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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. [ABSTRACT FROM AUTHOR]

This study describes and compares the possible effects of vitrification on the ultrastructural morphology of 20 human mature oocytes vitrified using two different supports, cryoleaf (n = 10) and cryoloop (n = 10). Fresh human mature oocytes (n = 15) were used as controls. Fresh and vitrified--warmed oocytes appeared rounded, with a homogeneous cytoplasm, an intact oolemma and a continuous zona pellucida. Sparse microvacuolization was only occasionally detected in fresh and vitrified--warmed oocytes, to the same extent. About 50% of the vitrified oocytes contained atypical, small and slender mitochondria--smooth endoplasmic reticulum aggregates, whereas a non-homogeneous microvillar pattern was observable in only 30% of the oocytes subjected to vitrification, regardless of the support utilized. Cortical granule content appeared generally reduced after vitrification, but cryoleaf-supported oocytes contained more cortical granules than cryoloop-supported oocytes (P < 0.05). Thus good overall preservation and virtual absence of cytoplasmic vacuolization seem to be the most relevant markers of quality in vitrified--warmed oocytes, using either support. In addition, cryoleaf-supported oocytes retained a higher number of cortical granules than cryoloop-supported oocytes. The variety of ultrastructural alterations recorded emphasizes the need for further studies aimed at assessing the actual tolerance of human oocytes to vitrification. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The aim of the present study was to assess the heterogeneity of cumulus cells that occurs in human cumuli associated with oviductal oocytes and fertilized eggs. Transmission electron microscopy was used to study the cumulus masses surrounding both unfertilized oocytes and fertilized eggs (a pronuclear and a 4-cell stage) recovered at different intervals after ovulation. The specimens were obtained by flushing the oviducts of normal cycling women who underwent surgical sterilization. The cumuli were expanded due to large and irregular intercellular spaces; small linear gap junctions were seen at cell contacts, whereas annular gap junctions were found only in the cytoplasm of some cells. Both types of junction were less abundant in fertilized specimens. Cells surrounding fertilized eggs projected numerous long, thin microvilli into the intercellular spaces. As a rule, the inner layer of the cumulus mass (corona cells) was composed of cells whose surface was relatively smooth. Cumulus cells showed oval nuclei with one or more nucleoli. The cytoplasm of most cells possessed abundant organelles typical of steroidosynthesis: (i) mitochondria with tubular or villiform cristae; (ii) a well-developed smooth endoplasmic reticulum; and (iii) electron dense lipid droplets often surrounded by a few concentric membranes of smooth endoplasmic reticulum and/or in close contact with microtubules and microfilaments. Microperoxisome-like structures were also present. After fertilization, an enhancement of the steroidosynthetic characteristics occurred in the outer layers of the cumulus mass, but not in the corona cells, which still appeared capable of protidosynthesis. Together, these morphological features support the hypothesis that the cumulus of oviductal oocytes and particularly of fertilized eggs, luteinizes like parietal granulosa cells, generating a steroid hormonal micro-environment in the oviduct which may affect fertilization and zygote segmentation. Cumulus cells showing spermiophagic activity, as well as activated macrophages, leukocytes and red blood cells, were also found in the cumulus mass. The macrophages may play a local role both by phagocytic activity and by modulating the steroid secretion of the neighbouring cumulus cells which occurs in the ovarian follicle and corpus luteum. In conclusion, the cumulus mass surrounding tubal oocytes and fertilized eggs appears to be a heterogeneous and dynamic system, in which the micro-environment for fertilization and early embryo development is provided by diverse cell populations in addition to the oviductal cells. [ABSTRACT FROM AUTHOR]

Ovarian damage, with consequent permanent infertility, is one of the more common side-effects which occurs during chemotherapeutic treatment of patients affected by Hodgkin's disease. This damage is morphologically represented by a marked loss of primordial and primary follicles. The administration of contraceptive drugs before starting chemotherapy enhances survival of a greater number of ovarian follicles, as revealed by morphometric analyses, nevertheless, total ovarian protection is not assured. This study evaluated the number and the morphology of ovarian follicles, by means of transmission electron microscopy, in patients with Hodgkin's disease treated with multi-drug chemotherapeutic protocols following the administration of medroxyprogesterone acetate. Ovarian biopsies were performed prior to any pharmacological treatment, after medroxyprogesterone therapy, and after this therapy plus chemotherapy. Particular attention was given to the ultrastructure of primordial follicles. After morphometric evaluation, primordial follicles were numerous in controls and medroxyprogesterone therapy (28.55 +/- 6.59/mm3 of ovarian cortex). After chemotherapy and medroxyprogesterone acetate, the number of follicles was slightly reduced (19.37 +/- 3.41/mm3 of ovarian cortex) in contrast to the dramatic loss usually observed when protection is not given, although more follicles were atretic. Medroxyprogesterone may protect follicles only from acute, toxic effects of chemotherapy, which dramatically reduce their number and lead to sterility. Nevertheless, the quality of follicles is still impaired, and many undergo atresia, resulting in a shortened fertility period. [ABSTRACT FROM AUTHOR]

This presentation, on both printed copy and CD-ROM, summarizes a series of original data on the ultrastructure of human reproduction produced by our research group. In particular, female germ cell behaviour at the time of migration and colonization of the gonad and germ-somatic cell interactions inside the developing ovary are reviewed from a morphodynamic point of view. The results mostly consist of black-and-white transmission and scanning electron microscopy (TEM and SEM) images. Artificially coloured SEM pictures, light microscopy images and drawings have also been selected for iconography to render complex microanatomical details and their morphofunctional relationships more comprehensible. In all, 35 images are presented in this article, each related to a concise text section and accompanied by a self-explaining caption. A list of pertinent references is also provided. [ABSTRACT FROM AUTHOR]

Vitrification currently offers interesting perspectives in the field of oocyte cryopreservation, being judged more tolerable than slow cooling by the oocyte. The present study was therefore aimed to investigate the possible effects of vitrification on the fine morphology of human oocytes at different stages of maturation. Immature (at the germinal vesicle -- GV -- stage and after GV breakdown) and mature oocytes were obtained from consenting IVF patients and vitrified according to the Cryotop method, using ethylene glycol and dimethylsulfoxide as cryoprotectants. After warming, the oocytes were fixed with glutaraldehyde and processed for light and transmission electron microscopy (LM and TEM) observations. By LM all vitrified-warmed oocytes appeared rounded in sections, measuring about 90-100 microns in diameter. All the observed oocytes displayed a homogeneous cytoplasm, an intact oolemma and a continuous zona pellucida. By TEM immature oocytes showed characteristic organelles such as dense, rounded mitochondria, isolated smooth endoplasmic reticulum (SER) membranes, dictyosomes belonging to the Golgi system, small mitochondria-vesicle complexes and scattered cortical granules (CGs), the latter found in subplasmalemmal areas or located deeply in the cytoplasm. In some cases, minute vacuoles were also present in the inner cytoplasm, particularly around the nucleus of the GV-stage oocytes. Mature oocytes showed rounded mitochondria with peripheral, arch-like cristae, typical mitochondria-SER aggregates in the cytoplasm and a rim -- often discontinuous -- of CGs just beneath the oolemma. A reduced complement of microvilli was also sporadically found in these oocytes. Isolated vacuoles of different sizes were sometimes detected, mostly in the cortical cytoplasm, irrespective of the maturational stage of the oocyte. In conclusion, the oocytes subjected to the described vitrification method show good overall preservation. However, the presence of alterations in CG and microvillar patterns in mature oocytes, as well as the finding of oocyte vacuolization emphasize the need of further ultrastructural studies on human vitrified oocytes, to confirm the actual safety and efficiency of this method. [ABSTRACT FROM AUTHOR]

Introduction: Several studies reported the effect of the number of oocytes retrieved on ART outcome. However, studies about the effect(s) of the number of immature oocytes retrieved on ART outcome are scarce. The goal was to evaluate the role of the number of retrieved immature oocytes on mature oocyte counts and. Materials and Methods: 101 ICSI cycles were included in this prospective evaluation. morphology, and also the rates of fertilization, embryo development, pregnancy and delivery rate in ICSI cycles. Patients were divided into 2 groups of A (⩽2 immature oocytes) and B (> 2 immature oocytes). In subanalysis, the impacts of the number of GV and MI oocytes were assessed on the rates of fertilization and embryo sub-analyzed development. Also, correlations between the numbers of immature and mature oocytes, as well as maternal age between two groups were analyzed. Assessments of oocyte morphology, fertilization, embryo quality and development were done accordingly. Results: There was no correlation between the immature oocytes pregnancy and delivery rate quantity with the number of mature ones. There were insignificant differences for embryo development between two groups, but fertilization rate was higher in group A (p=0.03). In sub-analysis, insignificant differences were observed between two groups of ⩽ and >2 GV and MI oocytes for rates of fertilization and embryo development. Also, the rates of clinical pregnancy and delivery were insignificant between groups. The rate of morphologically abnormal oocytes had no significant difference between two groups, except for wide perivitelline space (PVS) which was higher in group A (p⩽0.03). There was no significant difference for maternal age between two groups. Conclusion: In cases with few retrieved immature oocytes, rates of fertilization and incidence of wide PVS may increase, although immature oocytes may not have any negative impacts on early embryo development or the rates on number of mature oocytes. [ABSTRACT FROM AUTHOR]