 (4), 325-333 (2015).
  63. Cai, H., et al. Open versus closed vitrification system of human oocytes and embryos: a systematic review and meta-analysis of embryologic and clinical outcomes. Reproductive Biology & Endocrinology. 16 (1), 123(2018).
  64. Parmegiani, L., et al. Sterilization of liquid nitrogen with ultraviolet irradiation for safe vitrification of human oocytes or embryos. Fertility and Sterility. 94 (4), 1525-1528 (2010).
  65. Parmegiani, L., et al. Efficiency of aseptic open vitrification and hermetical cryostorage of human oocytes. Reproductive Biomedicine Online. 23 (4), 505-512 (2011).
  66. Cobo, A., et al. Storage of human oocytes in the vapor phase of nitrogen. Fertility and Sterility. 94 (5), 1903-1907 (2010).
  67. Eum, J. H., et al. Long-term liquid nitrogen vapor storage of mouse embryos cryopreserved using vitrification or slow cooling. Fertility and Sterility. 91 (5), 1928-1932 (2009).
  68. Fabozzi, G., et al. Which key performance indicators are most effective in evaluating and managing an in vitro fertilization laboratory. Fertility and Sterility. 114 (1), 9-15 (2020).
  69. Dessolle, L., et al. Learning curve of vitrification assessed by cumulative summation test for learning curve (LC-CUSUM). Fertility and Sterility. 92 (3), 943-945 (2009).
  70. Alpha Scientists In Reproductive Medicine. The Alpha consensus meeting on cryopreservation key performance indicators and benchmarks: proceedings of an expert meeting. Reproductive Biomedicine Online. 25 (2), 146-167 (2012).
  71. Edgar, D. H., Gook, D. A. A critical appraisal of cryopreservation (slow cooling versus vitrification) of human oocytes and embryos. Human Reproduction Update. 18 (5), 536-554 (2012).
  72. Edgar, D. H., Archer, J., Bourne, H. The application and impact of cryopreservation of early cleavage stage embryos in assisted reproduction. Human Fertility. 8 (4), 225-230 (2005).

Access restricted. Please log in or start a trial to view this content.

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Tags

Oocyte WarmingCryoprotectant ExposureOvarian Reserve EvaluationStimulation ProtocolOocyte RetrievalDenudation ProcedureVitrification SolutionThawing Solution

Related Articles