Kou Sueoka

Integrins and reproductive physiology: expression and modulation in fertilization, embryogenesis, and implantation

OBJECTIVE To review the available information regarding the role of integrins in reproductive physiology and to discuss their potential clinical implications. DESIGN Studies that specifically relate to the expression and modulation of integrins in fertilization, embryogenesis, and implantation were identified through the literature and Medline searches. RESULT(S) Integrins are a class of adhesion molecules that participate in cell-to-cell and cell-to-substratum interactions and are present on essentially all human cells. All mammalian eggs express integrins at their surface, and the integrin alpha 6 beta 1 serves as a sperm receptor that mediates sperm-egg binding. In addition, certain integrin moieties appear to be regulated within the cycling endometrium. Specifically, the expression of beta 1 integrins in the early proliferative phase is restricted to the glandular epithelium, whereas stromal cells also express beta 1 integrins in the midsecretory phase. The expression of beta 1 integrins increases at the time of implantation and remains elevated in the decidua during early pregnancy. A disruption of integrin expression is associated with certain types of infertility in women. The apical surface of the mural trophectoderm does indeed possess functional integrins, and trophoblast interactions with extracellular matrix proteins largely depend on the integrin family of adhesion receptors. CONCLUSION(S) Integrins play particularly important roles in both fertilization and embryogenesis, including the process of implantation.

The Effects of Age and Abnormal Sperm Count on the Nondisjunction of Spermatozoa

AbstractPurpose: The effect of paternal age on the nondisjunctionof sex chromosomes is controversial. Also, the prevalenceof chromosomal anomalies in infertile patients iscontroversial, it has been reported that the sex chromosomalaneuploidy rate following treatment with intracytoplasmic sperminjection (ICSI) is higher than in naturally conceivedpregnancies. We investigated the influence of paternal age andoligozoospermia on the nondisjunction of spermatozoa. Methods: We determined the rate of aneuploidy forgonosomes and autosomes, using two-color fluorescence in situhybridization (FISH) of the X and Y chromosomes andchromosomes 12 and 18 in 10 donors under 25 years of agewho had a normal sperm count (≤20 × 106/ml), 10 donorsover the age of 39 years with idiopathic infertility andnormozoospermia (≤20 × 106/ml), and 5 oligozoospermicdonors (<20 × 106/ml). Results: There was no obvious relationship betweenincreasing age and autosomal disomy (disomy 12 and disomy 18).Neither autosomal disomy nor diploidy was increased inany group. The frequency of X-, Y-, XX-, and YY-bearingsperm did not differ significantly among groups, but thefrequency of XY-bearing sperm was significantly higher inthe older infertile group than in the control donors. Conclusions: The incidence of nondisjunction of paternalsex chromosome in meiosis I was higher in older men withidiopathic infertility. The present results suggest that therisk of producing XXY fetuses is higher among men >39years of age with idiopathic infertility.

Assessment of the Dominant Abnormal Form Is Useful for Predicting the Outcome of Intracytoplasmic Sperm Injection in the Case of Severe Teratozoospermia

Purpose:Our purpose was to investigate the relation between the dominant sperm anomaly and sperm morphology and the outcome of intracytoplasmic sperm injection (ICSI).Methods:Two hundred ninety-five patients who underwent a total of 181 cycles of in vitro fertilization (n = 168) and/or 217 cycles of ICSI (n = 177) between July 1995 and May 1997 at Keio University Hospital were investigated.Results:The rates of fertilization and pregnancy were 63.3 and 27.8%, respectively, in ICSI cycles with ≤4% normal forms. When the percentage of strictly normal morphology was ≤4, the fertilization rate was lower in the case of severely tapered head (13.0%; n = 4) than in the cases of other deformities in ICSI. The acrosomal defect made no difference in the fertilization rate with ICSI.Conclusions:The predominant abnormal form affects the ICSI outcome in the case of ≤4% normal forms.

Interactions between insulin-like growth factor-I (IGF-I) and the renin-angiotensin system in follicular growth and ovulation.

The interactions between insulin-like growth factor-I (IGF-I) and the renin-angiotensin system (RAS) in follicular growth and ovulation were studied with the use of an isolated perfused rabbit ovary preparation. Ovulation failed to occur in either control ovaries or the experimental ovaries perfused with IGF-I in a concentration of 1, 10, or 100 ng/ml in the absence of gonadotropin. Exposure to IGF-I stimulated the secretion rate of angiotensin II-like immunoreactivity (Ang II-IR) in perfused rabbit ovaries in a dose-dependent manner. The percent increase in follicle diameter in ovaries perfused with IGF-I for 12 h was significantly correlated with the secretion rate of Ang II-IR at 12 h after exposure to IGF-I. The addition of IGFBP-3 to the perfusate did not induce ovulation in the absence of gonadotropin, but exposure to IGFBP-3 inhibited hCG-induced ovulation in a dose-dependent manner. In addition, IGFBP-3 significantly reduced the ovarian secretion rate of Ang II-IR and prostaglandins stimulated by hCG administration. Intrafollicular plasminogen activator (PA) activity significantly increased within 4 h after exposure to 100 ng/ml of IGF-I, compared with that in control ovaries perfused with medium alone. The concomitant addition of IGFBP-3 to the perfusate significantly reduced the IGF-I-stimulated PA activity in the preovulatory follicles at 4, 6, and 8 h after exposure to IGF-I. However, IGFBP-3 alone affected neither the ovarian secretion rate of Ang II-IR nor intrafollicular PA activity. Exposure to streptokinase, an exogenous PA, in vitro stimulated both follicular growth and the intrafollicular Ang II-IR content. In conclusion, IGF-I enhances both ovarian Ang II production and follicular development by stimulating intrafollicular PA activity.

Green tea extracts attenuate doxorubicin-induced spermatogenic disorders in conjunction with higher telomerase activity in mice

PurposeThe aim of this study was to investigate the protective effect of green tea extracts against doxorubicin-induced damage in the mouse testes correlating with telomerase activity.MethodsGreen tea extracts were administered orally. Doxorubicin was coadministered intraperitoneally. These testes were evaluated histologically and the telomerase activity was analyzed. Additional immunostaining was carried out.ResultsBoth the sperm density and sperm motility were significantly increased in green tea extracts coadministration groups as compared to the doxorubicin-treated groups. By histological analysis, germ cell damage was greatly attenuated by green tea extracts coadministration. Telomerase activity significantly increased in association with the coadministration of green tea extracts as compared to that of doxorubicin-only groups. In all groups, human telomerase reverse transcriptase signals were mainly observed in the spermatocytes and spermatids.ConclusionsThese findings suggest that green tea extracts exert protective effects against doxorubicin-induced spermatogenic disorders in conjunction with higher telomerase activity levels.

The development of novel quantification assay for mitochondrial DNA heteroplasmy aimed at preimplantation genetic diagnosis of Leigh encephalopathy

Purpose: To perform preimplantation genetic diagnosis (PGD) of Leigh encephalopathy, we developed a rapid and reliable quantification assay for the percentage of T8993G mtDNA mutation and analyzed various specimens. Methods: We prepared the standard curve by measuring serial proportion of 8993T/G cloned plasmid DNA using real-time PCR, and measured (1) mutant DNA (known proportions by PCR-RFLP), (2) single lymphocytes from 46% mutant carrier, (3) 123 blastomeres from 20 abnormal embryos. Results: (1) These were within −5∼+6% error range, (2) mean 44.3%(11–70%), (3) Five embryos harbored T8993G mutation (4–22%). Embryos from same person indicated different degrees of heteroplasmy, and blastomeres from same embryo demonstrated limited dispersion of heteroplasmy (2–11%). Conclusions: (1) This method provides rapid and reliable PGD for Leigh encephalopathy. (2) The variable heteroplasmy with somatic mitosis was suggested. (3) T8993G mutation was existed in undeveloped embryo, and the bottleneck theory was supported. The limited heteroplasmy dispersion of blastomeres from same embryo also supported reliability of PGD for T8993G mutation.

Platelet activating factor-induced early pregnancy factor activity from the perfused rabbit ovary and oviduct.

Early pregnancy factor, a glycoprotein, and platelet activating factor, a phospholipid, are early signals of pregnancy appearing during the preimplantation period. The aim of this study was to determine if the administration of platelet activating factor to rabbits would stimulate the production of early pregnancy factor and, if so, to ascertain the organs responsible. Early pregnancy factor was assayed after the administration of synthetic platelet activating factor to intact rabbits, rabbits that had undergone bilateral salpingo-oophorectomy, and isolated in vitro perfused rabbit ovary and/or oviduct. Although serum early pregnancy factor activity was detectable 30 minutes after platelet activating factor injection in intact rabbits, no activity was found in rabbits whose ovaries and oviducts had been removed. Perfusate early pregnancy factor activity was detectable only in the presence of both ovary and oviduct, and was first evident 120 minutes after platelet activating factor exposure. However, combined samples of perfusates from the ovary alone and oviduct alone expressed positive early pregnancy factor activity. Furthermore, the addition of platelet activating factor to either single or combined perfusates did not increase early pregnancy factor activity. Our data imply that platelet activating factor induces the production of ovary and oviduct early pregnancy factor components independently.

Guidelines for office gynecology in Japan: Japan Society of Obstetrics and Gynecology and Japan Association of Obstetricians and Gynecologists 2011 edition

Gynecology in the office setting is developing worldwide. Clinical guidelines for office gynecology were first published by the Japan Society of Obstetrics and Gynecology and the Japan Association of Obstetricians and Gynecologists in 2011. These guidelines include a total of 72 clinical questions covering four areas (Infectious disease, Malignancies and benign tumors, Endocrinology and infertility, and Healthcare for women). These clinical questions were followed by several answers, backgrounds, explanations and references covering common problems and questions encountered in office gynecology. Each answer with a recommendation level of A, B or C has been prepared based principally on evidence or consensus among Japanese gynecologists.These guidelines would promote a better understanding of the current standard care practices for gynecologic outpatients in Japan.

Role of β1 Integrins in Human Endometrium and Decidua during Implantation

The present study was undertaken to investigate the expression and function of β₁ integrins in human endometrium and decidua. Fluorescence-activated flow cytometry demonstrated the greater expression of the β₁, α₁, α₂, and α₅ subunits of the β₁ integrin family in cultured stromal cells from the midsecretory phase than in those of the early proliferative phase. The addition of estradiol (E₂) and progesterone (P) to cultured stromal cells in the early proliferative phase increased the expression of β₁ integrins in vitro. The immunohistochemical distribution of β₁ integrins demonstrated predominantly glandular epithelial staining in the proliferative phase, and stromal and glandular staining in the midsecretory phase. Flow cytometry also demonstrated the expression of β₁, α₁, α₂, α₃, α₅, and α₆ subunits of β₁ integrin family in cultured decidual cells. Immunohistochemistry confirmed the β₁ integrin cell surface phenotypes in cultured decidual cells observed by flow cytometry. In the subsequent experiment, the effects of antibodies against specific β₁ integrin heterodimers on mouse embryo attachment and spreading were tested to identify the role of β₁ integrins in early implantation. We developed assays for the attachment of mouse embryos and for trophoblastic spreading on cultured human decidual cells. The addition of antibodies directed against β₁ and α integrin subunits to cultured decidual cells did not affect the rates of hatching or attachment of the blastocysts, whereas the outgrowth of embryos on the decidual cells was inhibited by their antibodies in a dose-dependent manner. Thus, β₁ integrin in human endometrium and decidua may be important in mediating the organization of extracellular matrix proteins derived from embryos during the early stage of implantation.

Effects of systemic administration of indomethacin on ovulation, luteinization, and steroidogenesis in the rabbit ovary

Indomethacin blocks ovulation in human chorionic gonadotropin-stimulated rabbits. Experiments were done with an in vitro ovarian perfusion system to investigate whether indomethacin affects luteinization and steroidogenesis. Indomethacin (10 mg/kg) was administered in combination with human chorionic gonadotropin (100 IU) via a marginal ear vein, and a second dose of indomethacin was given 8 hours later. Control animals received vehicle in place of indomethacin. Laparotomy was performed 24 hours after the initial treatment. The presence of unruptured follicles and corpora lutea was recorded and the ovaries were perfused in vitro for 3 hours. Progesterone, prostaglandin F2 alpha, prostaglandin E2, and 6-keto-prostaglandin F1 alpha were measured in samples obtained at 0, 30, 60, 120, and 180 minutes from the circulating perfusion medium entering and exiting the ovary. At the end of the perfusion all ovaries (12 treated and 10 controls) were fixed for histologic analysis. Ovulation occurred in all control ovaries but in none of the indomethacin-treated ovaries. The mean number of unruptured follicles per ovary in the treated group was not significantly different from the number of corpora lutea plus unruptured follicles per ovary in the controls. Cells in both groups were qualitatively similar in ultrastructure; abundant lipid droplets, smooth endoplasmic reticulum, and mitochondria were seen. Secretion rates of progesterone and prostaglandin did not differ between the two groups during the 3-hour perfusion period. These results suggest that transformation of granulosa cells into fully functional luteal cells can occur in the absence of follicular rupture.