Successful pregnancy after ICSI with strontium oocyte activation in low rates of fertilization.

Fertilization failure (complete fertilization failure or low fertilization rates) after intracytoplasmic sperm injection (ICSI) can occur in rare cases. In the majority of these cases, the unfertilized oocytes are inactivated. Assisted oocyte activation was applied as a treatment option for a case of low fertilization rate as a clinical trial. A patient with a low fertilization rate (ranging from 0% to 33.3%; mean = 17.0%) after eight previous ICSI cycles at another hospital, was diagnosed with fertilization failure. The most likely cause of fertilization failure was failure of oocyte activation. Therefore, artificial oocyte activation by strontium treatment was combined with ICSI to achieve viable fertilized oocytes. Oocytes were stimulated with strontium (10 mM SrCl(2), 60 min) approximately 30 min after ICSl. Six injected oocytes were stimulated and all were then successfully fertilized. Two blastocysts were transferred into the uterus, resulting in a pregnancy and birth. A second pregnancy was achieved following implantation of two cryopreserved embryos (one blastocyst and one morula). In conclusion, strontium treatment was found to be an effective method for artificial oocyte activation in a case with a low fertilization rate after ICSI.

Influence of sperm immobilization on onset of Ca(2+) oscillations after ICSI.

Sperm immobilization prior to intracytoplasmic sperm injection (ICSI) is thought to be necessary for efficient fertilization. A variety of methods of sperm immobilization (pipetting, squeezing and piezo application) are currently employed in ICSI. The effect of differences in immobilization method on the timing of initial Ca(2+) oscillations of oocytes in ICSI was investigated. Motile spermatozoa were immobilized in eosin Y solution using pipetting, squeezing and piezo application. Complete staining of the sperm head was achieved after 220.7, 42.2 and 5.0 s respectively. Oscillations after ICSI were measured fluorometrically for each method. The onset of Ca(2+) oscillations was observed at 4.8 to 80.4 min after ICSI. Ca(2+) oscillations developed earlier with the piezo method (14.4 +/- 6.4 min) than other methods (pipetting, 43.1 +/- 20.2 min, P < 0.01; squeezing, 18.4 +/- 3.8 min, P = NS). The piezo method produced the earliest staining of the sperm head and may have caused the most damage to the sperm membrane. A more rapid onset of Ca(2+) oscillations was also observed with the piezo method. The method of sperm immobilization may be important for the rapid release of sperm factors that initiate oocyte activation. This study also showed that Ca(2+) oscillations develop earlier in human oocytes treated by ICSI than indicated in previous reports.

Comparison of oocyte activation and Ca2+ oscillation-inducing abilities of round/elongated spermatids of mouse, hamster, rat, rabbit and human assessed by mouse oocyte activation assay.

Oocyte activation and Ca2+ oscillation-inducing abilities of round spermatid (ROS) and elongated spermatid (ELS) of some rodents and human were assessed by their injection into mouse (B6D2F1) oocytes (mouse test). With mice (B6D2F1, ICR) and rat, ROS displayed no oocyte activation or Ca2+ oscillation-inducing abilities. Although ELS could induce activation at 87, 86 and 31% of injected oocytes respectively, most of the intracellular calcium concentration ([Ca2+]i) responses of ELS-injected oocytes did not show oscillation patterns; only several transient [Ca2+]i rises (transient pattern) were seen. Similarly, with hamster, rabbit and human, while ROS could induce oocyte activation efficiently (70, 71 and 52% respectively), most of the [Ca2+]i patterns of injected oocytes were transient patterns, and not oscillation patterns. When ROS nuclei only from these latter species were injected into mouse oocytes, most of the oocytes could not be activated. [Ca2+]i patterns of oocytes injected with immature sperm cells changed from transient pattern to oscillation pattern while the cells were maturing into spermatozoa. With hamster ROS, oocyte-activating factor was found to be distributed mainly in the cytoplasm. It was interesting that there is a dissociation between the timings of appearance of oocyte activation and that of Ca2+ oscillation of oocytes injected with developing immature sperm cells.

Oocyte activation induced by spermatids and the spermatozoa.

It has been reported that a sperm factor (SF) found in spermatozoa plays a critical role in fertilization. However, particulars of the oocyte-activating and Ca2+ oscillation (Ca-Os)-inducing abilities of this SF remain unknown. We examined these abilities of spermatids in mouse, hamster and human by a mouse test (injection of spermatids into mouse oocytes). In mice, the round spermatids (ROS), elongated spermatids (ELS) and spermatozoa activated 0%, 93% and 92% of the oocytes, respectively. ROS injection resulted in no Ca-Os (type C). ELS induced a normal oscillation (type A) at 0% and an abnormal oscillation (type B) at 94%. Mouse spermatozoa induced type A Ca-Os at 90%. For mice, oocyte-activating and Ca2+ oscillation-inducing ability arose in different phases of spermiogenesis. We also observed this differential timing for hamster spermatids. Hamster ROS activated 74% of oocyte (ELS: 90%, sperm: 86%). Human ROS activated 64% of oocytes (sperm: 100%), but only 35% of the oocytes showed type A Ca-Os. These results indicate that oocyte activation generally occurs between the ROS and ELS phases, although these phases differ among species. They also indicate that oocyte activation is not necessarily accompanied by Ca-Os. These findings suggest the existence of different thresholds at which the SF induces oocyte activation and Ca2+ oscillation, or of different factors that induce oocyte activation and Ca-Os. We found SF to be clinically impaired in 0.9% of ICSI patients. A combination of artificial oocyte activation and ICSI proved effective with such patients.

Successful fertilization and pregnancy following ICSI and electrical oocyte activation.

In a total of 1048 intracytoplasmic sperm injection (ICSI) cycles, motile spermatozoa from four out of 424 patients (0.9%) failed to fertilize oocytes, despite an apparently successful ICSI procedure. No activation was observed in these injected oocytes. The spermatozoa from three of the four patients were injected into unfertilized mouse oocytes by ICSI (mouse test) to evaluate their oocyte activating ability. The oocyte activation rate of the spermatozoa of patients A, B, and C in the mouse test was 46, 100, and 86% respectively (control: 100%). Simultaneous injection of two spermatozoa from patient A into the mouse oocytes increased the oocyte activating rate to 89% (sham control: 29%). 100% fertilization rates were obtained for patients A and B by combining ICSI and electrical stimulation, and this resulted in pregnancy and the birth of healthy twins for the partner of patient A. Thus, it is considered that the spermatozoa of these patients are not lacking sperm factors but that the activity of these factors is depressed. The combination of ICSI and electrical stimulation is effective in these cases.

The usefulness of a piezo-micromanipulator in intracytoplasmic sperm injection in humans.

Intracytoplasmic sperm injection (ICSI) has wide clinical application. In order to achieve good results with this method, it is important to restrict the possibility of oocyte injury as much as possible, and securely inject spermatozoa into the ooplasm. For this purpose, we clinically applied piezo-ICSI, which employs a micromanipulator with piezoelectric elements, to humans, and compared the results with those obtained by conventional ICSI. Conventional ICSI and piezo-ICSI were used in 279 cycles and 335 cycles respectively. Piezo-ICSI showed significantly more favourable results, with a survival rate of 88.1% (conventional ICSI: 81.4, P < 0.001), a fertilization rate of 79.4% (conventional ICSI: 66.4%, P < 0.001), and a pregnancy rate of 23.1% (conventional ICSI: 14.9%, P < 0.05). In piezo-ICSI, the needle used is not sharpened and has a flat tip. However, deformation of the oocyte during insertion of the needle is restrained by vibration of the piezo, and the oolemma is punctured readily and securely by the piezo pulse, at the site where the spermatozoon is injected. Piezo-ICSI is a promising new technique for human ICSI that should improve the survival, fertilization and pregnancy rates after ICSI.

Efficient injection of bull spermatozoa into oocytes using a Piezo-driven pipette.

Recently, mouse and human offspring have been successfully obtained from embryos developed after intracytoplasmic sperm injection(ICSI), using a Piezo micromanipulator. In this study, the Piezo-ICSI procedure was used with in vitro matured bovine oocytes known to be difficult to fertilize microsurgically. The efficacy of Piezo-ICSI versus conventional ICSI was examined after oocytes were activated and fertilized with or without calcium ionophore (A23187) exposure. In conventional ICSI, the rate of fertilization was 19% (11/59) with A23187 and 5% (2/38) without it. However, when the Piezo-ICSI procedure was performed, the fertilization rate was 72% (47/65) with A23187 and 72% (28/39) without it. The rate of oocyte survival after microinjection was nearly similar for both methods. We suggest that the bovine oocyte is successfully activated and fertilized when an immobilized spermatozoon is injected exactly into the ooplasm through the oolemma, perforated easily by the pulsation of the Piezo. Moreover, an activating procedure such as exposure of oocytes to A23187 is not necessary, because the so-called sperm factor (oocyte activating substances) is incorporated into the ooplasm along with a spermatozoon. In this respect, the Piezo-ICSI was more efficient than the conventional ICSI method for fertilizing and thus obtaining more bovine embryos.

Influence of oocyte preincubation time on fertilization after intracytoplasmic sperm injection.

During the intracytoplasmic sperm injection (ICSI) procedure, the collected oocytes are incubated until just before ICSI. The ideal preincubation time of oocytes was investigated in 544 treatment cycles. Oocyte retrieval was carried out 35 h after human chorionic gonadotrophin administration. Oocytes were cultured for between 1 and 11 h before ICSI. Embryo transfer was performed 48 h after oocyte collection. The survival, fertilization and cleavage rates of injected oocytes indicated no statistically significant differences between oocytes preincubated for different lengths of time. The proportion of good-quality embryos (grades 1 and 2) was lower at 9-11 h of preincubation time than for all the other preincubation times (P < 0.001). No statistically significant differences were detected in the pregnancy rate between each group (mean: 15.9%), although the pregnancy rate at 9-11 h of preincubation time appeared to be low (7.7%). These results suggest that the oocyte retained sufficient potential for fertilization between 1 and 9 h after oocyte collection in ICSI. For the researchers who practise more complex ICSI procedures than IVF, it would be convenient to be able to perform ICSI at any time between 1 and 9 h after oocyte collection.

Fertilization using male germ line-cells.

In recent years, favorable results have been achieved in patients suffering from azoospermia by microinsemination of spermatozoa taken from their testes. Microinsemination is being introduced in the treatment of patients who have no spermatozoa in their testes via their spermatid and spermatocyte. There are still doubts relating to immature male germ line-cells, such as whether they have, oocyte activating factors, the level of stability of DNA of cell nuclei, and the differences in chromosome numbers. The relatively few cases of gestation using the human spermatid treatment may be due to embryological problems resulting from the instability of nuclear DNA and the insufficiency of oocyte activating factors, which are the result of imperfect microinjection techniques. Improvements in techniques for the clinical application of spermatid and secondary spermatocyte, as well as the collection of basic data to confirm embryological safety are therefore necessary.

The relationship between acridine orange fluorescence of sperm nuclei and the fertilizing ability of human sperm.

OBJECTIVE: To determine whether the outcome of IVF can be predicted by acridine orange (AO) nuclear fluorescence of sperm. DESIGN: Based on the fact that AO nuclear fluorescence color after acid treatment reflects maturity (green fluorescence) or immaturity (yellow to red fluorescence) of spermatozoa, the relationships between sperm maturity and the outcome of IVF, subzonal insemination, or intracytoplasmic sperm injection (ICSI) were investigated. SETTING: The IVF program at the Obstetrics and Gynecology Hospital, Fukushima Medical College. PATIENTS: Sixty-eight patients undergoing 68 IVF treatment cycles. MAIN OUTCOME MEASURES: Acridine orange fluorescence of sperm nuclei and successful oocyte fertilization. RESULTS: conventional semen parameters (sperm concentration and percentages of motile or morphologically normal spermatozoa in semen) did not correlate with the incidence of spermatozoa with green AO fluorescent (mature) nuclei. When > or = 50% of spermatozoa in semen samples exhibited green AO nuclear fluorescence, IVF was always successful. When green AO nuclear fluorescence was < 50%, only 39% of IVF treatment cycles (13/33) were successful. Only green AO fluorescent spermatozoa were able to bind efficiently human zona pellucida. When the incidence of green AO fluorescent spermatozoa was < 50%, no pregnancy resulted even though an average of 26% of the oocytes could be fertilized by ICSI. CONCLUSIONS: The spermatozoa which fertilized oocytes in vivo and in IVF were limited to those whose nuclei exhibited green AO fluorescence. Intracytoplasmic sperm injection may be the method of choice when the incidence of green AO nuclear fluorescence is low regardless of the results of semen analysis.

Intracytoplasmic sperm injection using immobilized or motile human spermatozoon.

OBJECTIVES: To investigate the efficacy of the treatments for oocyte activation on the results of intracytoplasmic sperm injection using immobilized or motile human spermatozoa. DESIGN: The protocol of intracytoplasmic sperm injection was divided into four groups according to the states of sperm used for microinjection and the treatment for oocyte activation. In group A, immobilized sperm is used. The oocyte is activated merely by aspiration of the cytoplasm into the pipette. In group B, immobilized sperm is used. Microinjected oocyte is treated with A23187. In group C, immobilized sperm is used. Electroporation is performed on the microinjected oocyte. In group D, motile sperm is used. The oocyte is activated merely by aspiration of the cytoplasm into the pipette. SETTING: The Obstetrics and Gynecology Hospital, Fukushima Medical College. PATIENTS: The subjects are the cases that had failed fertilization in standard IVF, cases of severe oligozoospermia, and cases of severe asthenozoospermia. RESULTS: No difference was found between the groups as to the survival rate and fertilizing rates of oocytes after intracytoplasmic sperm injection. The cleavage rate of oocytes was high in order of group D, C, B, A. The cleavage rate for groups D, C, and B was significantly higher than group A. Cases of pregnancy were found in groups D and B. CONCLUSION: Using motile sperm rather than immobilized sperm can be expected to produce better results in human ICSI. Activating oocytes positively is needed when immobilized sperm is used.

Coculture of mouse embryos with cryopreserved human oviduct epithelial cells.

PURPOSE: The effect of coculturing mouse embryos with cryopreserved human oviduct epithelial cells was investigated. The cryopreserved cells in Cellbanker were thawed and cultured in Richard D. Goldsby culture medium to establish monolayers. Two-cell-stage mouse embryos were cultured alone (control group) or cocultured with monolayers established from cryopreserved cells (cryopreserved coculture group) or from fresh cells (fresh coculture group). The rates of embryo development and the qualities of the blastocysts in the three groups were compared. RESULTS: The two coculture groups had significantly higher blastocyst development rates (cryopreserved coculture group, 81.6%; fresh coculture group, 82.2%) than the control group (63.1%). The two coculture groups had significantly more blastomeres (cryopreserved coculture group, 108.3 +/- 25.9; fresh coculture group, 108.4 +/- 25.1) than the control group (87.7 +/- 31.9). CONCLUSION: The method of cryopreservation of human oviduct epithelial cells using Cellbanker is simpler than conventional cryopreservation methods. These cryopreserved human oviduct epithelial cells may provide a constant supply of cells for coculture for in vitro fertilization and embryo transfer.

Pronuclear formation and cleavage of mammalian eggs after microsurgical injection of freeze-dried sperm nuclei.

A study was conducted to evaluate the ability of mammalian oocytes to develop to the pronuclear stage and beyond if injected with freeze-dried sperm nuclei. The rate of development of hamster eggs to the pronuclear stage after microinjection of freeze-dried hamster sperm nuclei was 90%. The pronuclear formation rate of hamster eggs injected with freeze-dried human sperm nuclei was 85%. On the other hand, the rates for eggs injected with untreated sperm nuclei were 84% and 89% respectively. The survival rate of rabbit eggs microinjected with freeze-dried rabbit sperm nuclei was 64%, the fertilisation rate 56% and the cleavage rate 38%. The survival, fertilisation and cleavage rates of eggs injected with non-freeze-dried sperm nuclei were 78%, 45% and 34%, respectively. There was no difference between eggs injected with freeze-dried sperm nuclei and those injected with sperm nuclei that had not been freeze-dried. Of the viable rabbit eggs injected with freeze-dried rabbit sperm nuclei, 6% developed to the 6- to 8-cell stage 62 h after injection.

Fluorescence energy transfer shows that various physical and chemical treatments of human sperm induce unpacking of chromatin.

Fluorescence resonance energy transfer (FRET) was used to study the changes which human sperm chromatin went through after various physical and chemical treatments. This technique showed a dilatation of the spatial relationship among chromatin liner arrays, with UV and DNAse among the treatments that gave rise to the highest increase. FRET image analysis showed that the chromatin linear arrays after treatment reach a spatial disarrangement similar to that brought about by sperm decondensation. Comparison of these results with the ability of human treated sperm to form pronuclei after microinjection into hamster eggs, suggests that the highly condensed spatial organization of sperm chromatin arrays may not be a necessary prerequisite for pronucleus formation.

Moderate heat treatment increases the penetrability of zonae pellucidae of salt-stored mammalian oocytes by spermatozoa.

The zona pellucida of mammalian oocytes stored in highly concentrated solutions of neutral salts is known to retain its biological and biochemical properties. However, the zona may become resistant to sperm penetration as the storage period is increased. In cattle and hamsters, the penetrability of zonae of salt-stored oocytes was restored or increased by treating the oocytes with moderate heat without altering the gross morphology of the zona. Although this technique did not work for salt-stored human ova, this may have been due to the use of so-called inseminated-unfertilised ova which (1) may have been fertilised but failed to activate, or (2) were not fertilised because of functionally defective zonae.

The ability of dehydrated hamster and human sperm nuclei to develop into pronuclei.

To determine whether the nuclei of mature mammalian spermatozoa are resistant to dehydrated conditions, nuclei of hamster and human spermatozoa were freeze-dried or treated with various dehydrating agents before injection into hamster oocytes. Freeze-dried nuclei remained capable of developing into pronuclei even after 12 mo of storage at 4 degrees C. The level of DNA synthetic activity in the sperm (male) pronucleus was comparable to that in the egg (female) pronucleus. Sperm nuclei that had been stored in 100% ethanol, 100% methanol, or chloroform-methanol (2:1) mixture for 20 days were also capable of developing into pronuclei. Even the nuclei that had been dehydrated ("fixed") with Carnoy's fluid could develop into morphologically normal pronuclei. However, the level of DNA synthesis in the pronuclei derived from these chemically dehydrated nuclei was generally lower than that in the female pronuclei. Although the genetic integrity of the dehydrated sperm nuclei is yet to be determined, nuclei of mature hamster and human spermatozoa appear to be fairly resistant to dehydrated conditions.

Thiol-disulfide status and acridine orange fluorescence of mammalian sperm nuclei.

The relationship between thiol-disulfide status and acridine orange fluorescence of testicular, epididymal, and ejaculated spermatozoa in several mammalian species was investigated. Spermatozoa were fixed with acetic alcohol, stained with acridine orange, and examined with a fluorescence microscope. The majority of the nuclei of testicular spermatozoa of the hamster, mouse, and rabbit exhibited red acridine orange fluorescence. The proportion of sperm nuclei with red acridine orange fluorescence decreased as the spermatozoa descended the epididymis. Red acridine orange fluorescence was replaced by green acridine orange fluorescence. The site in the epididymis where 100% of the nuclei exhibited green fluorescence was the distal caput in the mouse, the corpus in the rabbit, and the proximal cauda in the hamster. In semen samples from men with proven fertility, normal semen parameters, or both, about 60% to 90% of the nuclei exhibited green acridine orange fluorescence. The proportion of sperm nuclei exhibiting green acridine orange fluorescence was higher in the spermatozoa pellet (containing highly motile spermatozoa) obtained by centrifugation through a Percoll gradient. From experiments using disulfide-reducing, thiol-oxidizing and thiol-detecting agents, we concluded that sperm nuclei fluoresce red when they are treated with acid while their DNA-associated protamines are poor in disulfides. Under such conditions, DNA is vulnerable to denaturation. Acridine orange binds to denatured (single-stranded) DNA as aggregates and emits red fluorescence. In contrast, when sperm nuclei are treated with acid while their DNA-associated protamines are rich in disulfides, DNA is resistant to denaturation. Acridine orange binds to native (double-stranded) DNA as a monomer and emits green fluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)