Laser-assisted hatching improves clinical outcomes of vitrified-warmed blastocysts developed from low-grade cleavage-stage embryos: a prospective randomized study.

The aim of this study was to evaluate the effects of quarter zona-pellucida (ZP) opening by laser-assisted hatching (QLAH) on the clinical outcomes following transfer of vitrified-warmed blastocysts developed from low-grade cleavage-stage embryos in patients with all high-grade and fair-grade cleavage-stage embryos transferred without achieving pregnancy. Patients were randomized into two groups: QLAH (n=101) and control (n=102). The implantation and clinical pregnancy rates were significantly higher in the QLAH group compared with the control group (P=0.021 and P=0.034, respectively). The live birth rate of the QLAH group was also higher, although not significantly. When the clinical outcomes according to the day of blastocyst vitrification were compared between the groups, the implantation, clinical pregnancy and live birth rates of the QLAH group were significantly higher (P<0.05) than those of the control group for day 6 blastocysts, but not for day 5 or day 5/day 6 blastocysts. These results suggest that QLAH improves the clinical outcomes of vitrified-warmed blastocysts, especially of day 6 vitrified blastocysts, developed from low-grade cleavage-stage embryos.

Structural chromosomal aberrations, aneuploidy, and mosaicism in early cleavage mouse embryos derived from spermatozoa exposed to γ-rays.

PURPOSE: To quantitatively and qualitatively investigate the changes in chromosomal aberrations during early cleavage in mouse embryos derived from γ-irradiated spermatozoa. MATERIALS AND METHODS: Mature males were exposed to 2 Gy or 4 Gy of ¹³7Cs γ-rays, and their spermatozoa were used to produce embryos via in vitro fertilisation (IVF). The metaphase chromosomes were prepared from one-cell, two-cell, and four-cell embryos. In the chromosome preparations from two-cell and four-cell embryos, the separation of the sister blastomeres was precluded by treatment of the embryos with concanavalin A. The incidence of embryos with structural chromosomal aberrations, aneuploidy, or mosaicism was estimated. The fates of the different types of γ-ray-induced structural chromosomal aberrations were also investigated in those embryos. RESULTS: The exposure of spermatozoa to 2 Gy or 4 Gy γ-rays caused structural chromosomal aberrations in 25.9% and 35.7% of the resultant one-cell embryos, respectively. At two-cell embryonic stage, the incidence of structural chromosomal aberrations was 17.4% in the 2 Gy group and 27.1% in the 4 Gy group. At the four-cell embryonic stage, although the incidence of control embryos with structural chromosomal aberrations was considerably high, the net incidence of embryos with radiation-induced structural chromosomal aberrations was similar to that at the one-cell stage. The incidence of aneuploidy was high in two-cell and four-cell embryos after both doses of γ-rays. The incidence of mosaicism increased significantly in dose- and embryonic-stage-dependent manners. Anaphase lag, and the degeneration and non-disjunction of the aberrant chromosomes were frequently observed in aneuploid and mosaic embryos. CONCLUSIONS: Mouse sperm DNA is highly vulnerable to γ-rays. The structural chromosomal aberrations of sperm origin are unstable in their behaviour and structure during cleavage, and therefore cause secondary aneuploidy and mosaicism in the early cleavage embryos.

Evaluation of the safety of time-lapse observations for human embryos.

PURPOSE: To assess the effects of light from an integrated optical microscope and evaluate the safety of time-lapse observations using a built-in microscope incubator. METHODS: We prospectively compared the fertilization rate and embryonic morphology after intracytoplasmic sperm injection between embryos cultured with time-lapse observations every 15 min in an incubator with an integrated optical microscope and embryos with intermittent observations (once a day) in conventional incubators. RESULTS: No significant differences were observed in the fertilization rate (57.5% vs. 57.5%) or the rate of excellent-good cleavage embryos (36.0% vs. 36.0%). CONCLUSIONS: These results suggest that time-lapse observations using an incubator with an integrated optical microscope may therefore be safely utilized in clinical practice.

Extremely low frequency electromagnetic field exposure affects fertilization outcome in swine animal model.

Modern society continuously exposes the population to electromagnetic radiation, the effects of which on human health, in particular reproduction, are still unknown. The aim of this research was to assess the effect of acute (1h) exposure of boar spermatozoa to a 50 Hz extremely low frequency electromagnetic field (ELF-EMF) on early fertility outcome. The effect of intensities ranging from 0 to 2 mT on morpho-functional integrity of capacitated spermatozoa was examined in vitro. The oviducts containing or without spermatozoa were then exposed to the minimum in vivo, TD(50,) and maximum intensities determined in vitro, 4h before ovulation. The effects of ELF-EMF on spermatozoa in terms of early embryo development were evaluated after 12h and 6 days. It was found that in vitro ELF-EMF > 0.5 mT induced a progressive acrosome damage, thus compromising the ability of spermatozoa to undergo acrosomal reaction after zona pellucida stimulation and reducing the in vitro fertilization outcome. These effects became evident at 0.75 mT and reached the plateau at 1 mT. Under in vivo conditions, the ELF-EMF intensity of 1 mT was able to compromise sperm function, significantly reducing the fertilization rate. In addition, the exposure of oviducts to fields > or = 0.75 mT in the absence of spermatozoa was able to negatively affect early embryo development. In fact, it was found to cause a slowdown in the embryo cleavage. In conclusion, it was demonstrated how and at which intensities ELF-EMF negatively affect early fertility outcome in a highly predictive animal model.

50-Hertz electromagnetic fields induce gammaH2AX foci formation in mouse preimplantation embryos in vitro.

Effects of electromagnetic fields (EMFs) on DNA damage in mammals are still controversial. In the present study, the effects of EMFs on DNA damage in preimplantation mouse embryos in vitro were investigated by using gammaH2AX foci formation, a new sensitive indicator for detecting DNA double-strand breaks (DSBs). The data obtained demonstrated that EMFs decreased the cleavage rate of preimplantation mouse embryos. This decreasing effect of EMFs was related to the DNA-damaging effect indicated by the induction of gammaH2AX foci formation in preimplantation mouse embryos. The inducing effects of EMFs on gammaH2AX foci formation could be inhibited by the treatment of noise MFs or wortmannin, a phosphatidylinositol 3-kinase (PI3K) family inhibitor. Furthermore, the data obtained also showed that EMFs could activate the DNA damage-repair mechanism by recruiting repair factor Rad50 to the damaged DNA sites to repair the corresponding DNA damage. These findings suggest that EMFs could cause DNA damage in preimplantation embryos in vitro and that the adverse effects of EMFs on development might at least partly act through DNA damage. The DNA damage induced by EMFs could be at least partly repaired by the natural activation of DNA damage-repair mechanism or prevented by the simultaneous treatment of noise magnetic fields.

Ultraviolet irradiation of eggs and blastomere isolation experiments suggest that gastrulation in the direct developing ascidian, Molgula pacifica, requires localized cytoplasmic determinants in the egg and cell signaling beginning at the two-cell stage.

Gastrulation in the maximum direct developing ascidian Molgula pacifica is highly modified compared with commonly studied "model" ascidians in that endoderm cells situated in the vegetal pole region do not undergo typical invagination and due to the absence of a typical blastopore the involution of mesoderm cells is highly modified. At the gastrula stage, embryos are comprised of a central cluster of large yolky cells that are surrounded by a single layer of ectoderm cells in which there is only a slight indication of an inward movement of cells at the vegetal pole. As a consequence, these embryos do not form an archenteron. In the present study, ultraviolet (UV) irradiation of fertilized eggs tested the possibility that cortical cytoplasmic factors are required for gastrulation, and blastomere isolation experiments tested the possibility that cell signaling beginning at the two-cell stage may be required for the development of the gastrula. Irradiation of unoriented fertilized eggs with UV light resulted in late cleavage stage embryos that failed to undergo gastrulation. When blastomeres were isolated from two-cell embryos, they developed into late cleavage stage embryos; however, they did not undergo gastrulation and subsequently develop into juveniles. These results suggest that cytoplasmic factors required for gastrulation are localized in the egg cortex, but in contrast to previously studied indirect developers, these factors are not exclusively localized in the vegetal pole region at the first stage of ooplasmic segregation. Furthermore, the inability of embryos derived from blastomeres isolated at the two-cell stage to undergo gastrulation and develop into juveniles suggests that important cell signaling begins as early as the two-cell stage in M. pacifica. These results are discussed in terms of the evolution of maximum direct development in ascidians.

Comparison of embryonic development in cleavage stage mouse embryo biopsy between acid Tyrode's solution and laser assisted techniques.

The study was carried out to determine the effectiveness and safety of the infrared 1.48 microm laser in cleavage stage mouse embryo biopsy, compared to the conventional acid Tyrode's solution. One hundred and thirty cryopreserved cleavage stage mouse embryos were included in the study. Fifty embryos were biopsied by acid Tyrode's solution. Forty-seven embryos were biopsied by the infrared 1.48 microm laser. Thirty-three embryos were incubated without biopsy as the control group. Thirteen of 50 embryos in the acid Tyrode's group and 16 of 47 in the laser assisted group became cavitating morulae on day 4, meanwhile 23 of 33 in the control group reached this stage. The blastocyst formation of acid Tyrode's, laser assisted and control group were 94.0, 97.8 and 100.0 per cent, respectively. The hatching rate of acid Tyrode's solution, laser assisted and control group were 78.7, 84.7 and 63.6 per cent, respectively. No significant difference in blastocyst formation and hatching rate was found. The percentage of grade I blastocysts in control group (96.9%) was significantly higher than those in acid Tyrode's solution (68.0%) and the laser assisted group (76.0%). There was no significant difference in the percentage of grade 1 blastocysts between the acid Tyrode's solution and the laser assisted group. In conclusion, the infrared 1.48 microm wavelength laser may be an alternative to acid Tyrode's solution in embryo biopsy.

Distribution of dorsal-forming activity in precleavage embryos of the Japanese newt, Cynops pyrrhogaster: effects of deletion of vegetal cytoplasm, UV irradiation, and lithium treatment.

Two types of axis-deficient embryos developed after deletion of the vegetal cytoplasm: wasp-shaped embryos and permanent-blastula-type embryos. In situ hybridization revealed that neither type of axis-deficient embryo expressed goosecoid or pax-6. brachyury was expressed in the constricted waist region of the wasp-shaped embryos but was not expressed in the permanent-blastula-type embryos. Further, we examined the effect of UV irradiation on Japanese newt embryos. Surprisingly, UV-irradiated Japanese newt eggs formed hyperdorsalized embryos. These embryos gastrulated in an irregular circular fashion with goosecoid expression in the circular equatorial region. At tailbud stage, these embryos formed a proboscis which is very reminiscent of that formed in hyperdorsalized Xenopus embryos. Transplantation of the marginal region of the UV-irradiated embryos revealed that the entire marginal zone had organizer activity. Thus we conclude that UV hyperdorsalizes Japanese newt embryos. Finally, lithium treatment of normal embryos at the 32-cell stage also resulted in hyperdorsalization. Lithium treatment of vegetally deleted embryos had two distinct results. Lithium treatment of permanent-blastula-type embryos did not result in the formation of dorsal axial structures, while the same treatment reinduced gastrulation and dorsal axis formation in the wasp-shaped embryos. Based on these results, we propose a model for early axis specification in Japanese newt embryos. The model presented here is fundamentally identical to the Xenopus model, with some important modifications. The vegetally located determinants required for dorsal development (dorsal determinants, DDs) are distributed over a wider region at fertilization in Japanese newt embryos than in Xenopus embryos. The marginal region of the Japanese newt embryo at the beginning of development overlaps with the field of the DDs. Gastrulation is very likely to be a dorsal marginal-specific property, while self-constriction is most probably a ventral marginal-specific property in Japanese newt embryos.

Dynamics of germ plasm localization and its inhibition by ultraviolet irradiation in early cleavage Xenopus embryos.

In the spawned Xenopus egg, germ plasm is found as cytoplasmic islands spread over a wide cortical region of the vegetal pole. However, by the blastula stage, the same material is found concentrated into a few large blastomeres at the floor of the blastocoel. Components of the germ plasm can be specifically labeled with a fluorescent dye, DiOC6(3), permitting the dynamic movements of germ plasm localization to be followed in live embryos via time-lapse confocal scanning optical microscopy. During the first cell cycle, the small islands initially appear to be fixed to the vegetal yolk mass and to move with it during the cortical rotation. After rotation, the islands appear to be released from the vegetal yolk mass and to begin fusing with one another. During early cleavages, the germ plasm aggregates into large islands at the vegetal pole in a movement dependent on microtubules. Two distinct actions can be discerned: (1) a continuous process of local fusing and (2) periodic surface contraction waves (SCWs) that gather the islands toward the vegetal pole. These SCWs are inhibited by ultraviolet irradiation of the vegetal pole. Near the vegetal pole, germ plasm patches ingress into the embryo's interior along the cleavage furrow in periodic movements contemporaneous with the SCWs.

Developmental anomalies derived from exposure of zygotes and first-cleavage embryos to mutagens.

Results of continuing studies indicate that the mouse zygote and two-cell embryo stages are a window of susceptibility in the experimental induction of congenital anomalies with certain mutagenic agents. The mechanisms by which the mutagens initiate the pathogenesis of these developmental defects are not known. However, in certain cases there is evidence that a nonconventional, perhaps epigenetic, mechanism is involved. Detailed characterization of the spectrum of anomalies induced and comparison of responses at the various stages exposed allowed classification of the mutagens generally into two groups. One group is characterized by being effective only in the early stages of zygote development and capable of producing a relatively high incidence of fetal death and hydrops. The other group affects all of the zygote stages studied as well as the two cell-embryo, but does not increase the incidence of fetal death and hydrops. Except for hydrops, chemicals in the two groups do not differ in terms of the types of anomalies present among malformed live fetuses, which bear a resemblance to a subset of common, sporadic human developmental anomalies that are of unknown etiology. This similarity raises the possibility that certain human developmental defects may have their origins in events that happen in the zygote and early pre-implantation stages.

The effects of tritium on embryo development: the embryotoxic effects of [3H]tryptophan.

The effects of tritiated amino acids on the development of the early mouse embryo in vitro are described. [3H]tryptophan was extremely toxic, compared with [3H]glutamic acid, and inhibited growth of the two-cell embryo to the blastocyst stage. The LD50 values for the two amino acids were, respectively, 1 and 296 kBq/ml of medium. The two-cell embryo was more sensitive to the irradiation from incorporated [3H]tryptophan is selectively incorporated into two chromosomal proteins in the two-cell embryo, and that those proteins were stage-specific markers of development.

The entire mesodermal mantle behaves as Spemann's organizer in dorsoanterior enhanced Xenopus laevis embryos.

The body plan of Xenopus laevis can be respecified by briefly exposing early cleavage stage embryos to lithium. Such embryos develop exaggerated dorsoanterior structures such as a radial eye and cement gland (K.R. Kao, Y. Masui, and R.P. Elinson, 1986, Nature (London) 322, 371-373). In this paper, we demonstrate that the enhanced dorsoanterior phenotype results from an overcommitment of mesoderm to dorsoanterior mesoderm. Histological and immunohistochemical observations reveal that the embryos have a greatly enlarged notochord with very little muscle tissue. In addition, they develop a radial, beating heart, suggesting that lithium also specifies anterior mesoderm and pharyngeal endoderm. Randomly oriented diametrically opposed marginal zone grafts from lithium-treated embryos, when transplanted into ultraviolet (uv)-irradiated axis-deficient hosts, rescue dorsal axial structures. These transplantation experiments demonstrate that the entire marginal zone of the early gastrula consists of presumptive dorsal mesoderm. Vital dye marking experiments also indicate that the entire marginal zone maps to the prominent proboscis that is composed of chordamesoderm and represents the long axis of the embryo. These results suggest that lithium respecifies the mesoderm of Xenopus laevis embryos so that it differentiates into the Spemann organizer. We suggest that the origin of the dorsoanterior enhanced phenotypes generated by lithium and the dorsoanterior deficient phenotypes generated by uv irradiation are due to relative quantities of organizer. Our evidence demonstrates the existence of a continuum of body plan phenotypes based on this premise.

Late division kinetics in relation to modification of protein synthesis in mouse eggs blocked in the G2 phase after X-irradiation.

Mouse zygotes (BALB/c) blocked in the G2 phase of the first cell cycle after X-irradiation were allowed to develop in culture medium. Delayed cleavage occurred at the same time in embryos exposed to 1 or 2 Gy and late division coincided with the second division in controls. Two-dimensional electrophoresis showed that blocked irradiated embryos underwent the same modifications in protein synthesis as control embryos of the same age, except during first mitosis, for three polypeptide sets of 30, 35 and 45 kilodaltons molecular weight. The most remarkable difference between them was the appearance in cleaving controls of three spots at 35 kilodaltons that were absent in blocked irradiated embryos. It is assumed that blocked embryos 'missed' some signal necessary for cell division, but remained ready to cleave when a second signal occurred. Eggs from the BALB/c strain were particularly susceptible to this effect of X-irradiation but it was also found in eggs from other strains, irradiated with much higher doses.

Functions of maternal mRNA as a cytoplasmic factor responsible for pole cell formation in Drosophila embryos.

Injection of mRNA extracted from Drosophila cleavage embryos or mature oocytes restored pole cell-forming ability to embryos that had been deprived of this ability by uv irradiation. However, mRNA extracted from blastoderms did not show the restoration activity. Pole cells thus formed in uv-irradiated embryos bear similarities to normal pole cells both in their morphology and their ability to migrate to the gonadal rudiments. But this mRNA does not appear to be capable of rescuing uv-induced sterility, or inducing pole cells in the anterior polar region.

The influence of G2 progression on X-ray sensitivity of two-cell mouse embryos.

Naturally synchronous, two-cell mouse embryos were X-irradiated in vitro. In experiment 1, irradiation was either in the early or in the late G2 phase, which lasts about 14 hours. In experiment 2, irradiation of all the embryos was in late G2 but embryos with different intervals between irradiation and the first mitosis after irradiation were separated and investigated independently. After 2 Gy the time interval between irradiation in late G2 and the first mitosis post-irradiation was on the average about 9 hours; after irradiation in the early G2 phase about 13.5 hours. Development (hatching of blastocysts) and cell proliferation (cell number per embryo at the stage of the hatched blastocyst) was most impaired and the frequency of micronuclei (determined in four- or eight-cell embryos) was highest in the case of a short interval between irradiation in G2 and the first mitosis post-irradiation. It is concluded that a longer interval allows a longer period of DNA repair. The results also demonstrate a positive correlation between the extent of chromosomal damage (micronuclei) and the extent of cell death as well as the impairment of the development of the whole biological system.

Effect of laser microbeam irradiation of the nucleus on the cleavage of mouse eggs in culture.

Two-cell mouse eggs were irradiated by a helium-cadmium laser on a spot of about 4 micron2 (d = 2.2 micron) in one or both nuclei either continuously or repeatedly at 0.36 erg micron-2 sec-1 and then cultured to observe cellular development. After exposing one nucleus to the microbeam to five or seven 1-sec pulses (1.80 or 2.52 ergs micron-2, respectively), about 45% developed to the 3-cell stage in 24 hr of culture. In overnight cultures of the 2-cell eggs in which both nuclei were irradiated for 9 or 20 sec continuously, 40 (9 sec) and 50% (20 sec) of the eggs remained at the 2-cell stage, while 45 (9 sec) and 25% (20 sec) developed to the 4-cell stage. Irradiating only one nucleus in a 2-cell egg by seven pulses in a spot of 4 micron2 amounting to 10 ergs reduced cleavage 45%. When both nuclei were each irradiated by a 9-sec continuous laser beam (totaling 13 ergs), about 40% of the embryos of the 2-cell stage did not divide. The effect of seven pulses on the blastomere cleavage of 2-cell mouse eggs appeared to be comparable to that of continuous 9-sec laser irradiation. Both pulse and continuous laser microirradiation methods may be developed for inactivation of the nucleus as a nonpipetting , less injurious method for enucleation of mammalian eggs.

Further studies on the effect of radiation during the storage of frozen 8-cell mouse embryos at -196 degrees C.

Frozen 8-cell mouse embryos were treated with radiation doses of 0, 10, 50, 100 or 200 cGy gamma-rays at a dose rate of congruent to 5 cGy/day. After thawing the embryos were scored for normal morphological appearance and for development to morulae and blastocysts after 24 h in culture. Embryos from each treatment were then separately transferred to the uteri of pseudopregnant foster mothers which were killed at Day 14 of pregnancy. There was no effect of radiation on morphological appearance, development to morulae and blastocysts, implantation rate, or on the ratio of live fetuses to the number of transferred embryos. As there appeared to be no detrimental effect of up to 200 cGy on frozen 8-cell mouse embryos and, as this is the equivalent of congruent to 2000 years of background radiation, it is concluded that normal levels of background radiation would not be a hazard to the long-term storage of mammalian embryos.

Suppression of first cleavage in the Mexican axolotl (Ambystoma mexicanum) by heat shock or hydrostatic pressure.

Androgenetic diploid axolotls were produced by ultraviolet inactivation of the egg pronucleus shortly after fertilization, followed by suppression of the first cleavage division by hydrostatic pressure or heat shock. After treatment at 14,000 psi for 8 minutes, diploidy was restored in 74% of the embryos, but only 0.8% survived to hatching. A 36-37 degrees C heat shock of 10-minutes duration, applied 5.5 hours after the eggs were collected, yielded a slightly lower percentage of diploids. However, the proportion surviving to hatching was significantly greater (up to 4.6%). A second generation of androgenetic diploids was produced from one of the oldest of the first generation males with a similar degree of success. The lack of significant improvement suggests that the low survival is due to the heat shock per se and not to the uncovering of recessive lethal genes carried by the parent.

A comparative study of the effects of egg rotation (gravity orientation) and UV irradiation on anuran vs. urodele amphibian eggs.

The response of various anuran and urodele eggs to a brief rotation prior to first cleavage were observed. In all instances, the dorsal lip formed on the side of the egg which opposed gravity during the rotation. The ability of egg rotation to prevent the series of developmental abnormalities associated with UV irradiation of the uncleaved egg was also examined. Anuran and urodele eggs responded to those manipulations in a similar fashion. These results should facilitate future studies on the mechanisms involved in the establishment of the dorsal/ventral polarity of the amphibian egg.