Shinji Iwasaki

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.

Traumatic bilateral testicular dislocation: a recovery of spermatogenesis by orchiopexy 15 years after the onset

OBJECTIVE To report a patient with azoospermia who achieved an induction of spermatogenesis after undergoing orchiopexy of long-standing bilateral traumatic dislocated testes. DESIGN Case report. SETTING Clinical. PATIENT(S) A 33-year-old man. INTERVENTION(S) Imaging studies, orchiopexy, testicular biopsy, and a review of similar cases. MAIN OUTCOME MEASURE(S) Recovery of spermatogenesis after orchiopexy. RESULT(S) A patient with azoospermia with bilateral traumatic dislocated testes due to a motorcycle accident 15 years previously. A bilateral testicular dislocation in the superficial inguinal pouch was diagnosed by palpation, imaging studies, and exploration. A recovery of spermatogenesis was obtained after performing orchiopexy of bilateral dislocated testes. CONCLUSION(S) The recovery of spermatogenesis in patients with azoospermia with a bilateral testicular dislocation therefore may be successfully obtained after appropriately performing orchiopexy.

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.

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.

Localization and gene expression of steroid sulfatase by RT-PCR in cumulus cells and relationship to serum FSH levels observed during in vitro fertilization

Background The purpose of this study was to localize the expression of steroid sulfatase (STS) in cumulus cells and to determine the relationship between STS mRNA expression and the serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol and progesterone. Methods The subject group included 49 women (29 to 44 years old) for whom in vitro fertilization treatment was indicated. All subjects gave informed consent. One hundred fourteen samples of cumulus-oocyte complex (COC) were obtained under microscopic observation. Part of the COC was stained by STS antibody. RNA was extracted by phenol-chloroform method and real-time PCR was performed. Serum of each patient was collected and was measured by ELISA. Results Some of the cumulus samples were stained by STS antibody. The expression of STS mRNA in all samples was confirmed by quantitative RT-PCR. Although there was no significant correlation between the level of STS mRNA and the serum levels of estradiol, progesterone and LH, there was a statistically significant negative correlation between the level of STS mRNA expression and the serum level of FSH (n = 105, p = 0.018, r = -0.22). Conclusion These results have demonstrated for the first time the expression of STS in cumulus cells by immunohistological stainings and real-time RT-PCR. STS expression in cumulus cells may be related to the control of the local steroidal environment in the oocyte. Serum FSH may control STS mRNA expression from the results of RT-PCR, although the correlation was low.

Sperm retention site and its influence on pronucleus stage evaluation following intracytoplasmic sperm injection

AimIt has been suggested that the position of the sperm after intracytoplasmic sperm injection (ICSI) has an effect on the development and quality of the embryo. In this study, we retrospectively examined whether pronucleus stage evaluation used through clinical studies in recent years has relevance with regard to sperm location.MethodsFrom 2003 to 2005, 1285 oocytes from 459 patients (average age: 36 years) were retrospectively analyzed. The 459 patients underwent ICSI because of fertilization disorders and oligozoospermia. Follicle stimulation was via either Clomid or the long protocol. Human chorionic gonadotropin was administered to induce ovulation and oocyte retrieval was conducted 35 h later. After confirming the presence of a polar body, we immobilized the ovum at the 6 o’clock position, introduced the injection pipette at the 3 o’clock position and carried out ICSI.ResultsWhen a sperm was located at a position that was opposite to the polar body, both classifications of Scott and Tesarik regarding embryo quality were distinctly low. Further-more, a good embryo classification ensued when the sperm was located adjacent to the polar body.ConclusionThe zone in which the sperm was located did not always correlate with embryo quality; however, our study suggested that sperm location affects the synchronization of the nudeolus. When carrying out ICSI, it is important to take into consideration the insertion point of the sperm.

Concentration of lactoferrin and interleukin-6 in cervical mucus from patients being treated for infertility

BackgroundThe concentrations of the iron-binding protein lactoferrin (LF) and interleukin-6 (IL-6) were measured in the cervical mucus of patients being treated for infertility throughout the menstrual cycle.MethodsA total of 251 cervical mucus samples were obtained from the patients throughout the menstrual cycle. One hundred and fifty samples were from primary infertility patients with unexplained infertility and 101 samples were from secondary infertility patients as a control. The concentrations of LF and IL-6 were measured by enzyme immunoassays. The standard curve of LF concentrations ranged from 1.6 to 50 ng/mL.ResultsThe mean LF and IL-6 concentrations in the cervical mucus of primary infertility patients were higher than that of the control patients (P = 0.04,P = 0.032, respectively) The LF and IL-6 concentrations were highly correlated (P < 0.0001).ConclusionElevated levels of IL-6 and LF in the cervical mucus were obtained from primary infertility patients. We speculate that LF might also be one of the causes of infertility and might play an important role in reproductive processes in the cervix.

Concentration of lactoferrin and interleukin-6 in cervical mucus from patients being treated for infertility: Lactoferrin and IL-6 in cervical mucus

Background:  The concentrations of the iron‐binding protein lactoferrin (LF) and interleukin‐6 (IL‐6) were measured in the cervical mucus of patients being treated for infertility throughout the menstrual cycle.

CAUSES AND TREATMENT OF IMPLANTATION FAILURE

Summary In recent years, a high fertilization rate has been achieved via the rapid evolution of assisted reproductive technology. However, the overall pregnancy rate remains the same. Implantation failure appears to be one factor. In preparation for implantation, the human endometrium undergoes dramatic proliferation and differentiation during the menstrual cycle in response to the rise and fall of ovarian steroids. The characteristic restructuring of the endometrium is thought to be effected not only by steroids but also by locally released factors. Elucidation of the implantation mechanism and treatment of implantation failure may improve the low pregnancy rate in in vitro fertilization-embryo transfer. In this review, the cause of and treatment for implantation failure are summarized.