A. Fukuda

Influence of water quality on in vitro fertilization and embryo development for the mouse.

Mouse in vitro fertilization and embryo culture were performed in media prepared with five different water preparations. The results of the experiments improved with the frequency of distillation. Each water preparation was analyzed by the measurement of the electrical conductivities and inorganic ion concentrations and by high-performance liquid chromatography to examine the mutual relation between water quality and the method of water purification. The best results were obtained with Milli-Q water, which had the lowest concentration of inorganicions and organic compounds. On the contrary, unexpected contamination by organic compounds and zinc ions occurred after multiple distillation, possibly leached from the glassware and silicon tube. The hatching rate seemed to be an appropriate indicator to assess the biological qualities of media for the development of embryos cultured in vitro.

Freeze–thaw programmes rescue the implantation of day 6 blastocysts

The developmental rate of a blastocyst is considered one of the main estimates for evaluating the implantation potential of embryos. Day 6 blastocysts have been reported to be much less viable than day 5 blastocysts. Regarding implantation, the implantation window is advanced due to a background of high sex hormones, and slower growing embryos may not implant because of possible desynchrony with the implantation window. The aim of this study was to investigate the efficacy of cryopreservation of such embryos and subsequent synchronization of embryo transfer with endometrial status. The results of 122 day 6 blastocysts transferred in the clinic were retrospectively examined. Pregnancy rates were compared between the stimulation cycle and hormone replacement cycle in terms of the method of endometrial preparation. Fifty-five day 6 blastocysts were transferred onto the stimulation cycle endometrium in 37 women, resulting in a 5.5% viable pregnancy rate. On the other hand, 67 day 6 blastocysts were transferred onto endometrium prepared by exogenous hormones in 40 women, resulting in a 26.9% viable pregnancy rate (P < 0.01). Consequently, the difference was highly significant. In conclusion, synchronous transfer of slow-growing embryos using the freeze-thaw technique contributes to a positive outcome.

Blocking effect of sperm immobilizing antibodies on sperm penetration of human zonae pellucidae

To investigate the mechanism of the blocking effect of sperm immobilizing antibodies on human fertilization, an in vitro zona penetration test was carried out using media containing the IgG fraction extracted from sperm immobilizing antibody-negative or-positive serum. The sperm penetration rate of the test was 100% (6/6) when spermatozoa were treated with the IgG fraction derived from sperm immobilizing antibody-negative serum, whereas it was only 17% (1/6) when spermatozoa were treated with the IgG fraction derived from sperm immobilizing antibody-positive serum. Electron microscopic observation of the sperm immobilizing antibody-negative and-positive serum-treated spermatozoa showed that the number of acrosome-reacted spermatozoa was significantly greater in the sperm immobilizing antibody-negative serum than in the antibody-positive serum. Therefore, it appears that one of the blocking mechanisms of the spermatozoal penetration of the zona pellucida by sperm immobilizing antibodies may be due to inhibition of the acrosome reaction in the spermatozoa.

Effect of aspiration vacuum on the developmental competence of immature human oocytes retrieved using a 20-gauge needle

In-vitro maturation (IVM) of immature oocytes has been proposed as a potential alternative to conventional IVF treatment following ovarian stimulation. However, the effects of the oocyte retrieval conditions on subsequent development have not been well understood. This study assessed the effects of different aspiration vacuums during oocyte retrieval on the developmental competence of immature oocytes following IVM, IVF and embryo transfer, retrospectively. Immature oocytes were aspirated with 20-gauge needles with a vacuum of 180 or 300 mmHg. Immature oocytes were cultured in IVM medium for 26 h. All mature oocytes were inseminated by intracytoplasmic sperm injection (ICSI). Embryo transfer was carried out 2 or 3 days after ICSI. The percentage of cumulus-cell enclosed oocytes and of transferable embryos per retrieved oocytes in 180 mmHg (69.7% and 23.8%, respectively) were significantly higher (P < 0.01) than those in 300 mmHg (46.2% and 12.8%, respectively). The ongoing pregnancy rate per retrieval cycle in 180 mmHg (30%) was higher (P < 0.01) than that in 300 mmHg (4.3%). The data indicate that lower pressure of vacuum aspiration with a 20-gauge needle improves the developmental competence of immature oocytes following IVM, IVF and embryo transfer.

Effects of prolactin during preincubation of mouse spermatozoa on fertilizing capacity in vitro

In the present study, some beneficial effects of mouse prolactin (PRL) on spermatozoa were demonstrated in mice. The motility rate of spermatozoa is well maintained during incubation in modified Krebs-Ringer bicarbonate solution (mKRB) containing PRL (10 or 100 ng/ml) for up to 120 min. When spermatozoa that had been preincubated in the same PRL-containing mKRB were incubated with oocytes in mKRB, significantly more spermatozoa attached to the zona pellucida of each oocyte. When spermatozoa that had been preincubated in mKRB with PRL (50 and 100 ng/ml) for only 15 or 30 min were used for in vitro fertilization (IVF), significantly higher fertilization rates were yielded by those spermatozoa than by control spermatozoa preincubated without PRL for the same periods. The prolongation of the preincubation period did not result in increased fertilization rates. Thus, PRL demonstrated biological effects on spermatozoa by shortening the optimal preincubation period for spermatozoa to acquire capacitation and by maintaining their motility and ability of the attachment to the oocyte during IVF. The results are relevant to clinical application of PRL for infertile patients with oligozoospermia or asthenozoospermia.

Effects of the supernatants of mixed lymphocyte cultures and decidual cell line cultures on mouse embryo development in vitro.

The effects of supernatants of human mixed lymphocyte cultures (MLC), with or without human decidual cell line culture extract (decidual factor; DCF), on F1-hybrid mouse embryo development in vitro from the two-cell stage were investigated. The development of mouse embryos from the two-cell stage through the expanded blastocyst stage was facilitated significantly by the addition of supernatants of not only MLC, but also MLC supplemented with DCF (MLCDCF) to the culture medium. Moreover, the supernatant of MLCDCF accelerated the attachment of the hatched blastocyst to the culture dish substratum and the outgrowth of trophoblasts in vitro. The findings indicate that the supernatant of MLCDCF facilitates the in vitro activity of mouse embryos for implantation and that the maternal immune response, along with the decidual tissue, contributes to the implantation processes.

Aspirin dose dependently inhibits the interleukin-1β–stimulated increase in inducible nitric oxide synthase, nitric oxide, and prostaglandin E2 production in rat ovarian dispersates cultured in vitro

OBJECTIVE Determine if aspirin inhibits the IL-1 beta-stimulated expression of inducible nitric oxide synthase (iNOS), nitric oxide (NO), and prostaglandin E(2) (PGE(2)) in rat ovarian dispersates cultured in vitro. DESIGN Prospective, controlled in vitro study. SETTING Academic research laboratory. ANIMALS Ovaries collected from immature rats. INTERVENTION(S) Ovaries were collected from immature rats and enzymatically dispersed. Ovarian dispersates were placed into plates containing media alone or media supplemented with IL-1 beta (100 U/mL) and varying concentrations of aspirin (0, 1, 3, 5 and 10 mM). Ovarian dispersates were cultured in a humidified environment of 5% CO(2) in air at 37 degrees C for 24 or 48 hours. MAIN OUTCOME MEASURE(S) Twenty-four- and 48-hour iNOS, nitrite (a stable metabolite of NO), and PGE(2) levels were determined from ovarian dispersates cultured in vitro. RESULT(S) Administration of IL-1 beta increased nitrite and PGE(2) levels over that observed in the control group after culture of ovarian dispersates for 24 and 48 hours. Aspirin dose dependently reduced the IL-1 beta-stimulated increase in nitrite production from ovarian dispersates after culture for 24 and 48 hours. Aspirin completely (24 hours) or dose dependently (48 hours) prevented the IL-beta-stimulated increase in PGE(2.) Coadministration of IL-1 beta and aspirin (10 mM) attenuates IL-1 beta-stimulated iNOS expression after culture for 24 and 48 hours. CONCLUSION(S) Aspirin significantly inhibits the IL-1 beta-stimulated expression of iNOS, NO, and PGE(2) in ovarian dispersates cultured in vitro.

Influences of prolactin upon spermatogenesis and spermatozoa during in vitro fertilization in mice

Hyperprolactinemia, induced by pituitary isografts for 20 weeks in male mice and confirmed by radioimmunoassay using anti-mouse prolactin serum, did not impair spermatogenesis in the testis and maturing processes of spermatozoa in the epididymis. Incubation of freshly obtained epididymal spermatozoa for 90 min in culture media containing various levels of mouse prolactin did not yield any adverse effects on percentage motility rates of epididymal spermatozoa. When the level of mouse prolactin in the preincubation medium for epididymal spermatozoa was 100 ng/ml, the rate of fertilization by these preincubated spermatozoa in the subsequent in vitro fertilization experiment was significantly lowered compared with that observed in controls. However, when the level of prolactin in preincubation media was 10 ng/ml, no significant reduction in the rate of fertilization occurred. The present experiments seem to indicate the existence of some differences in the effects of prolactin on male germ cells until they reach the tail of the epididymis and on the processes of capacitation and/or fertilization by epididymal spermatozoa after they leave the epididymis.

Ovum recovery and in vitro fertilization in crab-eating monkeys (Macaca fascicularis)

A total of 301 oocytes were recovered from crab-eating monkeys and subjected to insemination in vitro resulting in two fertilized ova. Sixteen monkeys in 24 cycles received 37.5 IU of hMG daily from the second day of the menstrual cycle for 7 to 10 days. Oocytes were recovered under laparotomy at 20 to 49 hr after administration of 1,000–1,500 IU of hCG. The maturation rate of the recovered oocytes was 24.2% as judged from morphological criteria under the light microscope. With additional maturation culture, the rate increased to 36.2%. The matured oocytes were inseminated at 3 to 4 hr after aspiration using homologous spermatozoa which had been capacitated in vitro. Two oocytes were judged as being fertilized based on the presence of 3 and 5 pronuclei, respectively, when examined 12 hr after the insemination. This is the first report of in vitro fertilized ova in nonhuman primates in Japan.

Effects of prolactin on gametes and zygotes during in vitro fertilization in mice

Hyperprolactinemia is one of the major causative factors of infertility. However, the effect of prolactin on gametes during in vitro fertilization has not been elucidated. In the present study, the effects of mouse prolactin on the motility of spermatozoa, in vitro fertilization, and in vitro development of the zygote were investigated in mice using media containing three different concentrations (10, 50, and 100 ng/ml) of mouse prolactin. The development of unfertilized and fertilized oocytes (zygotes) was not affected in vitro by prolactin regardless of the amount of prolactin added to culture media. However, the fertilizing capacity of the spermatozoa was suppressed when they were preincubated for 90 min in culture media containing prolactin at concentrations of 50 and 100 ng/ml. The motility of spermatozoa was not affected by prolactin regardless of the concentration of prolactin used for preincubation. The present study indicates that prolactin may have some effects on the capacitation process of spermatozoa in vitro. This result should be taken into consideration in in vitro fertilization and embryo transfer procedures in humans.