<meta charset="utf8"></head><body>Valutazione della vitalità follicolare nei tessuti della corteccia ovarica umana vitrificata

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		<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>
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			<td>Calcein AM</td>
			<td>Merck</td>
			<td>17783</td>
			<td></td>
		</tr>
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			<td>Collagenase type 1A</td>
			<td>Merck</td>
			<td>C2674</td>
			<td></td>
		</tr>
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			<td>Cryosure DMSO</td>
			<td>WAK Chemie</td>
			<td>WAK-DMSO-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Custodiol</td>
			<td>Dr. Franz K&#246;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>
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			<td>G-MOPS+</td>
			<td>Vitrolife</td>
			<td>10130</td>
			<td></td>
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			<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>
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vitrification of ovarian cortex tissue to achieve a glassy state of aggregation

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.

<meta charset="utf8"></head><body>Autore in primo piano: Promuovere la conservazione della fertilità nelle giovani pazienti oncologiche attraverso la vitrificazione del tessuto ovarico e la crescit

Vitrificazione del tessuto della corteccia ovarica per raggiungere uno stato vetroso di aggregazione

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

Research

JoVE Journal

Medicine

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

894 Views.  University Hospital of Bonn. Lo scopo del nostro gruppo di ricerca è la protezione della fertilità nelle giovani pazienti oncologiche. È importante migliorare i protocolli esistenti per aumentare le possibilità di una gravidanza successiva. Ciò include la vitrificazione del tessuto ovarico e anche la crescita in vitro dei follicoli Con la crescita in vitro del follicolo ovarico.Il punto cruciale è l'approccio alla coltura tissutale in più fasi prima della procedura di isola...

Video: Autore in primo piano: Promuovere la conservazione della fertilità nelle giovani pazienti oncologiche attraverso la vitrificazione del tessuto ovarico e la crescit

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.

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