Masahiko Hiroi

The incidence of apoptotic bodies in membrana granulosa can predict prognosis of ova from patients participating in in vitro fertilization programs

OBJECTIVE To investigate the relationship between the incidence of apoptotic bodies in membrana granulosa and follicular steroid concentrations in human follicles. DESIGN Case-controlled prospective study for 132 individual follicles. SETTING Procedures were performed in Yamagata University Hospital. PATIENT(S) Thirty-six normo-ovulatory women with tubal infertility underwent ovulation induction for IVF-ET with a conventional hyperstimulation method. INTERVENTION(S) Patients underwent follicle aspiration after the administration of hCG. MAIN OUTCOME MEASURE(S) The nuclei of recovered granulosa cells were examined by fluorescence microscopy, and the incidence of apoptotic bodies was tabulated. Intrafollicular steroids were evaluated mainly by RIA. These data were analyzed with respect to oocyte-retrieval, oocyte maturity, fertilization, and embryo quality. RESULT(S) Membrana granulosa cells in the follicles from which oocytes were subsequently fertilized showed a significantly lower incidence of apoptotic bodies than those in follicles from which the oocytes did not fertilize. Membrana granulosa cells in the follicles from which oocytes were developed into good quality showed a significantly lower incidence of apoptotic bodies than those in the follicles from which oocytes developed into fair and poor quality. The incidence of apoptotic bodies was significantly higher in the mural granulosa cell region than in the cumulus cell region in most cases. Intrafollicular E2, P, and free T levels were not different between the oocyte groups. CONCLUSION(S) These results indicate that lower incidence of apoptotic bodies in individual follicles is associated with better outcomes for oocytes. Also, mural granulosa cells and cumulus cell in each follicle may show differentiation during follicular maturation.

Incidence of apoptotic bodies in membrana granulosa of the patients participating in an in vitro fertilization program.

OBJECTIVE To investigate the incidence of apoptotic bodies in mural granulosa cell masses and cumulus cell masses. DESIGN Nonrandomized, prospective study. SETTING Department of Obstetrics and Gynecology, Yamagata University School of Medicine, Yamagata, Japan. PATIENT(S) One hundred twenty-nine normally ovulating women underwent ovulation induction for IVF-ET with GnRH analogue (GnRH-a) and gonadotropins. INTERVENTION(S) Patients underwent follicle aspiration after the administration of hCG. MAIN OUTCOME MEASURE(S) The nuclei of recovered granulosa cells were examined by fluorescence microscopy and the incidence of apoptotic bodies was tabulated. RESULT(S) The incidence of apoptotic bodies was significantly higher in mural granulosa cell masses than in cumulus cell masses in the entire group of 129 patients. Both incidence of apoptotic bodies of mural granulosa cell masses and cumulus cell masses were significantly higher in patients with less than six follicular oocytes compared with patients with six or more oocytes. Nonpregnant patients showed significantly higher incidence of apoptotic bodies in mural granulosa cell masses compared with pregnant patients. CONCLUSION(S) These results indicate that mural granulosa cell masses and cumulus cell masses may have different functions in follicular maturation. The incidence of apoptotic bodies in mural granulosa cell masses can be used as an indicator of success of IVF.

Moderate and severe endometriosis is associated with alterations in the cell cycle of granulosa cells in patients undergoing in vitro fertilization and embryo transfer.

OBJECTIVE To determine whether folliculogenesis is impaired in patients with endometriosis. DESIGN Prospective study. SETTING Yamagata University School of Medicine, Yamagata, Japan. PATIENT(S) Thirty women participating in an IVF program. INTERVENTION(S) The patients were divided into four groups according to the cause of their infertility: tubal factor (T), n = 7; male factor (M), n = 7; idiopathic (I), n = 7; and endometriosis (E), n = 9. Granulosa cells were obtained from the follicular fluid of each patient and analyzed by flow cytometry. MAIN OUTCOME MEASURE(S) The percentage of granulosa cells in each cell-cycle stage. RESULT(S) The mean (+/- SD) rate of apoptosis in the granulosa cells obtained from the patients with endometriosis was the highest among the four groups (T = 11.7% +/- 3.3%; M = 5.6% +/- 3.8%; I = 9.6% +/- 5.1%; and E = 18.6% +/- 9.6%). The percentage of S-phase granulosa cells was significantly higher in the patients with endometriosis than in all the other patients combined (E = 12.5% +/- 6%; T + M + I = 9.3% +/- 2.9%). The percentage of G2/M-phase granulosa cells was significantly lower in the endometriosis group than in the other three groups combined (E = 2.3% +/- 2.5%; T + M + I = 4.6% +/- 2.1%). CONCLUSION(S) Endometriosis impairs the cell cycle in granulosa cells. This phenomenon may have a detrimental effect on folliculogenesis.

Neonatal hyperbilirubinemia and a common mutation of the bilirubin uridine diphosphate-glucuronosyltransferase gene in Japanese

AbstractNeonatal hyperbilirubinemia, which is prevalent among Asian peoples, has been considered as a physiological phenomenon, and its metabolic basis has not been clearly explained. Gilbert syndrome is a common inherited disease of unconjugated hyperbilirubinemia due to decreased bilirubin uridine diphosphate-glucuronosyltransferase (B-UGT), and its role in neonatal jaundice has recently been considered. We have previously reported that the Gly71Arg mutation of the B-UGT gene associated with Gilbert syndrome is prevalent in Japanese, Korean, and Chinese populations and was more frequently detected in neonates with severe hyperbilirubinemia than in control subjects. We have studied 159 Japanese full-term neonates, evaluating the relationship between the B-UGT genotype and the severity of jaundice, as assessed with a transcutaneous bilirubinometer. The gene frequency of the Gly71Arg mutation in these neonates was 0.19, and neonates carrying the Gly71Arg mutation had significantly increased bilirubin levels on days 2–4, manifested in a gene dose-dependent manner. The frequency of the Gly71Arg mutation was 0.47 in the neonates who required phototherapy (i.e., those with more severe hyperbilirubinemia), significantly higher than 0.16 in the neonates who did not require the therapy. The gene frequency of the TA repeat promoter polymorphism, the (TA)7 mutation, was 0.07, and neonates carrying this mutation did not have an increase in bilirubin. These results suggested that the Gly71Arg mutation contributes to the high incidence of neonatal hyperbilirubinemia in Japanese.

Aging‐related changes in calcium oscillations in fertilized mouse oocytes

Aging of oocytes, being not fertilized after ovulation for a prolonged time, considerably affects normal development of the fertilized oocyte. We examined effects of the aging on a series of highly repetitive Ca2+ transients commonly seen in fertilized mouse oocytes (Ca2+ oscillations). Frequency of Ca2+ oscillations in the aged oocyte [20 hrs after induction of superovulation by i.p. human chorionic gonadotropin (hCG)] was significantly higher (34.1 ± 5.8 1/hr) than the fresh oocyte (14 hr post‐hCG, 21.8 ± 7.9 1/hr). Rates of rise and fall of individual Ca2+ transient in the aged oocyte were significantly slower than the fresh oocyte, whereas durations of individual Ca2+ transients were similar. When extracellular Ca2+ was raised from 2.04 mM to 5.00 mM, aged oocytes showed significant prolongation of the duration of individual Ca2+ transient, that resulted in a sustained elevation of intracellular Ca2+ ([Ca2+]i) in 33% of the aged oocyte. Transient increase in [Ca2+]i by photolysis of a caged Ca2+, Nitr‐5, injected into cytoplasm was completely restored in the fresh oocyte [fluorescence intensity of [Ca2+]i indicator dye Fluo‐3 (F480) returned to 97 ± 2% of the control level, time constant = 37 ± 9 sec]. In contrast, in the aged oocyte, restoration of F480 following Nitr‐5 photolysis was incomplete (115 ± 12% of the control) and slow (time constant = 64 ± 23 sec). Because inhibition of the Ca2+ pump of the endoplasmic reticulum (ER) by 5 μM thapsigargin almost completely inhibited restoration of F480 following Nitr‐5 photolysis in the fresh oocyte, we conclude that the aging‐related changes in Ca2+ oscillations may be accounted for by dysfunction of intracellular Ca2+ regulation, presumably of the Ca2+ pump of the ER. Mol. Reprod. Dev. 48:383–390, 1997.

Turner's Syndrome – Review of the Literature with Reference to a Successful Pregnancy Outcome

A 30-year-old Japanese female with Turners syndrome had become pregnant and delivered a normal male infant by cesarean section. Her menarche was at age 14, and secondary sex characteristics developed normally. She had some features of Turners syndrome, and cytogenetic studies from peripheral blood lymphocytes and several tissues revealed only 45,XO karyotype without evidence of mosaicism. To our knowledge, she is only the thirteenth case of monosomy X to achieve pregnancy. A review of the literature indicates a high incidence of fetal wastage and increased chromosomal errors in the offspring of women with a 45,XO cell line with or without mosaicism.

Effects of Gonadotropin-Releasing Hormone Agonist on the Incidence of Apoptosis in Porcine and Human Granulosa Cells

Gonadotropin-releasing hormone (GnRH) has been found to be expressed in ovaries of various species and to modulate cell differentiation in ovarian cells. GnRH agonists (GnRHa) are widely used in the stimulation protocols of assisted reproduction. In the present study, the direct effects of a GnRHa on the incidence of apoptosis and sterodogenesis in porcine in human granulosa cells were investigated. Cells were obtained from 6-month-old swine and in vitro fertilization patients, and cultured in minimum essential medium (MEM) supplemented with 5% fetal bovine serum for 24 h. The GnRHa (burserelin acetate) was added to the MEM at various concentrations (0, 5, 50, 500 pg/ml, and 5 ng/ml). The nuclei of granulosa cells were examined by fluorescence microscopy after Hoechst 33258 staining, and the incidence of apoptotic cells was tabulated. The GnRHa directly increased the incidence of apoptosis in both types of granulosa cells. The concentration of GnRHa in clinical use is much higher than the concentration which produced apoptosis in granulosa cells here. The clinical dosage of GnRHa should be reconsidered with regard to its apoptosis-inducing effect.

A monoclonal antibody reacting with the zona pellucida of the oviductal egg but not with that of the ovarian egg of the golden hamster

A monoclonal antibody (mAb) designated AZPO-8 was produced by hybridizing a mouse myeloma with spleen cells from BALB/c mice immunized with materials obtained from the hamster oviduct. With an immunofluorescence test, AZPO-8 reacted with the zona pellucida (ZP) of ovulated eggs in the oviduct (ZP-OVI) but not with the zona pellucida of eggs in the ovary (ZP-OVA). Using indirect enzyme immunostaining, this mAb reacted with epithelial cells of the oviduct, the uterus (especially the cervical epithelium) and the gastric mucosa, but not with other hamster tissues examined. The reactivity of antigen-positive tissues was abrogated by pretreatment of the tissues with periodic acid. Western blotting analysis revealed that AZPO-8 reacted with substances of broad molecular weight range, and the strongest reactivity was detected at a molecular weight of approximately 200,000 in both cases when extract of ZP-OVI or the hamster oviduct was applied on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel. AZPO-8 showed strong hemagglutination activity only to group A human red blood cells. These results indicated that (1) ZP-OVI had an antigen that was not detected on ZP-OVA, (2) ZP-OVI and the oviduct shared the same antigenicity, and (3) the antigenic determinant reactive with the mAb might be carbohydrate in nature. A possible role of this antigen in fertilization was discussed.

Hyaluronic Acid Inhibits Apoptosis in Granulosa Cells via CD44

AbstractPurpose: This study was designed to examine whether hyaluronicacid (HA) inhibits apoptosis in cumulus and muralgranulosa cells and to examine whether this effect of HAwas mediated through CD44. Methods: Mural and cumulus granulosa cells were obtainedfrom in vitro fertilization patients. The cells were culturedwith various concentrations of HA or HA plus variousconcentrations of anti-CD44 antibody without serum supplement.After 24 hr of culture, the cells were fixed and stainedwith Hoechst 33258. One thousand granulosa cells of eachconditions were observed by fluorescence microscopy. Results: HA inhibited apoptosis in both kinds of granulosacells, and anti-CD44 antibody prevented this effect of HA.Conclusions: The incidence of apoptotic granulosa cellswith fragmented condensed nuclei was reduced by HA viaCD44.

Do assisted reproductive technologies have effects on the demography of monozygotic twinning

This study examined the incidence of monozygotic twinning of one embryo with the use of cycles in which only one embryo, treated by various assisted reproductive technologies (ART), was transferred. It also examined the possibility of demographic alterations in the overall incidence of monozygotic twinning in Japan induced by ART. A total of 134 clinics or institutes participated in this study. Overall, it is noted that the incidence of monozygotic twinning in Japan has been almost constant (0.402%). In ART treatments, the monozygotic twinning rate was significantly higher than that of natural pregnancies (P = 0.0204). For in vitro fertilization (IVF) treatment cycles, the monozygotic twinning rate was also higher than that of natural pregnancies (P = 0.6285). However, this difference was not statistically significant because of the small number of IVF pregnancies. Moreover, in the context of microinsemination cycles, the monozygotic twinning rate was significantly higher than that of natural pregnancies (P = 0.0285) or IVF (P = 0.0006). In terms of the possible impact of ART on the demography of monozygotic twinning, adoption of mechanically assisted hatching for all embryos in Japan may alter the demography of monozygotic twinning.