Takakazu 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.

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.

Soluble CD44 in Human Ovarian Follicular Fluid

Purpose: In the present study, we investigated the existence of soluble CD44 (sCD44) in human follicular fluid, the relationship between the concentration of sCD44 and that of other hormonal parameters, and the prognostic value of sCD44 in follicular fluid in in vitro fertilization (IVF) programs.Methods: A total of 63 follicular fluid specimens from patients (n=30) participating in our IVF programs was analyzed by RIA and enzyme-linked immunosorbent assay (ELISA).Results: The mean concentration (±SE) of sCD44 in follicular fluid was 265.4 ± 7.8 ng/ml. The variation of the follicular fluid concentration of sCD44 was strictly associated with that of human chorionic gonadotropin (hCG) (r = 0.572, P < 0.0001). The mean concentration of sCD44 in follicular fluid was significantly higher in follicles containing subsequently unfertilized oocytes than that in those containing oocytes that had undergone fertilization (P = 0.0428). In the analysis of each follicle that contained an oocyte subsequently fertilized, the mean concentration of sCD44 was significantly higher in follicular fluid with the subsequently good-quality embryos than in that with the subsequently poor-quality embryos (P < 0.05).Conclusions: These results indicated that the concentration of sCD44 in follicular fluid reflects the development of embryos derived from the same follicle, so the sCD44 in human follicular fluid may be useful in the assessment of the prognostic value of IVF programs.