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: 3.02). 08/2010; 21(6):819-28. DOI: 10.1016/j.rbmo.2010.07.008
Source: PubMed


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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    • "After semithin sectioning, the 60 nm thick sections were collected on 200 mesh grids, stained with uranyl acetate followed by lead citrate and viewed using a Philips 410 T transmission electron microscope at 80 kV, in order to evaluate the ultrastructural features of follicles before and after vitrification/warming. Chromatin pattern, integrity of organelles, and membranes were carefully screened for oocytes, granulosa as well as stromal cells, according to previously reported subcellular criteria [5]. "
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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.
    Full-text · Article · Apr 2014
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    • "The remaining tissue was maintained in PBS + 10% HS solution until cryopreservation. Samples were cryopreserved using a slowfreezing/rapid-thawing protocol, according to Fabbri et al. [22]. At the end of the cooling program, the cryovials were transferred into liquid nitrogen and stored until thawing. "
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    ABSTRACT: 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.
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