Hidekazu Saito

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.

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.

Implications of assisted reproductive technologies on term singleton birth weight: an analysis of 25,777 children in the national assisted reproduction registry of Japan

OBJECTIVE To evaluate the implications of assisted reproductive technologies (ART) on neonatal birth weight. DESIGN A retrospective study using analysis of covariance and multiple logistic regression analysis of the Japanese ART registry. SETTING Japanese institutions providing ART treatment. PATIENT(S) A total of 25,777 singleton neonates reaching term gestation following ART during the years 2007-2008, with 11,374 achieved through fresh embryo transfers (fresh ET) and 14,403 achieved through frozen-thawed embryo transfers (FET). INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Birth weight. RESULT(S) The mean birth weight after FET was significantly higher compared with fresh ET and all Japanese births (3,100.7 ± 387.2 g, 3,009.8 ± 376.8 g, and 3,059.6 ± 369.6 g, respectively). The risk for low birth weight in FET was significantly lower compared with fresh ET. In fresh ET, ovarian stimulations were associated with about twofold risk of low birth weight compared with natural cycle. Regarding to the duration of embryonic culture, the risks resulting from a shorter culturing time were significantly higher compared with a longer culturing time in fresh ET. CONCLUSION(S) The best method of embryo transfer for fetal growth was FET after extended culturing until blastocyst stage. However, further investigations should be performed to understand the safety of ART treatment.

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.

The frequency of ovulation from the affected ovary decreases following laparoscopic cystectomy in infertile women with unilateral endometrioma during a natural cycle

PurposeTo evaluate the cystectomy-induced damage on the follicular growth and ovulation of an affected ovary during natural cycles.MethodsTwenty-eight infertile patients with unilateral ovarian endometriomas who underwent laparoscopic cystectomy were retrospectively evaluated. The ovulation rate of an affected ovary during natural cycles was compared before and after cystectomy in each patient, and it was also determined if ovulation from the affected ovaries resulted in pregnancy.ResultsAfter surgery, the ovulation rate was significantly lower than that before cystectomy (16.9 ± 4.5% vs. 34.4 ± 6.6%, P = 0.013). After surgery, 14 pregnancies were achieved without IVF treatment, and only 2 of them (14.3%) were achieved from an operated-side ovary. However, the pregnancy rate per ovulatory cycle of the operated-side ovary was not different from that of the intact ovary (8.8% vs. 5.8%, P = 0.750).ConclusionsLaparoscopic cystectomy is an invasive treatment in that it reduces the frequency of ovulation; however the pregnancy rate per ovulation did not deteriorate.

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.

Endometriosis and Oocyte Quality

Endometriosis is associated with marked subfertility and various causes for this subfertility have been previously studied. The poor quality of oocytes has been suggested as one possible cause. In this study, we evaluated the quality of oocytes by examining the status of granulosa cells surrounding the oocyte. For this purpose, we analysed the incidence of apoptosis, changes in cell cycle, and oxidative stress in the granulosa cells. Endometriosis patients had a higher apoptotic incidence, more alterations of the cell cycle, and a higher incidence of oxidative stress than patients with any of the other infertility causes (tube, male, and idiopathic factors). These changes might affect oocyte quality, and thus fertility in endometriosis patients.

Alteration of Glutathione Reductase Expression in the Female Reproductive Organs During the Estrous Cycle

Abstract The enzyme glutathione reductase (GR) recycles oxidized glutathione (GSSG) by converting it to the reduced form (GSH) in an NADPH-dependent manner. A specific antibody raised against recombinant rat GR was used to localize the protein in the female reproductive organs during the estrous cycle in the rat. In the ovary, the strongest reactivity to the antibody was observed in oocytes, followed by granulosa cells, corpus luteum, and interstitial cells. A strongly positive reaction was also observed mainly in the oviduct epithelia, uterine epithelia, and endometrial gland in the reproductive tract. Oviducts contained the highest GR activity. The GR activity of uterus during metestrus was about twice as high as that for other stages of the cycle. The levels of GR proteins in the tissues roughly matched the activities. The expression of the GR mRNA was highest during metestrus. Because GSH is known to increase gamete viability and the efficiency of fertility, GR, which is expressed in these tissues, is predicted to play a pivotal role in the reproduction process as a source of GSH.