Toxic effects of oxygen on human embryo development.

The toxic effects of oxygen on the embryos of various animal species are reviewed. Methodologies for assessing embryonic damage are discussed and possible ways of preventing the damage are explored. Three methods of potentially minimizing oxidative damage to human embryos were tested using gametes, zygotes, and embryos from a clinical IVF programme: (i) decreasing the oxygen tension in the gas phase used for culture during insemination, fertilization, and embryo growth; (ii) changing the formulation of culture media to include some components designed to protect against oxidative damage; and (iii) reducing the duration of insemination to minimize the effect of oxidative damage caused by spermatozoal metabolism. Fertilization, cleavage, embryo utilization, pregnancy, and embryo implantation rates were used to monitor these changes. Although all three methods gave an increase in success rates, there was still a dramatic decrease in success with patient age. It is suggested that, although the system of handling and culturing embryos can be optimized with respect to embryonic mitochondrial function, there are inherent age-related defects in oocytes and embryos that are still more fundamental than the environmental conditions of the embryo.

Time course of pronuclear formation and fertilisation after insemination in vitro and intracytoplasmic sperm injection of in vitro matured sheep oocytes.

The time course of sperm decondensation, oocyte activation, pronuclear formation and the possible causes of abnormalities after intracytoplasmic sperm injection (ICSI) and in vitro fertilisation (IVF) were examined. Frozen-thawed and pooled fresh semen from three different rams were washed and capacitated for ICSI or IVF. In vitro matured oocytes were cultured after sperm injection for 0.5, 0.75, 1, 2, 3, 4, 5, 6, 8, 18, 21 and 23 h, and oocytes were cultured after in vitro insemination for the same times other than 18 and 23 h. All oocytes were cultured in bicarbonate-buffered synthetic oviduct fluid medium (BSOF) supplemented with 2% oestrous sheep serum. A total of 746 metaphase II oocytes were injected with a single spermatozoon and 986 oocytes were inseminated for IVF. The earliest oocyte activation after ICSI was observed at 0.5 h, when 14.8% of oocytes were in anaphase II; this was earlier than after IVF, when only 6.4% of the oocytes exhibited anaphase II 1 h after insemination. Decondensing spermatozoa were first observed 1 h after ICSI and 3 h after insemination for IVF. The earliest female and male pronuclei after ICSI were observed at 2 and 3 h respectively, while the female and male pronuclei after IVF were observed at 4 h after insemination. The overall fertilisation rate was lower after ICSI (28.6%) than IVF (70.4%) but the percentage of abnormal fertilisation was not different between ICSI (8.7%) and IVF (15.2%). It was concluded that the fertilisation events were more advanced for ICSI than IVF, using injection and insemination time as reference points. The formation of male and female pronuclei were asynchronous after ICSI, in contrast to IVF when they appeared simultaneously at 4 h. Abnormalities found in fertilisation after ICSI may therefore be induced by the injection technique.

Sheep oocyte activation after intracytoplasmic sperm injection (ICSI).

The effect of calcium concentration on fertilization and activation was examined in oocytes injected in vitro with sperm. Oocytes were subjected to sperm injection, to sham injection or remained uninjected, and were then cultured for 19 h in bicarbonate-buffered synthetic oviduct fluid (BSOF) without calcium, or containing either calcium chloride or calcium ionophore. There was no difference in fertilization rates after ICSI when oocytes were cultured in vitro in media containing calcium chloride or calcium ionophore but the rate was lower in calcium-free media. There was also no difference in the fertilization rate after ICSI when oocytes were cultured in vivo compared with that observed in vitro in media containing calcium chloride or calcium ionophore. In calcium chloride-treated oocytes, activation was induced by mechanical injection, and in calcium ionophore-treated oocytes, by the ionophore. In uninjected oocytes, calcium itself did not cause oocyte activation. It is concluded that it is possible to induce activation by the injection process, but that manipulation alone is inadequate to cause proper oocyte activation unless calcium is also present. No difference in oocyte activation between ICSI and sham injection was found, indicating that the sperm may play no role in the early events of oocyte activation.

Cleavage, development and competence of sheep embryos fertilized by intracytoplasmic sperm injection and in vitro fertilization.

More abnormal fertilization has been found in sheep oocytes after intracytoplasmic sperm injection (ICSI) than after in vitro fertilization (IVF). Although the birth of a normal lamb has been reported, the efficiency of blastocyst production is low. We therefore evaluated the cleavage, development and viability of sheep embryos obtained from ICSI, IVF and sham injection. In vitro matured oocytes either injected or inseminated with spermatozoa were assessed for cleavage 1 and 4 d after injection or insemination, and for development to blastocyst after 7 d of culture. A total of 699 oocytes was injected (ICSI); 198 (30.6%) were activated and 55 (8.5%) developed to the blastocyst stage. Of the 17 recipient ewes with 1, 2, 3 or 4 embryos, 15 (88.2%) were pregnant on Day 18; of these 17 recipients, 7 (41.1%) and 6 (35.2%) ewes remained pregnant on Days 45 and 110, respectively. Two normal lambs were born, one ewe died on Day 110 with 2 normal male fetuses, another ewe aborted on Day 90 and 4 pregnancies were maintained. A total of 517 oocytes was inseminated (IVF); 296 (62%) were activated and 90 (18.8%) reached the blastocyst stage. A total of 19 ewes received 1, 2, 3 or 4 embryos; of these, 13 (68.4%) were pregnant on Day 18, 8 (42.1%) ewes remained pregnant on each of Days 45 and 110. Three ewes delivered 5 lambs. Five pregnancies were maintained. A total of 156 oocytes was sham injected, 38 (24.3%) were activated and no blatocysts were obtained after culture. The results of this study showed that blastocysts obtained after ICSI are potentially viable and are not a result of parthenogenesis.

Expression of immunoglobulin superfamily cell adhesion molecules on murine embryonic stem cells.

The expression of cell adhesion molecules of the Ig superfamily (Ig-CAM) were examined on embryonic stem (ES) cells during culture in vitro. ES cells maintained an undifferentiated phenotype when cultured in the presence of leukemia inhibitory factor (LIF) or with fibroblast feeder cells; > 90% of cells reacted positively to an antibody (ECMA-7) that marks undifferentiated ES cells. Using flow cytometry, high concentrations of ICAM-1, VCAM-1, and NCAM antigens were detected on undifferentiated ES cells, but their specific receptors, Mac-1, LFA-1, and VLA-4, were not detected. There was also no class I or II major histocompatibility complex (MHC) antigen expression. The ICAM-1 expressed was functional, since anti-ICAM-1 significantly (p < 0.0001) blocked ES cell-lymphocyte binding. Ig-CAM and MHC-1 expression on undifferentiated ES cells was not up-regulated by treatment of cells with interferon-gamma (IFN-gamma), tumor necrosis factor alpha, or flavivirus infection, agents that up-regulate these molecules in other embryonic cell types. Twelve hours after LIF withdrawal, ICAM-1 and NCAM expression decreased significantly, while VCAM-1 was undetectable. However, morphology and ECMA-7 expression remained unchanged. Similar patterns of expression were seen on ES cells maintained on fibroblast feeder cells. This suggests that LIF or other cytokines may maintain the expression of Ig-CAMs on undifferentiated cells. Differentiation was induced by dimethyl sulfoxide treatment for 14 days. Cells changed from a colony-forming to a monolayer morphology, and approximately 60% of the cell population no longer expressed ECMA-7. In these cells, VCAM-1 was undetectable and ICAM-1 and NCAM had declined to low levels. In these differentiated cells, ICAM-1 and MHC-1 were inducible by IFN-gamma. This study suggests that the pattern of expression of the Ig-CAMs in ES cells may have a role in defining the phenotype of differentiated and undifferentiated cells.

Effect of culture, incubation and acrosome reaction of fresh and frozen-thawed ram spermatozoa for in vitro fertilization and intracytoplasmic sperm injection.

This study evaluated different sperm treatments for fertilization of sheep oocytes by intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF). In Experiment 1, fresh and frozen semen was separated by Percoll centrifugation and incubated at 30 degrees C or 39 degrees C in HSOF or BSOF medium for 1 h before use for IVF or ICSI. For IVF, oocytes were inseminated and incubated with sperm for 30 min, 4 h and 19 h. Sperm were assessed for acrosome integrity after Percoll centrifugation and 1 h incubation, and those used for IVF were assessed after each period of exposure to the oocytes. Fertilization rates after ICSI were higher for fresh than for frozen-thawed sperm and were highest 19 h after IVF with fresh or frozen-thawed sperm in the presence of HSOF at 30 degrees C. In Experiment 2, fresh semen was separated by Percoll centrifugation and incubated for 5 h in HSOF, and the acrosome reaction was induced with lysophosphatidylcholine. Acrosome integrity was then assessed. Fertilization rates after ICSI were similar for acrosome-reacted and control spermatozoa. These results suggest that induction of the acrosome reaction in spermatozoa before ICSI is unnecessary, whereas a capacitating treatment of spermatozoa is required before IVF.

Birth of a male lamb derived from an in vitro matured oocyte fertilised by intracytoplasmic injection of a single presumptive male sperm.

The developmental competence of in vitro matured ovine oocytes, cytoplasmically injected with single male or female chromosome-bearing sperm, was investigated. Eighty-five unsorted, 92 'female-sorted' and 74 'male-sorted' ram sperm were injected into in vitro matured sheep oocytes and, two to four hours later, placed into the oviducts of 28 oestrous sheep. The sperm were separated according to sex by analysis of their DNA content with a flow cytometer. One pregnancy was diagnosed by ultrasound after 55 days and a 3 kg male lamb was born after 150 days gestation. This lamb was derived from an oocyte injected with 'male-sorted' sperm.

Comparative intracytoplasmic sperm injection (ICSI) in human and domestic species.

The current clinical use of intracytoplasmic sperm injection (ICSI) for the alleviation of male factor infertility has prompted a re-investigation of sperm injection techniques in a number of animal species. This report examines sperm injection of in vitro matured oocytes in the major domestic species and compares the results with the human. Ovine, bovine and porcine oocytes can undergo fertilization and at least limited development without exogenous activation either prior to or subsequent to injection. Porcine is temperature sensitive during fertilization and the early stages of embryo development. The oocytes of all three domestic species, particularly ovine, have a tendency to activate after the injection procedure regardless of the presence or absence of sperm. The implications for early development studies and the practical use of direct sperm injection for domestic species are discussed.

Clinical intracytoplasmic sperm injection (ICSI) results from Royal North Shore Hospital.

The technique of intracytoplasmic sperm injection (ICSI) was first introduced to the Royal North Shore Hospital in April 1993 as part of a controlled study of 100 patient cycles in which sibling oocytes were inseminated by either subzonal insemination (SUZI) or ICSI. This trial showed direct sperm injection to be superior in terms of fertilization. In that study, 58 embryo transfers of 101 ICSI-derived embryos resulted in 10 pregnancies. No miscarriages have occurred and a total of 10 fetal hearts (9.8% per embryo transferred) were detected on ultrasound. There have been 10 deliveries of 10 babies. Since the beginning of 1994, intracytoplasmic injection has been used exclusively for patients requiring micromanipulation to achieve fertilization. There have been 200 patient cycles with 1650 oocytes collected (8.8 oocytes per cycle). Of these oocytes, 1548 were mature (94%) and were subjected to ICSI, and normal fertilization occurred in 874 (56%) of the injected oocytes. The number of oocytes which cleaved and were suitable for fresh transfer or cryopreservation was 818 (94%). There have been 153 fresh embryo transfers of 326 embryos. Twenty-six pregnancies (17% per embryo transfer) have resulted, 22 of which proceeded to ultrasound examination in which 23 fetal hearts were detected (7% per embryo transferred). Three miscarriages have occurred, leaving 19 ongoing pregnancies. There have been 127 cryopreservation procedures involving 492 embryos. To date, there have been 47 embryo thaw cycles, and 93 of the 115 (81%) thawed embryos survived and were transferred. These 47 embryo transfers resulted in 10 pregnancies (21% per embryo transfer), one of which one has miscarried.(ABSTRACT TRUNCATED AT 250 WORDS)

Bovine embryo co-culture.

The three methods of culturing bovine zygotes; surrogate oviducts, co-culture and defined media are compared and contrasted. Surrogate oviducts provide the best environment and so provide a benchmark to compare in vitro culture systems. At present, co-culture systems provide a convenient method of reliably producing embryos suitable for transfer. This is important for commercial embryo production. The use of defined media is improving and can be as efficacious as co-culture. The advantage of defined culture systems is the ability to find the requirements of an early embryo and to tailor media to meet them. It is suggested that maturation, not zygote culture, may be the constraint in embryonic development.

Short-term corticosteroid treatment does not improve implantation for embryos derived from subzonal insertion of sperm.

A total of 111 patients undergoing treatment for male factor infertility using SUZI participated in the trial for one treatment cycle only. They were allocated to have either corticosteroid treatment to induce immunosuppression or placebo. An elevated beta-hCG was found in 25% (9 of 36) of patients having an ET and receiving treatment. The corresponding figure for the control group was 33% (10 of 30). On ultrasound examination, fetal hearts were found in 22% (8 of 36) and 20% (6 of 30), respectively, of patients having an ET. chi 2 analysis showed no difference in the results. When the results were analyzed on a per oocyte basis there was no significant difference in the implantation rate.

Injection of multiple sperm into the perivitelline space as a treatment of male infertility.

This paper reports the outcome of 274 treatment cycles using multiple injection of sperm into the perivitelline space as a treatment of male factor infertility. A total of 170 couples underwent this form of treatment; 59.1% of cycles had at least one oocyte normally fertilized with an overall normal fertilization rate of 17.2%. The development rate of normally fertilized embryos was high (98.5%) and resulted in a pregnancy rate (positive human chorionic gonadotrophin 18 days after embryo transfer) of 21.4% per embryo transfer procedure (a maximum of 3 embryos were transferred per procedure). The relationship between the number of sperm injected and the fertilization rate and other factors affecting the outcome are discussed.

Distribution of an endogenous beta-galactoside-specific lectin during foetal and neonatal rabbit development.

The tissue concentration of an endogenous beta-galactoside-specific lectin is developmentally regulated in rabbit tissues during foetal and neonatal development. Immunoassay data and localization studies both indicate that the lectin is particularly associated with late embryonic and early postnatal development. No lectin could be detected in embryos prior to 21 days of gestation, whereas many tissues showed an increase in lectin around the time of birth. It is likely that in most tissues the high concentrations of lectin are largely due to increased synthesis by fibroblasts, which are particularly abundant and active in connective tissue during the extensive tissue reorganization taking place at this time. The lectin appears to be synthesized by other differentiated cell types also, notably myoblasts, alveolar cells and erythroblasts. The peak of lectin concentration seen in foetal liver probably reflects lectin associated with foetal erythropoiesis. The rabbit lectin has a low specific activity but its tissue concentration is correspondingly higher than lectin concentrations in other mammals. This conservation of total lectin activity suggests a fundamental role for the lectin dependent upon its saccharide binding activity. This and other indirect evidence suggests that these lectins are involved in the synthesis, secretion or organization of extracellular matrix components.

Intra- and extracellular distribution of an endogenous lectin during erythropoiesis.

An endogenous beta-galactoside-specific lectin has previously been isolated from rabbit bone marrow. The quantification of extracted lectin now indicates that approximately 75% of the lectin is intracellular in marrow. Indirect immunofluorescence studies show the extracellular lectin is associated with the erythroblast cell surface and is also found in some acellular areas of the marrow stroma. At enucleation, lectin surrounds the extruded nucleus while some residual lectin is observed in the cytoplasm of circulating reticulocytes and erythrocytes.

Selective association of an endogenous lectin with connective tissues.

Using indirect immunofluorescence we have localized an endogenous beta-galactoside-specific lectin in resin-embedded rabbit tissue sections. The pattern of lectin distribution correlates well with biochemical estimations of lectin levels, being abundant in intestine, lung and heart tissue and relatively less abundant in skeletal muscle, liver and kidney. In all tissues lectin is found in connective tissue associated with fibroblasts and the extracellular matrix, and at the periphery of morphologically recognizable smooth muscle cells. The lectin is abundant in skin, intestine and blood vessels, where connective tissue forms the tissue architecture. It is also abundant in heart, where it is particularly associated with the capillaries and lung, where it is also found in alveolar cells. Discrete localization of lectin occurs in areas of connective tissue where epithelial elements are differentiating, such as the crypts of Lieberkuhns in the small intestine and hair follicles in the skin. From these observations we suggest that in cells of mesenchymal origin these endogenous lectins may play a role in the elaboration or organization of the extracellular matrix that regulates tissue differentiation in a number of embryonic and adult tissues.

Surface localization of an endogenous lectin in rabbit bone marrow.

A lectin, which may be involved in cell to cell adhesion during erythropoiesis in rabbit bone marrow, has been isolated and characterized. Several electron microscopical techniques have been used to investigate the cell surface distribution of this lectin in bone marrow utilizing colloidal gold conjugates of anti-lectin IgG or protein A. The lectin is present at the surface of erythroid cells at all stages of development but no lectin was detected on the surface of myeloid cells. The limitations and complementary nature of the techniques used are discussed.

Endogenous beta-galactoside-specific lectins in rabbit tissues.

Soluble beta-galactoside-specific lectins have been detected in many rabbits tissues and are relatively abundant in certain organs. These lectins are apparently identical to the lectin in rabbit bone marrow, which may be involved in inter-erythroblast associations during differentiation, and are very similar in structure and activity to lectins described in a number of species both in adult and embryonic tissues. The rabbit lectin is a monomer with a relative molecular mass of approximately 12 000 and has a low specific activity in the haemagglutination of trypsinized rabbit erythrocytes relative to lectins from other species, which are predominantly dimeric. In addition to the major antigen two minor isoforms of the rabbit galaptin were detected in all tissues. Since these lectins are widely distributed, it seems probable that they are involved in cellular functions that are prominent during development and also maintained in certain differentiated tissues. Experimental evidence currently available suggests that these lectins may be involved in the organization of extracellular matrix components.

Isoforms of an endogenous lectin in rabbit bone marrow.

A lectin which may mediate inter-erythroblast associations during red blood cell development in rabbit bone marrow has previously been purified and characterised. We have now detected different forms of this lectin in purified preparations and crude tissue extracts, by isoelectric focusing in agarose gels followed by rocket immunoelectrophoresis and by indirect antibody staining of focused proteins blotted onto nitrocellulose paper. These minor antigens are probably isoforms of the bone marrow lectin previously characterised.

Fertilization in brown algae. V. Further investigations of lectins as surface probes.

The binding of FITC- (fluorescein isothiocyanate), Au- and 125I-labelled lectins (conA, RCA120 (Ricinus communis agglutinin, MW 120 000) and FBP (fucose-binding protein from Lotus tetragonobolus)) to gametes of Fucus serratus and their physiological effects on fertilization have been studied. Results indicate that eggs strongly bind FITC- and Au-labelled conA and RCA120, whilst FITC-FBP binds strongly to sperm. All three iodinated lectins bound to eggs but this was apparently non-specific and similar in magnitude to the binding of iodinated bovine serum albumin. The results suggested the possibility of two distinct types of lectin receptor on egg surfaces: non-specific, highly abundant receptors and less abundant, specific receptors, possibly locally aggregated. All three lectins inhibit fertilization, FBP being the most effective.