We thank Cara F&#228;rber for proofreading; Katharina Wollersheim, Martin Mahlberg, Lea Korte, and Jasmin Rebholz for their technical assistance.

The study was approved by the ethics committee at University Hospital Bonn (007/09). Written informed consent was obtained from each patient. The study group included human ovarian tissue from 50 patients with an average age of 27.4 years prior to cryopreservation, as indicated in Figure 1. The reagents and equipment used in this study are listed in the Table of Materials.
1. Preparation of loading devices
<ol>
	<li>Prepare surgical scissors and metal meshes for customization. Cut the meshes into strips measuring 25 mm x 8 mm, as indicated in Figure 2A.</li>
	<li>Place the customized metal meshes in a sterilization container and autoclave for 2 h (Figure 2B). After autoclaving, place the sterilization container and 1.8 mL vials under the laminar flow bench.</li>
	<li>Turn on the ultraviolet (UV) irradiation of the laminar flow bench for 30 min for additional sterilization at 254 nm wavelength.</li>
	<li>Open the sterilization container, remove the metal grids, and fit them into the caps of the 1.8 mL vials, as indicated in Figure 2C. Close the 1.8 mL vials. 
	NOTE: All tissue culture work described above should be performed in a Class II laminar flow hood. Clean the laminar flow bench with a surface disinfectant while wearing disposable laboratory gloves; non-powdered gloves are recommended.</li>
</ol>
2. Preparation of vitrification media
<ol>
	<li>Prepare one serological 10 mL pipette, one electronic pipette aid, and three 50 mL tubes for Vitrification Solution 1 (VS1), Vitrification Solution 2 (VS2), and Vitrification Solution 3 (VS3), as well as for recording the date of preparation. Turn on the ultraviolet (UV) radiation of the laminar flow bench for additional sterilization.</li>
	<li>For VS1 (15 mL), pipette 12 mL of oocyte handling medium (supplemented with human serum albumin by the manufacturer), 1.5 mL of SSS (Serum Substitute Supplement), and 1.5 mL of ethylene glycol into one 50 mL tube.</li>
	<li>For VS2 (15 mL), pipette 10.5 mL of oocyte handling medium (supplemented with human serum albumin by the manufacturer), 3 mL of ethylene glycol, and 1.5 mL of SSS into one 50 mL tube.</li>
	<li>For VS3, pipette 8.5 mL of oocyte handling medium (supplemented with human serum albumin by the manufacturer), 5.25 mL of ethylene glycol, and add 2.57 g of sucrose and 0.75 g of Polyvinylpyrrolidone (PVP) into one 50 mL tube. See Figure 3 for details. Mix all solutions by vortexing at 3,000 rpm. 
	NOTE: All tissue culture work described above should be performed in a Class II laminar flow hood. Clean the laminar flow bench with a disinfectant while wearing disposable laboratory gloves; non-powdered gloves are recommended.</li>
</ol>
3. Vitrification preparation
<ol>
	<li>Clean the laminar flow bench with a surface disinfectant while wearing disposable laboratory gloves; non-powdered gloves are recommended.</li>
	<li>Prepare one 6-well plate, a cell strainer, a scalpel, tweezers, one 2 mm biopsy punch, and two 90 mm round-bottom dishes. Also, prepare media: preservation solution for tissue transplantation and VS1-3. Pipette 5 mL of each chilled solution (VS1-3) into separate wells and wait 30 min for the solutions to reach room temperature.</li>
	<li>Sterilize liquid nitrogen with an automated UV irradiation system; alternatively, use the UV irradiation of the laminar flow hood for 120 min.</li>
</ol>
4. Vitrification of tissue
<ol>
	<li>Prepare ovarian cortex tissue by removing the medulla. Since the cortex is usually harder than the medulla, they can be easily distinguished and separated.
	<ol>
		<li>Cut the ovarian cortex tissue into the desired shapes (10 mm x 5 mm for transplantation; 2 mm x 2 mm punches for viable follicle counting prior to and after vitrification), as indicated in Figure 4A. 
		NOTE: Avoid damaging the tissue by pressing too hard, and handle it carefully. Tissue samples may vary between patients.</li>
	</ol>
	</li>
	<li>Place a cell strainer into the first well of the 6-well plate with VS1. Equilibrate the ovarian cortex tissue for 5 min in 5 mL of VS1 in well 1 of the 6-well plate using the cell strainer.
	<ol>
		<li>Then, move the cell strainer with the cortex tissue to well 2 containing 5 mL of VS2, and equilibrate for 5 min. Finally, equilibrate for 6 min in 5 mL of VS3, as indicated in Figure 3 and Table 1.</li>
	</ol>
	</li>
	<li>Open the prepared cryovials. Fill the vials with sterilized liquid nitrogen and place them into the cryo dewar vessel filled with liquid nitrogen. Load the tissue samples onto the metal grid of the vitrification device within 1 min (Figure 4B,C).
	<ol>
		<li>Insert the tissue samples loaded on the metal grid into the liquid nitrogen in the prepared grid-based cryovials (Figure 4D). 
		NOTE: Rapid freezing of the tissue loaded on the metal grid is essential for the success of this method. All tissue culture work described above should be performed in a Class II laminar flow hood. Clean the laminar flow bench with a surface disinfectant while wearing disposable laboratory gloves; non-powdered gloves are recommended.</li>
	</ol>
	</li>
</ol>
5. Preparation of rapid warming media
<ol>
	<li>Prepare the solutions as noted in Figure 5 and Table 2. Weigh the sucrose for the rapid warming solution (RWS) and the equilibriation solution (ES) and transfer it to the tube. Add the oocyte handling medium (supplemented with human serum albumin by the manufacturer) using a serological pipette and a pipetting aid. Add SSS and sucrose as listed in Table 2.</li>
	<li>Close the tubes and let them agitate on a rolling shaker until fully dissolved. 
	NOTE: All tissue culture work described above should be performed in a Class II laminar flow hood. Clean the laminar flow bench with a surface disinfectant while wearing disposable laboratory gloves; non-powdered gloves are recommended.</li>
</ol>
6. Rapid warming preparation
<ol>
	<li>One day before rapid warming, prepare the consumables and decontaminate them using UV light in the laminar flow bench: Prepare the sample beaker for RWS, the 6-well plate for ES, RS1, and RS2, and the sample beaker for tissue transportation after rapid warming. Place sterile, disposable tweezers, pipetting equipment, and other consumables in the laminar flow bench.</li>
	<li>Preheat a heating plate to 37.2 &#176;C. Incubate RWS at 37 &#176;C for at least 1 h.</li>
	<li>Prepare a sterile 6-well plate with 6 mL of ES, 6 mL of RS for &#34;RS1,&#34; and another 6 mL of RS for &#34;RS2&#34; in the laminar flow bench. Refer to Figure 5 for a schematic illustration of the 6-well plate. Let these incubate at room temperature for 1 h before warming the tissue.</li>
	<li>Transfer RWS to a sterile sample beaker under sterile conditions and place it on the heating plate. The RWS should be maintained at 37 &#176;C. 
	NOTE: Rapid warming requires a fast warming rate. It is crucial to hold the RWS at 37 &#176;C to ensure the success of the first step.</li>
</ol>
7. Rapid warming of ovarian tissue
<ol>
	<li>Transport the cryopreserved vials containing vitrified ovarian cortical tissue in liquid nitrogen to the laminar flow bench. Open the vials while they are partially submerged under liquid nitrogen. Rapidly submerge the mesh with the vitrified tissue into the RWS and let the tissue sit there for 1 min at 37 &#176;C. 
	NOTE: This is the most critical step during rapid warming and must be conducted without any delay.</li>
	<li>Using sterile forceps, transfer the tissue to the ES and incubate it for 3 min while gently shaking it on a rocking shaker.</li>
	<li>Rinse the tissue at room temperature for 10 min each with RS1 and RS2 on a rocking shaker, as indicated in Figure 5.</li>
	<li>Transfer the warmed tissue to a sterile sample beaker containing 5 mL of preservation solution for tissue transplantation, which should be held at 4 &#176;C for transportation to the transplantation site. 
	NOTE: All tissue culture work described above should be performed in a Class II laminar flow hood. Clean the laminar flow bench with a surface disinfectant while wearing disposable laboratory gloves; non-powdered gloves are recommended.</li>
</ol>
8. Determination of follicular viability
<ol>
	<li>Bring dimethyl sulfoxide (DMSO) to room temperature overnight to obtain a liquid state. Pipette 100 &#181;L of DMSO into one vial of calcein and resuspend to dissolve the calcein. Pipette 3 &#181;L of the dissolved calcein into the bottom of one 1.5 mL tube and store the vials at -20 &#176;C.</li>
	<li>Place 0.007 g of collagenase in one 1.5 mL tube and store it at -20 &#176;C.</li>
	<li>Add 997 &#181;L of DPBS to the prealiquoted and frozen 3 &#181;L of calcein in one 1.5 mL tube, and resuspend to dissolve the calcein. Add 0.007 g of collagenase to obtain a 1000 &#181;L of working solution.</li>
	<li>Use 500 &#181;L of the working solution to digest two pieces of 2 mm ovarian cortex fragments for 90 min at 37 &#176;C, shielded from light, in a 4-well dish. After 60 min and 70 min, resuspend the solution repeatedly. Finally, determine the follicular viability via fluorescence microscopy, as shown in Figure 6.</li>
</ol>

Follicular Viability Assessment in Vitrified Human Ovarian Cortex Tissues

Here, a protocol for high-throughput vitrification of human ovarian cortex tissue, suitable for clinical routine, is presented. Similar to the vitrification of oocytes or embryos, the successful application of the procedure requires detailed adherence to the protocol concerning the temperature of the vitrification and warming solutions, as well as the equilibration period. Compliance with EU tissue directives37 regarding air quality and sterility is also essential.
The vitrification procedure results in a non-crystalline, amorphous, or glassy state. Overall, vitrification is a versatile process with significant implications in various scientific and technological domains. The primary benefit of vitrification is its ability to convert tissue into a glassy state, thereby preventing ice crystal formation38,39,40, which can negatively affect tissue integrity and its components.
Supplementation of cryoprotective agents (CPAs) with polyvinylpyrrolidone (PVP) allows for a reduction in CPA concentration without compromising the quality of the vitrification solutions41,42. Furthermore, the use of metal grids provides high thermal conductivity compared to plastic-based carrier systems. The grid structure also facilitates surface adhesion, ensuring the safe and secure cryopreservation of tissue samples and small cortex punches for quality measures. If cryovessels from other manufacturers are used, it is important to test the size of the metal grids beforehand to ensure stability and proper grip within the cryovessel lid, as well as a good fit into the vessel.
Critical steps to ensure successful vitrification include rapid vitrification by immersing the tissue in sterilized liquid nitrogen and performing rapid warming without delay to avoid adverse results. In terms of cost-effectiveness, tissue vitrification is less demanding compared to the slow freezing procedure, which may influence personnel deployment planning. Additionally, vitrification eliminates the need to purchase and service equipment required for slow freezing.
Biological measures and meta-analyses have demonstrated the comparability or even advantages of vitrification compared to slow freezing43. However, differences in results after vitrification may be attributed to the lack of standardization in both the vitrification device and the protocol, including the solutions used, which vary across studies. Future research should explore the potential for follicle culture from vitrified/rapid-warmed tissue to monitor growth in vitro, as successfully demonstrated in mouse ovarian tissue by several groups44,45,46,47,48,49,50.
In summary, vitrification of ovarian tissue is a significant alternative to the widely used slow freezing protocol, supported by five successful deliveries reported by Suzuki51 (Japan), Silber52 (USA), and S&#228;nger53 (Germany). In contrast to commercially available vitrification media and kits for cells, there are few FDA/CE-approved systems for ovarian tissue, which may limit their application in clinical settings. Therefore, the development of FDA/CE-approved kits and media for the vitrification and rapid warming of ovarian tissue is recommended30.

Cryopreservation of ovarian tissue is an important option for fertility preservation. Explanted tissue containing ovarian follicles, in which oocytes are embedded, is cryopreserved. After storage, the ovarian tissue can be thawed, warmed, and reimplanted in the patient. For viable cells or tissue, two cryopreservation methods are available: slow freezing and vitrification1.
Vitrification is used to preserve biological materials, such as embryos and oocytes, with superior survival rates compared to the slow freezing protocol1,2,3,4. Slow freezing has limitations, such as ice crystal formation, which can potentially damage cell and tissue structures. However, slow freezing is an important cryopreservation approach that facilitates the long-term storage of biological samples, and the functionality of this method has been widely proven5. Vitrification induces a glassy state of aggregation, preventing ice crystal formation6,7. On a technical level, vitrification significantly simplifies the cryopreservation procedure by reducing equipment maintenance, decreasing the likelihood of technical errors, and shortening the duration of the cryopreservation process8,9. In female fertility preservation, ovarian tissue cryopreservation is a decisive approach prior to cancer treatment10. Different groups have successfully demonstrated the concept of cryopreservation, thawing, and transplantation of tissue based on the slow freezing protocol11,12,13,14, which is currently regarded as the standard approach15.
Vitrification of ovarian tissue is regarded as a promising alternative method16,17,18,19,20,21, in terms of resource-saving22, follicular survival rates, DNA fragmentation levels, and balanced angiogenic potential23,24,25,26,27. This is substantiated by successful deliveries in Japan28, the USA29, and Germany30.
Comparing the two options for ovarian tissue cryopreservation (OTC)-vitrification versus the standard procedure of slow freezing-results are partially conflicting in current meta-analyses16. Several factors may have contributed to this, as current vitrification protocols vary greatly. These differences include the choice of cryoprotectant or combination of protectants, their concentration, the composition of the OTC media, the size of tissue fragments, and the device used as a tissue carrier. Accordingly, there is no standardized warming protocol.
As the authors found a method that yields convincing results in terms of handling, viability, apoptosis onset, release of angiogenic factors, and even a report of a birth after reimplantation9,27, a very detailed description of the protocol is provided. The described method offers a valid and effective protocol that may contribute to the standardization of vitrification of ovarian tissue.

<table><tbody><tr><td>1.8 mL vials</td><td>VWR International GmbH</td><td>479-6837</td><td></td></tr><tr><td>10 mL serological pipette</td><td>Sarstedt</td><td>86.1254.001</td><td></td></tr><tr><td>4 well plate</td><td>Gynemed</td><td>GYOOPW-FW04</td><td></td></tr><tr><td>50 mL Tube</td><td>Sarstedt</td><td>62.559.001</td><td></td></tr><tr><td>6 well plates</td><td>Sarstedt</td><td>83.3920</td><td></td></tr><tr><td>Bacillol AF</td><td>Hartmann</td><td>973385</td><td></td></tr><tr><td>Calcein AM</td><td>Merck</td><td>17783</td><td></td></tr><tr><td>Collagenase type 1A</td><td>Merck</td><td>C2674</td><td></td></tr><tr><td>Cryosure DMSO</td><td>WAK Chemie</td><td>WAK-DMSO-10</td><td></td></tr><tr><td>Custodiol</td><td>Dr. Franz K&amp;ouml;hler Chemie</td><td>00867288</td><td></td></tr><tr><td>DPBS CTS</td><td>Gibco Life technologies</td><td>A12856-01</td><td></td></tr><tr><td>ErgoOne pipette aid</td><td>Starlab</td><td>S7166-0010</td><td></td></tr><tr><td>Ethylene glycol</td><td>Sigma Aldrich</td><td>102466</td><td></td></tr><tr><td>Euronda sterilization container</td><td>euronda</td><td>282021</td><td></td></tr><tr><td>G-MOPS+</td><td>Vitrolife</td><td>10130</td><td></td></tr><tr><td>Metal meshes</td><td>Sigma Aldrich</td><td>S0770</td><td></td></tr><tr><td>Metzenbaum scissors</td><td>world precision instruments</td><td>501262102</td><td></td></tr><tr><td>N-Bath System</td><td>Nterilizer</td><td>N-Bath 3.0</td><td></td></tr><tr><td>Polyvinylpyrrolidone (PVP)</td><td>SAGE</td><td>ART-4005</td><td></td></tr><tr><td>Serum substitute supplement (SSS)</td><td>Fujifilm Irvine scientific</td><td>99193</td><td></td></tr><tr><td>Sterile cup</td><td>Sarstedt</td><td>75.562.105</td><td></td></tr><tr><td>Sterile forceps</td><td>Carl Roth</td><td>KL05.1</td><td></td></tr><tr><td>Sucrose</td><td>Merck</td><td>S0389</td><td></td></tr></tbody></table>

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.

This protocol presents the procedures for the preparation of vitrification media, loading devices, vitrification, preparation of rapid warming media, rapid warming, and determination of follicular viability. A direct comparison of follicular viability and angiogenic factors between slow freezing and vitrification has been validated and published31,32.
The overall success of the described vitrification protocol was assessed by comparing the vital follicle count before and after vitrification/rapid warming. The experimental setup is shown in Figure 1, and the results are presented in Figure 6. In 50 patients, a mean count of 77.98 vital follicles was observed before vitrification and 62.99 after vitrification/rapid warming, reflecting a survival rate of 80.8%. This was not significantly different according to the Wilcoxon test33.
Metal grids are individually customized with sharp scissors to a size of 25 mm x 8 mm, which fits into the caps of 1.8 mL cryovials, as shown in Figure 2A,C. After sterilization using steam autoclaving, the grids and caps of 1.8 mL vials are assembled under a laminar flow bench, as indicated in Figure 2B. This setup provides a secure hold in the cap without the need for additional actions and offers sufficient area for tissue of various sizes. Typically, tissue pieces of 5 mm x 10 mm for transplantation and 2 mm diameter pieces for assessing vital follicle count after rapid thawing are vitrified. Both sizes fit perfectly onto the metal grids.
Equilibration with vitrification solutions (VS1, VS2, and VS3) is conducted in a 6-well plate on a rocking shaker at room temperature under a laminar flow bench, as shown in Figure 3. The time frames in the scheme ensure the effective uptake of the cryoprotectant ethylene glycol. Using a 6-well plate over single vessels is recommended as it facilitates quick movement of the tissue between solutions and helps prevent mix-ups.
For rapid vertical vitrification in liquid nitrogen, ovarian cortex tissue is cut into suitable pieces (Figure 4A). Ovarian cortex samples are placed on loading devices (Figure 4B,C) and submerged vertically into liquid nitrogen (Figure 4D) to achieve a glassy state of aggregation through vitrification. The chosen metal grids allow for vertical handling of the tissue, as shown in Figure 4D. Additionally, the metal grids are highly thermally conductive, ensuring rapid cooling rates from 22 &#176;C to -196 &#176;C, which is a critically important step in the vitrification process.
For rapid warming, RWS is prepared in a sterile cup at 37 &#176;C. ES, RS1, and RS2 are prepared in a 6-well plate on a rocking shaker at room temperature, as shown in Figure 5. The high volume of pre-warmed RWS prevents the solution from cooling down excessively upon the addition of the vitrified tissue and ensures a consistently warm environment for the tissue throughout the rapid warming process.
To assess and ensure high quality standards, 2 mm x 2 mm biopsy punches are stained with calcein prior to vitrification and after rapid warming to determine follicular viability using fluorescence microscopy34,35 (Figure 6). Viable follicles emit green fluorescence at 495 nm after the intracellular uptake of calcein. Alternatively, follicular viability (Table 3) can be assessed using neutral red dyes36.
By following the described steps in this protocol, ovarian tissue is transformed into a glassy state of aggregation, which facilitates high survival rates after rapid warming, as confirmed by fluorescence microscopy.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66801/66801fig01.jpg" /> 
Figure 1: Study design. Ovarian cortical samples from 50 patients were examined before (fresh) and after vitrification and rapid warming for the number of viable follicles. For each group, two tissue pieces of 2 mm diameter were cultured for 24 h before follicle count assessment. The tissue was digested with collagenase and stained with calcein to assess viability. The number of viable follicles was determined using a microscope. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66801/66801fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66801/66801fig02.jpg" /> 
Figure 2: Preparation of loading devices. Metal grids were cut to a size of 8 mm x 25 mm (A). The customized metal grids were sterilized by autoclaving (B). The sterilized metal grids were then inserted into caps of 1.8 mL vials, ready for use (C). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66801/66801fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66801/66801fig03.jpg" /> 
Figure 3: Preparation of vitrification solutions. Vitrification solutions (VS) were prepared and transferred to a 6-well plate. The wells show the volume of each solution and the individual incubation times used in the vitrification protocol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66801/66801fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66801/66801fig04.jpg" /> 
Figure 4: Ovarian cortex processing and vitrification. Ovarian cortex tissue was processed for cryopreservation by removing the medulla and cutting the tissue into 5 mm x 10 mm pieces (A). After incubation in the vitrification solutions shown in Figure 3, the tissue was loaded onto the vitrification loading device (B,C). For rapid vertical vitrification of ovarian cortex samples, the caps with the loaded tissue were quickly inserted into sterilized liquid nitrogen (D). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66801/66801fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66801/66801fig05.jpg" /> 
Figure 5: Preparation of rapid warming solutions. Rapid warming solution (RWS), equilibration solution (ES), and rinsing solutions (RS) 1 and 2 were prepared and transferred to a 6-well plate. The vessels and wells show the volume of each solution and the corresponding incubation times. Note that RWS is maintained at 37.2 &#176;C on a heating plate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66801/66801fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66801/66801fig06.jpg" /> 
Figure 6: Viability count. To assess the number of viable follicles, tissue pieces of 2 mm diameter were digested with collagenase and stained with calcein. The representative images show (A) calcein staining of 3 recovered follicles prior to vitrification. (B) A recovered viable follicle after vitrification and rapid warming. (C) A recovered viable follicle prior to slow freezing. (D) Recovered viable follicles after slow freezing and thawing. Follicular viability is indicated by calcein, a green fluorescent dye that emits green fluorescence when enzymatically converted by viable cells at 495 nm. Scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66801/66801fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>VS 1 (15 mL)</td>
			<td>Ethylene glycol</td>
			<td>10%</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td></td>
			<td>SSS</td>
			<td>10%</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td></td>
			<td>G-MOPS+</td>
			<td></td>
			<td>12 mL</td>
		</tr>
		<tr>
			<td>VS 2 (15 mL)</td>
			<td>Ethylene glycol</td>
			<td>20%</td>
			<td>3 mL</td>
		</tr>
		<tr>
			<td></td>
			<td>SSS</td>
			<td>10%</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td></td>
			<td>G-MOPS+</td>
			<td></td>
			<td>10.5 mL</td>
		</tr>
		<tr>
			<td>VS 3 (15 mL)</td>
			<td>Ethylene glycol</td>
			<td>35%</td>
			<td>5.25 mL</td>
		</tr>
		<tr>
			<td></td>
			<td>SSS</td>
			<td>10%</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td></td>
			<td>Sucrose</td>
			<td>0.5 mol/L</td>
			<td>2.57 g</td>
		</tr>
		<tr>
			<td></td>
			<td>PVP</td>
			<td>5 % (w/v)</td>
			<td>0.75 g</td>
		</tr>
		<tr>
			<td></td>
			<td>G-MOPS+</td>
			<td></td>
			<td>ad 15 mL</td>
		</tr>
	</tbody>
</table>
Table 1: Composition of vitrification solutions (VS).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>RWS (30 mL)</td>
			<td>Sucrose</td>
			<td>0.8 mol/L</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>8.22 g</td>
		</tr>
		<tr>
			<td></td>
			<td>SSS</td>
			<td>10%</td>
			<td>3 mL</td>
		</tr>
		<tr>
			<td></td>
			<td>G-MOPS+</td>
			<td></td>
			<td>ad 30 mL</td>
		</tr>
		<tr>
			<td>ES (15 mL)</td>
			<td>Sucrose</td>
			<td>0.4 mol/L</td>
			<td>2.05 g</td>
		</tr>
		<tr>
			<td></td>
			<td>SSS</td>
			<td>10%</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td></td>
			<td>G-MOPS+</td>
			<td></td>
			<td>ad 15 mL</td>
		</tr>
		<tr>
			<td>RS 1&#38;2 (15 mL)</td>
			<td>SSS</td>
			<td>10%</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td></td>
			<td>G-MOPS+</td>
			<td></td>
			<td>ad 15 mL</td>
		</tr>
	</tbody>
</table>
Table 2: Composition of rapid warming solution (RWS), equilibration solution (ES), and rinsing solutions (RS). This table provides the components and concentrations for the rapid warming solution (RWS), equilibration solution (ES), and rinsing solutions (RS) used in the post-vitrification processing.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Parameter</td>
			<td>Fresh</td>
			<td>Interval</td>
			<td>Rapid warmed after vitrification</td>
			<td>Interval</td>
			<td>n</td>
			<td>*P-value</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>SD</td>
			<td></td>
			<td>SD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Follicular viability count [n]</td>
			<td>77.98</td>
			<td>0-386</td>
			<td>62.99</td>
			<td>0.5-349</td>
			<td>50</td>
			<td>0.130</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>77.95</td>
			<td></td>
			<td>80.02</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>*Wilcoxon test</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 3: Representative results of follicular viability. This table presents the results of follicular viability assessments before cryopreservation and after rapid warming. Two tissue pieces of 2 mm diameter per patient were used to count the number of viable follicles before and after vitrification/rapid warming. Paired tissue samples from 50 patients were analyzed using the Wilcoxon test.

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.

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 ...

vitrification-ovarian-cortex-tissue-to-achieve-glassy-state

Nanyang Technological University

Research

JoVE Journal

Medicine

1.2K Views.  University Hospital of Bonn. De reikwijdte van onze onderzoeksgroep is vruchtbaarheidsbescherming bij jonge vrouwelijke kankerpatiënten. Het is belangrijk om bestaande protocollen te verbeteren om de kans op een latere zwangerschap te vergroten. Dat omvat de vitrificatie van eierstokweefsel en ook de in vitro groei van follikels met de in vitro groei van ovariële follikels.Het cruciale punt is de meerstaps weefselkweekbenadering voorafgaand aan de folliculaire isolatieprocedure,...

Video: Auteur in de schijnwerpers: het bevorderen van vruchtbaarheidsbehoud bij jonge vrouwelijke kankerpatiënten door verglazing van eierstokweefsel en in vitro follicul

Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health disorders such as chronic wounds. We report the development of a dual-mode imaging system for non-invasive and non-contact imaging of cutaneous tissue oxygenation and vascular function. The imaging system integrated an infrared camera, a CCD camera, a liquid crystal tunable filter and a high intensity fiber light source. A Labview interface was programmed for equipment control, synchronization, image acquisition, processing, and visualization. Multispectral images captured by the CCD camera were used to reconstruct the tissue oxygenation map. Dynamic thermographic images captured by the infrared camera were used to reconstruct the vascular function map. Cutaneous tissue oxygenation and vascular function images were co-registered through fiduciary markers. The performance characteristics of the dual-mode image system were tested in humans.

A dual-mode imaging system was developed for non-contact assessment of cutaneous tissue oxygenation and vascular function.

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health ...

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

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 ...

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) ...

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 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.

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 ...

Laparoscopic Oocyte Retrieval and Cryopreservation during Vaginoplasty for Treatment of Mayer-Rokitansky-Kuster-Hauser Syndrome

In Mayer-Rokitansky-Kuster-Hauser Syndrome (MRKHS) patients who are scheduled for laparoscopic vaginoplasty and have a desire for biological motherhood, we propose that a concomitant laparoscopic oocyte retrieval for cryopreservation is performed. Oocyte retrieval is pursued at the beginning of the laparoscopy. Right and left 5 mm trocars are positioned, through which a 17 G ovum aspiration needle is used for puncture of the right and left ovaries, respectively. To facilitate exposure of the follicles, the ovaries are mobilized and held with laparoscopic forceps.
When aspirating multiple follicles near each other, the needle tip is retained in the ovary to reduce the number of times that the ovarian cortex is transfixed and due to the inherent risk of bleeding. Subsequent steps are unchanged compared to the Davydov laparoscopic modified technique for vaginoplasty. Prior to surgery, controlled ovarian stimulation is performed with a gonadotropin hormone-releasing hormone (Gn-RH) antagonist protocol, and the concomitant procedure of oocyte retrieval and vaginoplasty is scheduled 36 h after the final follicular maturation trigger. Follicular fluid is collected in the same 10 mL sterile tubes used during transvaginal oocyte retrieval and transferred in a warming block (37 &#176;C) to the assisted reproduction laboratory, where mature (metaphase II) oocytes are vitrified.
In this case, a series of 23 women with MRKH, oocytes were successfully retrieved and cryopreserved in all patients; vaginoplasty was subsequently conducted without modifications, and the inpatient and outpatient postoperative care (day of urinary catheter removal, day of hospital discharge, dilator use, and comfort at follow-up) remained unaffected. One postoperative complication occurred in one patient (fever developing on day 5 post surgery and intraperitoneal fluid detection on transabdominal ultrasound) and resolved after conservative treatment. Rather than performing surgical vaginoplasty and delaying oocyte retrieval in MRKH patients, this approach combines both procedures in a single laparoscopy, thereby minimizing surgical invasiveness and anesthesiologic risks.

A dedicated team can offer Mayer-Rokitansky-Kuster-Hauser patients the option to perform controlled ovarian stimulation and oocyte cryopreservation at the time of laparoscopic vaginoplasty.

In Mayer-Rokitansky-Kuster-Hauser Syndrome (MRKHS) patients who are scheduled for laparoscopic vaginoplasty and have a desire for biological motherhood, ...

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine system, but the complex structural relationships and three-dimensional (3D) vasculature architectures of the ovary are not well described. In order to visualize the 3D connections and architecture of blood vessels in the intact ovary, the first important step is to make the ovary optically clear. In order to avoid tissue shrinkage, we used the hydrogel fixation-based passive CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/ Immunostaining/In situ-hybridization-compatible Tissue Hydrogel) protocol method to clear an intact ovary. Immunostaining, advanced multiphoton confocal microscopy, and 3D image-reconstructions were then used for the visualization of ovarian vessels and follicular capillaries. Using this approach, we showed a significant positive correlation (P &#60;0.01) between the length of the follicular capillaries and volume of the follicular wall.

Here we present an adaptation of the passive CLARITY and 3D reconstruction method for visualization of the ovarian vasculature and follicular capillaries in intact mouse ovaries.

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine ...

Biology

Bovine Ovarian Cortex Tissue Culture

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors from somatic cells and cumulus cell-oocyte communication. Little is known about the ovarian microenvironment and how the cytokines and steroids produced in the surrounding milieu affect follicle progression or arrest. In vitro culture of ovarian cortex enables follicles to develop in a normalized environment that remains supported by adjacent stroma. Our objective was to determine the effect of nutritional Stair-Step diet on the ovarian microenvironment (follicle development, steroid, and cytokine production) through in vitro culture of bovine ovarian cortex. To accomplish this, ovarian cortical pieces were removed from heifers undergoing two different nutritionally developed schemes prior to puberty: Control (traditional nutrition development) and Stair-Step (feeding and restriction during development) that were cut into approximately 0.5-1 mm3 pieces. These pieces were subsequently passed through a series of washes and positioned on a tissue culture insert that is set into a well containing Waymouth&#39;s culture medium. Ovarian cortex was cultured for 7 days with daily culture media changes. Histological sectioning was performed to determine follicle stage changes before and after the culture to determine effects of nutrition and impact of culture without additional treatment. Cortex culture medium was pooled over days to measure steroids, steroid metabolites, and cytokines. There were tendencies for increased steroid hormones in ovarian microenvironment that allowed for follicle progression in the Stair-Step versus Control ovarian cortex cultures. The ovarian cortex culture technique allows for a better understanding of the ovarian microenvironment, and how alterations in endocrine secretion may affect follicle progression and growth from both in vivo and in vitro treatments. This culture method may also prove beneficial for testing potential therapeutics that may improve follicle progression in women to promote fertility.

In vitro culture of bovine ovarian cortex and the effect of nutritional Stair-step diet on ovarian microenvironment is presented. Ovarian cortex pieces were cultured for seven days and steroids, cytokines, and follicle stages were evaluated. The Stair-Step diet treatment had increased steroidogenesis resulting in follicle progression in culture.

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors ...

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

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