Ultrastructural markers of quality are impaired in human metaphase II aged oocytes: a comparison between reproductive and in vitro aging.

PURPOSE: Childbearing delay contributes to the increase of subfertile couples that require assisted reproductive technology (ART). Subfertility relates with reproductive aging (RA). In vitro aging (IvA) (due to extended culture) may also impair oocyte competence. Aims of this study were to evaluate and compare the oocyte ultrastructure after RA and IvA. METHODS: Cumulus-oocyte complexes (COCs) (n = 68), with metaphase II oocyte and expanded cumulus, from consenting patients (<35 years old and ≥35 years old, n = 36), were selected by phase contrast microscopy and fixed at pick up, or after 24 h culture. COCs (n = 44) were studied by light and qualitative/morphometric transmission electron microscopy. Two-way ANOVA, with age and culture as grouping factors, was applied for statistical analysis (p < 0.05). Metaphase II cumulus-free oocytes (n = 24) were selected for confocal microscopy observations. RESULTS: Significant decrease of mitochondria-smooth endoplasmic reticulum aggregates, increase of mitochondria-vesicle complexes size and amount, decrease of cortical granules and microvilli, and alterations of the spindle structure characterized both RA and IvA oocytes. These changes were significantly more evident in the RA oocytes submitted to IvA. RA oocytes also showed changes of the zona pellucida and occurrence of vacuoles after culture. Cumuli appeared re-compacted after culture, irrespective of the age of the patients. CONCLUSIONS: These data demonstrated that aging is related to decay of oocyte ultrastructural quality, and that oocytes from elder women are more sensitive to prolonged culture (IvA) than the oocytes from younger women. These morphological results should be considered when applying ART in aged patients, rescue ICSI, or artificial oocyte activation.

[Modification of in vitro insemination techniques in the treatment of severe male factors in assisted reproduction]. / Modificazione delle procedure di inseminazione in vitro nel trattamento dei gravi fattori maschili nella riproduzione assistita.

The aim of this study is to detect qualified in vitro insemination techniques in the treatment of the severe oligoasthenotheratospermia which is defined as total motile count in the pretreatment samples (< 5 x 10(6) with > 50% of abnormal morphology). These patients have taken part in the in vitro insemination program of the Assisted Reproduction Unit of the 2nd Department of Obstetrics and Gynecology at Rome University "La Sapienza" during a period between June and December 1995. Several modifications of the standard in vitro techniques have been developed such as: mechanical decumulation of the oocytes, reduction of the volume of culture medium, increase of spermatozoa and oocyte concentration at the moment of insemination. A good fertilization rate was achieved (33%) as regard to the semen sample and procedures utilized. Twelve Ets were performed and 4 clinical pregnancies (25% per patients and 33% per transfer) were achieved. These data demonstrate that by the modification of standard laboratory methods for in vitro insemination, a good fertilization rate and a high clinical pregnancy rate can be achieved in cases of severe male factor infertility without having to resort to micromanipulation techniques.

Heterogeneous distribution of fibronectin, tenascin-C, and laminin immunoreactive material in the cumulus-corona cells surrounding mature human oocytes from IVF-ET protocols--evidence that they are composed of different subpopulations: an immunohistochemical study using scanning confocal laser and fluorescence microscopy.

Monoclonal antibodies and immunofluorescence microscopy, including laser confocal microscopy, were used in this study to point out the production of fibronectin, tenascin-c, and laminin in the cumulus-corona (CC) cells surrounding mature human oocytes from IVF-ET protocols in view of their presumptive importance in the coordination of the processes leading to fertilization and early embryo cleavage, including the final maturation of the ovum, the sperm-egg interaction, and the "complex biochemical dialogue" between the gamete and the oviduct through the tubal luminal environment. One hundred fifty mature oocyte-CC complexes were obtained from IVF-ET protocols and fixed in 4.0% buffered paraformaldehyde. Specimens were incubated with a panel of primary monoclonal antibodies (mabs) recognizing different epitopes of fibronectin, tenascin-c, and laminin and then with fluorescein isothiocyanate-conjugated goat anti-mouse IgG. Observations were made by a scanning confocal microscope (Sarastro 2000) and a photomicroscope (Polyvar, Reichert-Jung) equipped with epifluorescence optics. The immunohistochemical data demonstrated that human CC cells are capable of producing fibronectin and tenascin-c but that their production is not homogeneous in the CC population. In fact, fibronectin immunoreactivity was shown mostly by inner CC cells (mainly corona cells), whereas tenascin was produced by some cells scattered in the entire cumulus mass. Moreover, fibronectin and tenascin-c immunoreactive material was observed in the intracytoplasmic areas, at the plasma membrane level as well as in the extracellular matrix. On the contrary, laminin immunofluorescent material was found around plasma membranes of almost all CC cells, but a clear intracytoplasmic reaction was never observed. This leads us to assume that laminin in the extracellular matrix remains entrapped once produced by granulosa follicular cells and that in the postovulatory period no active secretion occurs in CC cells. Even though the functional role of these extracellular matrix proteins remains still unclear, it is reasonable to suggest that they are necessary in various steps of the reproductive process, i.e., from the pick-up of the oocyte, its transport through the oviduct, and fertilization, up until the early cleavage of the embryo. Finally, functional differences between "corona radiata" and "cumulus" cells during the oocyte denudation may be accounted for particular distribution of these adhesive proteins.

In vitro sperm capacitation to treat antisperm antibodies bound to the sperm surface.

Our objective was to study antisperm antibody bound to the acrosome region during in vitro capacitation and to determine whether acrosome-antibody free sperm can be obtained from previously acrosome-antibody-coated sperm. The spermatozoa from a selected series of 14 patients were tested for sperm antibodies bound to the sperm surface using d-IBT and focusing on the acrosome positivity. The tests were carried out before the incubation and after 3, 6, 9, and 12 h of incubation in Tyrode's solution with 0.5% human serum albumin as the capacitation medium. Tests to evaluate acrosome region, sperm motion parameters, and zonae binding ability were carried out. In this way we were able to evaluate sperm function during capacitation protocol. The patients were 14 subjects selected according to good seminal characteristics, good post-rise sperm parameters, and high percentage of ASA bound to the sperm surface. In all cases the results showed that antisperm antibodies bound to the acrosome region were shed prior to the acrosome reaction. During sperm capacitation in human a modification, migration, or shedding of plasma membrane molecules takes place. The presence of antibodies in such an important area of the sperm head could certainly interfere in the fertilization process. Our data indicate that in vitro capacitation could provide an in vitro therapy capable of eluting antibodies from the acrosome region.

Human zona pellucida during in vitro fertilization: an ultrastructural study using saponin, ruthenium red, and osmium-thiocarbohydrazide.

The human zona pellucida (ZP) and its changes during in vitro fertilization in oocytes at different maturational stages and polypronuclear ova at one- to four-cells stages were studied by transmission electron microscopy (TEM) and correlative scanning electron microscopy (SEM). To define the microstructure of the ZP, its amorphous masking material was removed using a detergent (saponin), and its structural glycoproteins were stabilized with a cationic dye, ruthenium red, followed by osmium-thiocarbohydrazide treatment. These methods allowed in all samples the clear visualization of variously arranged networks of filaments composing the outer and inner surfaces of the ZP. These filaments were straight or curved, 0.1-0.4 microns in length and 10-14 nm thick as seen via TEM or 22-28 nm thick as seen via SEM (the difference in thickness was due to the presence of the metal coating for SEM). The filament arrangement was remarkably different between the inner and outer surfaces of the ZP and among the various stages studied. The filaments of the outer surface of the ZP were basically arranged in "large" and "tight" meshed networks. Mature oocytes and fertilized (polypronuclear) ova had a regular alternating pattern of wide and tight meshed networks of filaments. On the other hand, immature and atretic oocytes displayed almost exclusively a tight meshed network of filaments. The inner surface filaments of the ZP of unfertilized oocytes at any stage were arranged in repetitive structures characterized by numerous short and straight filaments anastomosing with each other and sometimes forming at the intersections small, rounded structures. After fertilization, the inner surface of the ZP displayed numerous areas where filaments fused together. Collectively, these data clearly reveal that oocyte maturation and fertilization in humans are accompanied by changes of ZP filaments arrangement, which may be relevant in the processes of binding, penetration, and selection of spermatozoa.

The ultrastructure of human cumulus-corona cells at the time of fertilization and early embryogenesis. A scanning and transmission electron microscopic study in an in vitro fertilization program.

We observed the ultrastructure of the cumulus-corona cells (CC cells) surrounding: 1) human preovulatory oocytes unfertilized after in vitro insemination and 2) in vitro-fertilized polypronuclear ova (PO) at the pronuclear stage (3 pronuclei) and during early cleavage, at the 3-8 cell stage (cleaving PO). All the samples were obtained from women who underwent pharmacological hormonal stimulation during in vitro-fertilization procedures. Both cell groups were composed of irregularly rounded CC cells, showing an oval nucleus with one or more reticular nucleoli. Spermatozoa in close contact with CC cells were also seen. Linear and annular gap junctions between neighbouring cells were present, particularly in Group 1. Lipid droplets were present in both groups, appearing slightly more numerous and electron-dense in Group 2. In Group 1, mitochondria were numerous, polymorphic, and provided with cristae varying from lamellar to tubular. In Group 2, mitochondria also showed polymorphism, with bacilliform organelles with tubular cristae being predominant. In both groups cisternae and associated vacuoles and vesicles belonging to the Golgi complex were scattered in the cytoplasm of CC cells. Similarly, tubular and vesicular profiles of smooth endoplasmic reticulum were abundant and uniformly distributed throughout the cytoplasm of CC cells of both groups. In contrast, the abundant rough endoplasmic reticulum in Group 1 was formed by parallel stacks of flattened cisternae, whereas it was less plentiful and not arranged in stacks in Group 2. The CC-cell surface appeared covered by numerous membrane expansions in both groups. The expansions in Group 1 were mainly composed of blebs of various sizes and a few short microvilli, whereas in Group 2 numerous microvilli covered the cell surface. These observations demonstrate that a gradual establishment and maintainance of a steroidosynthetic capability (luteinization) takes place in CC cells, particularly during and shortly after fertilization, as occurs contemporaneously in the granulosa cells of the postovulatory follicle. Our results may be considered as ultrastructural confirmation of the capability of the CC cells to produce small amounts of steroids (estrogens and mainly progesterone). These hormones, alone or together with other substances (proteins, nutrients, growth factors?), might--around the fertilization time--act positively upon the early embryo itself, as well as on the microenvironment in which the embryo develops, both in vivo and in vitro conditions.

The use of the Salt Stored Zona Assay in the study of the sperm function: methodological approach.

The authors mention many tests to study sperm function. On their opinion, the best model to evaluate spermo-oocyte interaction is the in vitro fertilization but it cannot be considered a test for ethical and practical problems. To overcome these problems they propose the Salt Stored Zona Assay to evaluate the spermatozoa.

Is the sperm-binding capability of the zona pellucida linked to its surface structure? A scanning electron microscopic study of human in vitro fertilization.

The structure of the zona pellucida and the early interactions between human oocytes and spermatozoa were investigated in an in vitro fertilization program. Thirty-five mature (preovulatory) oocytes, 10 immature oocytes lacking a germinal vesicle, and 11 atretic oocytes which had not undergone fertilization at 10-20 hr after insemination were studied by light and scanning electron microscopy. Observed through employment of these techniques, the zona pellucida showed two basically different patterns: a mesh-like, spongy structure having wide and/or close meshes; and a compact, smooth surface. The smooth-surfaced zona was most commonly seen in the cultured oocytes belonging to the immature and atretic groups. These observations seem to show that the spongy appearance of the zona pellucida is related mainly to oocyte development and maturity. In this study, greater numbers of penetrating spermatozoa were noted on oocytes showing the mesh-like zona, in contrast to the presence of a few sperm flattened against its surface or the frank absence of sperm associated with oocytes having the more compact, smooth zona. It is likely that the condensation of the outer aspect of the zona pellucida causes a disorientation of sperm-binding sites, which would probably result in markedly reduced binding and penetration capacity with spermatozoa. These changes might ultimately lead to impairment of in vitro oocyte fertilizability.

Prenatal diagnosis of hereditary tyrosinemia by determination of fumarylacetoacetase in cultured amniotic fluid cells.

Fumarylacetoacetase was assayed in cultured amniotic fluid cells from four pregnancies at risk for hereditary tyrosinemia and in 11 controls. The enzyme activity was normal in three of the pregnancies at risk for tyrosinemia and healthy children were born. In the fourth case the enzyme activity was deficient, indicating an affected fetus. As the pregnancy was very advanced it was continued, and the child has tyrosinemia. One parent in one of the four families is a compound heterozygote for the tyrosinemia gene and the recently reported "pseudodeficiency" gene for fumarylacetoacetase. This has important consequences for prenatal diagnosis in this family.

Transformation of human choroid cells in vitro by SV40. Ultrastructural and cytogenetic analysis of cloned cell lines.

Several clones were isolated from a Simian Virus 40-transformed human choroid cell line (HC/SV40) by the soft agar plating technique. Five of them which showed different morphology, growth capacity and cell density, were plated in monolayer and subjected to ultrastructural, immunological and cytogenetic analyses. The morphological features at both TEM and SEM were distinct for every examined clone but uniform in the cells of the same clone. In particular clone 3 (HC/SV40-C13) showed a greater increase of intracytoplasmic granules, a marker of all cells of choroid origin, while clone 2 (HC/SV40-C12) was totally devoid of them. No virus production was observed at the electron microscope. Immunofluorescence experiments revealed 100% positivity for nuclear T-antigen in all the clones, thus demonstrating the persistence of SV40 genome in these cells. Cytogenetic analysis showed a hyperdiploid number of chromosomes in clones 1 and 3, while clones 2 and 4 were clearly hypodiploid. All clones showed a general tendency to cytogenetic stabilization as compared to the karyotypes found in HC/SV40 parental cell line.

Regional mapping of hexokinase-1 within the short arm of chromosome 10.

Hexokinase-1 (HK1) activity was determined in red cells of 5 patients with partial duplications of chromosome 10, all of which involved the 10p region. In 4 patients the levels of HK1 activity were higher than the mean activity of controls, strongly indicating a triplex dosage effect. The most likely regional assignment for HK1 would appear to be 10p11.2.

Prenatal prediction of duplication 10q24 leads to qter by gene dosage of GOT1 on uncultured amniotic cells.

Glutamic-oxaloacetic transaminase (GOT1) gene dosage studies were performed on uncultured amniotic cells from a fetus at risk for duplication/deficiency of 10q24 leads to qter, due to maternal translocation t(9;10)(p24;q24). Previous investigations in the same pedigree had shown triplex dosage effect of GOT1 on red blood cells of a 10q24 leads to qter trisomic fetus monitored by midtrimester amniocentesis. In the present pregnancy, the GOT1 activity of amniotic cells exhibited a triplex gene dosage, suggesting duplication of region 10q24 leads to qter in the fetus. The biochemical prediction was confirmed two weeks later by cytogenetic analysis.

Cytogenetic findings in 4952 prenatal diagnoses. An Italian collaborative study.

The development of prenatal diagnosis in Italy was made difficult by the restrictions of the old abortion law and only in recent years has a consistent number of cases been investigated. We report the experience on prenatal chromosome diagnosis of ten Italian centers participating in a collaborative study on 4952 diagnoses performed from 1972 to 1980. The main indication groups were: advanced maternal age (2882 cases), previous child with chromosome anomaly from parents with normal karyotype (847 cases), and chromosome anomaly in one parent (97 cases). The other indications for amniocentesis, including cases without a cytogenetic risk, have been assembled into a "miscellaneous" group (1126 cases). We found 125 abnormal fetal karyotypes (2.5%) of which 89 were unbalanced (1.8%). The frequencies and types of chromosome anomalies are reported in detail for each indication group and are compared with the corresponding one from the European Munich Conference. The great majority of these Italian data were not included in the Munich report.

The possibility of prenatal diagnosis by gene dosage: confirmation of duplication 10q24 to qter from GOT-1 activity in fetal erythrocytes.

The activity of four enzymes, including GOT-1, has been investigated in he erythrocytes of a 10q to 24 qter trisomic fetus. Analyses have been performed on a feto-maternal blood mixture sampled by fetoscopy and on red cells obtained by cardiac puncture, following therapeutic abortion. The demonstration of a 40 per cent increase of GOT-1 activity, as compared to normal fetuses of similar gestational age, suggests that gene dosage studies may be a useful confirmatory technique in prenatal diagnosis of unbalanced chromosomal aberrations. Practical application of a similar diagnostic approach is conditioned by (1) precise characterization of fetal chromosome imbalance; (2) confirmed assignment of the gene locus coding for the gene product under investigation; (3) evidence of a linear proportionality between gene dose and concentration of the gene product in patients with the same chromosome imbalance detected in the fetus; (4) knowledge of the range of normal variation at different weeks of gestation of the enzyme activity to be tested in the fetus; (5) safety of fetal sampling procedure.