The use of near-infrared imaging with indocyanine green in the ovarian tissue transplantation: a case report.

The request for fertility preservation has consistently increased in recent years. To our knowledge this case report is the first to describe the application of near-infrared intraoperative imaging using indocyanine green (NIR-ICG) during ovarian tissue transplantation (OTT), to assist surgeon choosing the site of implantation of ovarian fragments. OTT was performed in a 42-year-old woman using NIR-ICG to evaluate the vascularisation of peritoneal area as the site of implantation for the ovarian graft. we believe this new approach could be useful in identifying the best reimplantation site.

Epigallocatechin-3-gallate inhibits doxorubicin-induced inflammation on human ovarian tissue.

Chemotherapy protocol can destroy the reproductive potential of young cancer patients. Doxorubicin (DOX) is a potent anthracycline commonly used in the treatment of numerous malignancies. The purpose of the study was to evaluate the ovarian toxicity of DOX via inflammation and the possible protective effect of the green tea polyphenol epigallocatechin-3-gallate (EGCG). Ovarian tissue of three patients was cultured with 1 µg/ml DOX and/or 10 µg/ml EGCG for 24 and 48 h. Levels of inflammatory factors were determined by quantitative Real-Time PCR, western blot, zimography, and multiplex bead-based immunoassay. Morphological evaluation, damaged follicle count and TUNEL assay were also performed. DOX influenced inflammatory responses by inducing a significant increase in the expression of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and cyclooxigenase-2 (COX-2), of inflammatory interleukins (IL), such as interleukin-6 (IL-6) and interleukin-8 (IL-8), and the inflammatory proteins mediators metalloproteinase-2 and metalloproteinase-9 (MMP2 and MMP9). IL-8 secretion in the culture supernatants and MMP9 activity also significantly raised after DOX treatment. Moreover, a histological evaluation of the ovarian tissue showed morphological damage to follicles and stroma after DOX exposure. EGCG significantly reduced DOX-induced inflammatory responses and improved the preservation of follicles. DOX-induced inflammation could be responsible for the ovarian function impairment of chemotherapy. EGCG could have a protective role in reducing DOX-mediated inflammatory responses in human ovarian tissue.

Successful achievement after heterotopic transplantations of long-term stored ovarian tissue in Hodgkin's lymphoma survivor.

In this case report, we describe the outcomes of two heterotopic transplantations of cryopreserved ovarian tissue performed in a patient with HL, after 11 and 15 years of storage. At the age of 30, the patient underwent laparoscopy to collect ovarian tissue for cryopreservation before chemotherapy and radiotherapy. Eleven years later she experienced premature ovarian failure (POF). As the patient was only interested in endocrine function recovery, two heterotopic ovarian tissue transplantations were performed in the abdominal wall above the rectus muscle respectively 11 and 15 years after cryopreservation. Before transplantation, ovarian samples were analyzed to assess neoplastic contamination and tissue quality. The analysis on thawed ovarian tissue did not reveal micrometastasis and it showed well-preserved follicles and stroma. After both ovarian tissue grafting, menopausal symptoms ceased. The patient had periods approximately every 30-days and hormonal levels were within the premenopausal range. The endocrine function lasted 3-years after the first heterotopic transplantation and is still ongoing after second transplantation. Cryopreservation of ovarian tissue should be proposed to HL patients, as the incidence of POF as a long-term complication is not negligible. In these patients heterotopic transplantation is a useful tool to eliminate menopausal symptoms, preventing osteoporosis and reducing cardiovascular risks.

Morphological, ultrastructural and functional imaging of frozen/thawed and vitrified/warmed human ovarian tissue retrieved from oncological patients.

STUDY QUESTION: Which is the best method for human ovarian tissue cryopreservation: slow freezing/rapid thawing (SF/RT) or vitrification/warming (V/W)? SUMMARY ANSWER: The conventional SF/RT protocol used in this study seems to better preserve the morpho-functional status of human cryopreserved ovarian tissue than the used open carrier V/W protocol. WHAT IS KNOWN ALREADY: Cryopreservation of human ovarian tissue is generally performed using the SF/RT method. However, reduction in the follicular pool and stroma damage are often observed. An emerging alternative procedure is represented by V/W which seems to allow the maintenance of the morphological integrity of the stroma. STUDY DESIGN, SIZE, DURATION: This is a retrospective cohort study including six patients affected by oncological diseases and enrolled from January to December 2014. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian tissue was laparoscopically harvested from the right and left ovaries and was cryopreserved using a routinary SF/RT protocol or a V/W method, involving tissue incubation in two solutions (containing propylene glycol, ethylene glycol and sucrose at different concentrations) and vitrification in an open system. For each patient, three pieces from each ovary were collected at the time of laparoscopy (fresh tissue) and after storage (SF/RT or V/W) and processed for light microscopy (LM) and transmission electron microscopy (TEM), to assess the morphological and ultrastructural features of follicles and stroma, and for laser scanning confocal microscopy (LSCM), to determine the functional energetic/redox stroma status. The preservation status of SF/RT and V/W ovarian tissues was compared with that of fresh ones, as well as between them. MAIN RESULTS AND THE ROLE OF CHANCE: By LM and TEM, SF/RT and V/W samples showed cryodamage of small entity. Interstitial oedema and increased stromal cell vacuolization and chromatin clumping were observed in SF/RT samples; in contrast, V/W samples showed oocyte nuclei with slightly thickened chromatin and irregular shapes. The functional imaging analysis by LSCM revealed that the mitochondrial activity and intracellular reactive oxygen species levels were reduced both in SF/RT and in V/W samples compared with fresh samples. The study also showed progressive dysfunction of the mitochondrial activity going from the outer to the inner serial section of the ovarian cortex. The reduction of mitochondrial activity of V/W samples compared with fresh samples was significantly higher in the inner section than in the outer section. LIMITATIONS, REASONS FOR CAUTION: The results report the bioenergetic and oxidative status assessment of fresh and cryopreserved human ovarian tissue by LSCM, a technique recently applied to tissue samples. The use of LSCM on human ovarian tissues after SF/RT or V/W is a new application that requires validation. The procedures for mitochondrial staining with functional probes and fixing are not yet standardized. Xenografting of the cryopreserved ovarian tissue in severe combined immunodeficient mice and in vitro culture have not yet been performed. WIDER IMPLICATIONS OF THE FINDINGS: The identification of a cryopreservation method able to maintain the morpho-functional integrity of the ovarian tissue and a number of follicles comparable with those observed in fresh tissue might optimize results in clinical practice, in terms of recovery, duration of ovarian function and increased delivery outcomes after replanting. The SF/RT protocol allowed better morpho-functional tissue integrity than the V/W procedure. STUDY FUNDING/COMPETING INTERESTS: Funding was provided by Fondazione del Monte di Bologna e Ravenna, Italy. Dr N.A.M. was granted by the project ONEV MIUR PONa3 00134-n.254/R&C 18 5 2011 and the project GR-2011-02351396 (Ministry of Health, Young Researchers Grant 2011/2012). There are no competing interests. TRIAL REGISTRATION NUMBER: Clinical trial 74/2001/0 (approved:13 2 2002): 'Pilot study on cryopreservation of human ovarian tissue: morphological and immunohistochemical analysis before and after cryopreservation'.

Cryopreservation of ovarian tissue in pediatric patients.

Cancer treatments improve the survival rate of children and adolescents; however chemo- and radiotherapy result in gonadal damage leading to acute ovarian failure and sterility. Ovarian tissue cryopreservation allows long-term storage of primordial follicles and represents the only possibility of preserving the potential fertility in prepubertal girls. The aim of the present study is to describe our experience in ovarian tissue cryopreservation in 45 pediatric patients. The number of follicles per square millimeter of the overall section area and follicle quality were evaluated histologically. A strong negative correlation was found between age and follicular density in patients both prior to and after chemotherapy (P < 0.0001). Damage in follicular quality, that is, increased oocyte vacuolization and detachment of the oocyte from granulosa cells, was found after chemotherapy. Ovarian tissue cryopreservation, preferably performed before initiation of chemotherapy, should be offered to pediatric patients, including prepubertal girls, at risk of sterility.