CYP2C76 deficiency is embryonic lethal in cynomolgus macaques: The potential role of CYP2C76 in early embryogenesis.

Cynomolgus macaques are an important primate species for drug metabolism studies; however cynomolgus CYP2C76, an important drug-metabolizing enzyme, accounts for drug metabolism differences to humans, so that CYP2C76-null animals might show drug-metabolizing properties more similar to humans. In this study, attempts were made to produce CYP2C76-null animals by assisted reproduction technology. Oocytes and sperm collected from the heterozygotes for the null allele (c.449TG > A) were subjected to intracytoplasmic sperm injection, and the embryos produced were cultured in vitro through the blastocyst stage. Preimplantation genetic diagnosis using a biopsied portion of the blastocyst revealed that none of the 32 blastocysts analyzed were homozygotes. In contrast, 2 of the 20 embryos analyzed were homozygotes at the 8-cell stage, indicating that CYP2C76-null embryos most likely stop developing between the 8-cell and blastocyst stage. By polymerase chain reaction, expression of CYP2C76 mRNA was detected in oocytes and blastocysts, but not in 2-, 4-, 8-, or 16/32-cell stage embryos. Metabolic assays showed that CYP2C76 metabolized progesterone. These results indicated that CYP2C76 null was likely embryonic lethal, suggesting its potential role during early embryogenesis in cynomolgus macaques.

Reproductive Outcome of Infertile Patients with Fibroids Based on the Patient and Fibroid Characteristics; Optimal and Personalized Management.

AIMS: To analyze the detailed clinical course of infertile patients with uterine fibroids and to identify optimal and personalized treatment based on the patient or fibroid characteristics. METHODS: Retrospective analysis of a case series was performed on 176 infertile patients with fibroids. The patients were classified into different groups according to different treatments (conservative infertility treatment, myomectomy and non-myomectomy surgery). Patient or fibroid characteristics for different groups were analyzed for a possible correlation with the reproductive outcome. RESULTS: The cumulative pregnancy rates by conservative treatment plateaued in 1 year. Myomectomy improved the reproductive outcome in patients who did not conceive with conservative infertility treatments. The most important determinant of the reproductive outcome in patients by conservative treatment prior to surgery was a past patient history of pregnancy. The most important determinant of the reproductive outcome after myomectomy was patient age. CONCLUSION: Myomectomy should be considered when infertile patients with fibroids do not conceive within 1 year of conservative infertility treatments. The most important determinant of reproductive outcome after myomectomy is patient age. Therefore, for patients younger than 40, the treatment schedule should be carefully considered so that the patients can sufficiently benefit from myomectomy and assisted reproductive technology.

Pregnancy following ovarian induction in a patient with premature ovarian failure and undetectable serum anti-Müllerian hormone.

We report the first case, to the best of our knowledge, of successful conception following ovarian induction in a patient with premature ovarian failure and undetectable serum anti-Müllerian hormone. A 34-year-old woman was referred because of ovarian amenorrhea. After endogenous gonadotrophins were normalized by hormone-replacement therapy and gonadotrophin-releasing hormone agonist, ovarian induction was performed using exogenous gonadotrophins. On ovarian induction day 8, one follicle had reached a mean diameter of 19.6 mm, the serum estradiol level had increased to 516 pg/mL, and human chorionic gonadotrophin (HCG) was injected. On HCG injection day 7, ultrasonography was unable to detect the follicle, and serum progesterone levels had increased to 6.1 ng/mL. One month after HCG injection, ultrasonography detected an intrauterine fetus with beating heart. Even with serum anti-Müllerian hormone levels below the threshold of detection, there is a chance for patients with premature ovarian failure.

The significance of laparoscopy in determining the optimal management plan for infertile patients with suspected tubal pathology revealed by hysterosalpingography.

The fallopian tube has numerous functions, including ovum pick-up, the place of fertilization of the ovum and cleavage of the embryo, and transfer of the embryo to the uterus. Tubal pathology impairs functions of the fallopian tube and reduces fertility. The degree of tubal pathology determines the possibility for fertility. The evaluation of the fallopian tube is necessary to determine the management plan of infertility. Hysterosalpingography (HSG) is often performed as a first line approach to assess tubal patency and the presence of adhesions; however, HSG has limitations in detecting tubal pathology. In the current study, we evaluated the significance of laparoscopy in determining the optimal management plan for infertile patients with suspected tubal pathology revealed by HSG. Between 1997 and 2009, 127 patients with suspected tubal pathology as demonstrated by HSG underwent laparoscopy at Kinki University Hospital, and a retrospective analysis was performed. Of 87 patients with unilateral tubal pathology revealed by HSG, 20 patients (23.0%) were given an indication for assisted reproductive technology (ART), based on the laparoscopic findings. Of 40 patients with bilateral tubal pathology revealed by HSG, 33 patients (82.5%) with bilateral tubal pathology detected by laparoscopy were given a high indication for ART. Laparoscopy enables exact evaluation of the fallopian tube and selection of the optimal management plan in infertile patients with suspected tubal pathology revealed by HSG. Therefore, laparoscopy should be performed in infertile patients with suspected tubal pathology revealed by HSG, as it is of diagnostic importance.

Evaluation of parametrial spread in endometrial carcinoma.

OBJECTIVE: To evaluate the detailed clinicopathologic characteristics of parametrial spread in uterine endometrial cancer. METHODS: We retrospectively identified 334 individuals with uterine endometrial cancer who had undergone radical hysterectomy between 1988 and 2007. Parametrial spread was determined by histopathological analysis of surgically resected specimens. RESULTS: Twenty-eight (8.4%) individuals had histopathologically confirmed parametrial spread, and lymphatic or blood vessel invasion (22 cases) was the most frequently observed type of parametrial spread; direct invasion to parametrial connective tissue (five cases) or cardinal lymph node metastasis (four cases) were less frequently observed. Parametrial spread occurred not only in individuals with cervical involvement but also in individuals with more than half myometrial invasion, retroperitoneal (pelvic, paraaortic, or both), lymph node metastasis, ovarian metastasis, positive peritoneal cytology results, and lymphovascular space invasion. Twenty-six individuals (92.9%) with parametrial spread showed more than one of these histopathological factors (median number of factors 3, range 1-6); the other two individuals had lymphovascular space invasion alone. In 10 individuals with parametrial spread (35.7%), the condition recurred during the median follow-up period of 49 months, and initial recurrence was observed in the lung in six individuals (60.0%). Although the long-term prognosis for those with parametrial spread was significantly poorer than that of those without parametrial spread, both among all individuals (P<.001) and among individuals with International Federation of Gynecology and Obstetrics stage III (P<.05), multivariate analysis showed that parametrial spread was not an independent prognostic factor for uterine endometrial cancer. CONCLUSION: Parametrial spread cannot be predicted by cervical involvement alone but may be predicted by various lymphovascular space invasion-related histopathologic factors. Further, parametrial spread may not be an independent prognostic factor in individuals with uterine endometrial cancer. LEVEL OF EVIDENCE: III.

A change in promoter methylation of hMLH1 is a cause of acquired resistance to platinum-based chemotherapy in epithelial ovarian cancer.

BACKGROUND: Acquired resistance to platinum-based chemotherapy (Pt-chemo) is a major problem for improving the prognosis for patients with advanced epithelial ovarian cancer (EOC). However, the molecular mechanism of acquired resistance to Pt-chemo is not well understood. MATERIALS AND METHODS: hMLH1 promoter methylation (hMLH1 MET) and hMLH1 protein expression was examined in 36 paired samples of primary and secondary resected tumors by methylation-specific polymerase chain reaction (PCR). RESULTS: No primary tumors exhibited hMLH1 MET, while 56.3% of secondary tumors showed hMLH1 MET. Moreover, no significant correlation was observed between hMLH1 MET and histological subtype, while hMLH1 MET was significantly greater (p < 0.001) in partially responsive secondary tumors compared with no change or progressive disease, and hMLH1 MET also occurred more frequently (p = 0.059) in tumors treated with four or more courses of Pt-chemo. CONCLUSION: A change in hMLH1 MET is a major molecular cause of acquired resistance to Pt-chemo in EOC.

Primate embryonic stem cells proceed to early gametogenesis in vitro.

Embryonic stem cells (ESCs) of nonhuman primates are important for research into human gametogenesis because of similarities between the embryos and fetuses of nonhuman primates and those of humans. Recently, the formation of germ cells from mouse ESCs in vitro has been reported. In this study, we established cynomolgus monkey ES cell lines (cyESCs) and attempted to induce their differentiation into germ cells to obtain further information on the development of primate germ cells by observing the markers specific to germ cells. Three cyESCs were newly established and confirmed to be pluripotent. When the cells are induced to differentiate, the transcripts of Vasa and some meiotic markers were expressed. VASA protein accumulated in differentiated cell clumps and VASA-positive cells gathered in clumps as the number of differentiation days increased. In the later stages, VASA-positive clumps coexpressed OCT-4, suggesting that these cells might correspond to early gonocytes at the postmigration stage. Furthermore, meiosis-specific gene expression was also observed. These results demonstrate that cyESCs can differentiate to developing germ cells such as primordial germ cells (PGCs) or more developed gonocytes in our differentiation systems, and may be a suitable model for studying the mechanisms of primate germ cell development.

Microtubule organization during rabbit fertilization by intracytoplasmic sperm injection with and without sperm centrosome.

Aim: In most mammalian fertilization, the sperm introduces the centrosome, which acts as a microtubule organizing center (MTOC) and is essential for pronuclear movement. In rabbit fertilization, biparental centrosomal contribution in microtubule organization has been suggested. Methods: To reveal the function and inheritance of the centrosome during rabbit fertilization, we compared microtubule organization and early embryonal development following intracytoplasmic sperm injection (ICSI) with and without sperm centrosome. Sperm centrosome was removed by sonication, and the isolated sperm head was injected by a Piezo-driven micromanipulator. Samples were studied by light microscope after immunocytological stain. Results: The sperm aster formation was observed 2-3 h after ICSI with intact sperm. In contrast, microtubules were organized between the male and female pronucleus without a nucleation site in the eggs after ICSI with an isolated sperm head. In the late pronuclear stage following ICSI with an isolated sperm head, microtubule organization was the same as in late pronuclear stage eggs after intact sperm injection. The first mitotic spindle was organized in eggs following ICSI with an isolated sperm head, as observed in eggs following ICSI with an intact sperm. Conclusions: These results indicate that the MTOC is in oocyte cytoplasm during fertilization and fulfils the function when the sperm centrosome is absent. (Reprod Med Biol 2005; 4: 169-178).

Activation with ethanol improves embryo development of ICSI-derived oocytes by regulation of kinetics of MPF activity.

Developmental potential of bovine embryos that are not artificially activated after intracytoplasmic sperm injection (ICSI) is generally very low. In this study, we investigated effects of artificial activation with ethanol on kinetics of maturation promoting factor (MPF) activity (p34(cdc2) kinase activity) and development of bovine oocytes following ICSI. Treatment of oocytes with ethanol at 4 h after ICSI improved their first cleavage and further preimplantation development (51% vs. 13%, 14% vs. 4%: treatment with vs. without ethanol, respectively). MPF activity of oocytes was lowered until at least 2 h after ICSI. In oocytes without activation after ICSI, MPF activity temporarily elevated at 6 h after ICSI, whereas this phenomena was not observed in the oocytes treated with ethanol. Furthermore, MPF activity was elevated 20 h after ICSI in oocytes activated with ethanol, whereas this elevation of MPF activity was not shown in oocytes without activation. These results indicate that the stimulus of sperm was sufficient to lower MPF activity of oocytes following ICSI, and moreover the activation treatment of bovine oocytes with ethanol after ICSI served to maintain the low levels of MPF activity until the next cell cycle started.