Atsushi Yanaihara

Detection of circulating urothelial cancer cells in the blood using the CellSearch System

Circulating tumor cells (CTCs) have been shown to aid in the therapeutic management of patients. But, only a few attempts have been made at the detection of urothelial cancer cells in the blood. The purpose of this study was to test the hypothesis that CTCs are detected in patients with urothelial cancers using newly developed CellSearch Assay.

Comparison in gene expression of secretory human endometrium using laser microdissection.

BackgroundThe endometrium prepares for implantation under the control of steroid hormones. It has been suggested that there are complicated interactions between the epithelium and stroma in the endometrium during menstrual cycle. In this study, we demonstrate a difference in gene expression between the epithelial and stromal areas of the secretory human endometrium using microdissection and macroarray technique.MethodsThe epithelial and stromal areas were microdissected from the human endometrium during the secretory phase. RNA was extracted and amplified by PCR. Macroarray analysis of nearly 1000 human genes was carried out in this study. Some genes identified by macroarray analysis were verified using real-time PCR.ResultsIn this study, changes in expression <2.5-fold in three samples were excluded. A total of 28 genes displayed changes in expression from array data. Fifteen genes were strongly expressed in the epithelial areas, while 13 genes were strongly expressed in the stromal areas. The strongly expressed genes in the epithelial areas with a changes >5-fold were WAP four-disulfide core domain 2 (44.1 fold), matrix metalloproteinase 7 (40.1 fold), homeo box B5 (19.8 fold), msh homeo box homolog (18.8 fold), homeo box B7 (12.7 fold) and protein kinase C, theta (6.4 fold). On the other hand, decorin (55.6 fold), discoidin domain receptor member 2 (17.3 fold), tissue inhibitor of metalloproteinase 1 (9 fold), ribosomal protein S3A (6.3 fold), and tyrosine kinase with immunoglobulin and epidermal growth factor homology domains (5.2 fold) were strongly expressed in the stromal areas. WAP four-disulfide core domain 2 (19.4 fold), matrix metalloproteinase 7 (9.7-fold), decorin (16.3-fold) and tissue inhibitor of metalloproteinase 1 (7.2-fold) were verified by real-time PCR.ConclusionsSome of the genes we identified with differential expression are related to the immune system. These results are telling us the new information for understanding the secretory human endometrium.

Regulation of estrogen activity in human endometrium: Effect of IL-1β on steroid sulfatase activity in human endometrial stromal cells

We investigated the effect of interleukin 1beta (IL-1beta) on steroid sulfatase (STS) activity and the expression of STS mRNA in human endometrial stromal cells. Endometrial tissue samples were obtained from patients undergoing hysterectomy to remove uterine fibroids. Stromal cells were isolated from the tissue preparation and cultured. IL-lbeta (1 approximately 100 ng/ml) was added into the culture medium and incubated for 24 h. The expression of STS mRNA was measured by competitive RT-PCR. The addition of IL-lbeta at 10 and 100 ng/ml suppressed STS mRNA expression to 55.2 +/- 12.8% and 25.1 +/- 10.9%, respectively, of the control sample to which no IL-lbeta had been added. STS activity was measured by radiolabelled steroid metabolite using thin layer chromatography, and this activity was also significantly suppressed in response to the administration of IL-lbeta in a dose-dependent manner. When IL-1 receptor antagonist (IL-1ra) was added together with IL-1beta to the culture medium, mRNA expression and STS activity were recovered. The present study is the first to demonstrate IL-1beta regulation of STS activity locally in human endometrium. IL-1beta suppressed mRNA and activity of STS in stromal cell culture. This initial demonstration of IL-1beta regulation of STS implies that IL-1beta may control the steroid microenvironment in human uterine endometrium by reducing biologic action of estrogen.

CLINICAL OUTCOMES OF TWO DIFFERENT ENDOMETRIAL PREPARATION METHODS FOR CRYOPRESERVED-THAWED EMBRYO TRANSFER IN PATIENTS WITH A NORMAL MENSTRUAL CYCLE

AimTo compare the clinical outcomes of cryopreserved-thawed embryo transfer among patients with a normal menstrual cycle who had natural or hormone-replacement cycles.MethodsFrom January 2004 to June 2006, cryopreserved embryos following conventionalin vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) were thawed and transferred in a total of 720 natural cycles and 136 hormone-replacement cycles.ResultsCryopreserved-thawed embryo transfer in patients who had a natural or hormone-replacement cycle resulted in clinical pregnancy in 43.1% and 40.4%, respectively; a rate of miscarriage of 14.5% and 23.6%, respectively; and a rate of ongoing pregnancy and delivery of 36.5% and 30.9%, respectively. None of these differences were statistically significant.ConclusionsPatients with a normal menstrual cycle who have natural or hormone-replacement cycles can be expected to have comparable clinical outcomes with cryopreserved-thawed embryo transfer.

Localization and expression of steroid sulfatase in human fallopian tubes

Localization of steroid sulfatase, a membrane-bound microsomal enzyme, in human fallopian tubes was immunohistochemically investigated, and expression of RNA was confirmed by competitive RT-PCR. Human fallopian tubes were obtained from 10 patients in follicular and early luteal phases during gynecological laparotomy. An anti-human rabbit polyclonal antibody was prepared against sulfatase protein purified from human placenta. Total RNA was isolated from epithelium of fallopian tubes. A heterologous RNA competitor was designed, and competitive RT-PCR was carried out. Steroid sulfatase was localized to the cytoplasm of epithelial cells. With respect to the positive staining of cells, the number of positive secretory cells was higher than that of ciliated cells. A significantly higher number of positive cells was found in tissue obtained from the early luteal phase than that found in tissue from the follicular phase. An abundant expression of sulfatase mRNA in early luteal phase was also observed. This study demonstrates, for the first time, that steroid sulfatase is localized to human epithelial cells and that steroid sulfatase staining and mRNA expression changes with the menstrual cycle. These results suggest that sulfatase in the fallopian tube may be involved in controlling the local steroid environment, which appears to regulate aspects of the physiological reproductive function of the fallopian tube.

The decrease of serum luteinizing hormone level by a gonadotropin-releasing hormone antagonist following the mild IVF stimulation protocol for IVF and its clinical outcome

PurposeWhile performing the mild ovarian stimulation protocol with a GnRH antagonist, the pregnancy rate was compared between the groups, which were divided by the degree that the luteinizing hormone (LH) level decreased.Materials and methodsPatients aged 27 to 42years (36.1 ± 3.79) underwent 308 IVF cycles who opted for IVF via the mild ovarian stimulation protocol began clomiphene citrate on day 3 and recombinant FSH on day 5. A GnRH antagonist was administered when the dominant follicle reached 14mm. Serum LH was measured at the time of GnRH antagonist administration and at the time of hCG injection. The pregnancy rate and implantation rate were compared between 50 cycles in which the LH level dropped less than one-third and the control (LH level within 1/3).Result(s)The pregnancy rate for the group in which the LH level fell less than one third was 18%. Conversely, the pregnancy rate for the control group was 39%. The implantation rate was 18% for the less than one-third group and 26% for the control group. Both the pregnancy rate and the implantation rate for the group in which the LH level fell less than one-third were significantly lower than that of control (p < 0.02).Conslusion(s)When performing the mild ovarian stimulation protocol, serum LH should be followed. If the serum LH level is less than one-third at the time of hCG injection, both the pregnancy rate and implantation rate are significantly lower.

Correlations between steroids concentration in follicular fluid, pronuclear morphology and embryo qualities in in vitro fertilization

BackgroundSeveral parameters of early embryo development are known as predictors of implantation success. Recently, zygote or embryo morphological assessments are thought to be a major method of selection in embryo transfer. We expected that the concentrations of the steroids in follicular fluid (FF) were associated with oocyte maturation and embryo quality. In the present paper, we evaluated the relationship of several parameters.MethodsWe investigated 105 samples of FF from 22 subjects byin vitro fertilization (IVF). We evaluated the correlations between the FF concentrations of estradiol (E2) and progesterone (P4), the diameter of the ovarian follicles, fertilization, and zygote assessment based on pronuclear morphology and day 3 embryo qualities (i.e. number of blastomeres and fragmentation rate).ResultsThere was a positive correlation between the E2 concentrations in FF and serum (r = 0.273,P < 0.01), but there was no correlation between follicular diameter and the FF concentration of each steroid. The concentration of E2 in FF containing fertilized oocytes was not significantly different from that in FF containing unfertilized oocytes. At the pronuclear stage, the concentration of either steroid in FF did not differ among the morphological groups. The concentration of P4 in FF was significantly lower in the group in which pronuclei were detected at 20 h after insemination than in the group in which pronuclei were not detected. The concentration of E2 in FF was significantly related to the number of blastomeres (r = 0.271,P < 0.05) and furthermore, was significantly higher in FF from which morphologically good embryos were obtained at day 3 (P < 0.05).ConclusionsThe FF concentrations of the steroids did not affect the pronudear pattern, but P4 production may play a role in reducing the potential of the oocyte to develop pronuclei and the concentration of E2 may predict the cleavage capability of the oocyte.

Mild stimulation with clomiphene citrate in combination with recombinant follicle-stimulating hormone and gonadotropin-releasing hormone antagonist and its influence on serum estradiol level and pregnancy rate

AimThe mild ovarian stimulation protocol for in vitro fertilization (IVF) is carried out to minimize adverse side-effects as well as cost. While performing mild ovarian stimulation with a gonadotropin-releasing hormone (GnRH) antagonist, the pregnancy rate was examined in cases that exhibited a serum estradiol (E2) drop down.MethodsIn this study, 174 patients who requested mild ovarian stimulation for IVF began clomiphene citrate on day 3 and recombinant follicle-stimulating hormone (FSH) on day 5 of their menstrual cycles. A GnRH antagonist was administered when the dominant follicle reached a diameter of 14 mm. Serum luteinizing hormone and estradiol were measured at the time of GnRH antagonist administration and at the time of human chorionic gonadotropin (hCG) injection. Pregnancy rates and implantation rates were compared between 24 cycles in which the E2 level fell at the time of hCG injection and 150 cycles in which it did not fall.ResultsThe pregnancy rate in the cases in which the E2 level fell (25% decrease) at the time of hCG injection was significantly lower than it was in the cases in which it did not fall (16.7 vs 41.0%). The implantation rate for the cases in which the E2 level fell was also lower than that of the control group (7.0 vs 31.0%). There was no significant difference in the number of good-quality embryos between the two groups.ConclusionWhen performing the mild ovarian stimulation protocol, serum E2 should be followed. It is prudent to avoid embryo transfer in the same cycle in cases that exhibit E2 drop down.

Sperm retention site and its influence on cleavage rate and early development following intracytoplasmic sperm injection.

Background: Intracytoplasmic sperm injection (ICSI) has risen to the forefront of reproductive technology. In the present study, the location of the sperm injection was noted, and a prospective study was conducted to evaluate the effect of the sperm retention site on cleavage rates and embryo quality after ICSI. Methods: This study involved 336 ICSI patients (age 27–44; average 37.4) where 1545 oocytes were observed. An oocyte was divided into nine sites and the sperm retention site was observed microscopically after injection. The polar body was placed at either the twelve or six o’clock position. The injection pipette was introduced at the three o’clock position and oolemma rupture was ascertained by mild suction. The main outcome measures were the relationship of sperm remaining in position in the oocyte to fertilization rate and embryo quality. Results: When the injection pipette was introduced at the three o’clock position, about 80% of the sperm remained in the center or left of center. The fertilization rate was significantly lower (p < 0.05) when the sperm remained near the site of introduction. Embryo quality was not significantly affected by the sperm retention site. Conclusions: About 12–14% of the spermatozoa remained near the introducing position, and in these cases the fertilization rate was low. However, once fertilization occurred, the sperm retention site had minimal impact on embryo quality. Injecting sperm near the spindle site may improve embryo quality.

Intracytoplasmic Sperm Injection: Technical Improvement

Summary Successful fertilization using intracytoplasmic sperm injection (ICSI) was first reported in 1988 and the first successful ICSI pregnancy was in 1992. Since then, this technology has quickly become an indispensable technique in reproductive medicine, and its validity has been verified through several clinical studies over the past 10 years. After several technologic improvements, the rate of ICSI fertilization is comparable to that of conventional in vitro fertilization. In this article, we discuss how ICSI has improved fertilization and pregnancy rates.