Optimising vitrification of human oocytes using multiple cryoprotectants and morphological and functional assessment.

Oocyte vitrification is a clinical practice that allows preservation of fertility potential in women. Vitrification involves quick cooling using high concentrations of cryoprotectants to minimise freezing injuries. However, high concentrations of cryoprotectants have detrimental effects on oocyte quality and eventually the offspring. In addition, current assessment of oocyte quality after vitrification is commonly based only on the morphological appearance of the oocyte, raising concerns regarding its efficiency. Using both morphological and functional assessments, the present study investigated whether combinations of cryoprotectants at lower individual concentrations result in better cryosurvival rates than single cryoprotectants at higher concentrations. Surplus oocytes from IVF patients were vitrified within 24h after retrieval using the Cryotop method with several cryoprotectants, either individually or in combination. The morphological and functional quality of the vitrified oocytes was investigated using light microscopy and computer-based quantification of mitochondrial integrity, respectively. Oocyte quality was significantly higher using a combination of cryoprotectants than vitrification with individual cryoprotectants. In addition, the quality of vitrified oocyte varied depending on the cryoprotectants and type of combination used. The results of the present study indicate that observations based purely on the morphological appearance of the oocyte to assess the cryosurvival rate are insufficient and sometimes misleading. The outcome will have a significant implication in the area of human oocyte cryopreservation as an important approach for fertility preservation.

Assessment of follicular development in cryopreserved primate ovarian tissue by xenografting: prepubertal tissues are less sensitive to the choice of cryoprotectant.

Improvements in cancer survival rates have renewed interest in the cryopreservation of ovarian tissue for fertility preservation. We used the marmoset as a non-human primate model to assess the effect of different cryoprotectives on follicular viability of prepubertal compared to adult ovarian tissue following xenografting. Cryopreservation was performed with dimethylsulfoxide (DMSO), 1,2-propanediol (PrOH), or ethylene glycol (EG) using a slow freezing protocol. Subsequently, nude mice received eight grafts per animal from the DMSO and the PrOH groups for a 4-week grafting period. Fresh, cryopreserved-thawed, and xenografted tissues were serially sectioned and evaluated for the number and morphology of follicles. In adult tissue, the percentage of morphologically normal primordial follicles significantly decreased from 41.2 ± 4.5% (fresh) to 13.6 ± 1.8 (DMSO), 9.5 ± 1.7 (PrOH), or 6.8 ± 1.0 (EG) following cryopreservation. After xenografting, the percentage of morphologically normal primordial (26.2 ± 2.5%) and primary follicles (28.1 ± 5.4%) in the DMSO group was significantly higher than that in the PrOH group (12.2 ± 3 and 5.4 ± 2.1% respectively). Proliferating cell nuclear antigen (PCNA) staining suggests the resumption of proliferative activity in all cellular compartments. In prepubertal tissues, primordial but not primary follicles display a similar sensitivity to cryopreservation, and no significant differences between DMSO and PrOH following xenografting were observed. In conclusion, DMSO shows a superior protective effect on follicular morphology compared with PrOH and EG in cryopreserved tissues. Xenografting has confirmed better efficacy of DMSO versus PrOH in adult but not in prepubertal tissues, probably owing to a greater capacity of younger animals to compensate for cryoinjury.

Measurement of skin-fold thickness in the guinea pig. Assessment of edema-inducing capacity of cutting fluids, acids, alkalis, formalin and dimethyl sulfoxide.

The rabbit has been used for decades for predictive testing of skin irritancy, but in recent years, the guinea pig has been suggested as an alternative, especially for assessment of one of the components of the irritant reaction: edema (fluid accumulation). A method based on skin-fold measurements with Harpenden calipers has been developed and modified. In previous papers, experience with sodium lauryl sulphate, nonanoic acid and industrial solvents was reported. The present results concern the use of cutting fluids, buffered and unbuffered acid and alkaline solutions, formalin and dimethyl sulfoxide. This inexpensive and comparatively unsophisticated method afforded clear dose-response relationships and good discriminating power. The only exception was the acid and alkaline solutions, where no changes in skin-fold thickness were observed despite their documented irritant potential. The appearance of erythema (visual scoring) and the increase in skin-fold thickness, and their relationship, are discussed with some illustrative examples. The method described is now well standardized and is suited for predictive testing of the edema-inducing capacity of chemicals and products.