Tsuyoshi Kasai

Oocyte mitochondria: strategies to improve embryogenesis.

Mitochondria play a central role to provide ATP for fertilization and preimplantation embryo development in the ooplasm. The mitochondrial dysfunction of oocyte has been proposed as one of the causes of high levels of developmental retardation and arrest that occur in preimplantation embryos generated using Assisted Reproductive Technology. Cytoplasmic transfer (CT) from a donor to a recipient oocyte has been applied to infertility due to dysfunctional ooplasm, with resulting pregnancies and births. However, neither the efficacy nor safety of this procedure has been appropriately investigated. In order to improve embryogenesis, we observed the mitochondrial distribution in ooplasma under the several conditions using mitochondrial GFP-transgenic mice (mtGFP-tg mice) in which the mitochondria are visualized by GFP. In this report, we will present our research about the mitochondrial distribution in ooplasm during early embryogenesis and the fate of injected donor mitochondria after CT using mtGFP-tg mice. The mitochondria in ooplasm from the germinal vesicle stage to the morula stage were accumulated in the perinuclear region. The mitochondria of the mtGFP-tg mouse oocyte transferred into the wild type mouse embryo could be observed until the blastocysts stage, suggesting that the mtGFP-tg mice oocyte is very useful for visual observation of the mitochondrial distribution in the oocyte, and that the aberrant early developmental competences due to the oocyte mitochondrial dysfunction may be overcome by transferring the “normal” mitochondria.

Cytoplasmic transfer in the mouse in conjunction with intracytoplasmic sperm injection.

Cytoplasmic transfer (CT) from a donor to a recipient oocyte has been used clinically to facilitate human pregnancies. Data reported here describe the first characterization of CT coincident with intracytoplasmic sperm injection in the mouse system. Sibling oocytes were used to transfer 2, 4, or 6 pl of ooplasm to a recipient egg along with a sperm head using piezo-actuated injection. Survival and fertilization after CT were comparable to controls at 2 pl and 4 pl, but survival was significantly reduced with 6 pl volumes. Development to the blastocyst stage was also inversely related to CT volume, with some decline beginning with the 4 pl CT group. However, some blastocysts did develop in all of the groups. The results are in contrast with human eggs, which tolerate larger CT volumes. Results indicate that the mouse system can be used to characterize the transfer of exogenous materials concomitant with sperm injection, provided that the CT volume is not excessive.

Mucinous Cystadenocarcinoma with Sarcoma‐like Mural Nodules Arising in a Mature Cystic Teratoma of the Ovary: A Case Report

Almost all cases with malignant transformation of a mature cystic teratoma of the ovary have been reported to be associated with squamous cell carcinoma. We describe a very rare case of mucinous cystadenocarcinoma with sarcoma‐like mural nodules arising in an ovarian teratoma.

Effect of Oocyte Retrieval from a Small Leading Follicle in Fixed-Schedule in vitro Fertilization Program

Objective: To evaluate the propriety of oocyte retrieval (OR) from a small leading follicle during a fixed‐schedule in vitro fertilization (IVF) program.

Life-table analysis of artificial insemination pregnancy rates for couples with male factor and idiopathic infertility

BackgroundIn the summer of 2002, standard guidelines for the application of assisted reproductive technology were reported by a research group of the Ministry of Health, Labor and Welfare. The present study aimed to examine the relationship between the number of cycles of artificial insemination and the cumulative pregnancy rates according to the cause of infertility.MethodsPatients who experienced their first cycle of artificial insemination during the period of January 1999-December 2002 were included in the study and were divided into a male factor infertility group and an idiopathic infertility group. Cumulative pregnancy rates resulting from artificial insemination with the husband’s semen were calculated by the life-table approach.ResultsDuring the study period, 139 couples entered the assisted reproduction program and underwent 581 cycles. Significant differences were observed in cumulative pregnancy rates between the two groups.ConclusionIt is recommended that couples with male factor infertility and who fail to conceive within six or seven cycles of intrauterine insemination, consider a modification of treatment strategy such asin vitro fertilization, because cumulative pregnancy rates of this group were reached at a plateau within six or seven cycles. In contrast, patients with idiopathic infertility, the cumulative pregnancy rates appeared to increase constantly with each subsequent cycle. It is important to consider modifications of treatment strategy in the light of the cause of infertility.

Delayed detection of ureteral thermal injury in laparoscopic surgery

The number of reports of ureteral injuries has increased with the number of laparoscopic surgeries. Some studies have claimed that the incidence of ureteral injury in laparoscopic surgery is 0.3e2.0%.1 About 50e70% of ureteral injuries are not diagnosed immediately postoperatively.2 Unexpected thermal injury during laparoscopic surgery is a possible cause of ureteral injury; however, it is rare. Ostrenski et al reported that among 70 cases of ureteral injury, the cause was thermal injury in only one case (1.4%).1 Here, we present a case in which ureteral thermal injury occurred despite perioperative discharge of indigo carmine dye from the ureteral orifice. A 70-year-old woman underwent right adnexectomy for an ovarian tumor. Because of strong adhesions of the ovarian tumor to the pelvic wall, we used a monopolar electric scalpel to remove the adhesions. After adhesiolysis, we coagulated and transected the right suspensory ligament using a BiClamp forceps, unintentionally exposing the right ureter (Figure 1). One month after surgery, the patient complained of right back pain. Computed tomography revealed right hydronephrosis and ureteral stenosis. Delayed ureteral thermal injury was suspected, and a ureteral 6 Fr double-J stent was placed. Up to the present day (2 years after surgery), the patient has visited our hospital every 4 months for ureteral stent exchange. Ostrenski et al reported that instruments used for electrocautery were identified as the leading cause of laparoscopic ureteral injury and accounted for 24.3% of cases.1 In the present case, we performed electrocautery and coagulation using a monopolar electric scalpel and a BiClamp forceps, which could cause ureteral thermal injury. The reported risk factors for ureteral injury are endometriosis, large uterus, extensive adhesions, malignancy, and previous surgery.2 However, these risk factors are not uncommon. Ureteral injury should be investigated in cases of acute abdominal pain after laparoscopic surgery even if it occurs long after surgery. This is not a novel case; therefore, we hope that this case encourages gynecologists to rethink the importance of early recognition of ureteral injury.

Abstracts of Symposium

S OF SYMPOSIUM “progress in ART and Future Reproductive Medicine”

Oxidative state and zona-binding ability in mouse spermatozoa treated with reduced glutathione

Background and AimsNuclear proteins in mature mammalian spermatozoa nuclei are oxidized to form numerous disulfide bonds. Reduced glutathione (GSH) in the oocyte has been linked to spermatozoan nuclear decondensation after fertilization. In this study, we analyzed whether GSH reduced protamines in sperm nuclei in vitro, and examined the zona-binding ability of treated nuclei.MethodsThree groups of mouse cauda epididymal sperma-tozoa were prepared. The first group was cultured in Chatot-Tasca-Ziomek (CZB; control group), the second in 10 mmol/L GSH (GSH group), and the third group was the GSH group recultured in CZB (re-cultured group). Each sperm was stained with acridine orange, and the oxidative and reductive state of nuclei was analyzed by using fluorescence microscopy. Furthermore, we examined the zona-binding ability for each group by insemination to mouse oocytes after exposure to hyaluronidase.ResultsAll sperm nuclei from the control group displayed an oxidized pattern (green), and those from the GSH group displayed a reduced pattern (red), attributable to reduced protamines. Sperm nuclei from the re-cultured group displayed the oxidized pattern. Although the zona-binding ability of the GSH group was deteriorated compared with the control group sperm, no significant differences were observed between the control and re-cultured groups.ConclusionFrom these results, in vitro reduced sperm were shown to be oxidized in CZB. A reduction of mouse spermatozoa in vitro by GSH is, therefore, reversible, and the oxidative state of sperm nuclei exerts an effect on zona-binding ability.

Effect of repeated administration of clomiphene citrate at two different times on the endometrium in patients undergoing intrauterine insemination

Background, Aims and MethodsClomiphene citrate (CC) has been widely used for induction of ovulation; however, despite the high rate of ovulation, the pregnancy rate is only 30%. The anti-estrogenic effect of CC on the endometrium is one explanation for this finding. It is well known that repeated administration of CC enhances its anti-estrogenic effect. To overcome this adverse affect, a number of techniques have been used. One technique is the early administration of CC, in an attempt to decrease the anti-estrogenic effects of CC on the endometrium. The aim of the present study was to retrospectively evaluate if repeated administration of CC at varying times may affect the endometrium during preovulatory period; pregnancy rates were compiled for patients with unexplained infertility undergoing intrauterine insemination (IUI). The patients were divided into four groups based on the number of repeat administrations and the time of CC administration.ResultsThe endometrial thickness at the day of human chorionic gonadotropin administration was greater in early administration within three consecutive CC cycles than the others.ConclusionWhen the endometrium is thin during classical administration of CC, it is worth attempting the early administration of CC in the CC/IUI treatment for patients with unexplained infertility. However, this effect disappeared over the duration of the three consecutive CC cycles.