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.
<ol>
	<li>Examine the medical history and previous documentation, and assess the patient&#39;s general health status.</li>
	<li>Record all information (including the oncologist&#39;s approval to undergo ovarian stimulation in case of cancer patients) in a relational database.</li>
	<li>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.</li>
	<li>Perform transvaginal ultrasound to obtain information on the ovarian reserve (i.e., AFC) and to assess the accessibility of the ovaries for egg collection.</li>
	<li>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).</li>
	<li>Request a cardiological evaluation (electrocardiogram).</li>
	<li>Recommend psychological counseling.</li>
</ol>
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.

<ol>
	<li>Tailor COS according to the patient&#39;s characteristics and ovarian reserve markers, mainly maternal age, FSH, AMH, and AFC.</li>
	<li>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&#39;s judgement.</li>
	<li>In patients with estrogen-sensitive diseases, include gonadotropins associated with aromatase inhibitors (letrozole) from day 1 of stimulation until day 7 after oocyte retrieval.</li>
	<li>Administer a fixed dose of gonadotropins for 4 days.</li>
	<li>Monitor follicular growth on day 5 and then every 2-3 days; eventually, adjust the gonadotropin dosage.</li>
	<li>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.</li>
</ol>
3. Oocyte retrieval
<ol>
	<li>Preparing for oocyte retrieval
	<ol>
		<li>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.</li>
		<li>Perform the oocyte retrieval procedure according to the recommendations of the ESHRE Working Group on Ultrasound in assisted reproduction technologies (ART)25.
		<ol>
			<li>Administer sedation or general anesthesia, and antibiotics for prophylaxis. 
			&#8203;NOTE: In this protocol, deep sedation was achieved by administrating propofol (whose dosage is adjusted according to the patient&#39;s weight) and 50-100 &#181;g of fentanyl, 1000 mg of paracetamol, and assisted mask ventilation with oxygen.</li>
			<li>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.</li>
			<li>Calibrate working surface temperature to ensure 37 &#176;C in the culture media. During the whole procedure, minimize oocyte exposure to even transient temperature that may affect their developmental competence.</li>
			<li>At the end of oocyte retrieval, observe the patient for 3-4 h before discharge.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Operation theater
	<ol>
		<li>Before entering the operating room, identify the patient and confirm the time of ovulation trigger.</li>
		<li>Have the patient lie down on the operating table in a gynecological position.</li>
		<li>Cleanse the vagina/cervix prior to oocyte retrieval to minimize bacterial contamination.</li>
		<li>Perform a preliminary transvaginal ultrasound to assess the position of the ovaries and the anatomical relationships with the various organs and blood vessels.</li>
		<li>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.</li>
		<li>Start aspiration from the closest follicle and moving on to the most distal ones.</li>
		<li>Puncture all follicles of a diameter larger than 11-12 mm, performing &#34;twisting movements&#34; 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.</li>
		<li>Immediately after retrieval, seal and label the tube with details of the patient&#39;s identity.</li>
		<li>Ensure that the nurse brings the tube to the laboratory, where it is immediately screened for the presence of cumulus-oocyte complexes (COCs).</li>
		<li>Instruct the embryologists to inform the clinician of the total number of COCs retrieved.</li>
		<li>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.</li>
		<li>After oocyte retrieval, evaluate any bleeding from the ovaries or blood vessels of the parametrium and free fluid in the pouch of Douglas. 
		&#8203;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.</li>
	</ol>
	</li>
</ol>
4. IVF laboratory
<ol>
	<li>The day before the oocyte retrieval procedure
	<ol>
		<li>Prepare oocyte collecting tubes (refer to the Table of Materials).
		<ol>
			<li>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. 
			&#8203;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.</li>
		</ol>
		</li>
		<li>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 &#176;C in a controlled atmosphere (6% CO2, 5% 02).</li>
	</ol>
	</li>
	<li>On the day of the oocyte retrieval
	<ol>
		<li>Prewarm the plastic supplies at 37 &#176;C (sterile culture dishes and Pasteur pipettes).</li>
		<li>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.</li>
		<li>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&#39;s information. Place the oocyte collection tubes in the block heater at 37 &#176;C.</li>
		<li>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.</li>
		<li>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 &#176;C in an a atmosphere of 6% CO2, 5% O2.</li>
		<li>Repeat steps 7 to 9 according to the number of oocytes retrieved. Update the laboratory sheet. 
		&#8203;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.</li>
		<li>Wipe down the working stage after completion of the procedure.</li>
	</ol>
	</li>
</ol>
5. Oocyte denudation
<ol>
	<li>Prewarm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered medium and hyaluronidase (refer to the Table of Materials) at 37 &#176;C at least 1 h before starting.</li>
	<li>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 &#176;C for at least 30 min.</li>
	<li>Label the oocyte denudation plate with details of the patient&#39;s identity.</li>
	<li>Immediately before starting the procedure, add hyaluronidase to the first well to obtain a final concentration of about 20 IU/mL.</li>
	<li>Place a limited number of COCs (up to 6) in the first well containing the enzyme to disperse the cumulus cells.</li>
	<li>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 &#956;m 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.</li>
	<li>Perform further denudation to remove the corona cells by using denuding pipettes with decreasing inner diameters (170-145 &#181;m). Use lower diameters (135&#181;m) only if strictly necessary.</li>
	<li>Carefully wash the denuded oocytes to wash out the enzyme.</li>
	<li>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).</li>
	<li>Move the MII oocytes to the vitrification area to immediately perform cryopreservation. Update the laboratory sheet.</li>
</ol>
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 &#181;m so as not to damage oocytes during manipulation.
<ol>
	<li>Approximately 30 min before the procedure, bring the equilibration solution (ES) and vitrification solution (VS) to RT (25-27 &#176;C).</li>
	<li>Properly label the cryodevices with the patient&#39;s name and ID, treatment ID, date of freezing, number of oocytes, and cryobarcode.</li>
	<li>Fill a disposable cooling rack up to the top with fresh liquid nitrogen, and start the sterilization process.</li>
	<li>Label the vitrification plate (refer to the Table of Materials) with the patient&#39;s name and ID. Ask a witness to check the correct ID of the cryodevices.</li>
	<li>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 &#181;L of HEPES-buffered medium (with 5% HSA) and one adjacent drop of 30 &#181;L of ES. 
	NOTE: Place drops just before use to limit medium evaporation.</li>
	<li>Using a 170 &#181;m 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).</li>
	<li>Incubate the oocytes in the first drop for 3 minutes. Add a third drop of 30 &#181;L 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.</li>
	<li>During the incubation, add one 30 &#181;L 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).</li>
	<li>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.</li>
	<li>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).</li>
	<li>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).</li>
	<li>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.</li>
	<li>Store the cryodevice in a visiotube previously labeled with the patient&#39;s information. Update the laboratory sheet.</li>
</ol>
7. Oocyte warming
<ol>
	<li>Approximately 30 min before the procedure, warm the thawing solution (TS), dilution solution (DS), and washing solution (WS) to RT (25-27 &#176;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 &#176;C for at least 1 h before starting the procedure.</li>
	<li>Label all plastic supplies with the patient&#39;s name and ID and the type of solution. Ask a witness to confirm the patient&#39;s information on the cryodevice.</li>
	<li>For each cryodevice to be warmed, prepare a 6-well plate with 200 &#181;L 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.</li>
	<li>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.</li>
	<li>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).</li>
	<li>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.</li>
	<li>Using a 170 &#181;m 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.</li>
	<li>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.</li>
</ol>

The authors have nothing to disclose.

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...

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&#39;s age at the time of oocyte vitrification is certainly the most important limiting factor15 as advanced maternal age (AMA; &#62;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-M&#252;llerian 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 &#176;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.

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

fertility preservation through oocyte vitrification: clinical and laboratory perspectives

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.

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...

Watch this Scientific Journal Video about Fertility Preservation Through Oocyte Vitrification: Clinical and Laboratory Perspectives at JoVE.com

Fertility Preservation Through Oocyte Vitrification: Clinical and Laboratory Perspectives

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.

Preserving female fertility is crucial in a multifunctional healthcare system that takes care of patients' future quality of life. Oocyte cryopreservation ...

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Research

JoVE Journal

Medicine

5.8K Views.  GeneraLife IVF. 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.

Video: Fertility Preservation Through Oocyte Vitrification: Clinical and Laboratory Perspectives

Modified MicroSecure Vitrification: A Safe, Simple and Highly Effective Cryopreservation Procedure for Human Blastocysts

Clinical embryo vitrification evolved with the development of unique vitrification devices in the 21st century and with the misconception that ultra-rapid cooling in an &#34;open&#34; system (i.e., direct LN2 contact) was a necessity to optimize vitrification success. The dogma surrounding the importance of cooling rates led to unsafe practices subject to technical variation and to the creation of vitrification devices that disregarded important quality-control factors (e.g., ease of use, repeatability, reliability, labeling security, and storage safety). Understanding the quality-control flaws of other devices allowed for the development of a safe, secure, repeatable, and reliable &#181;S-VTF method aimed to minimize intra- and inter-technician variation. Equally important, it combined the availability of two existing FDA-compliant devices: 1) a 0.3-mL ionomeric resin embryo straw with internalized, dual-colored, tamper-proof labeling with repeatable weld seal potential; and 2) shortened, commonly-used, 300-&#181;m ID sterile flexipettes to directly load the embryo(s) in order to create a highly-effective global vitrification device. Like other aseptic, closed vitrification systems (e.g., High Security Vitrification (HSV), Rapid-i, and VitriSafe) effectively used in reproductive medicine, microSecure Vitrification (&#181;S-VTF) has proven that it can achieve high post-warming survival and pregnancy outcomes with its attention to simplicity, and reduced technical variation. Although the 0.3-mL embryo straw containing an internal hydrophobic plug was commercially replaced with a standard semen straw possessing cotton-polyvinyl pyrrolidone (PVP) plugs, it maintained its ionomeric resin composition to ensure weld sealing. However, the cotton plugs can wick out the fluid-embryo contents of the flexipettes upon contact. A modified &#181;S-VTF method was adapted to include an additional internal weld seal before the plug on the device loading side. The added technical step to the &#181;S-VTF procedure has not affected its successful application, as high survival rates (&#62; 95%) and pregnancy rates continue today.

MicroSecure Vitrification was developed as a non-commercial, aseptic closed vitrification device system that is compliant with FDA good manufacturing and tissue-handling practices. Due to the withdrawal of hydrophobic plugged embryo straws from the industry, the vitrification procedure was modified to include an inner seal before the standard internal cotton plug.

Clinical embryo vitrification evolved with the development of unique vitrification devices in the 21st century and with the misconception that ultra-rapid ...

Developmental Biology

Clinical Assessment of Spatiotemporal Gait Parameters in Patients and Older Adults

Spatial and temporal characteristics of human walking are frequently evaluated to identify possible gait impairments, mainly in orthopedic and neurological patients1-4, but also in healthy older adults5,6. The quantitative gait analysis described in this protocol is performed with a recently-introduced photoelectric system (see&#160;Materials table) which has the potential to be used in the clinic because it is portable, easy to set up (no subject preparation is required before a test), and does not require maintenance and sensor calibration. The photoelectric system consists of series of high-density floor-based photoelectric cells with light-emitting and light-receiving diodes that are placed parallel to each other to create a corridor, and are oriented perpendicular to the line of progression7. The system simply detects interruptions in light signal, for instance due to the presence of feet within the recording area. Temporal gait parameters and 1D spatial coordinates of consecutive steps are subsequently calculated to provide common gait parameters such as step length, single limb support and walking velocity8, whose validity against a criterion instrument has recently been demonstrated7,9. The measurement procedures are very straightforward; a single patient can be tested in less than 5 min&#160;and a comprehensive report can be generated in less than 1&#160;min.

This protocol is used to evaluate spatial and temporal gait variables of neurological/orthopedic patients and older persons by means of a recently-introduced floor-based photocell system.

Spatial and temporal characteristics of human walking are frequently evaluated to identify possible gait impairments, mainly in orthopedic and ...

Minimum Volume Vitrification of Immature Feline Oocytes

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to promote biodiversity preservation. In felids, most species are threatened with extinction, and domestic breeds are used as a model to increase the efficiency of protocols for germplasm banking. Among oocyte cryopreservation techniques, vitrification is more and more popular in human and veterinary assisted reproduction. Cryotop vitrification, which was at first developed for human oocytes and embryos, has demonstrated to be well-suited for cat oocytes. This method offers several advantages, such as the feasibility in field conditions and the speed of the procedure, which can be helpful when several samples need to be processed. However, the efficiency is strongly dependent on the operator&#8217;s skills, and intra- and inter-laboratory standardization are needed, as well as personnel training. This protocol describes minimum volume vitrification of immature feline oocytes on a commercial support in a step by step field-friendly protocol, from oocyte collection to warming. Following the protocol, preservation of oocyte integrity and viability at warming (as high as 90%) can be expected, although there is still room for improvement in post-warming maturation and embryonic development outcomes.

This manuscript describes a protocol for the minimum volume vitrification of immature cat oocytes with laboratory-made media on commercial supports. It covers every step from oocyte isolation from ex vivo gonads to vitrification and warming.

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to ...

Vitrification of In Vitro Matured Oocytes Collected from Adult and Prepubertal Ovaries in Sheep

In livestock, in vitro embryo production systems can be developed and sustained thanks to the large number of ovaries and oocytes that can be easily obtained from a slaughterhouse. Adult ovaries always bear several antral follicles, while in pre-pubertal donors the maximal numbers of oocytes are available at 4 weeks of age, when ovaries bear peak numbers of antral follicles. Thus, 4 weeks old lambs are considered good donors, even if the developmental competence of prepubertal oocytes is lower compared to their adult counterpart. 
Basic research and commercial applications would be boosted by the possibility of successfully cryopreserving vitrified oocytes obtained from both adult and prepubertal donors. The vitrification of oocyte collected from prepubertal donors would also allow shortening the generation interval and thus increasing the genetic gain in breeding programs. However, the loss of developmental potential after cryopreservation makes mammalian oocytes probably one of the most difficult cell types to cryopreserve. Among the available cryopreservation techniques, vitrification is widely applied to animal and human oocytes. Despite recent advancements in the technique, exposures to high concentrations of cryoprotective agents as well as chilling injury and osmotic stress still induce several structural and molecular alterations and reduce the developmental potential of mammalian oocytes. Here, we describe a protocol for the vitrification of sheep oocytes collected from juvenile and adult donors and matured in vitro prior to cryopreservation. The protocol includes all the procedures from oocyte in vitro maturation to vitrification, warming and post-warming incubation period. Oocytes vitrified at the MII stage can indeed be fertilized following warming, but they need extra time prior to fertilization to restore damage due to cryopreservation procedures and to increase their developmental potential. Thus, post-warming culture conditions and timing are crucial steps for the restoration of oocyte developmental potential, especially when oocyte are collected from juvenile donors.

The protocol aims at providing a standard method for the vitrification of adult and juvenile sheep oocytes. It includes all the steps from the preparation of the in vitro maturation media to the post-warming culture. Oocytes are vitrified at the MII stage using Cryotop to ensure the minimum essential volume.

In livestock, in vitro embryo production systems can be developed and sustained thanks to the large number of ovaries and oocytes that can be easily ...

Functional Human Liver Preservation and Recovery by Means of Subnormothermic Machine Perfusion

There is currently a severe shortage of liver grafts available for transplantation. Novel organ preservation techniques are needed to expand the pool of donor livers. Machine perfusion of donor liver grafts is an alternative to traditional cold storage of livers and holds much promise as a modality to expand the donor organ pool. We have recently described the potential benefit of subnormothermic machine perfusion of human livers. Machine perfused livers showed improving function and restoration of tissue ATP levels. Additionally, machine perfusion of liver grafts at subnormothermic temperatures allows for objective assessment of the functionality and suitability of a liver for transplantation. In these ways a great many livers that were previously discarded due to their suboptimal quality can be rescued via the restorative effects of machine perfusion and utilized for transplantation. Here we describe this technique of subnormothermic machine perfusion in detail. Human liver grafts allocated for research are perfused via the hepatic artery and portal vein with an acellular oxygenated perfusate at 21 &#176;C.

We describe a method of ex vivo machine perfusion of human liver grafts at subnormothermic temperature (21 &#176;C).

There is currently a severe shortage of liver grafts available for transplantation.  Novel organ preservation techniques are needed to expand the pool of ...

Ovarian Tissue Oocyte-In Vitro Maturation for Fertility Preservation

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.

Ovarian Tissue Oocyte-In Vitro Maturation for Fertility Preservation

Presented here is ovarian tissue oocyte-in vitro maturation (OTO-IVM), an accessible technique within a medical assisted reproduction (MAR) laboratory offering realistic additional fertility preservation options to patients who need ovarian tissue cryopreservation.

Mature oocyte vitrification is the standard of care to preserve fertility in women at risk of infertility. However, ovarian tissue cryopreservation (OTC) ...

Methodology for Sputum Induction and Laboratory Processing

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which is interesting in various respiratory diseases like asthma, chronic obstructive pulmonary disease (COPD), chronic cough, or idiopathic pulmonary fibrosis. This technique is well tolerated, safe and non-invasive, but is currently limited to research services and specialized centers in clinical practice because it is technically demanding, time-consuming, and requires trained staff. The success rate of sputum induction and analysis is about 80%.
Here, we describe the induction and laboratory processing of sputum samples. Sputum is induced by inhalation of hypertonic or isotonic saline with salbutamol. For the processing, we use the whole sputum technique. Dithiothreitol (DTT) is used to allow mucolysis of sputum samples. The primary aim of sputum processing is to obtain a differential cell count to study the cell types present in the airway lumen. Additional analyses may also be performed on sputum supernatant and sputum cells, which may allow further investigation into inflammatory processes and immune mechanisms. Examples include studying mediators in sputum supernatant and performing a large spectrum of analysis on sputum cells such as flow cytometry, genomics, or proteomics.
Finally, representative results of sputum analysis in healthy controls, asthmatics, and COPD patients are presented.

Here we describe the technique of sputum induction. This protocol also explains the sputum processing to perform a differential cell count and to collect sputum supernatant and cells for further analysis.

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which ...

Cryopreservation of Oocytes Retrieved from Ovarian Tissue to Optimize Fertility Preservation in Prepubertal Girls and Women

Human ovarian tissue cryopreservation (OTC) is increasingly used worldwide to preserve female fertility in prepubertal girls and women at risk of premature ovarian insufficiency (POI) in the context of urgent gonadotoxic treatments or ovarian surgery. Fertility preservation is challenging because there is no consensus regarding patient management, preservation fertility strategies, or even technical laboratory protocols, which implies that each procedure must be adapted to the characteristics of the patient profile and its own risk-benefit ratio. During OTC, mature/immature oocytes can be aspirated directly from large/small antral follicles within ovarian tissue samples and/or be released into culture media from growing follicles during ovarian tissue dissection in prepubertal girls and women. In this manuscript, we present a protocol that combines ovarian tissue freezing with the cryopreservation of mature/immature oocytes retrieved from ovarian tissue samples, improving the reproductive potential of fertility preservation. Appropriate collection, handling, and storage of ovarian tissue and oocytes before, during, and after the cryopreservation will be described. The subsequent use and safety of cryopreserved/thawed ovarian tissue samples and oocytes will also be discussed, as well as the optimal timing for in vitro maturation of immature oocytes. We recommend the systematic use of this protocol in fertility preservation of prepubertal girls and women as it increases the whole reproductive potential of fertility preservation (i.e., oocyte vitrification in addition of OTC) and also improves the safety and use of fertility preservation (i.e., thawing of oocytes versus ovarian graft), maximizing the chance of successful childbirth for the patients at risk of POI.

We propose a protocol for fertility preservation in prepubertal girls and women at risk of premature ovarian insufficiency. It combines ovarian tissue freezing and cryopreservation of oocytes retrieved from ovarian tissue. This strategy improves the safety and optimizes the reproductive potential of fertility preservation, maximizing the chance of childbirth.

Human ovarian tissue cryopreservation (OTC) is increasingly used worldwide to preserve female fertility in prepubertal girls and women at risk of ...

Fertility Preservation in Patients with Severe Ovarian Dysfunction

Ovarian function progressively declines during aging and in some pathophysiological conditions including karyotype abnormality, autoimmune diseases, chemo- and radiation-therapies, as well as ovarian surgeries. In unmarried women with severe ovarian dysfunction, fertility preservation is important for future pregnancies. Although oocyte cryopreservation is an established method for fertility preservation, these patients could only preserve a limited number of oocytes even after ovarian hyperstimulation, leading to repeated stimulations to ensure sufficient oocytes to guarantee future pregnancy. To solve this issue, we have recently developed a drug-free in vitro activation (IVA) procedure, which enable us to stimulate early stages of ovarian follicles to develop to the preantral follicle stage. These preantral follicles can respond to the unique protocol of gonadotropin stimulation, resulting in increased number of retrieved oocytes per ovarian stimulation for cryopreservation. The drug-free IVA comprised from the surgical approach and ovarian stimulation. We removed a part of cortex from one or both ovaries from patients under laparoscopic surgery. The ovarian cortical tissues were cut into small cubes to disrupt the Hippo signaling pathway and stimulate the development of early stage follicles. These cubes were grafted orthotropically into remaining ovaries as well as beneath the serosa of both Fallopian tubes. We have already published the surgical procedure of the drug-free IVA and the protocol of subsequent ovarian stimulation, but herein we present the details of laboratory methods required for drug-free IVA.

We present details of laboratory procedures for drug-free in vitro activation (IVA) of ovarian follicles for patients with severe ovarian dysfunction. This method could increase the number of retrievable oocytes per ovarian hyperstimulation and benefit fertility preservation for those patients.

Ovarian function progressively declines during aging and in some pathophysiological conditions including karyotype abnormality, autoimmune diseases, ...

Follicular Viability Assessment in Vitrified Human Ovarian Cortex Tissues

Vitrification of Ovarian Cortex Tissue to Achieve a Glassy State of Aggregation

Ovarian tissue cryopreservation (OTC) is an important option for fertility preservation. For patients whose gonadotoxic treatments cannot be postponed or for pre-pubertal girls, it is often the only option for fertility protection. Cryopreservation can be performed either by vitrification or by slow freezing. Slow freezing is currently the standard approach. An increasing number of studies indicate that vitrification can replace slow freezing in the state-of-the-art in vitro fertilization (IVF) laboratories, significantly improving thawing survival rates and simplifying the technical aspects of cryopreservation. A metal grid-based, high-throughput protocol for rapid vitrification of ovarian cortex tissue, suitable for clinical routine, is described. The sterilization of metal grids and liquid nitrogen ensures high quality, meeting good manufacturing practice (GMP) standards. Vitrification was conducted to ensure ultra-rapid cooling rates. Instead of slowly thawing, samples were rapidly warmed. To assess follicular viability, calcein staining was performed both prior to cryopreservation and after rapid warming. The successful application of vitrification and rapid warming using metal grids is reported. No significant differences in follicular viability were observed prior to vitrification and after rapid warming. These results substantiate the high capacity of tissue vitrification for clinical routine applications as a potential substitute for the widely used slow-freezing method.

Author Spotlight: Advancing Fertility Preservation in Young Female Cancer Patients Through Ovarian Tissue Vitrification and In Vitro Follicular Growth

A protocol for the vitrification of ovarian tissue, as an alternative cryopreservation method to the widely used slow freezing protocol, is presented.

Ovarian tissue cryopreservation (OTC) is an important option for fertility preservation. For patients whose gonadotoxic treatments cannot be postponed or ...

Fertility Preservation Through Oocyte Vitrification: Clinical and Laboratory Perspectives

Summary

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Chapters in this video

Modified MicroSecure Vitrification: A Safe, Simple and Highly Effective Cryopreservation Procedure for Human Blastocysts

Clinical Assessment of Spatiotemporal Gait Parameters in Patients and Older Adults

Minimum Volume Vitrification of Immature Feline Oocytes

Vitrification of In Vitro Matured Oocytes Collected from Adult and Prepubertal Ovaries in Sheep

Functional Human Liver Preservation and Recovery by Means of Subnormothermic Machine Perfusion

Ovarian Tissue Oocyte-In Vitro Maturation for Fertility Preservation

Methodology for Sputum Induction and Laboratory Processing

Cryopreservation of Oocytes Retrieved from Ovarian Tissue to Optimize Fertility Preservation in Prepubertal Girls and Women

Fertility Preservation in Patients with Severe Ovarian Dysfunction

Vitrification of Ovarian Cortex Tissue to Achieve a Glassy State of Aggregation