Hiroaki Shibahara

Effects of Sperm Immobilizing Antibodies on Pregnancy Outcome in Infertile Women Treated With IVF‐ET

PROBLEM: Since it was found that anti‐sperm antibodies could impair in vitro development of fertilized eggs in the presence of complement in rats, the effects of sperm immobilizing antibodies on human pregnancy were examined in infertile women treated with IVF‐ET.

Mitochondrial DNA copy number in cumulus cells is a strong predictor of obtaining good-quality embryos after IVF

PurposeThe aim of this study was to establish a simple tool to predict good-quality embryos in in vitro fertilization (IVF) by using cumulus cells (CCs) or peripheral blood cells (PBCs).MethodsMitochondrial DNA was extracted from CCs and PBCs in patients undergoing IVF. Using real-time polymerase chain reaction, mtDNA copy number in a single cell was calculated. Embryo quality was assessed when it was transferred or frozen.ResultsCCs were obtained from 60 oocyte cumulus-cell complexes (OCCCs) in 30 women, and PBCs were collected from 18 women. For the 30 women in the study, the median age was 37xa0years old (range, 24–43), and the mean body mass index was 21.4 (standard error, 2.0). mtDNA content of CCs and PBCs was highly correlated (Pearson’s ru2009=u20090.900, pu2009<u20090.0001). The median mtDNA content of CCs for good- and poor-quality embryos was 140 and 57, respectively (pu2009<u20090.0001). The median mtDNA content of PBCs for good- and poor-quality embryos was 36 and 13, respectively (pu2009=u20090.604). The logistic regression model indicated that mtDNA content in CCs was the only parameter that predicted good-quality embryos (pu2009=u20090.020). The receiver operating characteristic curve for obtaining good-quality embryos by mtDNA copy number in CCs had an area under the curve of 0.823, and using a threshold of 86, positive and negative predictive values were 84.4 and 82.1xa0%, respectively.ConclusionsThe determination of mtDNA content in CCs can be used to predict good-quality embryos.

HLA-DR and HLA-DQ gene typing of infertile women possessing sperm-immobilizing antibody

Thirty-eight infertile women, possessing sperm-immobilizing antibody (SIA), were examined for their HLA-DR and -DQ types using DNA obtained from peripheral blood cells. The typing of HLA-DR and DQ was performed by polymerase chain reaction sequence specific oligonucleotide probes (PCR-SSOP) and PCR-restriction fragment length polymorphism (RFLP), respectively. In comparison to the normal Japanese population, the SIA positive patient population had higher genes frequencies in HLA-DRB1*0901 (26.3 vs. 13.6%, P<0.005), DQB1*0602 (13.2 vs. 6.2%, P<0.05) and -DQB1*0303 (26.3 vs. 14.8%, P<0.01), but not in any HLA-DQA1 gene types by chi2 test. After Bonferroni correction, the high frequency of HLA-DRB1*0901 remained significant (P<0.05) and HLA-DQB1*303 was slightly significant (P<0.07) but no other genes had a gene frequency significantly higher than that of the normal Japanese population. HLA-DRB1*0901 and HLA-DQB1*0303 are very rare among Caucasians but characteristically high among Japanese. The high frequency of HLA-DRB1*0901 and DQB1*0303 genes in the Japanese population may account for higher frequency of sperm-immobilizing antibody in Japanese compared to other ethnic groups.

Quantitative and qualitative changes of mitochondria in human preimplantation embryos.

PurposeThe oxygen consumption rates (OCRs) in mice and cattle have been reported to change during preimplantation embryogenesis. On the other hand, mitochondrial DNA (mtDNA) copy number has been shown to be unchanged in mice and changed in cattle and pigs. The interactions between mitochondrial functions and mtDNA copy numbers in human embryos during preimplantation development remain obscure.MethodsSixteen oocytes and 100 embryos were used to assess mtDNA copy numbers and OCR. Three oocytes and 12 embryos were used to determine cytochrome c oxidase activity. All specimens were obtained between July 2004 and November 2014, and donated from couples after they had given informed consent. Mature oocytes and embryos at 2–14-cell, morula, and blastocyst stages were used to assess their OCR in the presence or absence of mitotoxins. The mtDNA copy number was determined using the samples after analysis of OCR. The relationships between developmental stages and OCR, and developmental stages and mtDNA copy number were analyzed. Furthermore, cytochrome c oxidase activity was determined in oocytes and 4-cell to blastocyst stage embryos.ResultsThe structure of inner mitochondrial membranes and their respiratory function developed with embryonic growth and the mtDNA copy numbers decreased transiently compared with those of oocytes. The undifferentiated state of inner cell mass cells appears to be associated with a low OCR. On the other hand, the mtDNA copy numbers increased and aerobic metabolism of mitochondria increased in trophectoderm cells.ConclusionsThe mitochondrial respiratory function of human embryos developed along with embryonic growth although the copy numbers of mtDNA decreased transiently before blastulation. OCRs increased toward the morula stage ahead of an increase of mtDNA at the time of blastulation. Data regarding changes in mitochondrial function and mtDNA copy number during preimplantation development of human embryos will be useful for the development of ideal culture media.

Relationship Between the Sperm Motility Index Assessed by the Sperm Quality Analyzer and the Outcome of Intracytoplasmic Sperm Injection

Purpose:Intracytoplasmic sperm injection (ICSI) has been validated as a useful treatment in severe male-factor patients who could not achieve fertilization and live births by conventional in vitro fertilization treatment. To examine the impact of male factors on ICSI outcome, clinical laboratory data were retrospectively analyzed.Methods:One hundred two cycles of ICSI treatment indicated by severe male-factor infertility were entered into this study. Sperm parameters including sperm motility, sperm concentration, and sperm motility index assessed by the Sperm Quality Analyzer were evaluated.Results:Five hundred seventy-six metaphase II oocytes retrieved were manipulated. The normal fertilization (2 PN) rate per oocyte was 64.9 ± 26.0% (mean ± SD). Of the 99 transfers, 31 clinical pregnancies were obtained, yielding an average pregnancy rate of 31.3% per transfer. The mean sperm motility, sperm concentration, and sperm motility index were 20.3 ± 16.1% (range, 0 to 50%), 18.2 ± 25.1 × 106/ml (range, <1 to 150 × 106/ml), and 31.2 ± 45.0 (range, 0 to 220), respectively. Sperm concentration did not have a significant impact on fertilization rate by ICSI. In four cases, ICSI was performed using totally immotile sperm and the fertilization rate was 43.5%, which was significantly lower than that of some of the other sperm motility groups, and no pregnancy could be achieved. In 14 cases in which the sperm motility index assessed by the Sperm Quality Analyzer was 0, the fertilization rate (50.0%) was significantly lower than in most of the other sperm motility index groups.Conclusions:These findings suggest that in severe malefactor cases with totally immotile sperm or a sperm motility index of 0, the selection of good-quality sperm should be verified before injection.

Characterization of fertilization‐blocking monoclonal antibody 1G12 with human sperm‐immobilizing activity

A mouse hybridoma (1G12) producing sperm‐immobilizing MoAb to human sperm was established and characterized in order to study the antigens relevant to sperm immobilization by antibodies. MoAb 1G12 had strong sperm‐immobilizing and agglutinating activities and also showed a fertilization‐blocking activity on in vitro fertilization tests. The antibody absorption experiments showed that MoAb 1G12 reacted not only to ejaculated sperm but also human seminal plasma, suggesting that the corresponding antigen might be a sperm coating antigen. The MoAb also reacted with peripheral blood lymphocytes. In histochemical studies, the epithelia of corpus epididymis were most strongly stained. Ejaculated sperm were stained with a granular pattern for their entire surface by immunofluorescence. MoAb 1G12 recognized polymorphic glycoproteins of 15–25u2003kD in the ejaculated sperm extract in Western blot analysis. After deglycosilation of the sperm extract, only a single staining band of under 15u2003kD was detected by MoAb 1G12. This suggests that the antigen epitope recognized by MoAb 1G12 might be a peptide of the core portion of the glycoprotein. MoAb 1G12 might be a useful tool for studying the mechanism of egg–sperm interaction, and also be applied to identifying the corresponding antigen by using gene technology.

Characterization of anti-sperm antibodies and their coding cDNA sequences by Epstein-Barr virus transformed B cell lines from lymphocytes of infertile women possessing anti-sperm antibodies

Epstein-Barr virus (EBV)-transformed B cell lines that produce human antisperm antibodies were established using peripheral blood lymphocytes (PBLs) from infertile women with sperm immobilizing antibodies in their sera. We obtained three stable cell populations (designated B1, B2, D5) of transformed PBLs originating from three different patients. They produced IgM sperm-reacting antibodies directed against the tail of live, methanol-fixed and NaIO4-treated human spermatozoa. The established antisperm antibodies recognized noncarbohydrate sperm membrane antigens with different specificity and distribution in the male reproductive system. Antisperm antibody-B2 corresponding antigen appears to be specific for the male reproductive system. This antigen is excreted from the epithelial cells of the ductus epididymidis and bound to the spermatozoa in the lumen of the ductus. Antisperm antibodies B1 and D5 corresponding antigens were expressed on the spermatozoa in the seminiferous tubules and were common to the secretions of the ductus epididymidis, prostate and some other somatic organs. The cDNA of the immunoglobulin heavy chain genes were analyzed by reverse transcription polymerase chain reaction (RT-PCR) using RNA extracted from these clones. The immunoglobulin heavy chain cDNA sequences of these antisperm antibodies showed extremely high homology to previously reported immunoglobulin germline DNA sequences, implying that these antisperm antibodies might be natural autoantibodies rather than antibodies stimulated by external antigen.

Repurposing itraconazole as an anticancer agent (Review)

Itraconazole, a common anti-fungal agent, has demonstrated potential anticancer activity, including reversing chemoresistance mediated by P-glycoprotein, modulating the signal transduction pathways of Hedgehog, mechanistic target of rapamycin and Wnt/β-catenin in cancer cells, inhibiting angiogenesis and lymphangiogenesis, and possibly interfering with cancer-stromal cell interactions. Clinical trials have suggested the clinical benefits of itraconazole monotherapy for prostate cancer and basal cell carcinoma, as well as the survival advantage of combination chemotherapy for relapsed non-small cell lung, ovarian, triple negative breast, pancreatic and biliary tract cancer. As drug repurposing is cost-effective and timesaving, a review was conducted of preclinical and clinical data focusing on the anticancer activity of itraconazole, and discusses the future directions for repurposing itraconazole as an anticancer agent.

Feasibility and efficacy of intraperitoneal docetaxel administration as salvage chemotherapy for malignant gynaecological ascites

Abstract Ovarian and endometrial cancers diagnosed at advanced stages are often associated with malignant ascites. This study aimed to determine the safety, feasibility and efficacy of intraperitoneal (IP) docetaxel (TXT) for the treatment of ascites. A phase I study, including nine patients, was undertaken to determine the maximum tolerable dose. Efficacy was retrospectively assessed in 18 patients treated with 40–70 mg/m2 IP TXT between 2005 and 2012. In a phase I study, the dose was safely escalated to a maximum of 70 mg/m2, at which level no patients had grade –3 haematological adverse events. In a retrospective study of 18 patients, seven had an Eastern Cooperative Oncology Group performance status of 3; 16 had prior paclitaxel administration and two, with doses of 40 and 70 mg/m2, experienced a serological response and a decrease in paracentesis. Thus, palliative treatment of recurrent OC should be further studied with 40 mg/m2 among more patients, and 70 mg/m2 could be evaluated for first-line IP chemotherapy.

Benefit of palliative chemotherapy and hospice enrollment in late-stage ovarian cancer patients.

The ideal timing for transition to best supportive care (BSC) for ovarian cancer patients is not clear. We retrospectively assessed the survival benefit of continuing chemotherapy and hospice enrollment in late‐stage ovarian cancer patients.