Tomoko Kaneko

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.

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.

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.

Application of Vitrification to Human Embryo Freezing

To solve the problem of multiple pregnancies during the in vitro fertilization (IVF) and embryo transfer procedure, excess embryos must be cryopreserved for embryo transfer in future. We applied the vitrification method to cryopreservation of human embryos. A total of 31 frozen-thawed embryo transfer cycles were analyzed at the Yamagata University Hospital, Yamagata, Japan. The patients were introduced to IVF treatment and had an excess of valuable embryos to be frozen after the transfer of three fresh embryos that did not result in establishing a pregnancy. Excess human 8- to 16-cell stage embryos were exposed to vitrification solution and then frozen in liquid nitrogen. The cryoprotectant was removed by washing the embryos in media containing different concentrations of cryoprotectant. Three days after LH surge and/or 2 days after ultrasonographic ovulation the embryos were transferred. The rate of poor quality embryos significantly increased and the rate of good quality embryos decreased after thawing the embryos frozen by the vitrification method. In menstrual cycles with good quality embryo transfer, a higher rate of pregnancies was established than in the cycles in which fair or poor quality embryos were the highest grade of embryos transferred into the uterus. In total, 5 pregnancies were established from 31 embryo tansfers; 4 pregnancies were in cycles associated with the transfer of good quality embryos, and 1 pregnancy was in a cycle in which the highest grade of embryo was fair. When compared with slow embryo freezing methods, vitrification has marked advantages for clinical application in terms of cost and time. Vitrification will be an alternative method for embryo freezing.

Hyaluronan in follicular fluids and fertilization of oocytes

OBJECTIVE To determine the concentrations of hyaluronan, E(2), and progesterone in follicular fluids (FFs) and the incidence of apoptotic granulosa cells. Also, to examine the relationship between the concentration of hyaluronan and follicular steroids, the incidence of apoptotic cells, and the fertilizability of the oocyte in the same follicle. DESIGN Samples of 130 follicles were retrospectively analyzed for hyaluronan and steroids and the incidence of apoptotic cells. SETTING The reproductive center in Yamagata University Hospital. PATIENT(S) Forty women infertile because of tubal damage or unknown causes undergoing IVF treatment were selected. INTERVENTION(S) The samples were collected from follicle aspirations. MAIN OUTCOME MEASUREMENT(S) The concentrations of hyaluronan and steroids in FFs, the incidence of apoptotic granulosa cells, and oocyte fertilizability. RESULT(S) The levels of hyaluronan in FF were found to correlate positively with P (r=0.444, P<0.0001) and the incidence of apoptotic cumulus granulosa cells (r=0.387, P=0.002) and inversely with E(2) (r = -0.601, P<0.0001) and free T (r = -0.344, P=0.001). The concentration of hyaluronan in FFs containing a subsequently fertilized oocyte after insemination was significantly lower than that in FFs containing a subsequently unfertilized oocyte (P=0.0005) (fertilized, 50.0 +/- 2.6 ng/mL; triploidy, 59.1 +/- 6.8; and unfertilized, 66.9 +/- 5.9). CONCLUSION(S) The concentration of hyaluronan in FF is an indicator for estimation of oocyte viability for fertilization.

Effects of Aging on Ovarian Fecundity in Terms of the Incidence of Apoptotic Granulosa Cells

AbstractPurpose: The objective was to determine the effects of womensage on the ovarian fecundity as assessed by the incidenceof apoptotic granulosa cells. Methods: Twenty-eight normo-ovulatory women underwentovulation induction for standard IVF. The husbands of thesewomen showed severe male infertility factors. The womenwere divided into four groups according to their ages. Womenunderwent follicle aspiration after the administration ofhuman menopausal gonadotropin plus human chorionicgonadotropin. The nuclei of granulosa cells were examinedby using fluorescence microscopy, and the incidence of apoptotic granulosa cells was tabulated. Results: Granulosa cells in the older women revealed asignificant increase in the number of apoptotic cells. Thenumber of total oocytes and the number of mature oocytesobtained significantly decreased with age. However, endometrial thickness and follicular estradiol, progesterone, andfree testosterone levels were not significantly different amongfour different age groups. Conclusions: Age increases apoptotic changes in granulosacells and consequently decreases the ovarian fecundity.

Effects of Controlled Ovarian Hyperstimulation on Oocyte Quality in Terms of the Incidence of Apoptotic Granulosa Cells

AbstractPurpose: The aim was to investigate which ovarian hyperstimulation protocol performed in the same patients causes development of oocytes of good quality. Methods: Twenty normo-ovulatory women underwent three different controlled ovarian hyperstimulation protocols for in vitro fertilization–embryo transfer. Patients underwent follicle aspiration after administration of human chorionic gonadotropin (hCG). The total number of retrieved oocytes, the number of mature oocytes, and the rate of mature oocytes were examined. Recovered granulosa cells were stained with Hoechst 33258 and examined by fluorescence microscopy to estimate the incidence of apoptotic cells. Results: The total number of oocytes and the number of mature oocytes in gonadotropin-releasing hormone agonist (GnRHa) + human menopausal gonadotropin (hMG) + hCG and hMG + hCG cycles were higher than those in the natural cycle (P < 0.0001). The rate of mature oocytes in hMG + hCG cycle was the highest among the three protocols (P < 0.04). In the mural granulosa cells, the incidence of apoptotic cells in the GnRHa + hMG + hCG cycle was significantly higher than those of the natural (P < 0.002) and hMG + hCG cycles (P = 0.0002). The incidence of apoptotic cumulus granulosa cells in the GnRHa + hMG + hCG cycle was significantly higher than those of natural and hMG + hCG cycles (P < 0.002). Moreover, the incidence of apoptotic cumulus granulosa cells in the hMG + hCG cycle was significantly lower than that in the natural cycle (P < 0.01). Conclusions: These results indicated that hMG + hCG is the most appropriate controlled ovarian hyperstimulation protocol among the three examined with regard to oocyte quality.

Relatively poor oocyte quality is an indication for intracytoplasmic sperm injection

OBJECTIVE To determine the relation between the insemination method used and the quality of oocytes and embryos. DESIGN Prospective study. SETTING Assisted reproductive centers at Yamagata University Hospital and Kuramoto Womens Clinic in Yamagata, Japan. PATIENT(S) Forty patients undergoing IVF and 40 patients undergoing intracytoplasmic sperm injection (ICSI). INTERVENTION(S) To estimate oocyte quality, the granulosa cells surrounding the oocyte were fixed and stained with a commercial dye in both groups of patients. One thousand granulosa cells were examined under a fluorescence microscope. MAIN OUTCOME MEASURE(S) The incidence of apoptotic granulosa cells surrounding each oocyte. RESULT(S) The incidence of apoptosis in the granulosa cells enclosing the oocytes that were fertilized by IVF was significantly lower than that in the oocytes that were fertilized by ICSI. Moreover, the incidence of apoptosis in the granulosa cells enclosing the oocytes that grew into good-quality or fair-quality embryos was significantly lower after conventional IVF than after ICSI. With ICSI, the incidence of apoptosis was not significantly different among the granulosa cells surrounding the oocytes that were inseminated, were fertilized, or developed into good-quality or fair-quality embryos. With IVF, the incidence of apoptosis was highest in the granulosa cells surrounding the oocytes that were inseminated and lowest in the granulosa cells surrounding the oocytes that developed into good-quality and fair-quality embryos. CONCLUSION(S) A good-quality oocyte is necessary for the development of a good-quality embryo with IVF but not with ICSI. Thus, relatively poor oocyte quality is a good indication for the use of ICSI.