Fertilization, embryo quality, and cryosurvival in in vitro fertilization and intracytoplasmic sperm injection cycles.

PURPOSE: Our purpose was to investigate the influence of semen quality on fertilization, embryo morphology, cleavage, and cryosurvival in conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) programs. METHODS: A retrospective analysis of 513 couples undergoing IVF and 255 couples undergoing ICSI was done. RESULTS: Semen quality influenced fertilization in IVF and abnormal fertilization in IVF and ICSI, but no effects on the development, morphology, implantation capacity, or cryosurvival of embryos were found. Fertilization, embryo quality, and cryosurvival rates were similar after IVF and ICSI. The fertilization rate of mature oocytes in IVF was lower when cytoplasmic immaturity in the oocyte population was frequent. The speed of development of embryos was 2 hr faster after ICSI than after IVF. Two-cell-stage embryos survived best after cryopreservation with propanediol and sucrose on day 2. CONCLUSIONS: After fertilization, semen parameters had no effect on the quality or cryosurvival of embryos in either IVF or ICSI.

The ultrastructure of human endometrium is altered by administration of intrauterine levonorgestrel.

We studied the effect of intrauterine administration of levonorgestrel (LNG) on the ultrastructure of the endometrium. Twenty-one endometrial biopsy specimens, collected from nine fertile women during normal menstrual cycles and after 1, 3 or 6 months of use of a levonorgestrel-releasing intrauterine contraceptive system (LNG IUS), were studied using transmission and scanning electron microscopy. During the 6 month exposure to LNG IUS, changes took place in the endometrium. The glandular epithelial cells became lower. The junctional complexes between epithelial cells remained unchanged, whereas the lateral microvillar interdigitations became more prominent. The basal lamina under the epithelium became wavy but remained uniform and practically uninterrupted; only solitary epithelial cell protrusions through the basal lamina were seen. The stromal cells were largely decidualized. We conclude that in parallel with the generally known cellular effects, the use of the LNG IUS results in distinct changes in the basal lamina between the endometrial epithelial and stromal cells. The especially well-developed and uninterrupted basal lamina may be involved in the mechanism of the LNG IUS-induced endometrial suppression. Furthermore, the complex intercellular junctions between the epithelial cells, normally loosening around the time of implantation, persist during the local administration of levonorgestrel. This may have a pivotal role in the contraceptive effect of the LNG IUS.

PIP: The suppressive effect of intrauterine administration of levonorgestrel (LNG) on the ultrastructure of the endometrium was assessed through analysis of 21 endometrial biopsy specimens collected from 9 fertile women both during normal menstrual cycles and 1, 3, or 6 months after insertion of an LNG intrauterine system (LNG-IUS). After 6 months of LNG-IUS use, the morphology resembled that seen during the secretory phase of the normal menstrual cycle. Transmission and scanning electron microscopy revealed distinct changes in the basal lamina between the endometrial epithelial and stromal cells after LNG-IUS insertion. The glandular epithelial cells became thinner and the lateral microvillar interdigitations more prominent. The basal lamina under the epithelium became wavy but remained uniform and practically uninterrupted--a mechanism that may be salient to LNG-induced endometrial suppression. Only solitary epithelial cell protrusions through the basal lamina were seen. Stromal cells were largely decidualized. The junctional complexes between epithelial cells remained unchanged in contrast to the loosening normally seen around the time of implantation, which may have a key role in the contraceptive effect of the LNG-IUS.

A cloned human germ cell tumor-derived cell line differentiating in culture.

We have derived a clonal cell line (HGCT-1) from a lymph node metastasis of a primary testicular germ cell tumor (GCT). The tumor was negative for the embryonal carcinoma (EC) cell marker BerH2 but positive for vimentin, cytokeratin (CK) and desmin. Comparative genomic hybridization (CGH) revealed a high-level amplification at 12p that was observed in both the metastatic tumor and in the cultured HGCT-1 cells. In vitro, the phenotype of HGCT-1 cells was modulated by the culture conditions. In the presence of 10% fetal calf serum (FCS), the majority of HGCT-1 cells lacked CK and desmin. If cultured in 0.5% FCS, HGCT-1 cells acquired a uniform co-expression of vimentin, CK and desmin. Upon treatment with retinoic acid (RA), HGCT-1 cells lost the expression of desmin, but exhibited abundant CK filaments. Simultaneously, they started to express desmoplakin, form desmosomes and flatten on the culture substratum. The RA-induced changes were irreversible, whereas those following the culture in 0.5% FCS were at least partially reversible. When xenografted into an immunosuppressed rat, HGCT-1 cells formed a tumor consisting of epithelial- and mesenchymal-like structures. HGCT-1 cells thus represent a pluripotential cell system with a capacity for reversible phenotypic modulation and for irreversible differentiation into epithelial-type cells. The behavior of this novel cell line, distinct from established EC cell models, suggests a complex regulation of GCT cell differentiation.

Aluminium, lead and cadmium concentrations in seminal plasma and spermatozoa, and semen quality in Finnish men.

Aluminium, cadmium and lead concentrations in the spermatozoa and seminal plasma of 27 employees of two industrial companies, a refinery and a polyolefin factory, and 45 consecutive sperm donor candidates at a sperm bank were studied using atomic absorption measurements. The relationship between metal concentration and parameters of semen analysis was studied. A high concentration of aluminium in spermatozoa was correlated with decreased sperm motility. The concentrations of cadmium and lead were low and did not show any correlation with parameters of semen analysis. Aluminium may be one of the environmental pollutants causing impaired semen quality. The mean sperm concentrations were similar in the factory employees (96 x 10(6)/ml), in the sperm donor candidates of the comparison group (104 x 10(6)/ml) and in 352 donor candidates at the sperm bank of the Family Federation of Finland (107 x 10(6)/ml) between May 1993 and May 1995.

Pregnancy resulting from intracytoplasmic injection of spermatozoa from a frozen-thawed testicular biopsy specimen.

A testicular biopsy specimen was taken in connection with scrotal exploration of a healthy 35 year old man who had azoospermia. Bilateral severe scarring of unknown aetiology was found in the exploration, and no epididymal spermatozoa could be obtained. Spermatozoa from the fresh biopsy specimen were used for intracytoplasmic sperm injection (ICSI) on the same day. Two-embryo transfer resulted in biochemical pregnancy. The rest of the biopsy specimen was frozen as small pieces of tissue using glycerol as a cryoprotectant. ICSI was then performed with spermatozoa prepared from the frozen-thawed tissue. One embryo was obtained and transferred. The transfer resulted in pregnancy, and a living fetus was seen in ultrasound scans at the seventh and 16th weeks of pregnancy. It is possible to avoid repeated testicular biopsies by using cryopreservation of testicular tissue.

Preimplantation diagnosis by whole-genome amplification, PCR amplification, and solid-phase minisequencing of blastomere DNA.

We have developed a new method for preimplantation diagnosis of inherited diseases. Our procedure for the identification of point mutations in single cells combines whole-genome amplification using 15-mer random primers (primer extension preamplification, PEP) with a single locus-specific PCR amplification, followed by detection of the mutation by solid-phase minisequencing. The procedure was evaluated by detecting three disease-causing mutations and seven polymorphic nucleotides located on different human chromosomes from single granuloma and blastomere cells. The correct genotype of the cell was identified at 96% of the nucleotide positions analyzed, showing that a representative part of the genome is amplified during PEP. We estimate that PEP yielded at least 1000 copies of the genome. The quantitative nature of the solid-phase minisequencing method allowed us to notice that preferential amplification of one allele occurs at heterozygous loci during PEP, which is a potential problem in preimplantation diagnosis.

Vas deferens aspiration and intracytoplasmic injection of frozen-thawed spermatozoa in a case of anejaculation in a diabetic man.

By using aspiration from the vas deferens, apparently good quality spermatozoa can be obtained for in-vitro fertilization (IVF) in cases of non-treatable anejaculation. Being squeezed from the epididymis during aspiration, the spermatozoa may be immature and their fertilizing capacity lower than that of ejaculated spermatozoa. Our case report describes a couple who achieved pregnancy when intracytoplasmic sperm injection (ICSI) was carried out with frozen-thawed spermatozoa aspirated from the vas deferens of a man whose anejaculation was associated with diabetes mellitus. In the aspiration, 50 x 10(6) spermatozoa were obtained. One half of them was frozen, and the other half was used fresh for conventional IVF, resulting in total fertilization failure of all the oocytes. The second treatment was ICSI, in which eight out of 11 oocytes injected with frozen-thawed spermatozoa showed normal fertilization. The second frozen embryo transfer resulted in a normal pregnancy.

Treatment of severe male immunological infertility by intracytoplasmic sperm injection.

A total of 29 infertile couples (group A) with male antisperm antibodies detected by the mixed antiglobulin reaction (MAR) and partly by flow cytometry (n = 21) were treated using an intracytoplasmic sperm injection (ICSI) technique to assist fertilization. In all, 22 of them had shown a poor fertilization rate (6%) in previous in-vitro fertilization (IVF) treatments. The fertilization and cleavage rates in ICSI, 79 and 89% respectively, were similar to those in a MAR-negative group (group B; n = 20) injected because of male infertility (68 and 93% respectively). A third group (group C; n = 37) with male immune infertility was treated by conventional IVF. All these couples had at least one oocyte fertilized, but the overall fertilization rate (44%) in group C was significantly poorer (P < 0.001) than that in the two ICSI groups. However, the embryo quality was lower in group A compared with that in the other groups. A total of 13 pregnancies resulted in group A (46%), of which five ended in miscarriage. None of the six pregnancies (30%) in group B aborted during the first trimester. These results reveal, for the first time, that ICSI offers a good chance of fertilization for couples with male immunological infertility. However, post-fertilization events may compromise these results because of factors not yet clearly understood.

Testicular needle biopsy, open biopsy, epididymal aspiration and intracytoplasmic sperm injection in obstructive azoospermia.

Testicular or epididymal spermatozoa were obtained for in-vitro fertilization and intracytoplasmic sperm injection (ICSI) in 27 cycles out of 33 (in six men the azoospermia proved to have testicular causes). Testicular needle biopsy carried out in addition to surgical open biopsy proved to be an effective method to obtain spermatozoa for ICSI from patients with obstructive azoospermia. Thus it might be possible to replace scrotal operations by simple needle biopsies. Embryos resulting from ICSI with testicular spermatozoa were used in 19 transfers that resulted in six pregnancies. One pregnancy resulted from six embryo transfers from ICSI after microsurgical-epididymal sperm aspiration (MESA). The normal fertilization rates with testicular (37.3%) and MESA spermatozoa (53.7%) did not differ significantly from each other, but with testicular spermatozoa the rate was significantly lower than that obtained with ejaculated spermatozoa and ICSI (59.7%) in the matched couples. The abnormal fertilization of oocytes with one pronucleus was significantly higher with testicular spermatozoa than with ejaculated spermatozoa in the control couples.

The cytoskeleton and associated proteins during cleavage, compaction and blastocyst differentiation in the pig.

The organization of the cytoskeleton during early pig embryogenesis was investigated by using fluorescence and electron microscopy. The early morphogenesis of the pig embryo differed from that of the mouse, the standard model of the early mammalian development. In the pig, both compaction and polarization were gradual, and definitive polarization of cell surface microville occurred first shortly before blastocyst formation; the compaction and polarization of the mouse embryo are completed as early as at the 8 cell stage. Furthermore, the pig morula undergoes cycles of compaction and decompaction throughout its development. Distinct changes in the distribution of actin and the actin-associated proteins alpha-fodrin, vinculin and E-cadherin coincided with these events. In the pig, all these molecules were evenly distributed at all aspects of the blastomeres during early cleavage and then gradually accumulated in regions of intercellular contacts toward the blastocyst stage; microfilaments in trophectoderm cells formed a cortical meshwork associated with apical microvilli and adherent junctions (zonula adherens). In the mouse, the corresponding changes occur earlier, at the 8 cell stage. Microtubules formed a network-like cortical layer beneath the microvilli at the free outer surfaces of pig blastomeres. Cytokeratin bundles were not observed until the early blastocyst, where they characteristically associated with newly formed desmosomes. In both species a close correlation between morphologically defined developmental stages and the organization of the cytoskeleton: actin and actin-associated proteins are involved in polarization and compaction, whereas the appearance of intermediate filament bundles coincides with the building of the first epithelium, the trophectoderm; it is in the timing of events that a contrast between species is observed.

Maintenance of compaction and adherent-type junctions in mouse morula-stage embryos.

Timed morulae of different stages of development were exposed to cytochalasin B causing depolymerisation of microfilaments and to ECCD-1 antibodies interacting with Ca2(+)-dependent adhesion molecules or cultured in the absence of calcium. All three treatments decompacted mid-morula-stage embryos within one hour. Late morulae were resistant to ECCD-1 antibody treatment and relatively resistant to calcium-free cultivation, but not to cytochalasin B treatment. Scanning electron microscopy revealed that the decompacting treatments not only loosened the interblastomere contacts but also resulted in rearrangement of the cell surface microvilli. Transmission electron microscopy showed that normal, untreated embryos had specialized membrane junctions in the most apical regions of the interblastomere contacts. Immunoelectron microscopy revealed that these apical junction areas contained vinculin, a protein typical of adherent junctions. Upon decompaction the apical junctions disappeared completely. When transferred back to the normal medium, the embryos rapidly started to recompact. Simultaneously the apical junctions and cell surface microvilli reassumed the organization characteristic of the morula stage. Late morulae that were resistant to treatment had normal apical junctional areas. During subcultivation in the normal medium, the treated morulae developed into morphologically normal blastocysts. These data indicate that adherent-type junctions and cell surface microvilli participate in the initiation and maintenance of compaction of morula-stage embryos.

Cytoskeleton in preimplantation mouse development.

This paper reviews the constituents of the cytoskeleton in the cells of the preimplantation mouse embryo and how they change as the development proceeds. The cytoskeleton can be divided into two distinct groups, that in the cytosplasm and that associated with the membrane. The first and better-known group contains microfilaments, microtubules and intermediate filaments, the second such components of the cell and nuclear membrane as spectrin-like protein and nuclear lamin. The filamentous components of the cytoplasmic cytoskeleton adhere to the nuclear and cell membrane at attachment points where specific proteins such as vinculin may mediate the interaction. Each cell of the early embryo has all of these components, but their morphological organization and molecular constitution alter as the embryo develops. These modifications are especially pronounced when the cleavage-stage embryo compacts and when the blastocysts forms and differentiates. These events represent the most critical stages of morphogenesis and cytodifferentiation in the preimplantation embryo. The cytoskeleton may thus have an important role in the control of the early mammalian development.

Alternative complement pathway activation by Salmonella O polysaccharide as a virulence determinant in the mouse.

The quality of Salmonella O polysaccharide (the O antigen) is a virulence factor in mouse salmonellosis. It affects the rate by which these bacteria are phagocytosed and by which they activate the alternative complement pathway in a manner inversely proportional to their virulence, suggesting that the rate of complement activation is crucial for the fate of the bacteria in the mouse. The effector mechanism has, however, remained open since Salmonellae survive and multiply in the macrophages of the mouse. We show in this study that although the least virulent O-6,7 Salmonellae multiply in the liver macrophages they are rapidly killed in the peritoneal cavity by the local resident macrophages. Electron microscopy showed a striking morphological feature--a 35 nm thick homogenous electron-dense deposit--on all the bacteria found in association with the macrophages but absent from all non-cell-associated bacteria. A similar precipitate was formed by incubating the bacteria in fresh mouse serum and was dependent on heat-labile serum components and bound anti-C3. The least virulent O-6,7 bacteria acquired this deposit more rapidly and in a lower concentration of serum than the more virulent O-4,12 bacteria consistent with the previously demonstrated difference between these bacteria in their rate of complement activation via the alternative pathway. Preincubation of the O-4,12 bacteria in fresh mouse serum leading to complement deposition on 80% of the bacteria effectively opsonized them for rapid killing in the peritoneal cavity. These data for the first time demonstrate how the rate of complement activation determines the virulence of Salmonellae.

Changes in the distribution of vinculin during preimplantation mouse development.

It has been proposed that vinculin is a microfilament bundle-membrane linking cytoskeletal protein. We used double-fluorescence microscopy to study the distribution of vinculin and F-actin in mouse oocytes and preimplantation embryos. In oocytes and in the cells of cleavage- and blastocyst-stage embryos, vinculin exhibited a diffuse cytoplasmic distribution and was concentrated in a submembranous layer. The presence of vinculin in oocytes was confirmed by immunoblotting. In oocytes, a distinct concentration of actin was observed above the second metaphase spindle. During the 8-cell stage, compacting blastomeres exhibited partial polarization of cortical vinculin and actin toward their outward-facing surfaces. In precompaction-stage blastomeres, the submembranous layer of vinculin contained a ring-like concentration in the most peripheral region of each intercellular contact area. During later development, the amount of vinculin localized in the areas of intercellular contacts became modified. In embryos ranging from the compacted 8-cell stage to the mid-morula stage, the vinculin-specific fluorescence was only intense in some intercellular contacts, being indistinct in most contact areas. In late morulae, the flattened outer cells increasingly exhibited concentration of vinculin in contact areas. In contrast, actin-specific fluorescence was clearly evident in most intercellular contacts throughout the morula stage. At the early blastocyst stage, all contacts of the trophectoderm (TE) cells again regularly exhibited concentration of both components. At the late blastocyst stage, the staining pattern changed once again: the contact-associated concentration of vinculin-specific fluorescence was not observed in polar TE cells, while remaining clear in mural TE cells. In blastocyst outgrowths, TE cells displayed typical vinculin plaques at the peripheries of the cells. The continuous changes in the distribution of vinculin and actin suggest that these components are involved in the control of cellular relationships during early development. Immunoelectron microscopy and experiments using cytochalasin were performed in an attempt to relate the distribution of vinculin to the ultrastructural features of embryo cells.

Localization of nonerythroid spectrin and actin in mouse oocytes and preimplantation embryos.

Mouse oocytes, cleavage-stage embryos, and blastocyst-stage embryos were studied to show the distribution of both an immunoanalog to nonerythroid spectrin (p 230) and F-actin. Using antibodies to nonerythroid spectrin, diffuse, positive cytoplasmic fluorescence was regularly seen in oocytes and embryo cells. The presence of nonerythroid spectrin in oocytes was confirmed by immunoblotting. Oocytes usually exhibited an inconspicuous submembranous layer of nonerythroid spectrin, which was more pronounced in the area of the polar body. Oocytes regularly exhibited a peripheral concentration of actin. Throughout the cleavage and blastocyst stages, a cortical layer of nonerythroid spectrin and actin was usually observed in embryo cells. These submembranous layers on the outer surface of the embryo were relatively thin as compared to those in areas of intercellular contact. The contact areas regularly showed distinct positive staining, including a concentration of label at the most peripheral region of each contact area. This resulted in the presence of ring-like fluorescence around each blastomere. Nonerythroid spectrin and actin showed concentration to the contact area between the oocyte and the polar body. Although the general localization patterns of nonerythroid spectrin and actin were similar, double-staining experiments revealed that slightly different planes of focus were necessary to obtain sharp definition of the fluorescence of these components in areas of intercellular contact: the ring-like concentration of nonerythroid spectrin appeared to be localized more peripherally than that of actin. The cells of preimplantation embryos show motile features that include actual cell movements and striking changes in cell shape (e.g., during compaction). The submembraneous layers of nonerythroid spectrin and actin may contribute to the regulation of the deformability and thus the shape of embryo cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell relationships during aggregation between preimplantation embryos and teratocarcinoma-derived cells.

Cleavage-stage mouse embryos aggregate and form chimaeric blastocysts with embryonal carcinoma (EC) cells. We used scanning and transmission electron microscopy to study cell relationships during aggregate formation between 8-cell-stage embryos and F9 EC cells. Relations between heterotypic cells were similarly studied in aggregation experiments with embryos and teratocarcinoma-derived visceral (PSA5-E) and parietal (PYS-2) endoderm cells and in experiments with EC cells and endoderm cells. The embryos and F9 cells always adhered to each other and rapidly formed compacted aggregates. Numerous microvilli and cell processes, originating from both embryo and EC cells, extended between the two cell types during adhesion and early phases of aggregation. The aggregation process involved spreading of the blastomeres on the EC cells. Frequent adherent junctions and close contacts, including possible focal gap or tight junctions were observed between the embryo and F9 cells after 3 h of culture. Apparent gap or tight junctions were infrequent during the early phases of aggregation but during further culture, extensive typical gap junctions were also seen between embryo and EC cells. The embryos adhered only irregularly and loosely to PSA5-E and PYS-2 cells; this interaction never led to aggregate formation comparable to that seen in the experiments with embryos and EC cells. Close contacts but no gap or tight junctions could be observed between the embryo and endoderm cells. On the other hand, both PSA5-E and PYS-2 cells readily adhered to and aggregated with EC cells. The present results suggest that microvilli and cell processes mediate membrane interactions during adhesion and early phases of aggregation between embryos and EC cells. During aggregation, blastomeres spread over the EC cells, and rapid formation of adherent junctions and close contacts, including possible focal gap or tight junctions is involved during the early phases of this process. After this initial phase, typical gap junctions are also seen between the embryo and EC cells. Interestingly, adhesive properties of embryo and EC cells differ: the former aggregate only with EC cells, whereas the latter do so also with teratocarcinoma-derived visceral and parietal endoderm cells. Mechanisms operating in the morphogenetic movement of cells in this experimental setup may be involved also in the development of the blastocyst in vivo.