The morphological characteristics of frozen-thawed human mature oocytes (n = 12) were studied by light and transmission electron microscopy following cryopreservation using a slow cooling protocol including increasing concentrations of ethylene glycol (0.5-1.5 mol/l) and sucrose 0.2 mol/l in the freezing solution. Fresh human mature oocytes (n = 12) were used as controls. Fresh and frozen-thawed oocytes appeared rounded in section, with a homogeneous cytoplasm, an intact oolemma and a continuous zona pellucida. Disorganization of mitochondria-smooth endoplasmic reticulum aggregates and a decreased complement of microvilli and cortical granules were frequently observable in frozen-thawed oocytes. Increased density of the inner zona pellucida, possibly related to the occurrence of zona 'hardening', was sometimes found associated with a reduced amount of cortical granules. In addition, delamination of the zona pellucida was evident in some frozen-thawed samples. Finally, numerous vacuoles and secondary lysosomes were detected in the ooplasm of most frozen-thawed oocytes. In conclusion, frozen-thawed oocytes treated with ethylene glycol may show a variety of ultrastructural alterations, possibly related, at least in part, to the use of this cryoprotectant. Thus, the ethylene glycol-based protocol of slow cooling herein described does not seem to offer significant advantages in terms of oocyte structural preservation.
Vitrification is a new method that has been recently introduced in Assisted Reproduction Technique programs. The aim of this study was to design a new medium similar to normal human seminal fluid (SF), formulation artificial seminal fluid (ASF), and to compare the cryoprotective potency of this medium with SF and human tubal fluid (HTF) medium. Thirty normal ejaculates were processed with the swim-up technique and sperm suspensions were divided into four aliquots: (i) fresh sample (control); (ii) vitrification in HTF medium supplemented with 5 mg/mL human serum albumin and 0.25 mol sucrose (Vit HTF); (iii) vitrification with patients' SF (Vit SF); and (iv) vitrification in ASF (Vit ASF). After warming, sperm parameters of motility, viability, and morphology were analyzed using WHO criteria. Also, sperm pellets were fixed in 2.5% glutaraldehyde and processed for scanning electron microscopy and transmission electron microscopy observations. The results showed that progressive motility (46.09 ± 10.33 vs. 36.80 ± 13.75), grade A motility (36.59 ± 11.40 vs. 16.41 ± 11.24), and normal morphology (18.74 ± 8.35 vs. 11.85 ± 5.84) and viability (68.22 ± 10.83 vs. 60.86 ± 11.72) of spermatozoa were significantly higher in Vit ASF than in Vit HTF. All parameters were better in Vit ASF than in Vit SF, but only viability was significantly different (p = 0.006). After cryopreservation, deep invagination in cytoplasm and mechanically weak point sites and folded tail were commonly observed. But, this phenomenon was more significant in Vit HTF and Vit SF than in ASF (p < 0.05). In transmission electron microscopy evaluation, acrosome damage, plasma membrane loss, chromatin vacuolation, and disruption of mitochondria arrangement and structures were observed in all vitrified groups. Adherence of several tail sections together was also seen in all cryo groups. But this was seen more in Vit HTF and Vit SF than in ASF (p < 0.05). In conclusion, vitrification of human spermatozoa with ASF can effectively preserve the quality of sperm motility in comparison with Vit HTF.
The possible acute morphological changes induced by electrical transcranial unifocal stimulation (eTCS) in the rabbit extracerebral tissues were studied by light and scanning electron microscopy. In order to do this, a wide range of electric stimuli with respect to those employed in the clinical practice were utilized. Either surface electrodes were attached to the scalp, or needle electrodes were infixed in the subcutaneous tissue. Beneath the cathode a blood extravasation was constantly observed in the subcutaneous tissue of the scalp; the different electrode arrays produced either a large hemorrhagic lesion or a few petechiae. Beneath the anode, the damage was limited to the scalp, or reached the meninges when stimuli longer than 0.2 ms were used. Irrespective of the electrode arrays, the scalp and the dura mater displayed hemorrhagic petechiae over a limited area about 2–3 mm in extent. Moreover, the leptomeningeal membrane was microscopically disrupted over an area less than 1 mm large; therein the squamous, overlapping cells were transformed into fusiform or macrophage-like cells. Unduly intense eTCS produces evident hemorrhagic lesions in the scalp and in the dura mater, whereas it induces microscopic, reactive changes in the leptomeninx.
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