Optimization of protocols for human ovarian tissue cryopreservation with sucrose, 1,2-propanediol and human serum.

Human Reproductive Medicine Unit, S. Orsola-Malpighi Hospital, University of Bologna, via Massarenti 13, 40138 Bologna, Italy.
Reproductive biomedicine online (Impact Factor: 2.98). 08/2010; 21(6):819-28. DOI: 10.1016/j.rbmo.2010.07.008
Source: PubMed

ABSTRACT Chemotherapy and/or radiotherapy protocols have improved the long-term survival of cancer patients. Frequent consequences of antiblastic treatments, used to eradicate malignancies, are the partial loss of ovarian function, which in children and young women can result in permanent sterility. Ovarian tissue cryopreservation implemented before the beginning of treatment may potentially restore fertility. However, the physical effects of cryopreservation can damage oocyte survival and decrease follicular cell integrity and stromal preservation. The aim of this study was to examine the effects of different concentrations of 1,2-propanediol (PROH) and sucrose as cryoprotectants and human serum as protein support. Particular concentrations tested were 1.26, 1.5 and 1.08 mol/l PROH, 0.175, 0.2, 0.224 and 0.3 mol/l of sucrose and 20%, 30% and 40% human serum in the freezing solutions and normal or raised sucrose concentrations in the dilution solutions. Ovarian cortical slices from 13 patients, aged 5-38 years, were cryopreserved using slow freezing-rapid thawing. Tests were conducted using light and transmission electron microscopy. Cryo-damage occurred predominantly in the stromal and follicular cells. The best preservation of morphological characteristics was obtained using the freeze-thaw protocol in which concentrations of cryoprotectants were among the lowest (1.26 mol/l PROH+0.175 mol/l sucrose) with 30% human serum.

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    ABSTRACT: The aim of this study was to develop a vitrification procedure for human ovarian tissue cryopreservation in order to better preserve the ovarian tissue. Large size samples of ovarian tissue retrieved from 15 female-to-male transgender subjects (18-38 years) were vitrified using two solutions (containing propylene glycol, ethylene glycol, and sucrose at different concentrations) in an open system. Light microscopy, transmission electron microscopy, and TUNEL assay were applied to evaluate the efficiency of the vitrification protocol. After vitrification/warming, light microscopy showed oocyte nucleus with slightly thickened chromatin and irregular shape, while granulosa and stromal cells appeared well preserved. Transmission electron microscopy showed oocytes with slightly irregular nuclear shape and finely dispersed chromatin. Clear vacuoles and alterations in cellular organelles were seen in the oocyte cytoplasm. Stromal cells had a moderately dispersed chromatin and homogeneous cytoplasm with slight vacuolization. TUNEL assay revealed the lack of apoptosis induction by vitrification in all ovarian cell types. In conclusion after vitrification/warming the stromal compartment maintained morphological and ultrastructural features similar to fresh tissue, while the oocyte cytoplasm was slightly damaged. Although these data are encouraging, further studies are necessary and essential to optimize vitrification procedure.
    04/2014; 2014:673537. DOI:10.1155/2014/673537
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    ABSTRACT: Aim: To present preliminary results of autotransplantation of cryopreserved ovarian tissue performed at Sant'Orsola-Malpighi Hospital, Bologna, Italy. Materials & methods: Orthotopic transplantation was performed in two women with colorectal and breast cancer, and heterotopic transplantation was performed in one Hodgkin's lymphoma woman. The presence of micrometastasis in the ovarian tissue was checked, and morphological features of ovarian tissue were evaluated before transplantation. Ovarian function was monitored by hormonal and ultrasound-color Doppler examination after transplantation. Results: In all three women, no micrometastasis was found; light and transmission electron microscopy showed well-preserved thawed ovarian tissue. Ovarian function recovery was observed 2-4 months after transplantation. Spontaneous menstrual cycles occurred in two women with normal follicular densities. No periods occurred in the woman with low follicular density at the time of tissue collection. Conclusion: Ovarian tissue cryopreservation and transplantation is a promising approach for preserving ovarian function in women with cancer.
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    ABSTRACT: Purpose: To evaluate the effect of cryopreservation and thawing of ovarian tissue from oncological patients opting for fertility preservation on ovarian tissue viability. Methods: In this prospective cohort study, the ovarian tissue viability before and after cryopreservation and thawing was measured for 25 newly diagnosed oncological patients who had their ovarian tissue cryopreserved. Outcome measures were follicle integrity (histology), follicle viability (Calcein viability assay), steroid hormone production (estradiol and progesterone production in vitro) and overall tissue viability (glucose uptake in vitro). This study was conducted at a Cryobank for storage of ovarian tissue in a university hospital. Results: Cryopreserved/thawed ovarian tissue showed a de- creased glucose uptake when compared to tissue that had not been cryopreserved. In addition, a diminished E2 and P4 production was observed after cryopreservation and thawing, despite the fact that numbers of viable follicles as determined by the Calcein viability assay were comparable. Histological examination revealed a higher percentage of degenerated fol- licles after cryopreservation and thawing. Conclusions: Ovarian tissue cryopreservation and thawing im- pairs the viability of ovarian tissue in oncological patients opting for fertility preservation.
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