M. Maione

Ultrastructural markers of quality in human mature oocytes vitrified using cryoleaf and cryoloop

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.

Ultrastructure of human mature oocytes after vitrification

Since the introduction of human assisted reproduction, oocyte cryopreservation has been regarded as an attractive option to capitalize the reproductive potential of surplus oocytes and preserve female fertility. However, for two decades the endeavor to store oocytes has been limited by the not yet optimized methodologies, with the consequence of poor clinical outcome or of uncertain reproducibility. Vitrification has been developed as the promising technology of cryopreservation even if slow freezing remains a suitable choice. Nevertheless, the insufficiency of clinical and correlated multidisciplinary data is still stirring controversy on the impact of this technique on oocyte integrity. Morphological studies may actually provide a great insight in this debate. Phase contrast microscopy and other light microscopy techniques, including cytochemistry, provided substantial morpho-functional data on cryopreserved oocyte, but are unable to unraveling fine structural changes. The ultrastructural damage is one of the most adverse events associated with cryopreservation, as an effect of cryo-protectant toxicity, ice crystal formation and osmotic stress. Surprisingly, transmission electron microsco py has attracted only limited attention in the field of cryopreservation. In this review, the subcellular structure of human mature oocytes following vitrification is discussed at the light of most relevant ultrastructural studies.

Qualitative and morphometric analysis of the ultrastructure of human oocytes cryopreserved by two alternative slow cooling protocols

PurposeTo ascertain possible cell damage from cryopreservation, the ultrastructure of human oocytes cryopreserved by slow cooling was assessed.Materials and methodsCryopreservation was performed through two protocols with one-step or two-step propanediol. Fresh control oocytes were examined for comparison. Samples were processed for transmission electron microscopy analysis.ResultsBy light microscopy, both fresh and frozen-thawed oocytes appeared regularly rounded, with intact zona pellucida, and homogeneous cytoplasm. By electron microscopy observation, organelles were abundant and uniformly dispersed. Mitochondria-smooth endoplasmic reticulum associations appeared regular. However, both the amount and density of cortical granules appeared abnormally reduced in frozen-thawed samples. Slight to moderate vacuolization was also found in the ooplasm of oocytes of both frozen groups.ConclusionsSlow cooling ensures a good overall preservation of human oocytes. However, cytoplasmic vacuolization and cortical granule loss appears associated with cryopreservation, irrespective of the protocol used.

Ultrastructure of immature and mature human oocytes after cryotop vitrification

In vitro maturation of vitrified immature germinal vesicle (GV) oocytes is a promising fertility preservation option. We analyzed the ultrastructure of human GV oocytes after Cryotop vitrification (GVv) and compared it with fresh GV (GVc), fresh mature metaphase II (MIIc) and Cryotop-vitrified mature (MIIv) oocytes. By phase contrast microscopy and light microscopy, the oolemmal and cytoplasmic organization of fresh and vitrified oocytes did not show significant changes. GVv oocytes showed significant ultrastructural alterations of the microvilli in 40% of the samples; small vacuoles and occasional large/isolated vacuoles were abnormally present in the ooplasm periphery of 50% of samples. The ultrastructure of nuclei and mitochondria-vesicle (MV) complexes, as well as the distribution and characteristics of cortical granules (CGs), were comparable with those of GVc oocytes. MIIv oocytes showed an abnormal ultrastructure of microvilli in 30% of the samples and isolated large vacuoles in 70% of the samples. MV complexes were normal, but mitochondria-smooth endoplasmic reticulum aggregates appeared to be of reduced size. CGs were normally located under the oolemma but presented abnormalities in distribution and matrix electron density. In conclusion, Cryotop vitrification preserved main oocyte characteristics in the GV and MII stages, even if peculiar ultrastructural alterations appeared in both stages. This study also showed that the GV stage appears more suitable for vitrification than the MII stage, as indicated by the good ultrastructural preservation of important structures that are present only in immature oocytes, like the nucleus and migrating CGs.

Morphological expression of angiogenesis in the mammalian ovary as seen by SEM of corrosion casts

In the mammalian ovary, follicular and corpus luteum cycle is associated with intensive microvascular remodelling. The complex angiogenic dynamics are finely tuned by numerous regulatory factors acting as activators (up-regulators) or inhibitors (down-regulators) of angiogenesis. Alterations of such a tight modulation are involved in several pathologies, including infertility, polycystic ovarian syndrome, ovarian hyperstimulation syndrome and ovarian cancer. We have demonstrated in several experimental models that ovarian function is critically and specifically dependent on angiogenesis for follicular development, ovulation, and corpus luteum growth. The aim of this review is to summarize the results we have obtained on the morphodynamic remodelling of ovarian microvascularization, in polyovulatory (rat, rabbit and pig) and monovulatory species (cow), using scanning electron microscopy of vascular corrosion casts. The knowledge of the morphological expression of the up- and down-regulation of angiogenesis occurring in mono and polyovulatory animals might provide useful information to preserve fertility and to increase of the effectiveness of reproductive management in species of domestic interest.

Ultrastructural and morphometric evaluation of aged cumulus-oocyte-complexes

Maternal age is one of the most significant factors influencing oocyte quality (1). 35 years of age seems to be a watershed in reproductive potential. The aim of this study was to reveal the amount and distribution of specific ultrastructural organelles in human mature cumulus-oocyte-complexes belonging to women of different ages (<35 years old; ≥35 years old/ reproductive aging) and to evaluate their different response during 24 hours prolonged culture (defined as in vitro aging) (1). The samples were studied by light and transmission electron microscopy; a morphometric analysis of TEM data was performed (2). In all aged samples, the amount of mitochondria- smooth endoplasmic reticulum aggregates, cortical granules and microvilli decreased (p<0,05), while the amount of mitochondria-vesicle complexes increased up (p<0,05). Occasional vacuoles were found in oocytes from older women after in vitro aging. A significant (p<0,05) increase of zona pellucida thickness was linked to the donor age but not to in vitro aging. A re-compaction of cumulus cells was seen in in vitro aged samples. Morphometric data strongly confirmed our preliminary results (3) revealing that: i) reproductive aging and in vitro aging share specific ultrastructural features ii) In vitro aging can be consider a model for reproductive aging iii) young oocytes seem to be less sensitive to in vitro aging than older ones. The above results may represent a reliable background for further multidisciplinary studies regarding aged oocytes and may be also useful in clinical settings.

Ultrastructural features of human metaphase II oocytes subjected to slow freezing or vitrification in an IVF program: a comparative analysis

During the past two decades important advances have been made in the field of assisted reproduction by using oocyte cryopreservation. However, mature (metaphase II) oocytes are very susceptible to cryodamage. In order to contribute to the identification of a cryopreservation protocol with minimal side effects on the oocyte structure and function, we evaluated and compared the subcellular features of human oocytes cryopreserved either with slow (controlled rate) freezing or vitrification (ultrarapid freezing). Supernumerary human metaphase II oocytes were donated by consenting patients enrolled in an IVF program. The age of these women ranged from 27 to 32 years old. The eggs were cryopreserved using slow freezing with 1.5M propanediol + 0.2M sucrose concentration or a closed vitrification system (Cryotip Irvine Scientific CA). Fresh oocytes were used as controls. Samples were fixed and prepared for light and transmission electron microscopy (LM and TEM) observations. By LM, all the oocytes were generally rounded, 90-100 microns in diameter, with regular ooplasm showing uniform distribution of organelles. By TEM, mitochondria-smooth endoplasmic reticulum (M-SER) aggregates were the most common structures found in all the oocytes fixed or cryopreserved within 3-4 hours after the retrieval. M-SER aggregates appeared instead partially replaced by mitochondria-vesicle complexes when oocytes were maintained in culture for a prolonged period of time. A slight to moderate vacuolization was found in the cytoplasm of the oocytes subjected to slow freezing. Slight microvacuolization was also found in vitrified oocytes, whereas vacuoles were almost completely absent in fresh controls. Amount and density of cortical granules (CGs) appeared abnormally reduced in all cryopreserved oocytes, irrespective of the protocol applied. In conclusion, it has been evidenced that prolonged stay in culture induces an intracellular membrane “recycling” in the oocytes, that causes the transformation of slender, anastomosed SER tubules into rounded vesicles surrounded by mitochondria, whose role is still uncertain. In addition, even though all cryopreservation protocols studied ensured a good overall preservation of the oocyte, vacuolization appears as a recurrent form of cell damage. This happens both during slow freezing and, at a lesser extent, during vitrification using a closed device. In addition, premature CG exocytosis was observed in both groups.

Morphofunctional aspects of in vitro matured oocytes from prepubertal and adult sheep

The aim of this study was to analyze the morphofunctional aspects of prepubertal and adult sheep oocytes subjected to in vitro maturation (IVM). The structural and ultrastructural morphology was examined by light and transmission electron microscopy (LM and TEM) while the metabolic competence, as determined by the distribution of active mitochondria, was assessed by confocal microscopy (CM). Cumulus-oocyte complexes underwent IVM in standard condition for 24 h. Half of the mature metaphase II (MII) stage oocytes were processed for LM and TEM observations. The other half was subjected to immunostaining with MitoTracker Red (to stain mitochondria with functionally active membrane potential), SNARF-1 (a pH sensitive fluoroprobe), Hoechst 33342 (to label DNA) and analyzed by CM. Immature germinal vesicle (GV) stage oocytes, retrieved at 0 h IVM, were used as controls. By LM and TEM all the oocytes were regularly rounded, covered by microvilli and surrounded by an intact zona pellucida. Numerous rounded, oval or hooded mitochondria appeared either isolated or grouped in the ooplasm. The GV was usually rounded in prepubertal oocytes. In the adult, the GV often appeared flattened against the oolemma, with a crescent-shaped outline (an early sign of meiotic resumption). Scattered cortical granules (CGs) were rarely found in the ooplasm of both prepubertal and adult GV oocytes. After 24 h of IVM, CGs became abundant and distributed in a single row under the oolemma, particularly in adult oocytes. CM showed a homogeneous fine-to-granular mitochondrial distribution in prepubertal GV and MII oocytes. In adult GV oocytes, the mitochondrial distribution pattern was granular while in MII oocytes mitochondria were arranged in heterogeneous clusters. Thus, both prepubertal and adult oocytes completed maturation after 24 h in culture and showed an overall good preservation after IVM. However, the diverse distribution patterns of mitochondria in prepubertal oocytes reflect their low developmental competence.