Effects of oxygen at varied concentrations on in vitro maturation of human immature oocytes and early-stage development of embryos / 实用医学杂志

Objective To determine the effects of oxygen at varied concentrations on in vitro maturation (IVM)of oocytes,and subsequent fertilization,early-stage development of embryos by collecting the human imma-ture oocytes from assisted reproduction treatment. Methods Immature oocytes were randomly allocated to be cul-tured with oxygen at a lower or higher concentration. Intracytoplasmic sperm injection of mature oocytes after IVM , the rates of maturation,fertilization,embryo cleavage and high quality embryo were investigated. Results The GV oocytes cultured with oxygen at the lower concentration yielded higher maturation and fertilization rates than those at the higher concentration(P<0.05). There were no significant differences in the rates of embryo cleavage and high quality embryo between two groups(P > 0.05). For MI oocytes,the maturation rate of oocytes cultured with oxygen at the lower concentration was higher as compared to that at the higher concertration (P < 0.05). There were no significant differences in the rates of fertilization ,embryo cleavage and high quality embryo between the two groups(P > 0.05). Conclusions Oxygen at a lower concentration is beneficial to IVM of human imma-ture oocytes and it also improves the fertilization of GV oocytes after IVM. Oxygen at a lower concentration has no beneficial effect on the embryo cleavage rate and high quality embryo rate.

Effects of oxygen at varied concentrations on in vitro maturation of human immature oocytes and early-stage development of embryos / 实用医学杂志

Objective To determine the effects of oxygen at varied concentrations on in vitro maturation (IVM)of oocytes,and subsequent fertilization,early-stage development of embryos by collecting the human imma-ture oocytes from assisted reproduction treatment. Methods Immature oocytes were randomly allocated to be cul-tured with oxygen at a lower or higher concentration. Intracytoplasmic sperm injection of mature oocytes after IVM , the rates of maturation,fertilization,embryo cleavage and high quality embryo were investigated. Results The GV oocytes cultured with oxygen at the lower concentration yielded higher maturation and fertilization rates than those at the higher concentration(P<0.05). There were no significant differences in the rates of embryo cleavage and high quality embryo between two groups(P > 0.05). For MI oocytes,the maturation rate of oocytes cultured with oxygen at the lower concentration was higher as compared to that at the higher concertration (P < 0.05). There were no significant differences in the rates of fertilization ,embryo cleavage and high quality embryo between the two groups(P > 0.05). Conclusions Oxygen at a lower concentration is beneficial to IVM of human imma-ture oocytes and it also improves the fertilization of GV oocytes after IVM. Oxygen at a lower concentration has no beneficial effect on the embryo cleavage rate and high quality embryo rate.

Cryotop no desenvolvimento de oócitos bovinos imaturos vitrificados / Cryotop and development of vitrified immature bovine oocytes

The effectiveness of different cryodevices (open-pulled straw (OPS), electron microscopy grid (EMG), and Cryotop was evaluated for vitrification of immature bovine oocytes. Polar body, metaphase II stage (MII), survivability, and subsequent developmental rates were determined. Only oocytes with four or five layers of cumulus cells were used. Oocytes were equilibrated in two vitrification solutions - 1: 10 percent DMSO + 10 percent ethylene glycol (EG) for 30-45sec and 2: 20 percent DMSO + 20 percent EG +0.5M sucrose for 25sec -, mounted on one of the cryodevices and directly plunged into liquid nitrogen for 10 days. Immature vitrified oocytes using Cryotop showed the highest rates of polar body extrusion (PB) and nuclear maturity (MII); 41 and 58 percent respectively. Vitrified oocytes using OPS and EMG showed 26 and 32 percent; and 35 and 46 percent of PB and MII rates, respectively. The highest survivability resulted from Cryotop and EMG groups and no significant difference was found between them. Vitrified oocytes using Cryotop had the highest cleavage and blastocyst rates. All of the mean rates for vitrified immature oocytes were significantly lower than that of control group (P<0.05). The results of this study showed the superiority of Cryotop device for vitrification of immature bovine oocytes.

Avaliou-se a eficácia de diferentes dispositivos de congelamento (envasamento em palhetas (EP), microscopia eletrônica de grade (MEG) e Cryotop) para vitrificação de ovócitos imaturos de bovinos. Para tal, foram determinados o corpo polar, a metáfase II (MII), a viabilidade e as subsequentes taxas de desenvolvimento. Foram utilizados somente ovócitos com quatro ou cinco camadas de células do cumulus. Os ovócitos foram equilibrados em duas soluções de vitrificação - 1: DMSO (10 por cento) + etilenoglicol (EG; 10 por cento) por 30 a 45 segundos e 2: DMSO (20 por cento) + EG (20 por cento) + sacarose (0,5M) por 25 segundos -, transferidos para os dispositivos de congelamento e mantidos, por 10 dias, em nitrogênio líquido. Imediatamente após serem retirados do nitrogênio, os ovócitos foram removidos dos dispositivos e processados para maturação, fertilização e cultivo in vitro. Os ovócitos vitrificados com o Cryotop apresentaram as maiores taxas de extrusão do corpo polar (CP) e de maturidade nuclear (MII), 41 e 58 por cento, respectivamente. Para os ovócitos vitrificados com EP e MEG, as taxas de CP e as de MII foram, respectivamente, de 26 e 32 por cento e de 35 e 46 por cento. As taxas de viabilidade não diferiram entre os grupos Cryotop e EMG. Os ovócitos vitrificados com Cryotop apresentaram as maiores taxas de clivagem e de blastocisto. Para todas as variáveis estudadas, as taxas para os ovócitos vitrificados foram significativamente menores do que as do grupo-controle (P<0,05). Os resultados deste estudo mostraram a superioridade do dispositivo Cryotop para vitrificação de ovócitos imaturos de bovinos.

Expression of interferon regulatory factor-1 in the mouse cumulus-oocyte complex is negatively related with oocyte maturation / 대한생식의학회지

OBJECTIVE: We found previously that interferon regulatory factor (Irf)-1 is a germinal vesicle (GV)-selective gene that highly expressed in GV as compared to metaphase II oocytes. To our knowledge, the function of Irf-1 in oocytes has yet to be examined. The present study was conducted to determine the relationship between retinoic acid (RA) and RA-mediated expression of Irf-1 and the mouse oocyte maturation. METHODS: Immature cumulus-oocyte-complexes (COCs) were collected from 17-day-old female mice and cultured in vitro for 16 hours in the presence of varying concentrations of RA (0-10 microM). Rate of oocyte maturation and activation was measured. Gene expression was measured by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) and cytokine secretion in the medium was measured by Bio-Plex analysis. Apoptosis was analyzed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. RESULTS: The rates of oocyte maturation to metaphase II and oocyte activation increased significantly with RA treatment (10 nM-1 microM). With 100 nM RA treatment, lowest level of Irf-1 mRNA and cumulus cell's apoptosis was found. Among 23 cytokines measured by Bio-Plex system, the substantial changes in secretion of tumor necrosis factor-alpha, macrophage inflammatory protein-1beta, eotaxin and interleukin-12 (p40) from COCs in response to RA were detected. CONCLUSION: We concluded that the maturation of oocytes and Irf-1 expression are negatively correlated, and RA enhances the developmental competence of mouse immature oocytes in vitro by suppressing apoptosis of cumulus cells. Using a mouse model, results of the present study provide insights into improved culture conditions for in vitro oocyte maturation and relevant cytokine production and secretion in assisted reproductive technology.

Serum Anti-Mullerian Hormone and Inhibin B Levels at Ovulation Triggering Day Can Predict the Number of Immature Oocytes Retrieved in In Vitro Fertilization Cycles

The aim of this study was to investigate whether serum levels of anti-Mullerian hormone (AMH) and inhibin B at ovulation triggering day correlate with the number of immature oocytes obtained from stimulated in vitro fertilization (IVF) cycles. Fiftynine consecutive cycles of ovarian hyperstimulation and IVF were selected from 45 women who had tubal (n=18) or unexplained infertility (n=27) and obtained at least one oocyte. Serum levels of AMH and inhibin B at ovulation triggering day were measured by enzyme-linked immunosorbent assay (ELISA). Univariate analysis and multiple regressions revealed that serum AMH or inhibin B levels were significantly correlated with immature oocyte count and the correlation coefficients were higher compared to the mature oocyte count. Serum AMH and inhibin B levels on triggering day seems to be more closely related with the immature oocyte count and thus could be good predictors to determine the immature oocyte count in IVF cycle.

Comparison of two culture media on in vitro maturation and fertilization of immature oocytes obtained from stimulated cycles / 대한산부인과학회지

OBJECTIVE: The optimal culture conditions for human immature oocytes are still investigating. We compared retrospectively the efficacy of two culture media (pyruvate-dominant YS media vs glucose-dominant G2 media) on in vitro maturation and fertilization rate of immature oocytes obtained from stimulated IVF cycles. METHODS: One hundred forty-three immature oocytes (including GV and metaphase I) were obtained from 67 cycles of IVF-ET (52 patients). The mean age of female was 32.5+/-3.7 years. Ovarian hyperstimulation was performed using hMG or rFSH with GnRH antagonist and then ovulation was triggered by urinary or rhCG. Immature oocytes were cultured for 4-24 hrs and then fertilized by ICSI. We used YS media between July 2003 and September 2004, and G2 media between October 2004 and August 2005. Each media was prepared containing 30% of patients' follicular fluid, rFSH (1 IU/mL), rLH (1 IU/mL) and rEGF (10 ng/mL). RESULTS: The in vitro maturation rate of immature oocytes (67% vs 66%) and their fertilization rate (61% vs 61%) was not different between YS and G2 media. CONCLUSION: Immature oocytes seem to have a similar developmental competency in each of pyruvate dominant YS and glucose dominant G2 media containing human follicular fluid. Further investigations will be warranted to find the optimal media which can increase the developmental potential of immature oocytes.

Effects of Age on in vitro Maturation and Fertilization of Immature Oocytes from Stimulated Cycles in Human IVF-ET Program / 대한불임학회지

OBJECTIVE: To investigate the effects of female age on in vitro maturation and fertilization of immature oocytes from controlled ovarian hyperstimulation (COH) in human IVF-ET program. METHOD: A total of 96 immature oocytes (GV & metaphase I) obtained from 40 cycles of IVF-ET (29 patients). The mean age of female patients was 31.8+/-3.1 years. Ovulation was triggered by urinary or recombinant hCG. Immature oocytes were cultured with YS medium containing 30% of patients' human follicular fluids, LH (1 IU/mL), FSH (1 IU/mL) and EGF (10 ng/mL), and then matured oocytes were fertilized by ICSI. In vitro maturation and fertilization of immature oocytes were analyzed according to age of female ( or = 34 years). RESULTS: The maturation rate was similar between two groups (68% vs 64%). The fertilization rate of in?vitro-matured oocytes was higher in patients < 34 years old, but there was no statistical significance (64% vs 50%, p=0.347). The fertilization rate of in-vitro-matured oocytes was significantly lower compared with those of in-vivo-matured oocytes in both age groups (64% vs 79%, p=0.035, 50% vs 86%, p=0.007). CONCLUSION: In older female group, fertilization rate of in-vitro-matured oocytes seems to be decreased. Further investigations should be warranted to increase fertilization potential of in-vitro-matured oocytes.

Study on In Vitro Maturation and Culture of Immature Oocytes Collected from Ovaries of Infertile Women / 대한불임학회지

OBJECTIVE: This study was performed to examine the maturation and the development to the blastocyst stage of immature oocytes collected from patients with high risk of ovarian hyperstimulation syndrome (OHSS). MATERIALS AND METHODS: Cumulus-oocyte complexes (COCs) were collected following only HCGpriming for non stimulated IVF-ET cycles of the patients. At the time of oocyte collection, COCs were classified into three groups in accordance with their appearance (Group I: oocytes with dispersed cumulus cells; Group II: oocytes with compacted cumulus cells; Group III: oocytes with sparse cumulus cells). The in vitro maturation and blastocyst development rates of the COCs were compared among these groups. From August 2001 to June 2002, 48 IVM/IVF-ET cycles from 42 patients (mean age: 32.4+/-3.8 years) were performed. To prevent the occurrence of OHSS, the patients were primed with 10,000 IU HCG alone 36 h before oocyte collection without gonadotropin stimulation. Oocytes were aspirated on cycle days from 7 to 13. The normal COCs were classified into three groups according to their appearance. The aspirated immature oocytes were cultured in YS maturation medium containing 30% (v/v) human follicular fluid (HFF), 1 IU/ml FSH, 10 IU/ml HCG and 10 ng/ml rhEGF. Fertilization was induced by intracytoplasmic sperm injection (ICSI). All zygotes were co-cultured with cumulus cells in 10 mul YS medium containing 10% HFF until day 7 after oocyte collection. Blastocyst transfer was performed on day 5 after ICSI. RESULTS: The mean number of oocytes cultured in the IVM/IVF cycles was 24.7+/-10.6. Of 1185 COCs, those assigned to Group I, II and III were 470 (39.7%), 414 (35.0%) and 301 (25.4%), respectively. The maturation rate (94.5%, 444/470, p<0.05) in Group I was significantly higher than those of Group II (62.8%, 260/414) and Group III (73.1%, 220/301). Especially, 30.9% of COCs in Group I (145/470) was matured on the day of oocyte aspiration. There were no differences in the rates of fertilization and cleavage among the three groups. The development rate to the blastocyst stage in Group I (54.6%, 206/377, p<0.05) was also significantly higher than those in Group II (33.0%, 68/206) and Group III (30.1%, 52/173). Twenty-four clinical pregnancies (50.0%) was obtained and 22 pregnancies (45.8%) are ongoing. Implantation rate in the present study was 24.6%. CONCLUSION: These results suggest that there is a positive correlation between the appearance of COCs and the developmental competence of the immature oocytes in non stimulated IVM/IVF cycles.