Koichi Takebayashi

Mutation analysis of the growth differentiation factor-9 and -9B genes in patients with premature ovarian failure and polycystic ovary syndrome

OBJECTIVES To investigate whether growth differentiation factor (GDF)-9 or GDF-9B/bone morphogenetic protein (BMP)-15 mutation is present in Japanese women with premature ovarian failure (POF) and polycystic ovary syndrome (PCOS). DESIGN Clinical and molecular studies. SETTING Outpatient clinic in a university hospital. PATIENT(S) Fifteen women with POF, 38 women with PCOS, and 3 normal fertile controls. INTERVENTION(S) Extraction of DNA from blood samples for subsequent polymerase chain reaction (PCR). MAIN OUTCOME MEASURE(S) Amplified DNA was analyzed by denaturing gradient gel electrophoresis (DGGE) and/or by direct sequencing. RESULT(S) No missense mutation was found in any exons of the GDF-9 gene and the GDF-9B/BMP-15 gene in patients with POF and PCOS. CONCLUSION(S) The missense mutation in the GDF-9 gene or the GDF-9B/BMP-15 gene is uncommon in anovulatory Japanese women with POF and PCOS.

Follicle-stimulating hormone receptor gene mutations are rare in Japanese women with premature ovarian failure and polycystic ovary syndrome

OBJECTIVE To determine whether the known inactivating FSH receptor gene mutations are present in Japanese women with secondary amenorrhea because of premature ovarian failure (POF) and polycystic ovary syndrome (PCOS). DESIGN Clinical and molecular studies. SETTING An outpatient clinic in a university hospital. PATIENT(S) Fifteen women with idiopathic POF, 38 women with PCOS, and three normal controls. INTERVENTION(S) Extraction of DNA from blood samples for subsequent polymerase chain reaction (PCR). MAIN OUTCOME MEASURE(S) PCR fragments digested with MunI, BsmI, and HhaI were compared in patients and controls. PCR fragments were also analyzed by denaturing gradient gel electrophoresis (DGGE) and direct sequencing. RESULT(S) No inactivating mutations reported thus far in exons 6, 7, 9, and 10 of the FSH receptor gene were identified in Japanese women with POF and PCOS. DGGE analysis of PCR fragments of exon 10 also revealed no FSH receptor gene mutations in this region. CONCLUSION(S) Although we cannot exclude the presence of point mutations in other regions of the FSH receptor gene, the described FSH receptor mutations may be uncommon in Japanese patients with POF and PCOS.

Successful intrauterine treatment with radiofrequency ablation in a case of acardiac twin pregnancy complicated with a hydropic pump twin

Twin reversed arterial perfusion sequence is a serious complication of monochorionic twin pregnancy, as the pump twin that perfuses blood to the acardiac twin may experience heart failure and fetal hydrops resulting in a poor perinatal outcome. A woman with an acardiac twin pregnancy complicated by a hydropic pump twin underwent intrauterine treatment with radiofrequency ablation (RFA) at 27 weeks of gestation. Obliteration of blood flow to the acardiac twin from the pump twin was successful. Fetal hydrops resolved by the time of delivery at 32 weeks of gestation, in spite of transient deterioration, and a good postnatal outcome was achieved for the pump twin. We found that RFA was an effective intrauterine treatment for acardiac twin pregnancy and suggest that it could be introduced in cases complicated by a hydropic pump twin. Copyright

Mutational analysis of the PTEN gene in women with premature ovarian failure

Recent studies in mammals have suggested that the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) – phosphatidylinositol‐3, 4, 5‐trisphosphate pathway in oocytes might be related to the pathogenesis of premature ovarian failure (POF). The aim of this study was to investigate whether mutations of the PTEN gene are present in women with POF. We analyzed the coding region of the PTEN gene in 20 women with idiopathic POF and 20 normal controls. The PTEN gene was amplified by the polymerase chain reaction using genomic DNA isolated from blood samples. Amplified DNA was analyzed by denaturing gradient gel electrophoresis and direct sequencing. No causative mutation was detected in the coding regions of this gene. Although we found a point variation in exon 7 of one POF patient, this was a single nucleotide polymorphism that has already been reported.

Fetal hemolytic disease due to anti-Rh17 alloimmunization

Objective: To delineate clinical features of a case of fetal hemolytic disease due to anti-Rh17, along with a review of relevant studies published in English and Japanese. Methods: We present clinical features of a –D-/-D- phenotype woman with anti-Rh17 alloimmunization during pregnancy. Relevant English literature in the MEDLINE database was reviewed, while Japanese studies were searched in the Japana Centra Revuo Medicina database. Results: A Japanese –D-/-D- woman with anti-Rh17 (Hro) was treated during pregnancy. Serial ultrasonography, antibody titers, amniocenteses, and cordocenteses were conducted for perinatal management. Amniocentesis results demonstrated a high delta optical density level of 450 in the amniotic fluid, while cordocentesis revealed alloimmunization between the mother and the fetus as well as fetal hemolytic anemia. Blood flow velocity in the middle cerebral artery indicated a rapid development of fetal anemia. The newborn demonstrated severe anemia and hyperbilirubinemia, which were successfully treated with exchange transfusions. Two cases of prenatally diagnosed fetal hemolytic disease due to anti-Rh17 were found published in English and 5 in Japanese. Conclusion: A –D-/-D- phenotype patient with anti-Rh17 was successfully managed during pregnancy and a good outcome for the neonate was achieved. Our results and a review of related literature led to the following suggestions. The first pregnancy in a –D-/-D- woman may be affected, an anamnestic immune response can easily occur during pregnancy, the level of anti-Rh17 titer is indicative of the degree of fetal hemolysis, and appropriate intrauterine intervention is warranted for achievement of a good outcome.

Protein S gene mutation in a young woman with type III protein S deficiency and venous thrombosis during pregnancy.

AbstractBackground: We attempted to identify a gene defect in a young woman with type III protein S deficiency and venous thrombosis during pregnancy. Methods: Measurements of total and free PS antigen levels in plasma were carried out using an enzyme-linked immunosorbent assay. Plasma PS cofactor activity was determined by a clotting assay using activated factor V as the substrate. Genomic DNA prepared from peripheral blood was amplified by polymerase chain reaction (PCR) with PROS1-specific oligonucleotide primers. PCR products were sequenced on both strands using specific oligonucleotide primers. Results: Plasma PS cofactor activity was undetectable in every measurement at 36 weeks of gestation, as well as at 2 weeks and 4 months after delivery. Plasma total PS antigen levels were 70% and 67% at 2 weeks and 4 months after delivery, respectively. Free PS antigen level was 24% at 4 months after delivery. Of all exons analyzed, codon 295 of GGC in exon 10 was substituted for AGC. This missense mutation predicted an amino acid change of glycine to serine. Conclusions: Measurements of total and free PS antigen levels along with PS activity indicated that this was a case of type III PS deficiency. DNA analysis identified a heterozygous missense mutation of codon 295 in the PS gene, substituting glycine for serine.

Mutational analysis of the follistatin-related gene in women with premature ovarian failure and polycystic ovary syndrome

Follistatin-related gene (FLRG) protein, also called follistatin-like 3 (FSTL3), is a recently described member of the follistatin family and is widely expressed, including in human ovary (1) and endometrium. Like follistatin, FLRG protein binds activins that appear to play a crucial role in ovarian follicle development, however, follistatin and FLRG protein have distinct intracellular localization and differentially regulate activins (2). Urbanek et al. recently studied 37 possible candidate genes that may be involved in the inheritance of polycystic ovary syndrome (PCOS) and demonstrated that the strongest evidence for genetic linkage in PCOS patients was with the follistatin gene (3), however, the conclusion is still controversial. On the other hand, preliminary analysis of FLRG transgenic female mice showed that their ovaries contained fewer antral follicles and more apparent follicular atresia, suggesting that FLRG protein regulates follicular development and function through inhibition of the paracrine activity of activin and/or other related factors, such as bone morphogenetic proteins (BMPs) (4). Therefore we focused on this new follistatin member, FLRG , and speculated that impaired FLRG protein signaling might cause ovarian dysfunction in humans. The aim of this study was to search for mutations in the coding regions of the FLRG in Japanese women with 46,XX premature ovarian failure (POF) and PCOS. This study was approved by the Institutional Review Board of Shiga University of Medical Science. Informed consent was obtained from all patients and controls. Fifty-three unrelated women with a history of infertility were recruited for this study. The age at presentation ranged from 20 to 37 years. Of these 53 patients, 16 patients were diagnosed as idiopathic POF, defined as secondary amenorrhea before 40 years of age with elevated gonadotropins on at least two occasions (with follicle-stimulating hormone (FSH): 96.99/8.9 mIU/ml and LH: 38.99/15.3 mIU/ml; mean9/SD). The reference range for FSH is 2.0 14.8 mIU/ml, and values above 40 mIU/ml are indicative of ovarian failure. Each patient’s medical history was taken and each underwent a physical examination to rule out any identifiable cause of POF. Thirty-seven patients were diagnosed as PCOS according to the following criteria of the Japan Society of Obstetrics and Gynecology: 1. amenorrhea or oligomenorrhea without specific underlying diseases of the adrenal or pituitary glands; 2. a high basal LH level and a high LH:FSH ratio; and 3. bilaterally enlarged ovaries with multiple small