Yasuhisa Araki

Birth of a healthy baby following vitrification of human blastocysts

OBJECTIVE To assess vitrification of human blastocysts. DESIGN Retrospective study of blastocyst vitrification. SETTING A private clinic. PATIENT(S) Twenty couples with different types of infertility. INTERVENTION(S) Blastocysts were frozen with rapid vitrification and then transferred after thawing. We vitrified blastocysts using a modification of Ishimoris vitrification solution of ethylene glycol and dimethyl sulfoxide (VSED). MAIN OUTCOME MEASURE(S) After thawing, survival was defined by the embryos development morphology after 6 hours or overnight culture. RESULT(S) Eighteen of 20 patients underwent treatment. Of 45 vitrified blastocysts, 36 survived, for a survival rate of 80% (36 of 45). The implantation rate was 21.9% (7 of 32), and the pregnancy rate (per embryo transfer cycle) was 33.3% (6 of 18). One of the pregnancies resulted in the delivery of a healthy baby. CONCLUSION(S) Supernumerary embryos were grown in culture to blastocysts, and the survival rate of vitrified-thawed blastocysts was the same as that for slow freezing of early stage embryos. Blastocyst vitrification should prove effective for clinical treatment. The present results strongly suggest that this rapid and successful vitrification procedure will replace conventional cryopreservation in the future.

Intracytoplasmic injection with late spermatids: A successful procedure in achieving childbirth for couples in which the male partner suffers from azoospermia due to deficient spermatogenesis

OBJECTIVE To investigate the feasibility of using late spermatids in intracytoplasmic sperm injection (ICSI) for patients in which there is a total lack of normal and testicular sperm. DESIGN Clinical study. SETTING Private fertility center with adjacent laboratory facilities. PATIENT(S) Thirty-six males diagnosed with azoospermia underwent testicular biopsy and the resulting spermatids from nine patients were used to fertilize oocytes retrieved from their respective wives by ICSI. INTERVENTION(S) Intracytoplasmic spermatid injection of oocytes with late spermatids obtained by testicular biopsy. MAIN OUTCOME MEASURE(S) Fertilization, pregnancy, and delivery rates. RESULT(S) Three of the cases resulted in pregnancies with childbirth occurring in all three, identical twins in one, and individual singletons in the other two cases. CONCLUSION(S) It is possible to achieve successful levels of pregnancy and childbirth in cases of azoospermia by intracytoplasmic injection of late spermatids.

In Vitro Murine Spermatogenesis in an Organ Culture System

Achieving mammalian spermatogenesis in vitro has a long history of research but remains elusive. The organ culture method has advantages over the cell culture method, because germ cells are in situ albeit the tissue as a whole is in vitro. The method was used in the 1960s and 1970s but encountered difficulties in inducing complete meiosis, i.e., in getting meiosis to proceed beyond the pachytene stage. In the present study, we reevaluated the organ culture method using two lines of transgenic mice, Acr-GFP and Gsg2 (haspin)-GFP mice, whose germ cells express green fluorescent protein (GFP) at the mid and end stages of meiosis onward, respectively. Immature testicular tissues from these mice, ranging from 4.5 to 14.5 days postpartum, were cultured on the surface of the medium, providing a liquid-gas interface. Culturing testicular tissues of all ages tested resulted in the expression of both Acr- and Gsg2-GFP. Round spermatids were identified by a combination of Gsg2-GFP expression, cell size, and the presence of a single nucleus with a dot stained by Hoechst. In addition, the chromosome number of one of such presumptive spermatids was found to be 20 by the premature chromosome condensation method. As our semiquantitative assay system using GFP expression grading was useful for monitoring the effects of different environmental factors, including temperature, oxygen concentration, and antiretinoic molecules, further improvement of the culture conditions should be possible in the future.

Seven pregnancies and deliveries from non-mosaic Klinefelter syndrome patients using fresh and frozen testicular sperm.

AbstractPurpose: The aim of this study was to investigate the feasibility of using frozen-thawed testicular sperm as well as the timing of testicular sperm extraction (TESE) in patients with non-mosaic Klinefelter syndrome. Methods: Intracytoplasmic sperm injection (ICSI) was performed in six of 17 (35%) patients whose sperm was recovered by TESE. Multiple biopsies of both testes were performed on the day of oocyte retrieval in all but one of the six patients. Results: Seven pregnancies and deliveries were achieved in five couples, and one couple was unsuccessful. Five pregnancies were achieved using fresh motile sperm, and two were achieved using frozen-thawed sperm. Sperm cryopreservation was not possible in one of the five couples because of the small number of recovered sperm, and possible in four other couples for subsequent ICSI. One woman whose husband had TESE performed prior to ovarian stimulation did not become pregnant. This may be due to the attainment of only a few immotile sperm following the frozen-thawed procedure. Conclusion: The outcome of ICSI using fresh or frozen-thawed testicular sperm in patients with non-mosaic Klinefelter syndrome was identical; however, TESE should be performed on the day of oocyte retrieval until such time as a procedure with a higher sperm yield from TESE is available. Moreover, an improved recovery procedure after cryopreservation-thawing of a single spermatozoon must be developed.

Birth of healthy twins from in vitro development of human refrozen embryos.

OBJECTIVE To avoid the risk of ovarian hyperstimulation syndrome and promote pregnancy rate by freezing pronuclear embryos at the oocyte retrieval and using developed refrozen supernumerary embryos in the event of failed pregnancies. DESIGN A case study. SETTING Private IVF center. PATIENT(S) Healthy infertile woman undergoing IVF treatment. INTERVENTION(S) Slowly frozen and rapidly refrozen embryos at varying stages of embryonic development were transferred. MAIN OUTCOME MEASURE(S) Survival rate of refrozen and thawed embryos; safety of refrozen embryo transference. RESULT(S) Fertilized pronuclear embryos that were frozen, thawed, and developed to morula embryos were subsequently vitrified, rethawed, and cultured until blastocyst formation before transfer. Healthy fraternal twins were born by caesarian section. The result of transfer was the novel finding of a successful delivery after the use of refrozen embryos. CONCLUSION(S) The transfer of refrozen supernumerary embryos resulted in successful pregnancy and delivery. We will continue to investigate the safety and efficacy of the elective freezing of all embryos as well as their refreezing so that supernumerary embryos can be reused to avoid ovarian hyperstimulation syndrome or in cases of repeated failed pregnancies.

Chromosomal diagnosis in each individual blastomere of 5- to 10-cell bovine embryos derived from in vitro fertilization

Chromosomal normality and sex were diagnosed in each blastomere of bovine embryos derived from in vitro fertilization (IVF). Bovine embryos developing to the 5- to 10-cell stage were separated into individual blastomeres with 0.5% protease. After treatment with 100 ng/mL vinblastine sulfate for 8 to 10 h, they were prepared for chromosome samples. In total, 33 bovine embryos and 185 blastomeres were examined. Chromosomal normality was analyzed in 43.8% (81/185) of the blastomeres and 60.6% (20/33) of the embryos; while chromosomal anomalies were found in 16 (80%, 16/20) of the embryos, 5 haploid embryos and 11 mosaic (n/2n) embryos. Mosaicism characteristic of the opposite sex in X-and Y-chromosomes was found in 2 haploid embryos, and that of a Y-chromosome and of XX chromosomes in 1 n/2n embryo. Various sex-chromosome compositions were also observed in the other 10 chromosomal mosaic n/2n embryos.

Use of mouse oocytes to evaluate the ability of human sperm to activate oocytes after failure of activation by intracytoplasmic sperm injection

The objective of the present study was to investigate the nuclei of human sperms that failed to fertilize human oocytes after intracytoplasmic sperm injection (ICSI). The sperms were injected into mouse oocytes by a piezo-micromanipulator, and some of these oocytes were artificially activated with strontium chloride (SrCl2) after ICSI. The oocytes were fixed, stained, and subjected to chromosomal analysis. The survival rate of mouse oocytes injected with infertile human sperms was 92.0% (46/50), while that of the control mouse oocytes injected with fertile human sperms was 73.6% (81/110). The rate of two pronuclei (2PN) formation was 0 (0/46) by the infertile sperms and 81.5% (66/81) by the fertile ones, a significant difference (p < 0.01). Sperm chromosomes in non-activated oocytes were present as premature chromosome condensation (PCC). Artificial activation after ICSI increased the 2PN formation rate in the infertile group to 90.3% (28/31). The results of the present study suggest that infertile sperms have a low potential to spontaneously activate oocytes and to form pronuclei. Thus, artificial activation after ICSI may rescue oocytes fertilized with infertile human sperms that do not produce 2PN. The present study proved the usefulness of mouse oocytes as specimens in evaluating the oocyte-activating capacity of objective human sperms prior to ICSI treatment.

The efficacy of the transfer of twice frozen-thawed embryos with the vitrification method

OBJECTIVE To confirm the clinical benefits of recryopreserved, twice-thawed embryo transfer (ET). DESIGN Retrospective study. SETTING Private fertility clinic. PATIENT(S) Forty-nine women whose embryos had been refrozen after a previous frozen-thawed ET. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Comparison of implantation and pregnancy rates of twice-cryopreserved, twice-thawed embryos versus once-cryopreserved, once-thawed embryos. RESULT(S) The pregnancy rate per ET cycle was 27.8% in the refrozen group and 25.9% in the control group (no statistically significant difference). The implantation rate was 25.0% in the refrozen group and 19.3% in the control group (no statistically significant difference). CONCLUSION(S) The refreezing of supernumerary embryos can prevent ovarian hyperstimulation syndrome in stimulated patients and in those who have experienced repeated failed pregnancies. If unexpected supernumerary embryos are available for recryopreservation after frozen-thawed ET, these embryos may be revitrified for a future transfer.

Perinatal outcome of twice-frozen-thawed embryo transfers: a clinical follow-up study

We evaluated our clinical data on refrozen-thawed ETs (92 cycles) and found that human embryos were capable of withstanding two freeze-thaw cycles, resulting in normal live births after transfer at a rate similar to that of primary frozen-thawed embryos. This is the first follow-up study to present perinatal outcomes of children born after embryo re-cryopreservation, and our results should encourage clinicians to explore the possibility of performing the refreezing procedure.

Evaluating the Quality of Human Embryos with a Measurement of Oxygen Consumption by Scanning Electrochemical Microscopy

ABSTRACT Morphological evaluation has been widely used to evaluate embryo quality because it is non-invasive and useful in predicting pregnancy rate. However, morphological evaluations are subjective and categorization standards often vary between investigators. The respiration rate of embryos is a useful parameter for evaluating embryo quality. The scanning electrochemical microscopy (SECM) measuring system provides a non-invasive, simple, accurate, and consistent measurement of the respiration activity of human embryos. After morphological evaluation by Veecks method, oxygen consumption by individual human embryos was quantified by SECM. Fundamentally, the maturation of mitochondria correlated with an increase in oxygen consumption during the development of embryos. The development of mitochondria may be an important factor in embryo quality, because mitochondria provide ATP for embryonic development by metabolism of nutrients in the cytoplasm. The respiration rates on the day 3 after in vitro fertilization (IVF) were measured and significant differences in oxygen consumption were registered even among embryos with the same morphological classification. There were no significant differences between the mean rates of oxygen consumption at each cleavage stage, however, there was considerable variation in respiration rate within embryos of the same morphological grade. The safety of SECM is assured as the embryos which were examined by SECM for oxygen consumption showed the same development levels as the control group. These results support the hypothesis that measuring embryonic respiration provides additional and valuable information about embryo quality.