The effect of melatonin on in vitro maturation fertilization and early embryo development of mouse oocytes and expression of HMGB1 gene in blastocysts

Antioxidants are commonly used for maturation, fertilization and early development of embryos. Melatonin as an antioxidant have been recently proven to be useful for the assisted reproductive technology. In the present study, we evaluated the roles of melatonin in the in vitro maturation, fertilization, development and also the gene expression of high mobility group box-1 (HMGB1) in the blastocysts. The immature oocytes of BDF1 mice were transferred to the media containing different doses of melatonin (10-6, 10-9, 10-12 M). The blastocysts that developed under in vitro fertilization from each group were stained to determine the cell number of embryos and analyzed to determine the expression level of HMGB1 by real-time PCR. The most effective doses of melatonin for maturation of oocytes were 10-6 and 10-12M (P<0.05). Fertilization rate, early development and the cell number of blastocysts were significantly higher in the group that treated with 10-12 M of melatonin comparing to the other groups. The HMGB1 expression decreased in groups that treated with 10-6M and 10-9M of melatonin and increased in the group that treated with 10-12 M of melatonin, but did not show a significant difference (p˃0.05). From the results, it may be concluded that the melatonin could be effective when the embryos undergo maturation, fertilization and early developmental processes. The HMGB1 expression, as a marker of early development in mice embryos, increased in the groups that treated with low doses of melatonin

Avaliação da técnica de vitrificação de ovários de camundongos da linhagem B6D2F1, pertencentes ao Instituto de Ciência e Tecnologia em Biomodelos - ICTB/Fiocruz-RJ / Evaluation of ovary vitrification technique from B6D2F1 strain belonging to the Institute of Biomodels Science and Technology ICTB / Fiocruz-RJ

Objetivou-se testar a vitrificação de ovários de camundongos do ICTB/Fiocruz. Inicialmente, fez-se coleta e maturação in vitro dos oócitos de ovários a fresco e vitrificados, bem como avaliação de estruturas no cultivo embrionário, pós-fertilização in vitro. Fêmeas B6D2F1 foram eutanasiadas para remoção dos ovários (n=60) e divididas em três grupos: grupo 1 (n=30 animais) - oócito de ovários vitrificados, maturados e fertilizados in vitro (120 fragmentos); grupo 2 (n=15) (controle 1) - oócitos coletados a fresco, maturados e fertilizados in vitro; e grupo 3 (n=15) (controle 2) - oócitos maturados in vivo e fertilizados in vitro. A técnica foi verificada no desenvolvimento embrionário in vitro, que foi avaliado pelo teste de qui-quadrado (BioStat 5.0). Recuperaram-se 123, 224 e 328 oócitos nos G1, G2 e G3, respectivamente. Observaram-se diferenças significativas nas taxas de clivagem às 24 horas (embriões ≥ 2 células) entre G1 (8%) e G2 (32%) (P<0,1) e G1 e G3 (49%) (P<0,05), mas não entre G2 e G3 (P>0,05). Para blastocistos, às 96 horas, os grupos G1, G2 e G3 apresentaram, respectivamente, 6%, 11% e 46%, diferindo significativamente entre eles (P<0,05). A vitrificação de ovários, a maturação oocitária e a fertilização in vitro são alternativas para a produção de embriões de camundongos in vitro.(AU)

This work aimed test ovarian vitrification of hybrid mouse from ICTB/Fiocruz. Protocol collection and oocyte in vitro maturation from fresh and vitrified ovaries was established and embryos were evaluated after fertilization. B6D2F1 females were euthanized for ovarian removal (n= 60) and divided into 3 groups: G1 (n= 30) - ovaries fragmented (n= 120), vitrified, matured and fertilized; G2 (n= 15) - in vitro fertilization of oocytes matured in vitro from fresh ovaries; G3 (n= 15) - ampulla region oocytes in vitro fertilizated. Viability was verified by thawing, oocyte in vitro maturation and fertilization. In vitro embryo development of each group was evaluated by Chi-square test (BioStat 5.0). 123, 224 and 328 oocytes were recovered from G1, G2 and G3, respectively. Significant differences were observed in cleavage rates at 24 hours (embryos with 2 cells or more) between G1 (8%) and G2 (32%) (P< 0.1) and G1 and G3 (49%) (P< 0.05) but not between G2 and G3 (P> 0.05). Blastocysts at 96 hours presented 6%, 11% and 46%, respectively for G1, G2 and G3, differing significantly (P< 0.05). Ovary vitrification, oocyte in vitro maturation and in vitro fertilization were available for the production of in vitro mouse embryos.(AU)

Various macromolecules in in vitro growth medium influence growth, maturation, and parthenogenetic development of pig oocytes derived from small antral follicles / 대한수의학회지

This study was performed to examine the effects of various macromolecules in in vitro growth (IVG) media on the growth, maturation, and parthenogenesis (PA) of pig oocytes derived from small antral follicles (SAF). Immature oocytes were cultured for two days in IVG medium supplemented with 10% (v/v) fetal bovine serum (FBS), 10% (v/v) pig follicular fluid (PFF), 0.4% (w/v) bovine serum albumin (BSA), or 0.1% (w/v) polyvinyl alcohol (PVA) and then maintained for 44 h for maturation. After IVG, the mean diameters of the SAF treated with FBS, PVA, and no IVG-MAF (113.0–114.8 µm) were significantly larger than that of no IVG-SAF (111.8 µm). The proportion of metaphase II oocytes was higher in PFF (73.6%) than in BSA (43.5%) and PVA (53.7%) but similar to that in the FBS treatment (61.5%). FBS and PFF increased cumulus expansion significantly compared to PVA and BSA while the intraoocyte glutathione content was not influenced by the macromolecules. Blastocyst formation of PA oocytes treated with FBS (51.8%), PFF (50.4%), and PVA (45.2%) was significantly higher than that of the BSA-treated oocytes (20.6%). These results show that the PFF and FBS treatments during IVG improved the growth, maturation, and embryonic development of SAF.

Modulatory Effects of Single and Complex Vitamins on the In Vitro Growth of Murine Ovarian Follicles

BACKGROUND: Vitamin is a well-known co-factor for many metabolic processes and its roles in fertility and follicular growth have been studied. Vitamin supplementation is frequently achieved by daily ingestion in the form of a complex capsule. However, the role of single and complex vitamins in in vitro maturation of murine follicles is not fully elucidated. METHODS: In this study, we evaluated the effects of two forms of vitamins. Pure L-ascorbic acid, and multi-vitamin (vitamin C+vitamin B complex) was treated at two different concentrations (50 and 100 µg/ml), to pre-puberty murine follicles during in vitro maturation. To determine the specific stage of growth that is affected by treatment with vitamins, the vitamins were treated from day 0, 4, 9, and 13. Growth of each follicle was assessed by measuring diameters of whole expanded area and of the granulosa cells. Expression of follicular and oocyte growth-related genes and the effect of vitamin on the viability of follicles was assessed using senescence associated β-galactosidase staining. RESULTS: Treatment with vitamins promoted the in vitro growth of murine follicles and the upregulated the expression of granulosa cell- and oocyte-specific genes such as BMP15, Fsh receptor, and GDF9. The proliferation of the granulosa cells was enhanced by the treatment of vitamin. Fifty µg/ml concentration vitamin showed greater effects compared to higher concentration. The viability of in vitro grown follicles was also significantly improved in vitamin-treated follicles. The effects of single L-ascorbic acid and complex vitamin were not significantly different to those of day 4 and day 9 follicles. Vitamins promoted murine follicle development in vitro with different effects on specific growth stage. CONCLUSION: Supplementation of vitamins during in vitro maturation of murine follicles is an efficient strategy for in vitro expansion of follicular cells. These results could be customized to the sophisticated culture of follicles retrieved from aged or cancer-survived female that contain smaller number of follicles with reduced potential to develop into mature follicles.

Growth differentiation factor 9 and cumulus cell supplementation in in vitro maturation culture media enhances the viability of human blastocysts / 대한생식의학회지

OBJECTIVE: In vitro maturation (IVM) of immature oocytes can be useful for some infertile patients. In IVM programs, the rates of embryo formation and pregnancy are low. Therefore, it is essential to recognize the main factors involved in regulating oocyte maturation in vitro. The purpose of this study was to investigate the effects of growth differentiation factor 9 (GDF9) and cumulus cell (CC) supplementation in IVM medium on the rates of embryo formation and viability of human blastocysts.METHODS: A total of 80 germinal vesicle oocytes from stimulated cycles underwent an IVM program. The oocytes were divided into four groups, where group I consisted of IVM media only and served as the control, group II consisted of IVM+CCs, group III consisted of IVM+GDF9 (200 ng/mL), and group IV consisted of IVM+CCs+GDF9 (200 ng/mL). Intracytoplasmic sperm injection was performed on the IVM oocytes, and the cleavage embryos that were generated were vitrified. Following thawing, the embryos were cultured for 3 additional days, and the viability rates of the developed blastocysts were determined.RESULTS: The maturation rate of the oocytes did not differ significantly across the four groups. The fertilization rate in group II was significantly higher than that in the control group (76.5% vs. 46.2%). Embryo formation was significantly more frequent in all experimental groups than in the control group, while blastocyst formation did not show significant differences in the three experimental groups compared to the control. The mean viability rates in groups II, III, and IV were 58.16%, 55.91%, and 55.95%, respectively, versus 37.78% in the control group (p<0.05).CONCLUSION: Supplementation of IVM culture media with GDF9 and CCs enhanced the fertilization, embryo formation, and viability rates of blastocysts generated from vitrified cleavage embryos.

Ovarian Stimulation and In Vitro Maturation for Fertility Preservation in Female Cancer Patients / 中山大学学报(医学科学版)

@#【Objective】To determine the effect of ovarian stimulation or in vitro maturation for fertility preservation in female cancer patients. 【Methods】 A retrospective study was conducted in 27 females who underwent fertility preservation procedures in our center.【Results】Female patients were included in this study with an average age of 27.1. Patients spent on average for 6.8 d to retrieve oocytes since their attendance day. Total amount of Gn was on average 910 U per patient and for patients with breast cancer，the average estrogen level on trigger day reached 360 pg/mL. The maturation rate of oocytes from ovarian stimulation cycles was 82.6% ，which of that in emergency in- vitro maturation cycles was 38.1%.【Conclusion】The development capability of oocytes from cancer patients are comparable with those of other infertility patients. Peak estradiol levels were controlled by the administration of letrozole. In vitro maturation of oocytes performed at random time of the menstrual cycle may result in a lower maturation rate ，which is associated with the time limit of the follow- up cancer treatment. In conclusion，clinicians should consider a more holistic approach for female cancer patients，which focuses not only on the characteristic of the primary cancer but also on the phase of the menstrual cycle at their attendance day.

Assessment of mouse oocytes ultrastructure following vitrification before and after in vitro maturation / Evaluación de la ultraestructura de los ovocitos de ratón después de la vitrificación antes y después de la maduración in vitro

SUMMARY: Vitrification is a physical process in which the concentrated cryoprotectant solution after exposure to extreme cold without ice crystal formation in living cells to be converted glassing state. In this study, maturation rate and ultrastructure of mouse oocytes followed by vitrification before or after in-virto maturation (IVM) were evaluated. A total of 373 germinal vesicle oocytes were obtained from ovaries and divided into three fresh IVM, IVM vitrified, vitrified IVM groups. Ten metaphase II oocytes were obtained from uterine tubes and considered as the control group. Oocytes in vitrified groups were vitrified by Cryotop using vitrification medium and kept in liquid nitrogen. The maturation media was a-MEM supplemented with rFSH + hCG. After 24-48 h of incubation, the oocytes were investigated for nuclear maturation and ultrastructural changes using transmission electron microscopy (TEM). The oocyte maturation rate in vIVM group was significantly lower than IVMv group, when the two groups were compared with vIVM had the highest maturity. The evaluation ultrastructure of the four groups showed that the number of cortical granules, microvilli and mitochondria-SER aggregates in vIVM group were lowest and the highest amongst the number of vacuoles. Zona pellucida was darker than the control group in two freeze groups vIVM and IVMv. Most similar groups to the control group were group vIVM, Group IVMv and ultimately vIVM group, respectively. According to the results, IVM procedure is more efficient when it is performed before oocyte vitrification.

RESUMEN: La vitrificación es un proceso físico en el que la solución concentrada de crioprotectores, después de la exposición al frío extremo sin formación de cristales de hielo en las células vivas, se convierte en estado de cristal. En este estudio, se evaluaron la velocidad de maduración y la ultraestructura de los ovocitos de ratón seguidos por la vitrificación antes o después de la maduración in vitro (IVM). Se obtuvieron un total de 373 ovocitos, de vesículas germinales de ovarios, y se dividieron en tres grupos de IVM vitrificados, IVM e IVM frescos. Diez ovocitos metafase II se obtuvieron a partir de tubas uterinas y se consideraron como el grupo de control. Los ovocitos en grupos vitrificados fueron vitrificados por Cryotop usando medio de vitrificación y mantenidos en nitrógeno líquido. El medio de maduración fue a-MEM suplementado con rFSH + hCG. Después de 24-48 h de incubación, fueron observados en los ovocitos la maduración nuclear y cambios ultraestructurales utilizando microscopía electrónica de transmisión (MET). La tasa de maduración de los ovocitos en el grupo vIVM fue significativamente más baja que en el grupo IVM v, cuando los dos grupos se compararon con los que tenían la mayor madurez. La evaluación de la ultraestructura de los cuatro grupos mostró que el número de gránulos corticales, microvellosidades y acúmulos de mitocondrias-SER en el grupo vIVM fue el más bajo y el más alto entre el número de vacuolas. La zona pelúcida fue más oscura en dos grupos de congelación vIVM e IVMv, que en el grupo control. La mayoría de los grupos, similares al grupo de control, fueron los grupos vIVM, IVMv y,finalmente, el grupo vIVM, respectivamente. De acuerdo con los resultados, el procedimiento de IVM es más eficiente cuando se realiza antes de la vitrificación de ovocitos.

Developmental competence of in vitro-matured human oocytes obtained from pregnant and non-pregnant women / 대한생식의학회지

OBJECTIVE: The aim of this study was to compare the rate of maturation, fertilization, and embryo development of in vitro-matured human oocytes derived from pregnant and non-pregnant women. METHODS: Immature oocytes were obtained by needle aspiration from 49 pregnant women (group 1) who underwent a cesarean section at term and 77 non-pregnant women (group 2) who underwent a gynecological operation during the same period (8 months). Healthy immature oocytes (530 in group 1 and 539 in group 2) were cultured and assessed for maturation 36 hours later. Mature oocytes were inseminated by intracytoplasmic sperm injection and cultured up to 144 hours. RESULTS: The percentage of degenerated oocytes was significantly higher (12.1% vs. 6.3%; p < 0.001) in group 1 than in group 2. There was no significant difference in the maturation rate (66.8% vs. 68.1%; p=0.698), fertilization rate (66.7% vs. 67.6%; p=0.857), or the rate of formation of good-quality blastocysts (46.2% vs. 47.2%; p=0.898) in oocytes obtained from pregnant and non-pregnant women. CONCLUSION: The developmental competence of immature oocytes did not differ between pregnant and non-pregnant women.

Effect of seasonality on the quality of bovine oocytes selected by the brilliant cresyl blue method / Efecto de la estacionalidad en la calidad de los ovocitos seleccionados por el método de azul cresil brillante / Efeito da sazonalidade na qualidade dos ovócitos bovinos selecionados pelo método azul cresil brilhante

Abstract Background: The brilliant cresyl blue (BCB) staining is a non-invasive test to select the best-suited oocytes for embryonic development. This makes it a useful tool to select best-quality oocytes at the times of the year when there is forage restriction. Objective: To evaluate the effect of seasonality on the nuclear maturation and quality of oocytes selected by the BCB test. Methods: The cumulus-oophorus complexes (COCs) were obtained in summer and winter of 2010 and 2011. Selected COCs were maintained for 90 min at 38.5 °C in a CO2 incubator, in TCM 199 medium containing 10% fetal bovine serum and antibiotics, and supplemented with 26 µM brilliant cresyl blue. Afterwards, they were divided according to the ooplasm staining (BCB+ -blue; BCB− -unstained). Subsequently, COCs were matured for 22 h. Nuclear maturation was evaluated at 22 h of culture. Results: The proportion of BCB− oocytes was higher in the winter of 2010, but there was no increase in this group in the winter of 2011. The percentage of oocytes that reached metaphase II was higher in control and BCB+ groups in relation to oocytes from BCB− group. Conclusion: The season of the year influences the percentage of oocytes best suited for embryonic production in situations in which oocyte donors receive pasture-based feeding, since the method was effective in determining the effect of seasonality on the competence of bovine oocytes to reach nuclear maturation.

Resumen Antecedentes: La tinción con azul cresil brillante (BCB) es un método no invasivo para seleccionar ovocitos aptos para el desarrollo embrionario. Por tanto, es una herramienta útil para selecionar los ovocitos de mejor calidad en temporadas de restricción de forraje. Objetivo: Evaluar el efecto de la estacionalidad sobre la maduración nuclear y calidad de los ovocitos seleccionados por el test BCB. Métodos: Los complejos cumulus-oophorus (CCOs) fueron obtenidos durante el verano y el invierno de 2010 y 2011. Los CCOs seleccionados se mantuvieron durante 90 min a 38,5 oC en una incubadora de CO2 en un medio TCM 199 con 10% de suero fetal bovino y antibióticos, suplementado con 26 µM de azul cresil brillante. Luego se separaron según el color del citoplasma (BCB+ -azul y BCB− -incoloro). Posteriormente, los CCOs se maduraron durante 22 h. La evaluación de la maduración nuclear se realizó a las 22 h de cultivo. Resultados: La proporción de ovocitos BCB− fue mayor en el invierno de 2010, pero no hubo un aumento de ese grupo en el invierno de 2011. El porcentaje de ovocitos que alcanzaron la etapa de metafase II fue mayor en el grupo control y BCB+ con respecto al grupo BCB−. Conclusión: La estación del año influye en el porcentaje de ovocitos más aptos para la producción de embriones en situaciones donde las donadoras de ovocitos reciben alimentación a base de pastos, ya que este método fue eficaz para determinar el efecto de la estacionalidad en la competencia de ovocitos bovinos en alcanzar la maduración nuclear.

Resumo Antecedentes: O método do azul cresil brilhante (BCB) não é invasivo e seleciona ovócitos mais aptos ao desenvolvimento embrionário. Portanto é ferramenta útil para selecionar ovócitos de melhor qualidade em épocas do ano que ocorre restrição de pastagem. Objetivo: Avaliar o efeito da sazonalidade sobre a maturação nuclear e a qualidade dos ovócitos selecionados pelo teste BCB. Métodos: Os complexos cumulus oophorus (CCOs) foram obtidos no verão e inverno de 2010 e 2011. Os CCOs selecionados foram mantidos por 90 min, a 38,5 °C, em incubadora de CO2, em meio TCM 199 contendo 10% de soro fetal bovino e antibióticos, e suplementado com 26 µM de azul cresil brilhante. Em seguida, estes foram divididos de acordo com a coloração do citoplasma (BCB+ -azuis e BCB− -incolores). Então os CCOs foram maturados durante 22 h. A avaliação da maturação nuclear foi realizada às 22 h de cultivo. Resultados: A proporção dos ovócitos BCB− foi maior no inverno de 2010, mas não houve aumento desse grupo no inverno de 2011. O percentual de ovócitos que atingiu o estágio de metáfase II foi maior no controle e no grupo BCB+ em relação ao grupo BCB−. Conclusão: A estação do ano influencia o percentual de ovócitos mais aptos a produção de embriões, em situações onde as doadoras de ovócitos recebem alimentação baseada em pastagens, já que o método se mostrou efetivo para determinação do efeito da sazonalidade sobre a competência de ovócitos bovinos em atingirem a maturação nuclear.

Research progress of in vitro maturation of Beagle dog oocytes / 中国实验动物学报

Sufficient embryos are needed for the preservation of Beagle dogs germplasm resources and the prepara-tion of gene?modified human disease animal models. Up to now, the induced ovulation technique has no effect on dogs,it is hard to obtain mature oocytes in vivo, although the scientists try a lot in many aspects, but still could not make a break?through. The in vitro maturation rate is too low to support the preservation of germplasm resources, application in gene?modified disease models and biomedical research. Aiming to provide useful information on breakthrough in dog oocytes mat?uration, this review will summarize the effect of different age and reproductive stage,different morphology and size of the oo?cytes and lipid droplet on the in vitro maturation of dog oocytes.

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.

The effects of different types of media on in vitro maturation outcomes of human germinal vesicle oocytes retrieved in intracytoplasmic sperm injection cycles / 대한생식의학회지

OBJECTIVE: Optimizing in vitro maturation (IVM) media to achieve better outcomes has been a matter of interest in recent years. The aim of this prospective clinical trial was to investigate the effects of different media on the IVM outcomes of immature oocytes at the germinal vesicle (GV) stage. METHODS: A total of 400 immature oocytes at the GV stage with normal morphology were retrieved from 320 infertile women aged 31±4.63 years during stimulated intracytoplasmic sperm injection (ICSI) cycles. They were divided into groups of homemade IVM medium (I, n=100), cleavage medium (II, n=100), blastocyst medium (III, n=100), and Sage IVM medium (IV, n=100) and cultured for 24 to 48 hours at 37℃. ICSI was performed, and the rates of fertilization and embryo formation were compared across the four groups. RESULTS: In the 400 retrieved GV oocytes, the total maturation rates showed significant differences in groups I to IV (55%, 53%, 78%, and 68%, respectively, p<0.001). However, there were no significant differences in the fertilization, embryo formation, or arrest rates of metaphase II oocytes across these groups. In all groups, GV maturation was mostly completed after 24 hours, with fewer oocytes requiring 48 hours to mature (p<0.01). Moreover, the rate of high-quality embryos was higher in group IV than in the other groups (p=0.01). CONCLUSION: The quality of the IVM medium was found to affect clinical IVM outcomes. Additionally, blastocyst medium may be a good choice in IVM/ICSI cycles as an alternative IVM medium.

Vitrificación de ovocitos bovinos parcialmente madurados de la raza frisón rojo chileno / Vitrification of partially matured bovine oocytes of the Chilean red friesian breed

Se evaluó la tasa de maduración in vitro post descongelación de ovocitos bovinos, de la raza Frisón Rojo Chileno, parcialmente madurados y vitrificados. Complejos Cúmulus-ovocito fueron obtenidos por aspiración folicular, clasificados morfológicamente y aleatoriamente cultivados in vitro en TCM199 (10 % Suero Fetal Bovino (SFB), 50 mg/mL gentamicina, 0,2 mM piruvato de sodio, 0,08 µg/mL FSH, 1 µg/mL LH y 1 µg/mL estradiol) en los grupos: a) control (n= 137), madurados por 24 h a 38,5 C, 5 % CO2 y 99 % humedad y, b) tratamiento (n= 156), madurados por 6 h, parcialmente denudados, e incubados hasta completar 20 h, para luego ser vitrificados por el método Open Pulled Straws (OPS). Los ovocitos fueron expuestos a la solución de vitrificación uno (SV1) (Buffer Fosfato Salino (PBS), 10 % Etilenglicol (EG), 10% DMSO) por 30 s, posteriormente traspasados a la SV2 (PBS, 20 % EG, 20 % DMSO) por 25 s. Inmediatamente los ovocitos fueron cargados en pajuelas francesas estiradas (OPS) y sumergidos en nitrógeno líquido. Las ovocitos fueron descongelados introduciéndolos en una secuencia de soluciones con concentraciones decrecientes de sucrosa (0,3; 0,15 y 0 M respectivamente). Finalmente, los ovocitos continuaron con la maduración por 4 h adicionales. Posterior al periodo de maduración, los ovocitos de ambos grupos fueron fijados, teñidos y evaluados. Las proporciones de ovocitos en Metafase I (MI), Metafase II (MII) y degenerados fueron comparadas mediante el test de Chi cuadrado. La vitrificación aumentó (p 0,05) el porcentaje de pérdida y de ovocitos dañados en comparación al control. Además, aumentó (p 0,05) la tasa de ovocitos en MI y el número de ovocitos degenerados, y redujo el porcentaje de ovocitos MII, en comparación al control. Por tanto, la vitrificación por el método Open Pulled Straw de ovocitos parcialmente madurados in vitro es una alternativa viable para la conservación de material genético de hembras Frisón Rojo Chileno.

Post thawing in vitro maturation rate was evaluated for partially matured vitrified oocytes from Chilean Red Friesian cattle. Cumulus-Oocytes Complexes were obtained by follicular aspiration, classified by morphology and randomly in vitro matured in TCM199 (10 % Bovine Fetal Serum (BFS), 50 mg/mL gentamicine, 0.2 mM sodium piruvate, 0.08 µg/ml FSH, 1 µg/mL LH and 1 µg/mL estradiol) in the following groups: a) control (n= 137), matured for 24 h at 38.5 C, 5 % CO2 y 99 % humidity, and b) treatment (n= 156), matured for 6 h, partially denuded, and incubated until completion of 20 h. Then, oocytes were vitrified by the Open Pulled Straws (OPS) method. Oocytes were exposed to vitrification solution one (VS1) (Phosphate Buffered Saline (PBS), 10 % Ethylen glycol (EG), 10 % DMSO) for 30 s, then they were exposed to VS2 (PBS, 20% EG, 20% DMSO) for 25 s. Afterwards, oocytes were loaded into open pulled straws and submerged into liquid nitrogen. Oocytes were thawed by exposure to sequential solutions with decreasing concentrations of sucrose (0.3; 0.15 y 0 M respectively). Finally, oocytes continued the in vitro maturation for 4 additional hours. After completion of maturation period oocytes from both groups were fixated, stained and evaluated. The proportion of lost and damaged, MI, MII, and degenerate oocytes were compared between groups by Chi square test. Vitrification procedure increased (p 0.05) the percentage of oocytes lost and damaged when compared to control group. Additionally, vitrification increased (p 0.05) the proportion of MI and degenerated oocytes, and decreased the proportion of MII oocytes. Therefore, vitrification by the OPS method of partially matured bovine oocytes is a reliable alternative for the conservation of germinal cells from Chilean Red Friesian females.

Application of serum anti-Müllerian hormone levels in selecting patients with polycystic ovary syndrome for in vitro maturation treatment / 대한생식의학회지

OBJECTIVE: The purpose of this study was to identify useful clinical factors for the identification of patients with polycystic ovary syndrome (PCOS) who would benefit from in vitro maturation (IVM) treatment without exhibiting compromised pregnancy outcomes. METHODS: A retrospective cohort study was performed of 186 consecutive patients with PCOS who underwent human chorionic gonadotropin-primed IVM treatment between March 2010 and March 2014. Only the first IVM cycle of each patient was included in this study. A retrospective case-control study was subsequently conducted to compare pregnancy outcomes between IVM and conventional in vitro fertilization (IVF) cycles. RESULTS: Through logistic regression analyses, we arrived at the novel finding that serum anti-Müllerian hormone (AMH) levels and the number of fertilized oocytes in IVM were independent predictive factors for live birth with unstandardized coefficients of 0.078 (95% confidence interval [CI], 1.005-1.164; p=0.037) and 0.113 (95% CI, 1.038-1.208; p=0.003), respectively. Furthermore, these two parameters were able to discriminate patients who experienced live births from non-pregnant IVM patients using cut-off levels of 8.5 ng/mL and five fertilized oocytes, respectively. A subsequent retrospective case-control study of patients with PCOS who had serum AMH levels ≥8.5 ng/mL showed that IVM had pregnancy outcomes comparable to conventional IVF, and that no cases of ovarian hyperstimulation syndrome were observed. CONCLUSION: Serum AMH levels are a useful factor for predicting pregnancy outcomes in PCOS patients before the beginning of an IVM cycle. IVM may be an alternative to conventional IVF for PCOS patients if the patients are properly selected according to predictive factors such as serum AMH levels.

Effect of transport temperature of ovaries on in vitro maturation of canine oocytes collected in different stages of the estrous cycle / Influência da temperatura de transporte de ovários na maturação in vitro de oócitos caninos coletados em diferentes estágios do ciclo estral

This study evaluated the influence of estrous cycle stage and transport temperature of ovaries on in vitro maturation of canine oocytes. The bitches were categorized into two groups based on stage of estrus cycle: diestrus or anestrus. One ovary of each pair collected was transported in saline solution at 4°C while the other was transported at 37°C. Thus, ovarian tissue was sliced in PBS to release cumulus oocyte complexes (COCs). A total of 345 COCs (n = 186 oocytes from ovaries of bitches in anestrus and 159 in diestrus) were cultivated in TCM 199 supplemented with HEPES, sodium pyruvate, cysteine, follicle stimulating hormone (FSH), human chorionic gonadotropin (hCG), estrogen (E2) and epidermal growth factor (EGF). After 72h of maturation, the COCs were denuded, fixed and stained to assess nuclear maturation. The Fisher test was applied to examine the differences between the groups. The significance level adopted was 0.05. The oocytes obtained from the ovaries from bitches in diestrus transported at 4°C shown increased frequency of oocytes in metaphase II stage than those maintained at 37°C (p 0.01). Similarly, there was increased frequency of oocytes in metaphase II (11.1%) stage from the ovaries of bitches in anestrus and transported at 4°C, than those maintained at 37°C (p 0.05). It was concluded that transport temperature influences the results of canine oocyte viability and in vitro maturation, regardless of reproductive stage of the female.

Foi avaliada a influencia do ciclo estral e temperatura de transporte de ovários na maturação in vitro de oócitos caninos. As cadelas foram categorizadas em dois grupos baseados no estagio do ciclo estral anestro ou diestro. Um ovário por par coletado foi transportado em solução fisiológica 0,9% a 4°C enquanto o outro foi transportado a 37°C. Então, os ovários foram seccionados em PBS para a liberação dos complexos cumulus oocito (COCs). Um total de 345 COCs (n = 186 oocitos obtidos de cadelas em anestro e 159 em diestro) foi cultivado em TCM 199 suplementado com HEPES, piruvato de sódio, cisteina, hormônio folículo estimulante (FSH), gonadotrofina coriônica humana (hCG), estrógeno (E2) e fator de crescimento epidermal (EGF). Apos 72h de maturação, os COCs foram desnudados, fixados e corados para avaliação da maturação nuclear. O teste de Fisher foi utilizado para avaliar as diferenças entre os grupos. O nível de significância adotado foi de 0,05. Os oócitos obtidos de cadelas em diestro transportados a 4°C apresentaram maior frequência de oócitos no estagio de metáfase II (21,1%) que os mantidos na temperatura de 37°C (p 0,01). De forma similar, houve maior frequência de oócitos nos estágios de metáfase II (11,2%) nos ovários obtidos de cadelas em anestro e transportados a 4°C que nos ovários mantidos a 37°C (p 0,05). Concluiu-se que a temperatura de transporte influencia os resultados de viabilidade oocitária canina e a maturação in vitro, independentemente do estagio reprodutivo da fêmea.

Pregnancy and fertilization potential of immature oocytes retrieved in intracytoplasmic sperm injection cycles / 대한생식의학회지

OBJECTIVE: The goal of this study was to evaluate the pregnancy potential of immature (metaphase I or germinal vesicle stage) oocytes retrieved in intracytoplasmic sperm injection (ICSI) cycles. METHODS: A total of 1,871 couples with infertility underwent 2,984 ICSI cycles. Cycles in which three or fewer oocytes were retrieved were included in this study in order to evaluate the pregnancy potential of immature oocytes. Cycles were divided into five groups (group I-V), according to the maturation status of the oocytes at the time of cumulus cell removal and ICSI. The fertilization and pregnancy rates after ICSI were analyzed and compared among the study groups based on the maturation status of the retrieved oocytes. RESULTS: The retrieval of only immature oocytes was associated with a significant decrease in the fertilization rate (76.1%+/-37.3% vs. 49.0%+/-49.1%, 66.7%+/-48.7%; group I vs. group II, group III, respectively) and the average number of transferred embryos (1.5+/-0.7 vs. 1.1+/-0.4, 1.1+/-0.6). The cycle cancellation rate was significantly higher when only immature oocytes were retrieved. The clinical pregnancy rate decreased significantly when the transferred embryos had originated from immature oocytes (16.9% vs. 10.3%, 1.2%). CONCLUSION: In ICSI cycles, the fertilization potential and pregnancy potential of the immature oocytes retrieved in ICSI cycles were inferior to those of mature oocytes. Therefore, increasing the number of injectable oocytes and transferrable embryos by using immature oocytes after their spontaneous in vitro maturation does not necessarily improve pregnancy outcomes.

Efeitos da redução ou substituição do soro fetal bovino por outros compostos na maturação in vitro de oócitos bovinos / Effects of reduction or replacement of fetal calf serum by other compounds during in vitro maturation of bovine oocytes

A utilização do soro fetal bovino (SFB), embora bastante disseminada na produção in vitro (PIV) de embriões bovinos, apresenta limitações por ser um meio indefinido e por causar efeitos que prejudicam a qualidade desses embriões. Por esse motivo, nos últimos anos, grande parte das pesquisas relacionadas à PIV está voltada para a substituição do SFB por outros compostos nos meios de cultura. No presente estudo, foram utilizados como compostos protéicos a albumina sérica bovina livre de ácidos graxos (BSA-FAF) e um produto comercial denominado fluido embriônico (FE) de maneira isolada ou em diferentes combinações e concentrações, com objetivo de substituir ou diminuir a concentração do SFB durante a maturação in vitro (MIV). [...] Ademais, o G3 também apresentou diminuição na taxa de maturação nuclear quando comparado ao G4. Quanto à maturação citoplasmática, nos grupos G2, G7, G6 e G3, houve redução (p<0,05) das taxas para 43,9por cento, 43,2 por cento, 43,1 por cento e 36,5 por cento, respectivamente, quando comparadas ao meio controle (G1), que permitiu a obtenção de valores médios de 62,4 por cento. Por outro lado, nos grupos G8, G4 e G5, a taxa de maturação citoplasmática não foi afetada com a redução do SFB, onde 59,3 por cento, 51,3 por cento e 50,8 por cento dos oócitos apresentaram os GC dispostos na periferia, respectivamente. Os resultados obtidos pelo teste de contrastes ortogonais complementam os obtidos na avaliação da maturação nuclear e migração de grânulos corticais, mostrando a necessidade do SFB durante a MIV, mesmo que em baixas concentrações, e a possibilidade de diminuir a sua concentração associando-o a BSA-FAF e/ou FE. Dessa forma, conclui-se que é possível reduzir a concentração de SFB no meio de MIV para até 3,5% sem prejuízo significativo aos índices de maturação nuclear e citoplasmática.

The use of fetal calf serum (FCS), although widely employed during in vitro production (IVP) of bovine embryos, has limitations. FCS is an undefined media and may have harmful effects on the quality of embryos. For this reason, in recent years, research efforts aimed at improving IVP of bovine embryos, have focused at the replacement of FCS by alternative compounds in culture media. In this study, fatty acid free bovine serum albumin (BSA-FAF) and embryonic fluid (EF) were used separately or in combination, in different concentrations, to replace or reduce the concentration of FCS during in vitro maturation (IVM). [...] Moreover, G3 also showed inferior nuclear maturation rate when compared to G4. Regarding cytoplasmic maturation, the rates were reduced to 43.9 percent, 43.2 percent, 43.1 percent and 36.5 percent in G2, G7, G6 and G3 groups, respectively, compared to the control group (G1; 62.4 percent). On the other hand, in the groups G8, G4 and G5, maturation rates were not affected by reduction of FCS, where 59.3 percent, 51.3 percent and 50.8 percent of the oocytes displayed CG arranged peripherally, respectively. The results obtained by the orthogonal contrast test are in accordance with the ones from the evaluation of the nuclear maturation and cortical granules migration. These data show the need of FCS on the MIV, even in low concentrations, and the possibility of decrease its concentration by associating it with BSA-FAF and/or EF. Therefore, we concluded that it is possible to reduce the concentration of FCS in IVM medium to a concentration of 3.5 percent without affecting nuclear and cytoplasmic maturation rates.

The seasonal and ovarian status effects on in vitro production of domestic cat embryos between Equator and Tropic of Capricorn / Efeitos da sazonalidade e da condição ovariana sobre a produção in vitro de embriões de gatos domésticos em uma região entre o Equador e o Trópico de Capricórnio

From the Tropic of Capricorn to Equator, the seasonality of domestic cat is known to be absent, i.e., these animals are considered non-seasonal breeders at these regions. We hypothesized that this particularity might have some influence on in vitro embryo production. The aim of this experiment was to determine the percentage of cleavage and morulae and blastocyst formation produced from oocytes recovered from queen ovaries of three distinct status - follicular, luteal or inactive - during two different reproductive seasons experienced by cats in southeast of Brazil (22°53'09" S and 48°26'42" W) - non breeding season (NBS), comprehending January to March; and breeding season (BS), August to October. Thirty queens were neutered. [...] During NBS, from a total of 272 (inactive), 162 (luteal) and 134 (follicular) fertilized oocytes, the percentage of cleaved zygotes, morulae and blastocysts derived from inactive ovaries were 24.63, 16.54 and 8.09 respectively; for those derived from luteal ovaries, the percentage was 21.6, 12.96 and 8.64, and for those from follicular ovaries, they were 24.62, 16.41 and 8.21. Considering BS, from a total of 102 (inactive), 198 (luteal) and 86 (follicular) fertilized oocytes, the relative frequency (%) of cleaved zygotes, morulae and blastocysts derived from inactive ovaries were 64.7, 41.17 and 23.53 respectively; for those derived from luteal ovaries, the percentage was 64.14, 40.41 and 23.73, and for those from follicular ovaries, they were 63.95, 39.54 and 24.41. The results of this experiment demonstrate that no statistically significant difference (P<0.05) was verified in the frequency of cleaved embryos and morulae and blastocyst formation when comparing the three ovarian conditions in the same season. However the breeding season presented better results considering cleavage and morulae and blastocyst formation.

Do Trópico de Capricórnio ao Equador, sabe-se que a sazonalidade no gato domestico é ausente, i.e., estes animais são considerados reprodutores não sazonais nestas regiões. [...] O objetivo deste experimento foi determinar a porcentagem de clivagem e formação de mórulas e blastocistos produzidos a partir de oócitos recuperados de ovários de gatas em três condições - folicular, lútea ou inativa - durante duas estações reprodutivas pelas quais gatas passam na região sudeste do Brasil (22°53'09" S e 48°26'42" O) - estação não reprodutiva (ENR), que compreende os meses de janeiro a março; e estação reprodutiva (ER), agosto à outubro. Trinta gatas foram castradas. [...] Durante a ENR, de um total de 272 (inativo), 162 (lútea) e 134 (folicular) oócitos fertilizados, a porcentagem de clivagem de zigotos, formação de mórulas e de blastocistos derivados de ovários inativos foi 24,63, 16,54 e 8,09 respectivamente; para aqueles oriundos de ovários na condição lútea, a porcentagem foi de 21,6, 12,96 e 8,64, e para aqueles provenientes de ovários na fase folicular, foi de 24,62, 16,41 e 8,21. Considerando a ER, de um total de 102 (inativo), 198 (lútea) e 86 (folicular) oócitos fertilizados, a frequência relativa (%) de zigotos clivados, mórulas e blastocistos derivados de ovários na condição inativa foi de 64,7, 41,17 e 23,53 respectivamente; para aqueles oriundos de ovários na condição lútea, a porcentagem foi de 64,14, 40,41 e 23,73, e para aqueles provenientes de ovários na fase folicular, foi de 63,95, 39,54 e 24,41. Os resultados deste experimento demonstraram que não houve diferença estatística significante (P < 0.05) na frequência de embriões clivados e na formação de mórulas e blastocistos quando comparadas as três condições ovarianas dentro da mesma estação. Entretanto, a ER apresentou resultados melhores considerando as taxas de clivagem e formação de mórula e de blastocisto se comparada à ENR.

Efeito do fator de crescimento epidermal (EGF) na maturação in vitro de oócitos caninos / Effect of epidermal growth factor (EGF) on in vitro maturation of canine oocyte

Este trabalho leve o objetivo de avaliar a influência do EGF na maturação in vitro (MIV) de cadelas. Realizou-se o transporte dos ovários em solução de cloreto de sódio 0,9%, que foram seccionados em solução salina fosfato temponado (PBS) e os complexos cumulus oócitos (COCs) selecionados em meio de cultura de tecidos (TCM) 199 suplementado com HEPES. Foram coletados 405 oócitos grau 1 de cadelas em anestro e diestro. Os oocitos provenientes das duas fases do ciclo estral foram submetidos a dois tratamentos: meio com adição de 10ng/mL do fator de crescimento epidermal (EGF) (T) e meio sem suplementação (C). Depois de 72 horas de maturação, os COCs foram desnudados, fixados e corados com HOESCHT 33342 para avaliação da maturação nuclear. Os oócitos obtidos dos ovários da fase de diestro do grupo T demonstraram maior porcentagem (18,8%) de oócitos no estágio de metáfase II (M-II), em relação ao grupo C (1,3%) (p < 0,01). Nos oócitos obtidos da fase de anestro houve diferença (p < 0,05) entre os grupos, observando-se menor parcela (4,1%) de oócitos no estágio de vesícula germinativa (VG) no grupo T, quando comparado ao grupo C (16,1%). Comparando-se as diferentes fases reprodutivas, em meios suplementados com EGF, foi observada diferença (p < 0,05) apenas nos oócitos obtidos da fase de diestro em relação ao estágio de M-II. Dessa maneira, a suplementação no meio de maturação na concentração de 10 ηg/mL, neste estudo, exerceu influência positiva apenas na MIV de oócitos caninos oriundos da fase de diestro, incrementando os índices de M-II nessa espécie.

This work aimed to evaluate the influence of EGF on canine oocyte in vitro maturation (IVM). We carried out the transport of the ovaries in sodium chloride 0.9% solution, which were cut into phosphate-buffered saline (PBS) and the cumulus oocyte complexes (COCs) selected in tissue culture medium (TCM), supplemented with 199 HEPES. A total of 405 grade 1 oocytes were collected from bitches in anestrus and diestrus. Oocytes from the two phases of estrous cycle were subjected to two treatments: medium with addition of 10 ηg/mL epidermal growth factor (EGF) (T) and medium without supplementation (C). After 72 h of maturation, COCs were denuded, fixed and stained with Hoechst 33342 to assess nuclear maturation. The oocytes obtained from the ovaries of the diestrus phase of the T group demonstrated higher percentage (18.8%) of oocytes at metaphase II stage (M-II) than C group (1.3%) (p < 0.01). The oocytes from anestrus phase demonstrated difference (p < 0.05) between the groups, observing smaller proportion (4.1%) of oocytes at the stage of germinal vesicle (GV) in group T compared to group C (16.1%). Comparing the different reproductive status in media supplemented with EGF difference (p < 0.05) was observed only in oocytes diestrus phase relative to the stage of M-II. Thus, supplementation on maturation medium at a concentration of 10 ηg/mL of EGF in this study had positive influence only on IVM of canine oocytes obtained from diestrus phase, increasing the levels of M-II in this species.