An improved fixation technique for fluorescence in situ hybridization for preimplantation genetic diagnosis.

OBJECTIVE: To improve existing preimplantation genetic diagnosis fixation techniques. DESIGN: Prospective randomized in vitro study. SETTING: Academic medical center. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The intensity and clarity of fluorescence in situ hybridization (FISH) signals and the percentage of successfully fixed blastomeres. RESULT(S): The described fixation technique resulted in 100% fixation and 100% adequate FISH signals. Two conventional techniques resulted in 94% and 87% fixation, and in adequate FISH signals in 81% and 87%, respectively. CONCLUSION(S): This newly developed fixation technique simplifies the process of fixation of blastomeres for preimplantation diagnosis while essentially eliminating the possibility of losing a cell during fixation. It will hopefully allow more IVF programs to offer their patients preimplantation genetic diagnosis using the FISH technique.

Nucleoli in a pronuclei-stage mouse embryo are represented by major satellite DNA of interconnecting chromosomes.

OBJECTIVE: To investigate the arrangement of chromosomes within pronuclei-stage mouse zygotes. DESIGN: In vitro study. SETTING: Academic medical center. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Location of major alpha-satellite DNA, centromeres, and telomeres, and relative location of chromosomes. RESULT(S): Chromosomes appeared to be oriented inward by centromeres and to be interconnected by major alpha-satellite DNA, which appeared to be the sole DNA component of the nucleoli. This chromosomal arrangement persisted throughout interphase. Chromosomal painting failed to identify chromosomal ordering within pronuclei. CONCLUSION(S): Pronuclear nucleoli are represented by alpha-satellite sequences of interconnecting chromosomes that hold all chromosomes together during interphase. Chromosomes within the pronucleus are randomly positioned relative to each other.

Culture media and their components differ in their ability to scavenge reactive oxygen species in the plasmid relaxation assay.

OBJECTIVE: To investigate the modulation of DNA-damaging effects of reactive oxygen species by media composition. DESIGN: In vitro study. SETTING: Academic medical center. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Plasmid relaxation. RESULT(S): Ham's F-10 medium, 1% Percoll, superoxide dismutase (1, 10, or 100 IU), and synthetic serum substitute did not affect DNA damage by reactive oxygen species and did not have any effect on plasmid DNA damage. Plasmid DNA damage was partially inhibited in the presence of P-1 and human tubal fluid media. Human serum albumin, phenol red, glucose, polyvinyl alcohol, polyvinylpyrrolidone, sucrose, and HEPES also were found to protect DNA from damage. CONCLUSION(S): In vitro fertilization media and their components vary widely in the way they affect DNA damage by reactive oxygen species.

Intracytoplasmic sperm injection in the rat.

We applied intracytoplasmic sperm injection (ICSI) to the rat comparing three different sperm injection techniques: conventional setup with a sharp needle bearing a spike (method 1), combination of partial zona dissection (PZD) needle and blunt pipette (method 2) and piezo-injection using a blunt pipette (method 3). We also investigated the timing of sperm pronuclear formation after injection. Survival rates after injection were 8%, 24% and 71% for the methods 1, 2 and 3, respectively. All surviving oocytes formed pronuclei by about 6 h after injection. Although the survival and activation rates following sperm injection using piezo-injection were high, the incidence of normal fertilisation, as evidenced by second polar body extrusion and formation of two pronuclei, was only 10%. The vast majority of the zygotes were multinucleated, although most of them subsequently underwent cleavage. Fixation and staining of injected oocytes at different times after injection revealed that replacement of sperm nuclear protamines by histones takes place by 15 min after injection, sperm head swelling occurs within 0.5-1 h after injection and pronuclei become fully developed by 7 h after injection. Although the rate of normal fertilisation in the rat following ICSI was low under the present experimental conditions, the results indicated that direct ICSI using a piezo-driven pipette would be a potentially valuable method of producing rat offspring.

The impact of cellular fragmentation induced experimentally at different stages of mouse preimplantation development.

It has been demonstrated previously that removal of acellular debris from the preimplantation mouse embryo is beneficial for subsequent development to the hatched blastocyst stage. We have studied the impact of cellular fragmentation induced in the mouse embryo during the late pronuclei and 8-cell stages on the hatching frequency and total cell number at the blastocyst stage. At the late pronuclei stage about one-quarter of the cytoplasm was removed from embryos in the experimental group, in four to six steps, thus creating four to six cytoplasts that were subsequently returned as anucleated fragments under the zona pellucida. Embryos with one-quarter of the cytoplasm removed and with intact cytoplasm after partial zona dissection (PZD) served as controls. At the 8-cell stage, embryos with their nucleoplast removed from two blastomeres served as an experimental group. Groups of embryos with part of the cytoplast removed from two blastomeres (nucleated fragments), embryos with two blastomeres removed and embryos after PZD alone served as controls. After manipulation all embryos were left in culture and analysed at about 100 h after human chorionic gonadotrophin administration. Fragments induced at the late pronuclei stage did not participate in compaction and were often spontaneously expelled from the embryo during hatching. Neither embryo hatching rate nor total cell number was affected when compared with zygotes with reduced cytoplasm. Although both nucleated and anucleated fragments induced at the 8-cell stage participated in recompaction, hatching was not compromised and there was no interference in further development as assessed by the cell number or hatching rate at the blastocyst stage, as compared with embryos with blastomeres removed. We conclude that anucleated cellular fragments formed in an otherwise healthy embryo, both before and after acquisition of the ability for compaction, are benign and that their removal provides no benefit for embryo development, at least to the hatched blastocyst stage.

Sperm-associated oocyte-activating factor is released from the spermatozoon within 30 minutes after injection as a result of the sperm-oocyte interaction.

We have previously shown that sperm plasma membrane damage makes the sperm plasma membrane permeable and the sperm nucleus accessible for low molecular weight molecules such as eosin and dithiothreitol. In the present study, we investigated whether this damage is associated with a passive release of the sperm-associated oocyte activating factor (SAOAF) from the spermatozoon and, if so, its time sequence. In a first study, human oocytes remaining unfertilized after conventional in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) and freshly ovulated mouse oocytes were injected with a whole spermatozoon or a sperm head respectively. They were randomly allocated to one of three groups: oocytes in group 1 were injected with a spermatozoon immobilized or sperm head detached immediately prior to the injection; oocytes in group 2 were injected with a spermatozoon immobilized or sperm head detached 2-4 h before injection; oocytes in group 3 were injected with a spermatozoon or sperm head that had been subjected to heat treatment. The activation rate of oocytes injected with a spermatozoon or sperm head was the same for groups 1 and 2, and significantly higher than in group 3 (P < 0.001). In a second series of experiments, human oocytes remaining unfertilized after IVF or ICSI were injected with a sperm head that was subsequently removed from the ooplasm 20-30 min after injection. The activation rates were compared to that of oocytes injected with heat-treated spermatozoa which subsequently were removed from the ooplasm. We found that the removal of the spermatozoon 30 min after injection did not prevent oocyte activation. Our data indicate that the initial damage to the sperm plasma membrane induced at immobilization, although essential for the onset of sperm nuclear swelling after ICSI, does not by itself lead to the release of SAOAF from the spermatozoon. We postulate, however, that SAOAF is released during the sperm nuclear swelling phase, which is induced by the so-called sperm nucleus decondensing factor (SNDF) of the oocyte.

Analysis of the oocyte activating capacity and chromosomal complement of round-headed human spermatozoa by their injection into mouse oocytes.

Intracytoplasmic sperm injection (ICSI) in the human is a very effective procedure which allows the fertilization of the majority of oocytes even in cases of extreme oligoasthenoteratozoospermia. Round-headed acrosomeless human spermatozoa, however, form an exception to this rule, because in about half of the couples with globozoospermia all oocytes remain unfertilized after injection. The incapacity of the spermatozoon to activate the oocyte following injection of round-headed spermatozoa could be the underlying mechanism. To investigate this hypothesis, activation rates of mouse oocytes injected with spermatozoa from a patient with globozoospermia were compared with those obtained after injection with normal spermatozoa. Of mouse oocytes surviving the injection with donor spermatozoa, 95% underwent activation, compared to none of the 88 mouse oocytes surviving the injection with round-headed spermatozoa. After fixation, prematurely condensed sperm chromosomes were found in these oocytes. Parthenogenetic activation of mouse oocytes (8% ethanol at 40 min after injection) injected with round-headed spermatozoa led to the activation of 96% of oocytes. These oocytes developed normally to the first mitosis and were fixed for analysis of the sperm karyotypes. The incidence of chromosomal abnormalities of round-headed spermatozoa (6%) was similar to that in spermatozoa from a fertile donor (9%). These data provide further information on the basic defect in cases of globozoospermia and demonstrate that globozoospermia is not associated with sperm karyotype abnormalities.

Factors affecting the role of the spindle during early response of mouse oocytes to ethanol stimulation.

Winston et al. ([1995] J. Cell Sci., 108:143-151) have shown recently that short (6 min) exposure of spindle intact oocytes from Swiss mice to 8% ethanol induced activation of most oocytes, while disruption of the spindles in these oocytes by nocodazole, before and during ethanol exposure, completely inhibited oocyte activation. We compared the activation rates (ARs) of nocodazole-treated and intact oocytes recovered from SJL and B6D2 F1 hybrid mice under the same experimental conditions. The difference between the ARs of nocodazole-treated and intact SLJ oocytes was about the same as reported for Swiss oocytes (2% vs. 82%, respectively). In contrast, this difference was minor for B6D2 oocytes (87% vs. 100%, respectively). Moreover, 41% of these oocytes underwent activation when the spindle was absent, not only before and during, but also 2 h after ethanol exposure. Shortened exposure (2 min) of B6D2 oocytes to ethanol, however, increased the difference in the ARs of nocodazole-treated and intact oocytes (18% vs. 67%, respectively). We conclude that at least two parameters affect the necessity of the presence of the spindle during ethanol exposure for the activation of mouse oocytes. They are the genotype of the oocytes and the duration of exposure to ethanol. Under one set of these parameters the presence of the spindle is absolutely necessary, while under the other the appearance of the spindle a few hours after ethanol exposure is sufficient to allow the activation of some oocytes.

Transport intracytoplasmic sperm injection (ICSI): a cost-effective alternative.

PURPOSE: Transport in vitro fertilization (IVF) programs are operational in a lot of countries and especially popular in The Netherlands, where IVF activities are strictly regulated. Since the introduction of intracytoplasmic sperm injection (ICSI) in the IVF laboratory, many laboratories are now setting up this new technique, which necessitates major investments in terms of infrastructure and specialized personnel. METHODS: We present a cost effective alternative, consisting of patient selection, preparation, and oocyte retrieval at one center and transport of oocytes to a second center, where the ICSI procedure and embryo transfer are performed. Since early 1994 several Dutch centers have a transport ICSI program running with the Gent University Infertility Center, and we wish to present the results of our cooperation with two major centers, comparing them to our local results, for the first 10 months of 1994. Patient selection was similar at all three centers: only couples with previously failed in vitro fertilization or having been refused for routine IVF were enrolled in the program. Stimulation schemes and follow-up of the stimulation were different at all three centers. Transport of oocytes was carried out in a transport box or by attaching the closed tubes containing the follicular aspirates to the chest of the husband. Transport times varied between 1.5 and 3 hr, depending on traffic conditions. RESULTS: Up to November 1, 1994, a total of 77 transport ICSI cycles and 294 own ICSI cycles were carried out. Although locally significantly more oocytes were retrieved and thus available for ICSI than in transport cycles, fertilization and pregnancy rates were not different between the two groups. CONCLUSIONS: These results suggest that long-distance transport of human oocytes seems not to be harmful to their capacity to be successfully injected and to further embryonic development and their implantation potential. Transport ICSI seems to be a valuable and cost-effective approach to treat high numbers of patients at a restricted number of highly specialized IVF laboratories, especially in countries where ICSI is not commonly available.

Oocyte morphology does not correlate with fertilization rate and embryo quality after intracytoplasmic sperm injection.

The fertilization rates and further development of 528 human metaphase II oocytes directly injected by a single spermatozoon were analysed with respect to their morphological features at the light microscopy level at the time of retrieval. The deviations of oocyte morphology which were most frequently observed, after removal of cumulus cells, were dark incorporations, dark zona pellucida, large perivitelline space, spots, vacuoles, refractile bodies and irregular shape. These deviations correlated neither with the fertilization rate nor with the embryo quality score, as compared to 'ideal' oocytes. Since the majority of oocytes displayed deviations from the 'ideal' morphotype but were still fertilized and developed in culture at a normal rate, they were probably as normal as 'ideal' oocytes. Since some of these morphotypes, such as refractile bodies, have been shown to be associated with failure of fertilization, it seems that intracytoplasmic sperm injection may be an appropriate method of treatment for couples in whom repeated failure of in-vitro fertilization is associated with the retrieval of dysmorphic oocytes in the presence of normal semen characteristics.

Does assisted hatching improve implantation rates after in vitro fertilization or intracytoplasmic sperm injection in all patients? A prospective randomized study.

PURPOSE: Preliminary data from some research centers indicate that assisted hatching might be of value to increase embryo implantation rate in the human, at least in selected cases. It is not clear, however, whether this technique would be of benefit for all patients undergoing an embryo transfer. We therefore performed a prospective randomized study to evaluate the effect of assisted hatching on the implantation rate in our in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) program. METHODS: In total, 120 couples undergoing an embryo transfer were randomized between two groups: in one group no assisted hatching was performed (AH-), whereas in the other group the embryos selected for transfer were subjected to partial zona dissection (PZD) immediately prior to the transfer (AH+). Using a computer-generated minimization procedure, patients were allocated to one of the two groups according to four pre-selected criteria: the number of embryos transferred, the cumulative score of transferred embryos, the age of the patient, and the use of ICSI. RESULTS: Pregnancy and implantation rates in the AH+ and AH- groups were, respectively, 42.1 versus 38.1% and 17.9 versus 17.1%. CONCLUSIONS: From our data we conclude that assisted hatching through partial zona dissection prior to embryo transfer does not improve pregnancy and embryo implantation rates in unselected patients undergoing IVF or ICSI.

Sperm plasma membrane damage prior to intracytoplasmic sperm injection: a necessary condition for sperm nucleus decondensation.

In the present study we investigated the relevance of sperm immobilization prior to intracytoplasmic sperm injection (ICSI) in the fertilization process. Using supravital staining of the spermatozoa with eosin and studying sperm decondensation with 2 mM dithiothreitol (DTT) in conditions imitating sperm handling during ICSI, we demonstrated that immobilization of the spermatozoon by squeezing its tail between the glass pipette and the bottom of the dish damages the sperm plasma membrane. Polyvinylpyrrolidone (PVP), which is usually present in the drop with the spermatozoon to facilitate its handling, was found to impede the access of both eosin and DTT to the sperm nucleus. We conclude that (i) sperm immobilization prior to ICSI damages the sperm plasma membrane, that (ii) this damage is sufficient for thiol-reducing agents to gain access to the sperm nucleus, and finally that (iii) PVP possibly interferes with sperm nucleus decondensation.

Timing of sperm and oocyte nuclear progression after intracytoplasmic sperm injection.

We investigated the time course of human oocyte activation after intracytoplasmic sperm injection (ICSI) by observing the oocyte chromosome configuration at different times after injection. One day old human oocytes were injected with spermatozoa and subjected to cytogenetic analysis at 2, 3, 4 and 5 h after injection. We found that anaphase is initiated in the vast majority of the oocytes between 2 and 3 h after injection, and that by 4-5 h after injection most of the oocytes have reached the chromatin mass stage. Two distinguishable stages of sperm nucleus transformation were observed. The first phase-swelling-was reached within 2 h after the injection and was independent of oocyte activation. The second phase-the "brush'-like stage or decondensed chromatin stage-was found only in activated oocytes. Moreover, this stage was not reached before the chromatin mass stage (late telophase) of the oocyte. The same proportion of metaphase II oocyte chromosome configurations and unchanged sperm nuclei was found at any given time after injection. We conclude that: (i) ICSI allows users to obtain an almost synchronized population of activated oocytes; (ii) anaphase II is initiated in the majority of oocytes not later than 2-3 h after injection and telophase II is reached approximately 5 h after injection; and (iii) there are two distinguishable phases of sperm nucleus transformation after ICSI: oocyte activation-independent swelling of the sperm head and oocyte activation-dependent chromatin decondensation which is coupled to the beginning of oocyte chromosome decondensation.

Intracytoplasmic injection of human spermatozoa into mouse oocytes: a useful model to investigate the oocyte-activating capacity and the karyotype of human spermatozoa.

When intracytoplasmic sperm injection (ICSI) is performed, it is important to know the capacity of sperm cells to activate the oocytes, although knowledge of their ability to fuse with the oocytes is not vital. Hamster oocytes are not suitable for this purpose because they are easily activated by the injection procedure Itself. We therefore investigated whether mouse oocytes could be used to assess the activation properties of human spermatozoa. Mouse oocytes were randomized for injection with initially motile spermatozoa, medium, heat-treated or salt-extracted spermatozoa, and the survival and activation rates were examined. About half of the mouse oocytes survived the intracytoplasmic injection of a human sperm cell. Unlike hamster oocytes, the rate of activation provoked by the injection procedure itself was acceptably low (20%), resembling in this respect the behaviour of human oocytes. Following the injection of initially motile human spermatozoa, all mouse oocytes were activated. The injection of heat-treated or salt-extracted human spermatozoa resulted in activation rates of 14 and 15% respectively, comparable with the results following sham ICSI. These data support the hypothesis of a sperm-associated oocyte activation factor. In most activated oocytes, the human sperm nucleus decondensed to form a male pronucleus. Cytogenetic analysis at the first metaphase revealed that human sperm chromosomes were able to undergo replication in a heterologous environment.(ABSTRACT TRUNCATED AT 250 WORDS)

Human oocyte activation following intracytoplasmic injection: the role of the sperm cell.

The aim of this study was to investigate whether the human spermatozoon participates in the activation of human oocytes following intracytoplasmic sperm injection (ICSI) and if so, by what mechanism. In the first series of experiments, we randomized human oocytes which had remained unfertilized after in-vitro fertilization (IVF) or ICSI, for intracytoplasmic injection with live spermatozoa, spermatozoa presumed to be dead and no spermatozoa. Secondly, unfertilized human oocytes and freshly ovulated mouse oocytes were randomized for intracytoplasmic and sub-zonal injection with human sperm cytosolic fraction (CF) before and after heat treatment. We found that oocyte injection with initially motile spermatozoa induces human oocyte activation at a significantly higher rate than injection with dead spermatozoa (61 versus 0%; P < 0.001) or injection without a spermatozoon (61 versus 14%; P < 0.001). Intracytoplasmic injection of CF activated both human and mouse oocytes at the same rate as sperm injection of human oocytes (activation rates of 70 and 65% respectively). This effect was greatly reduced by heat treatment of the CF. From these experiments we conclude firstly that the human spermatozoon injected intracytoplasmically contributes to human oocyte activation and secondly that the spermatozoon releases into the oocyte a heat-sensitive, intracellularly active factor, which is not species-specific.

Behaviour of spermatozoa in human oocytes displaying no or one pronucleus after intracytoplasmic sperm injection.

The behaviour of sperm cells after intracytoplasmic sperm injection (ICSI) was investigated by analysing 192 unfertilized and 37 one-pronuclear (1PN) oocytes following ICSI. Eighty-two unfertilized oocytes were directly fixed whereas 110 were first parthenogenetically activated by puromycin. In contrast to the findings in unfertilized oocytes after in-vitro fertilization, most unfertilized oocytes after ICSI (n = 76) contained evidence of the presence of spermatozoa in the cytoplasm. Few oocytes (n = 6) contained prematurely condensed sperm chromosomes (PCC), whereas the majority contained either intact sperm heads (n = 31) or swollen sperm nuclei (n = 39) along with metaphase II chromosomes of the oocyte. Following activation by puromycin, swollen sperm nuclei and PCC were no longer observed, whereas unchanged sperm heads persisted in 12 oocytes displaying a single pronucleus. A non-decondensed sperm nucleus along with decondensed maternal chromatin were also discovered in 32 out of 37 oocytes displaying a single pronucleus after ICSI. The findings in unfertilized and 1PN oocytes after ICSI indicate that successful sperm injection, even followed by oocyte activation, is not sufficient to guarantee normal fertilization. It seems that partial sperm membrane damage prior to injection is also required to ensure normal sperm decondensation.

Cytogenetic analysis of human oocytes parthenogenetically activated by puromycin.

PURPOSE: Treatment of aged human oocytes by puromycin allows a high rate of parthenogenetic activation and development until the first cleavage division. This technique was used for the study of the chromosome complement of oocytes which remained unfertilized after in vitro fertilization. Three hundred four unfertilized oocytes were treated with 10 micrograms/ml puromycin for 6-8 hr and further cultured for 12-15 hr. RESULTS: Activation occurred in 90.5% of the oocytes. Heterozygous diploids with two pronuclei predominated (61%), which is in contrast to the mouse, where the majority of oocytes activated by puromycin are uniform haploids (89%). CONCLUSIONS: Therefore we conclude that puromycin treatment induces retention of the second polar body in human oocytes, unlike in mouse oocytes treated in the same way. Chromosome analysis performed on 182 oocytes suggested a nondisjunction (ND) rate for the second meiotic division of 12.7%. This is a low figure considering the fact that puromycin itself has been reported to induce nondisjunction. For the first meiotic division a ND rate of only 5.6% was found. This rate is lower than the one found in metaphase II arrested oocytes and we believe that this difference is due to the technical differences between the study of meiotic and that of mitotic chromosomes.

Visualization and cytogenetic analysis of second polar body chromosomes following its fusion with a one-cell mouse embryo.

PURPOSE: This study was designed to visualize the second polar body (2PB) chromosomes using its electrofusion with a one-cell-stage mouse embryo to approach preconception diagnosis of chromosomal disorders. RESULTS: Eighty to 90% hybridization efficiency has been achieved by electrofusion of 2PB with mouse zygotes. 2PB chromosomes were visualized in 40-50% of hybrids. Sixty-five percent of 2PB chromosomes were visualized when fused with the cytoplast obtained microsurgically by removing pronuclei from a one-cell embryo. As much as 33-43% of these resulting metaphases appeared to contain chromosomal aberrations. The follow-up of the development of the reconstructed one cell-stage hybrids in vitro revealed a significant decrease in their viability. The hybrid embryos resulting from 2PB electrofusion with enucleated zygotes did not develop beyond the two-cell stage. CONCLUSION: Electrofusion is an efficient approach for hybridization of 2PB with a one-cell mouse embryo and may be useful for visualization and cytogenetic analysis of 2PB chromosomes. The visualization rate of 2PB chromosomes is higher if 2PB is fused with enucleated zygotes. However, the method induces over 30% of chromosomal aberrations and may lead to a significant decrease in the viability of the resulting one-cell embryos.