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Transmission electron microscopy of human cryopreserved ovarian tissue after long-term storage. A primordial/primary follicle: the oocyte shows a nucleus (n) having finely dispersed chromatin and cytoplasm (oc) rich in organelles; a layer of flattened/cuboidal granulosa cells (gc) surrounding oocyte cytoplasm; B perinuclear aggregates of rounded mitochondria (m) having moderate electron dense matrices, ( ) nuclear pores; C Golgi apparatus; D annulate lamellae consist of membrane-bound cisternae traversed by pore complexes; E granulosa cells with oval shaped nuclei (n); f stromal cells with oval shaped nuclei (n) and slight band of peripherically condensed chromatin (pch). Empty interstitial areas are detectable ( )
Source publication
Background
Ovarian tissue cryopreservation is an emerging technique, also addressed to very young cancer patients, for whom it is not possible to perform an ovarian stimulation for oocytes freezing, before gonadotoxic treatment. In this cases, ovarian tissue must be cryopreserved for a long period of time and it is very important to know if it main...
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Objective
This retrospective study compared the effect of the luteal phase ovarian stimulation protocol (LP group) with the gonadotrophin-releasing hormone (GnRH) antagonist protocol (AN group) in women with poor ovarian responses.
Methods
Ovarian stimulation was initiated with 225 IU of human gonadotrophin (hMG) daily. When the dominant follicle...
Citations
... Furthermore, two independent and complementary systematic reviews and meta-analyses have recently been published supporting the safety of the long-term cryostorage of human embryos at both the cleavage and blastocyst stages (Canosa et al., 2022;Ma et al., 2021). There is also reassuring evidence concerning frozen-thawed ovarian tissue transplantation after a prolonged storage time (Andersen et al., 2012;Fabbri et al., 2016). ...
Among the wide range of procedures performed by clinical embryologists, the cryopreservation of reproductive cells and tissues represents a fundamental task in the daily routine. Indeed, cryopreservation procedures can be considered a subspecialty of medically assisted reproductive technology (ART), having the same relevance as sperm injection or embryo biopsy for preimplantation genetic testing. However, although a great deal of care has been devoted to optimizing cryopreservation protocols, the same energy has only recently been spent on developing and implementing strategies for the safe and reliable storage and transport of reproductive specimens. Herein, we have summarized the content of the available guidelines, the risks, the needs and the future perspectives regarding the management of cryopreservation biorepositories used in ART.
... Fresh and frozen/thawed tissue samples were analyzed according to Fabbri et al. (17). Briefly, the strips were embedded in paraffin blocks and nine serial sections 4 mm thickness per block were obtained: the first, third and seventh were stained with hematoxylin and eosin (Merck, Darmstadt, Germany), to assess the morphological characteristics of follicles and stromal cells. ...
... Usually, the ovarian tissue of these patients remains frozen for a long period of time. We recently demonstrated that ovarian tissue stored for 18 years maintained its morpho-functional integrity (17). In addition, we recently reported ovarian function recovery after heterotopic OTT stored for 11 and 15 years, providing strong evidence that time has no impact on tissue preservation and ovarian function recovery (28). ...
Objective
To report the 20-year experience in ovarian tissue cryopreservation (OTC) and ovarian tissue transplantation (OTT) of the Bologna clinical center (Bologna, Italy).
Design
Retrospective cohort study.
Patients
1026 pediatrics and women aged between 2 and 38 years who underwent OTC and OTT between January 2002 to January 2022.
Results
Of the 1026 patients, 238 (22.8%) were pediatrics (≤ 17 years, Group 1) and 788 (77.2%) were adult women (range 18-38 years, Group 2). In Group 1, 184 (77.3%) patients had malignant diseases and 54 (22.7%) had non-malignant diseases. In Group 2, 746 (94.7%) patients had malignant diseases and 42 (5.3%) had non-malignant diseases. No real complications were observed during surgery. In all the samples analyzed most of the follicles were in the resting stage, while only a few follicles were growing. In both fresh and thawed samples, follicular density was higher in Group 1 than in Group 2 (p < 0.01). Regardless of age, good preservation of follicles and stroma was observed in fresh and thawed ovarian tissue by histological and immunohistochemical analyses (estrogen and progesterone receptors; Ki67 and Bcl2 markers; TUNEL). To date, out of 1026 total women, 812 (79.1%) had their tissue stored. Sixty-eight (6.6%) patients died from their primary disease. Twenty-four (2.3%) women performed 33 OTTs between December 2011 and January 2022. Restoration of menstruation was observed in 15 out of 17 menopausal women. Six pregnancies were achieved, two hesitated in abortion and four in the birth of healthy babies.
Conclusion
OTC is the only fertility preservation technique applicable in pre-pubertal/pediatrics and in adult patients when stimulation for oocytes/embryos cryopreservation is not possible. The reported data can help future patients and physicians in their discussions and decisions about the need and possibilities of preserving ovarian function.
... The expression of surface markers as well as in vitro differentiation potential was not significantly different from fresh control. In reproductive medicine, Fabbri et al. found no negative impact of long-term storage (18 years in liquid nitrogen) of cryopreserved ovarian tissue on its viability and ultrastructure (Fabbri et al. 2016). The results of van der Ven et al. indicated a pregnancy rate to be around 30% in 74 women arising from transplantation of cryopreserved ovarian tissue which had been stored for 4 years in liquid nitrogen (van der Ven et al. 2016). ...
... The expression of surface markers as well as in vitro differentiation potential was not significantly different from fresh control. In reproductive medicine, Fabbri et al. found no negative impact of long-term storage (18 years in liquid nitrogen) of cryopreserved ovarian tissue on its viability and ultrastructure (Fabbri et al. 2016). The results of van der Ven et al. indicated a pregnancy rate to be around 30% in 74 women arising from transplantation of cryopreserved ovarian tissue which had been stored for 4 years in liquid nitrogen (van der Ven et al. 2016). ...
... Therefore, accurate histological evaluation of ovarian tissue is critical. Ovarian tissue analysis involves classifying follicles according to their developmental stage in addition to assessing follicle health on fixed sections, both of which are employed by many research groups studying ovarian function (Fenwick and Hurst, 2002;Pangas et al., 2007;Chambers et al., 2010;Fenwick et al., 2013;Kim et al., 2015;Fabbri et al., 2016;Stefansdottir et al., 2016;Chiti et al., 2017;McLaughlin et al., 2018;Lee et al., 2019;Winship et al., 2019). ...
Study question:
Can ovarian tissue morphology be better preserved whilst enabling histological molecular analyses following fixation with a novel fixative, neutral buffered formalin (NBF) with 5% acetic acid (referred to hereafter as Form-Acetic)?
Summary answer:
Fixation with Form-Acetic improved ovarian tissue histology compared to NBF in multiple species while still enabling histological molecular analyses.
What is known already:
NBF fixation results in tissue shrinkage in various tissue types including the ovary. Components of ovarian tissue, notably follicles, are particularly susceptible to NBF-induced morphological alterations and can lead to data misrepresentation. Bouin's solution (which contains 5% acetic acid) better preserves tissue architecture compared to NBF but is limited for immunohistochemical analyses.
Study design, size, duration:
A comparison of routinely used fixatives, NBF and Bouin's, and a new fixative, Form-Acetic was carried out. Ovarian tissue was used from three different species: human (n = 5 patients), sheep (n = 3; 6 ovaries; 3 animals per condition) and mouse (n = 14 mice; 3 ovaries from 3 different animals per condition).
Participants/materials, setting, methods:
Ovarian tissue from humans (aged 13 weeks to 32 years), sheep (reproductively young i.e. 3-6 months) and mice (10 weeks old) were obtained and fixed in 2 ml NBF, Bouin's or Form-Acetic for 4, 8, and 24 h at room temperature. Tissues were embedded and sectioned. Five-micron sections were stained with haemotoxylin and eosin (H&E) and the percentage of artefact (clear space as a result of shrinkage) between ovarian structures was calculated. Additional histological staining using Periodic acid-Schiff and Masson's trichrome were performed on 8 and 24 h NBF, Bouin's and Form-Acetic fixed samples to assess the compatibility of the new fixative with stains. On ovarian tissue fixed for both 8 and 24 h in NBF and Form-Acetic, immunohistochemistry (IHC) studies to detect FOXO3a, FoxL2, collagen IV, laminin and anti-Müllerian hormone (AMH) proteins were performed in addition to the terminal deoxynucleotidyl transferase nick end labelling (TUNEL) assay to determine the compatibility of Form-Acetic fixation with types of histological molecular analyses.
Main results and the role of chance:
Fixation in Form-Acetic improved ovarian tissue morphology compared to NBF from all three species and either slightly improved or was comparable to Bouin's for human, mouse and sheep tissues. Form-Acetic was compatible with H&E, Periodic acid-Schiff and Masson's trichrome staining and all proteins (FOXO3a, FoxL2, collagen IV and laminin and AMH) could be detected via IHC. Furthermore, Form-Acetic, unlike NBF, enabled antigen recognition for most of the proteins tested without the need for antigen retrieval. Form-Acetic also enabled the detection of damaged DNA via the TUNEL assay using fluorescence.
Large scale data:
N/A.
Limitations, reasons for caution:
In this study, IHC analysis was performed on a select number of protein types in ovarian tissue thus encouraging further studies to confirm the use of Form-Acetic in enabling the detection of a wider range of protein forms in addition to other tissue types.
Wider implications of the findings:
The simplicity in preparation of Form-Acetic and its superior preservative properties whilst enabling forms of histological molecular analyses make it a highly valuable tool for studying ovarian tissue. We, therefore, recommend that Form-Acetic replaces currently used fixatives and encourage others to introduce it into their research workflow.
Study funding/competing interest(s):
This work was supported by the Oxford Medical Research Council Doctoral Training Programme (Oxford MRC-DTP) grant awarded to B.D.B. (Grant no. MR/N013468/1), the Fondation Hoffmann supporting R.A. and the Petroleum Technology Development Fund (PTDF) awarded to B.V.A.
... 33 In one study spanning 18 years, three cryopreserved human ovaries were evaluated for tissue quality, maintenance of ovarian potential, and markers of viability. The markers were similar in tissue samples that were analyzed immediately after slow cryopreservation, after 120 days, and after 18 years, 34 suggesting that long-term storage did not adversely affect function. ...
Intensive treatments necessary to treat some childhood malignancies and other conditions, as well as certain anatomic variations, may lead to infertility in adulthood. Until recently, no fertility preservation options for prepubertal females were available. However, ovarian tissue cryopreservation has emerged as a safe and effective option for these children. In the next several years, it is likely that more pediatric patients, their families, and medical teams will pursue an ovarian cryopreservation protocol at their institutions. Patient selection, consenting, and laparoscopic oophorectomy can be done at many centers. Then, the ovarian tissue is initially processed and transported to a specialized center for processing for cryopreservation. The cryopreservation techniques are best performed at appropriately certified centers processing high volumes of reproductive cells/tissues with expert personnel and specialized equipment. This article aims to provide an overview for pediatric biobank professionals who may be called to participate in this or similar protocols.
... Because antral follicles are very large and surrounded by multiple layers of cuboidal granulosa cells, they could not tolerate the freezing damage and maintain their structural integrity during vitrification [11]. Nevertheless, preantral follicles can maintain their follicular structure and survive the transplantation procedure [10,33]. Vitrification is a kind of technology that is easy to perform for the simple and quick preservation of ovaries. ...
Background
After ovarian tissue transplantation, ischemia-reperfusion injury and free radicals cause follicle depletion and apoptosis. Therefore, the use of antioxidants to reduce the production of free radicals is an important method to address the consequences of ischemia-reperfusion injury. Resveratrol is a natural active polyphenol compound with anti-inflammatory, antitumor, strong antioxidant and anti-free radical properties. The aim of this study was to investigate whether resveratrol could improve the effect of autologous ovarian transplantation after cryopreserve-thawn mouse ovarian tissue.
Methods
Whole-ovary vitrification and autotransplantation models were used to investigate the effects of resveratrol. Six-week-old female mice from the Institute of Cancer Research (ICR) were subjected to vitrification. All ovaries were preserved in liquid nitrogen for 1 week before being thawed. After thawing, ovarian tissues were autotransplanted in the bilateral kidney capsules. Mice ( n = 72) were randomly divided into four groups to determine the optimal concentration of resveratrol (experiment I).
Treatments were given as follows: saline, 5 mg/kg resveratrol, 15 mg/kg resveratrol and 45 mg/kg resveratrol, which were administered orally for one week. Grafted ovaries were collected for analysis on days 3, 7, and 21 after transplantation. Ovarian follicle morphology was assessed by hematoxylin and eosin staining. Serum FSH and E 2 levels were measured to estimate the transplanted ovarian reserve and endocrine function. Other mice were randomly divided into two groups—saline and 45 mg/kg resveratrol to further evaluate the effect of resveratrol and explore the mechanisms underlying this effect (experiment II). Ovarian follicle apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays. Immunohistochemistry, qRT-PCR and western blotting (MDA, SOD, NF-κB, IL-6 and SIRT1) were used to explore the mechanisms of resveratrol. Moreover, oocytes derived from autotransplanted ovaries at 21 days were cultured and fertilized in vitro.
Results
The proportions of morphologically normal (G1) follicles at 3, 7 and 21 days were significantly higher in the 45 mg/kg resveratrol group than in the saline group. The TUNEL-stained follicles (%) at 7 days were significantly decreased in the 45 mg/kg resveratrol group compared with the saline group. Western blot analysis revealed that SOD2 and SIRT1 levels were significantly higher in the 45 mg/kg resveratrol group than in the saline group at day 7 and that MDA and NF-κB levels were lower in the saline group on day 3. Likewise, IL-6 was lower in the saline group on day 7. These results are basically consistent with the qRT-PCR results. In addition, the mean number of retrieved oocytes and fertilization and cleavage were significantly increased in the 45 mg/kg resveratrol group compared with the saline group.
Conclusions
Administration of resveratrol could improve the quality of cryopreserved mouse ovarian tissue after transplantation and the embryo outcome, through anti-inflammatory and antioxidative mechanisms.
... Because antral follicles are very large and surrounded by multiple layers of cuboidal granulosa cells, they could not tolerate the freezing damage and maintain their structural integrity during vitri cation [11]. Nevertheless, preantral follicles can maintain their follicular structure and survive the transplantation procedure [10,34]. Vitri cation is a kind of technology that is easy to perform for the simple and quick preservation of ovaries. ...
Background
After ovarian tissue transplantation, ischemia-reperfusion injury and free radicals cause follicle depletion and apoptosis. Therefore, the use of antioxidants to reduce the production of free radicals is an important method to address the consequences of ischemia-reperfusion injury. Resveratrol is a natural active polyphenol compound with anti-inflammatory, antitumor, strong antioxidant and anti-free radical properties. The aim of this study was to investigate whether resveratrol could improve the effect of autologous ovarian transplantation after cryopreserve-thawn mouse ovarian tissue.Methods
Whole-ovary vitrification and autotransplantation models were used to investigate the effects of resveratrol. Six-week-old female mice from the Institute of Cancer Research (ICR) were subjected to vitrification. All ovaries were preserved in liquid nitrogen for 1 week before being thawed. After thawing, ovarian tissues were autotransplanted in the bilateral kidney capsules. Mice (n=72) were randomly divided into four groups to determine the optimal concentration of resveratrol (experiment I). Treatments were given as follows: saline, 5 mg/kg resveratrol, 15 mg/kg resveratrol and 45 mg/kg resveratrol, which were administered orally for one week. Grafted ovaries were collected for analysis on days 3, 7, and 21 after transplantation. Ovarian follicle morphology was assessed by hematoxylin and eosin staining. Serum FSH and E 2 levels were measured to estimate the transplanted ovarian reserve and endocrine function. Other mice were randomly divided into two groups—saline and 45 mg/kg resveratrol to further evaluate the effect of resveratrol and explore the mechanisms underlying this effect (experiment II). Ovarian follicle apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays. Immunohistochemistry, qRT-PCR and western blotting (MDA, SOD, NF-κB, IL-6 and SIRT1) were used to explore the mechanisms of resveratrol. Moreover, oocytes derived from autotransplanted ovaries at 21 days were cultured and fertilized in vitro. ResultsThe proportions of morphologically normal (G1) follicles at 3, 7 and 21 days were significantly higher in the 45 mg/kg resveratrol group than in the saline group. The TUNEL-stained follicles (%) at 7 days were significantly decreased in the 45 mg/kg resveratrol group compared with the saline group. Western blot analysis revealed that SOD2 and SIRT1 levels were significantly higher in the 45mg/kg resveratrol group than in the saline group at day 7 and that MDA and NF-κB levels were lower in the saline group on day 3. Likewise, IL-6 was lower in the saline group on day 7. These results are basically consistent with the qRT-PCR results. In addition, the mean number of retrieved oocytes and fertilization and cleavage were significantly increased in the 45 mg/kg resveratrol group compared with the saline group. Conclusions
Administration of resveratrol could improve the quality of cryopreserved mouse ovarian tissue after transplantation and the embryo outcome, through anti-inflammatory and antioxidative mechanisms.
... Each fragment can be cryopreserved individually for long-term storage using slow-freezing or vitrification techniques (these options are discussed in detail below). Analysis of tissue stored for 18 years showed that long storage did not affect follicular morphology and survival (Fabbri, et al., 2016a). Cryopreserved ovarian tissue using the slow-freezing procedure and stored for more than 14 years has been transplanted with success (Gellert, et al., 2018). ...
The guideline is aimed at healthcare professionals who are involved in information provision and
decision making with women scheduled to undergo gonadotoxic treatments or women and
transmen considering fertility preservation for other reasons. This includes, but is not limited to,
reproductive medicine specialists, endocrinologists, oncologists and oncological surgeons and
gynaecologists, paramedical and reproductive biologists (including embryologists), and
geneticists.
For the benefit of patient education and shared decision-making, a patient version of this guideline
will be developed.
... Among the other techniques aiming to preserve the reproductive potential in oncological patients, ovarian tissue cryopreservation is currently considered an innovative or established method [10][11][12] , representing an option for patients who require immediate gonadotoxic treatment of aggressive malignancies when there is not enough time to allow the woman to undergo ovulation induction, oocytes retrieval and cryopreservation of oocytes and/or embryos. Moreover, ovarian tissue cryopreservation is the only option available for fertility preservation in young girls in prepubertal age or in women affected by hormone-sensitive malignancies 1,4,13,14 . Other potential indications for this fertility preservation technique are related to patients with genetic mutations that pose a high risk for premature ovarian failure and are not eligible for other fertility preservation approaches 4,7 . ...
Recently, the interest of the scientific community is focused on the application of tissue engineering approach for the fertility restoration. In this paper innovative patterned electrospun fibrous scaffolds were fabricated and used as 3D system for porcine follicles culture. The obtained scaffolds demonstrated to be a suitable support which did not alter or interfere with the typical spherical follicles morphology. The fibrillar structure of the scaffolds mimics the morphology of the healthy native tissue. The use of porcine follicles implied many advantages respect to the use of mouse model. Relevant results showed that more than the scaffold pattern and struts dimension, the selection of proper biomaterials improve the follicles adhesion and development.
