BackgroundIn recent years, autotransplantation of cryopreserved ovarian tissue became a promising approach to preserve female fertility. The slow freezing is the most effective technique which resulted in greater live birth incidence so far. Despite that, interest to vitrification of the ovarian tissue is swiftly growing, thereby undermining the necessity for further improvements in the technique. In present study, we evaluated possibilities to increase follicle survival rates adopting innovative multi-protectoral vitrification protocols, applied to the slivers of ovarian cortex or isolated early-antral follicles, frozen individually. These experimental protocols have been compared with with validated vitrification and slow freezing ones, clinically used for female fertility preservation.ResultsThe results showed that third tested variation of experimental vitrification protocol, with four cryoprotectants in relatively low concentrations and applied to pieces of ovarian tissue at 0 °C during equilibration, increased survival rate of ovine ovarian tissue and improved results in comparison with conventional vitrification method. This variation of experimental protocol showed significant increase in percentage of follicles with good morphology (69,3%) in comparison with only commercially available vitrification protocol for ovarian tissue (62,1%). Morphology results were confirmed by TUNEL assay. Analysis of estradiol and progesterone production by cultured individual follicles after freezing/thawing revealed that steroids secretion remained significantly higher after multi-protectoral vitrification and slow freezing protocol, when follicles after standard vitrification protocol demonstrated decline in steroidogenic activity.ConclusionsThe multi-protectoral approach represents a workable solution to improve vitrification outcome on ovarian tissue and isolated follicles. The reduction of individual cryoprotectants concentrations, while maintaining their sufficient cumulative level in the final freezing solution, helps to increase efficiency of the procedure. Moreover, equilibration with lower temperatures helped to decrease even further the toxic effects of cryoprotectants and preserve original quality of ovarian tissue. Therefore, multi-protectoral vitrification can be suggested as an improved method for the clinical cryopreservation of ovarian tissue.

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The ovarian function during reproductive life is related to a normal development of blood vessel neoformation and consequently to the expression of several factors that may upregulate/downregulate follicle and corpus luteum angiogenesis [1,2]. Follicle activation involves vascular endothelial growth factor production and increases blood vessels extension. Numerous morphological studies, mainly performed by means of vascular casting media, analysed the distribution and cyclical rearrangement of ovarian blood vessels in several mammals, in different experimental conditions [3]. Despite the wide literature on the blood vessel distribution in the mammalian ovary, only a few information is available on ovarian angiogenic morphological aspects of developing and atretic follicles, especially in domestic animals, that shows morphofunctional characteristics of the ovarian follicle more similar to humans than rodent models. In addition, it is not yet clear which morphological aspects are related to a correct angiogenesis and capillary regression, especially when evaluated in the light of species differences. Therefore, in order to better understand the morphological mechanisms regulating the microvascular changes occurring in the theca layer before ovulation of larger follicles, we have reviewed by scanning electron microscopy (SEM) of vascular corrosion casts (VCC) the ovarian microvasculature in different experimental conditions, in rodents and domestic animals.Rat, rabbit, pig, sheep, and cow ovaries, at different reproductive stages and or/subjected to different protocols of hormonal stimulation were studied. Rat model was used as control. Rabbit was chosen as a model for reflex ovulation [4]; pig [2,5,6] and sheep [7] as multiovulatory species and cow [8] as a monovulatory model similar to humans. SEM of CC was adopted because it is one of the technique of choice for 3D visualization of blood vessels and capillary network arrangements [4]. Methods and hormonal protocols were previously described [2,[4][5][6][7][8][9]].An intensive functional and morphological remodeling of cortical venules and arterioles accompanied follicle development [9]. Follicle angiogenesis was demonstrated during follicular maturation in all species studied [3]. SEM of VCC demonstrated the differentiation of several microstructures, namely buds, sprouts (sprouting angiogenesis) and intussusceptions (non sprouting angiogenesis) that allowed firstly the growth, then the duplication and multiplication of the preexisting capillaries. The gradual formation of a dense sinusoidal network supplying the theca layer of fully developed follicles (dominant) was also clearly shown. Atretic and subordinated follicles presented degenerative microvascular structures such as thinned capillaries and avascular areas. Species differences were related to follicle size and to mechanisms of follicle selection. In rodents, significant angiogenic morphological figures were mainly confined to the capillaries of the inner thecal layer. In larger follicles of domestic ani...