Use of Triggers on in vitro Fertilization and Evaluation of Risk Factors for Sub-Optimal Maturation Rate / Uso de gatilhos na fertilização in vitro e avaliação dos fatores de risco para taxa de maturação subótima

Abstract Objective To compare the oocyte maturation rate in the treatment of in vitro fertilization (IVF) in terms of the use of human chorionic gonadotropin (hCG), agonist gonadotropin-releasing hormone (GnRH) and dual trigger and to evaluate the associated risk factors for sub-optimal maturation rates. Methods A retrospective cohort study with 856 women who underwent IVF. They performed oocyte retrieval and were classified into 3 groups (1 - hCG, 2 - GnRHagonist, 3 - dual trigger). The primary outcome was maturation rate per trigger, and the secondary outcomes were the pregnancy rate per oocyte retrieval and the correlations between low maturation rate as well as the clinical and treatment characteristics of women. Results The maturation rate was 77% in group 1; 76% in group 2, and 83% in group 3 (p=0.003). Group 2 showed women with better ovarian reserve, greater number of oocytes collected, and more mature oocytes and embryos compared with the other groups (p<0.001). The cumulative clinical pregnancy rate was no different between the groups (p=0.755). Low ovarian reserve and low doses of follicle-stimulating hormone (FSH) administered during the stimulus were associated with a higher chance of null maturation rate. Conclusion The oocyte maturation rates and IVF results were similar in all groups. Low ovarian reserve is associated with the worst treatment results.

Resumo Objetivo Comparar a taxa de maturação oocitária no tratamento de fertilização in vitro (FIV) emrelação so o uso de gonadotrofina coriônica humana (hCG), agonista de hormônio liberador de gonadotrofina (GnRH), e gatilho duplo e avaliar os fatores de risco associados a taxas de maturação subótimas. Métodos Estudo de coorte retrospectivo com 856 mulheres submetidas à FIV. Elas foram classificadas em 3 grupos (1 - hCG, 2 - GnRH agonista, 3 - gatilho duplo). O desfecho primário foi a taxa de maturação por gatilho, e os desfechos secundários foram a taxa de gravidez por recuperação de oócitos e as correlações entre a baixa taxa de maturação bem como as características clínicas e do tratamento das mulheres. Resultados A taxa de maturação foi de 77% no grupo 1; 76% no grupo 2, e 83% no grupo 3 (p=0,003). O grupo 2 apresentou mulheres com melhor reserva ovariana, maior número de oócitos coletados, oócitosmaduros, e embriões, emcomparação aos demais grupos (p<0,001). A taxa cumulativa de gravidez clínica não foi diferente entre os grupos (p=0,755). Baixa reserva ovariana e baixas doses de hormônio folículoestimulante (FSH) administradas durante o estímulo foram associadas a uma maior chance de taxa de maturação nula. Conclusão As taxas de maturação oocitárias e os resultados de FIV foram semelhantes em todos os grupos. A baixa reserva ovariana está associada aos piores resultados do tratamento.

Influence of different protein supplements on the recovery and in vitro maturation of bovine oocytes / Influencia de diferentes suplementos proteicos en la recuperación y maduración in vitro de ovocitos bovinos / Influência de diferentes suplementações proteicas sobre a recuperação e maturação in vitro de oócitos bovinos

Abstract Background: Oocyte quality and maturation are influenced by protein supplementation. Objective: To evaluate the influence of fetal bovine serum (FBS) and bovine serum albumin (BSA) concentrations on the recovery and in vitro maturation (IVM) of bovine oocytes. Methods: The study was divided into Stage 1 (oocyte recovery), and Stage 2 (IVM). In the first stage, three experiments were conducted according to the recovery (R) medium used: (R1) 10 vs. 20% FBS; (R2) 5 vs. 10% BSA; and (R3) the best results from R1, R2, and the combination of FBS+BSA (5+5%). Within the second stage, the maturation medium was supplemented according to three experiments: (M1) 5 vs. 10% FBS; (M2) 0.4 vs. 0.8% BSA; and (M3) better results of M1, M2, and the combination of FBS+BSA (5+0.8%). Results: In Stage 1 (R1 and R2), the media with 10% FBS and 10% BSA showed better oocyte quality results and were defined for experiment R3. In R3, the 10% FBS and the combination of FBS+BSA (5+5%) allowed recovery of better-quality oocytes. In Stage 2 (M1 and M2), media with both levels of FBS (5 and 10%) and 0.8% BSA were defined as better according to the maturation and viability rates of cumulus cells, so they were defined for experiment M3. In M3, no difference was noted among the supplements. Conclusions: For oocyte recovery, 10% FBS and the combination of FBS+BSA (5+5%) can be used to obtain immature oocytes. For the in vitro maturation, FBS (both levels, 5 and 10%) and BSA (0.8%) can be used alone or in combination.

Resumen Antecedentes: La calidad y maduración de los ovocitos son influenciados por la suplementación proteica. Objetivo: Evaluar la influencia de concentraciones de suero fetal bovino (FBS) y albúmina sérica bovina (BSA) en la recuperación y maduración in vitro (IVM) de ovocitos bovinos. Métodos: El estudio se dividió en Etapa 1 (recuperación de ovocitos) y Etapa 2 (IVM). En la primera etapa, tres experimentos se realizaron de acuerdo con el medio de recuperación: (R1) 10 vs. 20% FBS; (R2) 5 vs. 10% BSA; y (R3) los mejores resultados de R1, R2 y la combinación de FBS+BSA (5+5%). En la segunda etapa, el medio de maduración fue suplementado para tres experimentos: (M1) 5 vs. 10% FBS; (M2) 0,4 vs. 0,8% BSA; y (M3) mejores resultados de M1, M2 y la combinación de FBS+BSA (5+0,8%). Resultados: En la Etapa 1 (R1 y R2), los medios con 10% FBS y 10% BSA mostraron mejores resultados de calidad oocitaria y fueron definidos para el experimento R3. En R3, 10% FBS y la combinación de FBS+BSA (5+5%) permitieron la recuperación de ovocitos de mejor calidad. En la Etapa 2 (M1 y M2), los medios con ambos niveles de FBS (5 y 10%) y 0,8% de BSA se definieron como mejores de acuerdo con las tasas de maduración y viabilidad de las células del cumulus, por lo que se definieron para el experimento M3. En M3, no se observó diferencia entre los suplementos. Conclusiones: Para la recuperación de ovocitos, se puede utilizar 10% de FBS y la combinación de FBS+BSA (5+5%) para obtener ovocitos inmaduros. Para la maduración in vitro, FBS (ambos niveles, 5 y 10%) y BSA (0,8%) se pueden usar solos o en combinación.

Resumo Antecedentes: A qualidade e a maturação de oócitos são influenciadas pela suplementação proteica. Objetivo: Avaliar a influência de concentrações de soro fetal bovino (FBS) e albumina sérica bovina (BSA) sobre a recuperação e maturação in vitro (IVM) de oócitos bovinos. Métodos: O estudo foi dividido em Etapa 1 (recuperação de oócitos) e Etapa 2 (IVM). Na primeira etapa, três experimentos foram realizados de acordo com o meio de recuperação: (R1) 10 vs. 20% FBS; (R2) 5 vs. 10% BSA; e (R3) melhores resultados de R1, R2 e a combinação de FBS+BSA (5+5%). Na segunda etapa, o meio de maturação foi suplementado de acordo com três experimentos: (M1) 5 vs. 10% FBS; (M2) 0,4 vs. 0,8% BSA; e (M3) melhores resultados de M1, M2 e a combinação de FBS+BSA (5+0,8%). Resultados: Na Etapa 1 (R1 e R2), os meios com 10% FBS e 10% BSA mostraram melhores resultados de qualidade oocitária e foram definidos para o experimento R3. Em R3, 10% FBS e a combinação de FBS+BSA (5+5%) permitiram a recuperação de oócitos de melhor qualidade. Na segunda etapa (M1 e M2), meios com ambos os níveis de FBS (5 e 10%) e 0,8% BSA foram definidos como os melhores de acordo com as taxas de maturação e viabilidade de células do cumulus, então foram definidos para o experimento M3. No M3, não houve diferença entre os suplementos. Conclusões: Para a recuperação oocitária, 10% FBS e a combinação de FBS+BSA (5+5%) podem ser usados para obter oócitos imaturos. Para maturação in vitro, FBS (ambos os níveis, 5 e 10%) e BSA (0,8%) podem ser usados sozinhos ou em combinação.

Técnica de doble disparo (doubble trigger) en pacientes normorrespondedoras con bajo porcentaje de ovocitos maduros / Doubble Trigger in patients with a normal response with a low percentage of mature oocytes

Resumen OBJETIVO: Evaluar el efecto del doble disparo en pacientes con un ciclo previo con menos de 65% de ovocitos maduros respecto de los ovocitos capturados, en una población con respuesta normal a la inducción de la ovulación con hCG recombinante o urinaria. MATERIALES Y MÉTODOS: Estudio de cohorte, prospectivo, efectuado en pacientes con diagnóstico de infertilidad, en tratamiento con fertilización in vitro, evaluadas en el Centro Mexicano de Fertilidad (Hospital Ángeles Lomas) entre 2017 y 2019. El tratamiento se llevó a cabo en la misma paciente, en cuyo ciclo previo convencional con esquema de antagonista e inducción de la ovulación con hCG tuvo respuesta ovárica subóptima y captura ovocitaria con menos de 65% en fase M2 (Grupo 1). Posteriormente se les indicó un segundo ciclo con el mismo esquema de gonadotropinas e inducción de la ovulación con doble disparo: acetato de triptorelina 1 mg + 5000 UI de hCG urinaria 40 y 34 horas previas a la captura (Grupo 2). Se evaluaron el porcentaje y la cantidad de ovocitos capturados en fase M2. RESULTADOS: Se registraron 34 pacientes en quienes se llevaron a cabo 68 ciclos. La cantidad de ovocitos capturados fue mayor en el grupo 2 (agonista de GnRH + hCG urinaria; p = 0.03). El doble disparo aumentó el porcentaje de ovocitos maduros (65.4 ± 21.3 vs 74.6 ± 20.2; p = 0.07). CONCLUSIONES: La técnica de doble disparo es valiosa para el tratamiento de pacientes con captura de ovocitos deficiente, aun con desarrollo folicular normal y concentraciones de estradiol adecuadas y óptimas de hCG el día de la captura. Se requieren estudios prospectivos de gran tamaño para dilucidar la recomendación mencionada de la técnica de "doble disparo".

Abstract OBJECTIVE: To evaluate the effect of double trigger in patients with a previous cycle with less than 65% of mature oocytes compared to the captured oocytes, in a normorresponding population with induction of ovulation with recombinant or urinary hCG. MATERIALS AND METHODS: A prospective cohort study, conducted in patients diagnosed with infertility, treated with in vitro fertilization, evaluated at the Mexican Fertility Center (Hospital Angeles Lomas) between 2017 and 2019. The treatment was carried out in the same patient, in whose previous conventional cycle with antagonist scheme and induction of ovulation with hCG had suboptimal ovarian response and oocyte capture with less than 65% in M2 phase (Group 1). Subsequently, a second cycle was performed with the same scheme of gonadotropins and induction of ovulation with double shot: 1 mg triptorelin acetate + 5000 IU of urinary hCG 40 and 34 hours prior to capture (Group 2 or double trigger). Percentage and quantity of oocytes captured in M2 phase were evaluated. RESULTS: 34 patients were registered, in whom 68 cycles were performed. The number of oocytes captured was greater in group 2 (agonist of GnRH + urinary hCG; p = 0.03). The double shot increased the percentage of mature oocytes 65.4 ± 21.3 vs 74.6 ± 20.2 (p = 0.07). CONCLUSIONS: The double trigger technique is valuable for the treatment of patients with poor oocyte capture, even with normal follicular development and adequate and optimal hCG estradiol concentrations on the day of capture. Large prospective studies are required to elucidate the aforementioned recommendation of the "double shot" technique.

Prediction of efficacy of gonadotropin releasing hormone agonist trigger for final oocyte maturation through post-trigger 12-hour luteinizing hormone, follicle stimulating hormone and progesterone levels in COS: a prospective study

Background: Circulating levels of luteinizing hormone (LH), follicle stimulating hormone (FSH) and progesterone (P4) in serum after administration of gonadotropin releasing hormone agonist (GnRHa) trigger for final oocyte maturation are found to be predictive of oocyte maturity. This prospective study was conducted at a tertiary care centre to evaluate relationship between serum LH, FSH and P4 levels at 12-h post-trigger and oocyte maturity rate and to predict which hormone has maximum sensitivity and specificity for appropriate oocyte maturation.Methods: Women at risk of ovarian hyper-stimulation syndrome who underwent either autologous or donor IVF cycle treated with flexible GnRH antagonist protocol were taken as participants of the study. GnRHa as trigger for final oocyte maturation was given. After 12 hours of agonist trigger, blood sample was drawn to assess LH, FSH and P4 levels in serum. Continuous variables were expressed as mean±SD. Independent sample t test was used for continuous variables which were normally distributed and Mann-Whitney U test for data not normally distributed. Main outcome measures were number of oocytes retrieved, oocyte maturity rate, fertilization rate, cleavage rate and grade of embryos.Results: There was a statistically significant reduction in number of retrieved oocytes, maturity rate, fertilization rate and grade 1 embryos with a concentration of serum LH and P4 less than the cut off value (p < 0.05).Conclusions: Serum LH and P4 level less than the cut off value at 12-hour post-trigger with GnRHa is associated with a dramatically less oocyte maturity rate and fertilization rate.

Oocyte maturation under a biophoton generator improves preimplantation development of pig embryos derived by parthenogenesis and somatic cell nuclear transfer

This study was conducted to determine the effects of biophoton treatment during in vitro maturation (IVM) and/or in vitro culture (IVC) on oocyte maturation and embryonic development in pigs. An apparatus capable of generating homogeneous biophoton energy emissions was placed in an incubator. Initially, immature pig oocytes were matured in the biophoton-equipped incubator in medium 199 supplemented with cysteine, epidermal growth factor, insulin, and gonadotrophic hormones for 22 h, after which they were matured in hormone-free medium for an additional 22 hr. Next, IVM oocytes were induced for parthenogenesis (PA) or provided as cytoplasts for somatic cell nuclear transfer (SCNT). Treatment of oocytes with biophoton energy during IVM did not improve cumulus cell expansion, nuclear maturation, intraoocyte glutathione content, or mitochondrial distribution of oocytes. However, biophoton-treated oocytes showed higher (p < 0.05) blastocyst formation after PA than that in untreated oocytes (50.7% vs. 42.7%). In an additional experiment, SCNT embryos produced from biophoton-treated oocytes showed a greater (p < 0.05) number of cells in blastocysts (52.6 vs. 43.9) than that in untreated oocytes. Taken together, our results demonstrate that biophoton treatment during IVM improves developmental competence of PA- and SCNT-derived embryos.

Effects of sperm insemination on the final meiotic maturation of mouse oocytes arrested at metaphase I after in vitro maturation / 대한생식의학회지

OBJECTIVE: The aims of this study were to investigate whether fertilization could induce the resumption of meiosis in mouse oocytes arrested at metaphase I (MI) after in vitro maturation (IVM), and to investigate the effect of Ca²⁺ chelator treatment at the time of fertilization on the transition from MI to metaphase II (MII). METHODS: MII-stage and arrested MI-stage mouse oocytes after IVM were fertilized, and then embryonic development was monitored. Blastocysts from each group were transferred into 2.5 days post-coitum pseudo-pregnant ICR mice. MI oocytes after IVM were treated with a Ca²⁺ chelator to investigate the effect of Ca²⁺ oscillations on their maturation. RESULTS: As insemination time increased, the number of oocytes in the MI group that reached the MII stage also increased. The blastocyst rates and total cell numbers in the MII group were significantly higher than in the MI group. No pregnancy occurred in the MI group, but 10 pregnancies were achieved (10 of 12) in the MII group. The proportion of MI oocytes that matured to MII oocytes after fertilization was significantly higher in the non-treated group than in the Ca²⁺ chelator-treated group. CONCLUSION: The findings that a higher proportion of MI-arrested oocytes progressed to MII after fertilization and that the MI-to-MII transition was blocked by Ca2+ chelator treatments before fertilization indicate that the maturation of MI oocytes to MII oocytes is associated with intracellular Ca²⁺ oscillations driven by fertilization.

Maduración gonadal de la cucha mariposa, Glyptoperichthys gibbiceps (Kner, 1854) (Pisces: Loricariidae), en el río Guaviare, Colombia

Glyptoperichthys gibbiceps, es uno de los loricáridos de gran tamaño (hasta 50 cm LT), presentes en la cuenca del río Orinoco en Colombia, de la cual se sabe poco en el medio natural. Para conocer del ciclo reproductivo, fueron capturados bimensualmente durante doce meses, en una madre vieja del río Guaviare (N 02°37' 52.7", W 072° 42' 28,08", 196 msnm), especímenes adultos los cuales fueron sacrificados in situ, previa anestesia, registrándose las medidas morfométricas y peso, luego eviscerados, identificando el sexo y estado de desarrollo gonadal y retiradas las gónadas las cuales fueron pesadas. Una muestra de la porción media de cada gónada fue fijada en formol buferado y transferida a las 24 horas a etanol 70% donde se conservó para procesamiento histológico estándar (H-E). Un total de 26 hembras con peso de 495.7±52.9 g y longitud total de 36.6±4.1 cm y 9 machos con peso de 676.7±192.8 g y longitud total de 36.6±4.1 cm, proporción sexual 3 hembras: 1 macho, fueron estudiados describiéndose la escala de maduración gonadal en cuatro estados (reposo, en maduración, maduro y un estado juvenil pre-reproductivo). En reposo los ovarios y testículos se presentan reducidos traslúcidos, de difícil diferenciación. En estado de maduración los ovarios son diferenciables ligeramente globosos y de color amarillo traslucido con ovocitos con diámetros de 2768 ± 373µm. En el estado maduro los ovarios son saculares de color amarillo intenso y brillante y los ovocitos tienen diámetros de 4155 ± 96 µm; los testículos se presentan blancos cuando alcanzan el punto máximo de madurez. Hembras y machos adultos reproductivamente activos maduran sus gónadas una vez al año de manera sincrónica, con desove total en aguas subiendo. Se estimó una fecundidad media de 1483 ± 380 óvulos / hembra madura. Se describe el ciclo ovocitario y espermático de la especie como similar al de otros loricáridos.

Glyptoperichthys gibbiceps (sailfin pleco), is one of the largest loricariids (up to 50 cm TL) in the Orinoco River basin in Colombia of which little is known of their natural history. To know the reproductive cycle, bimonthly collections for twelve months of adult specimens were made in a section of the Guaviare River (N 02 ° 37 '52.7 "W 072 ° 42' 28.08", 196 m). Specimens were killed on site, after anesthesia, recording morphometric and weight measurements, and then eviscerated. The gonads were weighed, and the sex and stage of sexual maturity of individual gonads were identified. A sample of the middle portion of each gonad was fixed for 24 h in buffered formalin and transferred to 70% ethanol until processed for standard histological analysis of tissues stained with H&E. A total of 26 females weighing 495.7 ± 52.9 g and 36.6 ± 4.1 cm TL, and 9 males of 676.7 ± 192.8 g and 36.6 ± 4.1 cm TL, (sex ratio 3 females to 1 male) were studied and four main stages were used to describe gonadal development (resting stage , developing or maturing, ripe or matured, and pre-reproductive juveniles). At rest the ovaries and testicles were small translucent and difficult to differentiate. The maturing ovaries were slightly differentiated, globular, translucent yellow containing oocytes with diameters of 2768 ± 372µm; the testicles had a rosy coloration. The mature ovaries were globular of an intense brilliant yellow, and the oocytes attained diameters of 4155 ± 96µm, and were visible to the naked eye; the testicles were white and apparent. The pre-reproductive juveniles or individuals with immature gonads were found throughout the year. In the reproductively active females and males the gonads mature synchronously once a year and spawning activity occurred during periods when water levels were rising. The average fecundity was estimated at 1483 ± 380 eggs / mature female. The oocyte and sperm cycles were very similar to those described for other Loricariids.

Glyptoperichthys gibbiceps (Bode-cacunda) é um dos loricaridos de maior tamanho (até 50 cm CT), na bacia do rio Orinoco na Colômbia, de quem pouco se conhece de sua vida na natureza. Para estudar seu ciclo reprodutivo, foram capturados espécimes adultos bimensalmente, durante doce meses em uma várzea do rio Guaviare (N 02°37' 52.7", W 072° 42' 28,08", 196 msnm), os quais foram sacrificados in situ, previa anestesia, registrando suas medidas morfométricas e peso, logo foram eviscerados, identificado o sexo, estádio de crescimento gonadal e peso da gônada. Uma amostra da porção media de cada gônada foi fixada em formol tamponado e transferida às 24 horas em etanol 70%, onde se conservo para processamento histológico padrão (H- E). Um total de 26 fêmeas (com peso de 495.7±52.9 g com comprimento total de 36.6±4.1 cm) e 9 machos (com peso de 676.7±192.8 g e comprimento total de 36.6±4.1 cm), com proporção sexual 3 fêmeas: 1 macho, foram estudados descrevendo sua escala de maduração gonadal em quatro estados (repouso, em maduração, maduro e um estado juvenil pre-reprodutivo). Em repouso os ovários e testículos se apresentam reduzidos translúcidos, de difícil diferenciação. Em estado em maduração os ovários são diferenciáveis ligeiramente globosos, de cor amarelo translúcido e ovócitos com diâmetros de 2768 ± 373µm; os testículos se apresentam rosáceos. No estado maduro os ovários são globosos de cor amarelo intenso e brilhante, os ovócitos conseguem diâmetros de 4155 ± 96 µm e são visíveis ao olho nu; os testículos são brancos, diferenciáveis. Um estado juvenil pre-reprodutivo o de indivíduos com gônadas imaturas se apresentou durante o ano inteiro. Fêmeas e machos adultos reprodutivamente ativos maduram suas gônadas uma vez ao ano de maneira sincrônica, com desove total em águas subindo. Estimou-se uma fecundidade media de 1483 ± 380 óvulos / fêmea madura. Descreve-se o ciclo ovocitario e espermático da espécie como similar ao de outros loricaridos.

Maduración in vitro de ovocitos con cultivo extendido hasta estadio de blastocisto: Primer nacimiento reportado en el Perú / In vitro oocyte maturation combined with extended blastocyst embryo culture: Report of the first baby born in Peru

La maduración in vitro de ovocitos (MIV) es una técnica de reproducción asistida muy poco difundida entre los centros de reproducción asistida, debido al bajo éxito en obtener embarazos. Sin embargo, en los últimos años, diferentes estrategias empleadas han demostrado tasas de embarazo similares a las técnicas convencionales de fecundación in vitro (FIV). En el presente reporte, describimos el caso clínico del primer nacido vivo usando MIV en combinación del cultivo extendido hasta estadio de blastocisto.

In vitro oocyte maturation is not yet considered a well-established technique in in vitro fertilization (IVF) laboratories. This is due to a lower pregnancy rates. However in the last few years, reports have shown similar pregnancy rates compared to the conventional IVF techniques. The current report describes the first baby born after an IVM treatment in combination with extended blastocyst culture in Peru.

Cryopreservation of in vitro matured oocytes after ex vivo oocyte retrieval from gynecologic cancer patients undergoing radical surgery / 대한생식의학회지

OBJECTIVE: The aim of this study was to report a case series of in vitro matured (IVM) oocyte freezing in gynecologic cancer patients undergoing radical surgery under time constraints as an option for fertility preservation (FP). METHODS: Case series report. University-based in vitro fertilization center. Six gynecologic cancer patients who were scheduled to undergo radical surgery the next day were referred for FP. The patients had endometrial (n=2), ovarian (n=3), and double primary endometrial and ovarian (n=1) cancer. Ex vivo retrieval of immature oocytes from macroscopically normal ovarian tissue was followed by mature oocyte freezing after IVM or embryo freezing with intracytoplasmic sperm injection. RESULTS: A total of 53 oocytes were retrieved from five patients, with a mean of 10.6 oocytes per patient. After IVM, a total of 36 mature oocytes were obtained, demonstrating a 67.9% maturation rate. With regard to the ovarian cancer patients, seven IVM oocytes were frozen from patient 3, who had stage IC cancer, whereas one IVM oocyte was frozen from patient 4, who had stage IV cancer despite being of a similar age. With regard to the endometrial cancer patients, 15 IVM oocytes from patient 1 were frozen. Five embryos were frozen after the fertilization of IVM oocytes from patient 6. CONCLUSION: Immature oocytes can be successfully retrieved ex vivo from macroscopically normal ovarian tissue before radical surgery. IVM oocyte freezing provides a possible FP option in patients with advanced-stage endometrial or ovarian cancer without the risk of cancer cell spillage or time delays.

The potential role of granulosa cells in the maturation rate of immature human oocytes and embryo development: A co-culture study / 대한생식의학회지

OBJECTIVE: In order to increase the number of mature oocytes usable for intracytoplasmic sperm injection (ICSI), we aimed to investigate the effect of co-culturing granulosa cells (GCs) on human oocyte maturation in vitro, the fertilization rate, and embryo development. METHODS: A total of 133 immature oocytes were retrieved and were randomly divided into two groups; oocytes that were cultured with GCs (group A) and oocytes that were cultured without GCs (group B). After in vitro maturation, only oocytes that displayed metaphase II (MII) underwent the ICSI procedure. The maturation and fertilization rates were analyzed, as well as the frequency of embryo development. RESULTS: The mean age of the patients, their basal levels of follicle-stimulating hormone, and the number of oocytes recovered from the patients were all comparable between the two study groups. The number of oocytes that reached MII (mature oocytes) was 59 out of 70 (84.28%) in group A, compared to 41 out of 63 (65.07%) in group B (p=0.011). No significant difference between fertilization rates was found between the two study groups (p=0.702). The embryo development rate was higher in group A (33/59, 75%) than in group B (12/41, 42.85%; p=0.006). The proportion of highest-quality embryos and the blastocyst formation rate were significantly lower in group B than in group A (p=0.003 and p<0.001, respectively). CONCLUSION: The findings of the current study demonstrate that culturing immature human oocytes with GCs prior to ICSI improves the maturation rate and the likelihood of embryo development.

Rapamycin treatment during in vitro maturation of oocytes improves embryonic development after parthenogenesis and somatic cell nuclear transfer in pigs

This study was conducted to investigate the effects of rapamycin treatment during in vitro maturation (IVM) on oocyte maturation and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in pigs. Morphologically good (MGCOCs) and poor oocytes (MPCOCs) were untreated or treated with 1 nM rapamycin during 0-22 h, 22-42 h, or 0-42 h of IVM. Rapamycin had no significant effects on nuclear maturation and blastocyst formation after PA of MGCOCs. Blastocyst formation after PA was significantly increased by rapamycin treatment during 22-42 h and 0-42 h (46.6% and 46.5%, respectively) relative to the control (33.3%) and 0-22 h groups (38.6%) in MPCOCs. In SCNT, blastocyst formation tended to increase in MPCOCs treated with rapamycin during 0-42 h of IVM relative to untreated oocytes (20.3% vs. 14.3%, 0.05 < p < 0.1), while no improvement was observed in MGCOCs. Gene expression analysis revealed that transcript abundance of Beclin 1 and microtubule-associated protein 1 light chain 3 mRNAs was significantly increased in MPCOCs by rapamycin relative to the control. Our results demonstrated that autophagy induction by rapamycin during IVM improved developmental competence of oocytes derived from MPCOCs.

Signaling Pathways in Insulin- and IGF-I Mediated Oocyte Maturation in Lower Vertebrates.

The endocrine control of oocyte maturation in fish and amphibians has proved to be a valuable model for investigating the rapid and non-genomic steroid actions at the cell surface. Considerable progress has made over the last decade in elucidating signaling pathways in steroid-induced oocyte maturation. In addition to steroids, various growth factors have also been reported to be involved in this process and progress being made to elucidate their mechanism of actions. Exposure of fully-grown oocytes to steroids or growth factors (insulin/IGFs) initiates various signaling cascade, leading to formation and activation of maturation-promoting factor (MPF), a key enzyme that catalyzes entry into M-phase of meiosis I and II. Whereas the function of MPF in promoting oocyte maturation is ubiquitous, there are differences in signaling pathways between steroids- and growth factors-induced oocyte maturation in amphibian and fish. Here, we have reviewed the recent advances on the signaling pathways in insulin- and IGF-I-induced oocyte maturation in these two groups of non-mammalian vertebrates. New findings demonstrating the involvement of PI3 kinase and MAP kinase in induction of oocyte maturation by insulin and IGF-I are presented.

Growth differentiation factor 9 and bone morphogenetic protein 15 expression in previtellogenic oocytes and during early embryonic development of Yellow-tail Kingfish Seriola lalandi

BACKGROUND: During fish oocyte maturation, specific molecules are expressed and accumulated within oocyte until fertilization and embryo development. Special attention have been paid in members of the transforming growth factor (TGF-ß) superfamily; growth differentiation factor 9 (GDF9/gdf9) and bone morphogenetic protein 15 (BMP15/bmp15), which exert regulatory functions during oocyte maturation and follicle development. However, little attention has been paid to the involvement of these molecules during embryogenesis considering its importance for the formation of a good quality egg and subsequent embryo survival. The purpose of this study was to analyze the expression of gdf9 andbmp15 in previtellogenic oocytes and during early embryonic development in Seriola lalandi, a pelagic fish with increasing prospect for its aquaculture development, which however, show high mortality at embryo and larval stages. RESULTS: Through RT-qPCR it was found that gdf9 expression was higher in previtellogenic oocytes decreasing after ovulation. This expression profile agrees with its participation in early stages of the follicular development. The transcripts for bmp15 also showed the highest levels in previtellogenic oocytes, however this expression was lower than obtained with gdf9. Conversely, in recently spawned oocytes mRNA bmp15 levels were highest than observed to gdf9. This, is consequent with the main role proposed for this growth factor at the final fish oocyte maturation: avoid the ovulation of an immature oocyte. During embryo development, low levels of mRNA were detected to gdf9, with an increase in 48 H post-fertilization embryos. The bmp15 expression did not change throughout development and was higher than gdf9 at 16 cells, blastula and appearance embryos stages. CONCLUSIONS: Both (gdf9 and bmp15) expression profiles in previtellogenic oocytes and newly spawned eggs are consistent with the described functions for these growth factors in vertebrate ovarian physiology in early and late stages of the follicular development. So, these genes could be considered as quality biomarkers at these stages. However, further studies of these proteins throughout folliculogenesis, are necessaries to fully understand their functions during the oocyte formation. In addition, the persistent expression of these growth factors during development, allows us to speculate possible roles in embryonic processes, which must also be addressed.

Comparision of in vitro maturation applied in PCOS and non-PCOS patients undergo stimulated and unstimulated protocols / 中华妇产科杂志

Objective To compare the laboratory and clinical results between unstimulated in vitro maturation (IVM) and IVM converted from in vitro fertilization (IVF) in polycystic ovarian syndrome (PCOS) and non-PCOS patients.Methods We divided 591 IVM cycles in the First Affiliated Hospital of Wenzhou Medical Univesity from Jan.2008 to Dec.2013 into 4 groups:group A1B1,PCOS patients underwent unstimulated IVM protocol,240 cycles; group A1B2,PCOS patients underwent IVM converted from conventional stimulated IVF protocol,153 cycles; group A2B1,non-PCOS patients underwent unstimutlated IVM protocol,103 cycles; group A2B2,non-PCOS patient underwent IVM converted from conventional stimulated IVF protocol,95 cycles.Multiple linear regression method and binary logistic regression method were used to assess the influence of PCOS and protocols for IVM on laboratory and clinical outcomes.Results The mean number of oocytes retrieved was positively related with PCOS [partial regression coefficient (B)=3.37,P＜0.01].The maturation rate of oocytes was positively related with hCG-prime prior to oocyte aspiration (B=0.05,P=0.010).High-quality embryo rate was positively related with PCOS and IVM converted from IVF (B=0.08,P=0.010; B=0.09,P=0.001),as well as implantation rate related with them (B=0.07,P=0.010; B=0.10,P＜0.01).PCOS and IVM converted from IVF improved hCG positive (hCG＞10 U/L) rate (OR=1.636,95％CI:1.113-2.204,P＜0.05; OR=1.861,95％CI:1.307-2.649,P＜0.05) and the clinical pregnancy rate (OR=1.507,95％CI:1.041-2.240,P＜0.05; OR=1.881,95％CI:1.312-2.696,P＜0.05).IVM converted from IVF protocol decreased the spontaneous abortion rate (OR=0.490,95％CI:0.245-0.978,P＜0.05).Multiple gestation rate and ectopic pregnancy rate were not affected by PCOS condition and protocol used (P＞0.05).Conclusions PCOS and IVM converted from IVF protocol improved the high-quality embryo rate,implantation rate,hCG positive rate and clinical pregnancy rate.IVM converted from IVF protocol reduced the spontaneous abortion rate.PCOS patients may be more suitable for the IVM treatment.No matter PCOS or non-PCOS patients,IVM converted from IVF protocol had better pregnancy outcome than that of unstimulated cycle.

Preservação da fertilidade em mulheres com câncer: atualização e perspectivas / Fertility preservation in women with cancer: update and perspectives

Com o aumento do diagnóstico de câncer em mulheres jovens e os avanços no seu tratamento, muitas pacientes que poderão ter sua fertilidade comprometida com a quimioterapia têm manifestado desejo de engravidar futuramente. O congelamento de embriões, após fertilização in vitro, para preservar a fertilidade está bem estabelecido. A criopreservação de oócitos por vitrificação evoluiu bastante nos últimos anos, deixando de ser experimental. Até 2009 nasceram mais de 900 crianças a partir de oócitos criopreservados, sem aumento do risco de anomalias congênitas. O uso de análogos do GnRH para a supressão ovariana durante a quimioterapia na tentativa de prevenir a falência ovariana prematura apresenta resultados incertos. Outras técnicas ainda são consideradas experimentais, como a criopreservação e posterior autotransplante de tecido ovariano. Já foram relatados 24 nascimentos com o seu uso, persistindo, entretanto, dúvidas que motivam o seu estudo. A maturação de folículos ovarianos in vitro é alternativa promissora para preservação da fertilidade nessas pacientes e tem apresentado resultados positivos em roedores, macacos e humanos. Muita cautela deve ser tomada com o uso de técnicas experimentais, especialmente quando oferecidas para pacientes diante de fragilidade emocional. Por isso, é importante transmitir corretamente informações sobre chances de gravidez com tratamentos existentes e as limitações das técnicas experimentais...

With the increased number of cancer diagnoses among young women and the advances in treatment, many patients who may have had their fertility compromised by chemotherapy express desire to become pregnant in the future. Freezing embryos for later IVF so as to preserve fertility is a well established process. Oocyte cryopreservation by vitrification has evolved greatly in recent years and is no longer considered experimental. By 2009 more than 900 children were born from cryopreserved oocytes, without increased risk of congenital anomalies. The preventive use of GnRH analogues for ovarian suppression during chemotherapy to avoid premature ovarian failure has uncertain outcomes. Other techniques such as cryopreservation of ovarian tissue for later autograft are still considered experimental. Although use has already been reported in 24 births, doubts still persist and motivate further study. In vitro maturation of ovarian follicles is a promising alternative for preserving patient fertility and has shown positive results in rodents, monkeys, andhumans. Caution should be used with experimental techniques, especially when offered to emotionally fragile patients. Therefore it is important to thoroughly convey information on the chances of pregnancy with existing treatments and the limitations of experimental techniques...

Desempeño reproductivo del bocachico Prochilodus magdalenae inducido dos veces en un mismo año / Reproductive performance of bocachico Prochilodus magdalenae induced twice in the same year

Objetivo. Evaluar el desempeño reproductivo del bocachico (Prochilodus magdalenae) sometido a dos inducciones hormonales con extracto pituitario de carpa (EPC) en un mismo año. Materiales y métodos. 23 hembras, en fase de maduración final, fueron inducidas con 5mg EPC/Kg de peso vivo, en dos aplicaciones, inicialmente 10% y doce horas después 90% restante. A los machos fue aplicado 80% de la dosis total de las hembras. Resultados. Después de la primera inducción hormonal, las hembras estuvieron aptas nuevamente a los 97.6±12.4 días. El índice de ovulación en ambas inducciones fue de 0.91 (21/23). La fecundidad absoluta (g-ovocitos/hembra) no presentó diferencias significativa entre la primera y segunda inducción (36.2 y 44.6 g-ovocitos/hembra, respectivamente) (p>0.05); pero cuando se expresó en número de ovocitos/hembra se observó diferencia significativa (p<0.05), siendo mayor en la primera inducción (53535 ovocitos/hembra) que en la segunda (40658 ovocitos/hembra). La fecundidad relativa, expresada tanto en g-ovocitos/Kg-hembra como ovocitos/Kg-hembra, mostraron diferencia significativa, siendo mayor en la primera inducción (p<0.05). La tasa de fecundación (73.9±19.6%) y eclosión (56.9±17.9%) fueron mayores en la primera inducción, comparadas con la segunda (55.6±21.1% y 35.6±20.7%, respectivamente) (p<0.05). Conclusiones. Después de una inducción hormonal se requiere de aproximadamente tres meses para que una hembra de bocachico alcance nuevamente la maduración final y esté apta para una nueva inducción hormonal. El índice de ovulación no fue afectado por una segunda inducción en un mismo año, pero la fecundidad absoluta y relativa puede disminuirse entre 24 y 66% y las tasas de fecundación y eclosión entre 23 y 36%.

Objective.To evaluate the reproductive performance of hormonally induced bocachico with carp pituitary extract (CPE) twice in the same year. Materials and methods. 23 females, in their final maturation phase, were induced with 5 mg EPC/Kg of body weight in two applications, initially 10% and 90% twelve hours later. Males was applied 80% of the total female dose. Results. After the first hormonal induction, females were fit again at 97.6±12.4 days. Ovulation rate in both hormonal inductions was 0.91 (21/23). Absolute fecundity (g-oocytes/female) showed no significant differences between the first and second induction (36.2 and 44.6 g oocytes/female, respectively) (p>0.05), but when expressed in number of oocytes per female there was a significant difference (p<0.05), the fertility rate being higher in the first induction (53535 oocytes/female) compared with the second induction (40658 oocytes/female). Relative fecundity, expressed in both g-oocytes/Kg-female and oocytes/Kg-female showed a significant difference, being higher in the first induction (p<0.05). Fertilization rate (73.9±19.6%) and hatching (56.9±17.9%) were higher in the first induction, compared to the second one(55.6±21.1% and 35.6±20.7%, respectively) (p<0.05). Conclusions. After hormonal induction a period of approximately three months is required for a female bocachico to reach final maturation and be suitable for a new hormonal induction. The ovulation rate was not affected by a second induction in the same year, but the absolute and relative fertility can be reduced between 24 and 66% and fertilization and hatching rates between 23 and 36%.

Cessation of gonadotropin-releasing hormone antagonist on triggering day in flexible multiple-dose protocol: A randomized controlled study / 대한생식의학회지

OBJECTIVE: To investigate outcomes of stimulated IVF cycles in which GnRH antagonist was omitted on the ovulation triggering day. METHODS: A total of 86 women who underwent controlled ovarian hyperstimulation with recombinant FSH and GnRH antagonist flexible multiple-dose protocols were recruited and prospectively randomized into the conventional group (group A) or cessation group (group B). The GnRH antagonist, 0.25 mg/day of cetrorelix, was started when the leading follicle reached 14 mm in diameter and was continuously administered until the hCG triggering day (group A, 43 cycles) or until the day before hCG administration (group B, 43 cycles). The maturity of oocytes, fertilization rate, embryo quality, and implantation and clinical pregnancy rates were evaluated. RESULTS: The duration of ovarian stimulation, total dose of gonadotropins, serum estradiol levels on hCG administration day, and number of oocytes retrieved were not significantly different between the two groups. The total dose of GnRH antagonist was significantly lower in group B than group A (2.5+/-0.9 vs. 3.2+/-0.8 ampoules, p<0.05). There was no premature luteinization in any of the subjects. The proportion of mature oocytes and fertilization rate were not significantly different in group B than group A (70.7% vs. 66.7%; 71.1% vs. 66.4%, respectively). There were no significant differences in the implantation or clinical pregnancy rates. CONCLUSION: Our prospective randomized study suggested that cessation of GnRH antagonist on the hCG administration day during a flexible multiple-dose protocol could reduce the total dose of GnRH antagonist without compromising its effects on pregnancy rates.

Fertilization and pregnancy potential of immature oocytes from stimulated intracytoplasmic sperm injection cycles / 대한생식의학회지

OBJECTIVE: We evaluated the fertilization potential of immature oocytes obtained from controlled ovarian hyperstimulation cycles of patients undergoing ICSI. METHODS: We retrospectively analyzed 463 ICSI cycles containing at least one immature oocyte at oocyte denudation. ICSI was performed on mature oocytes at oocyte denudation (metaphase-II [MII] oocytes) and the oocytes that extruded the first polar body between oocyte denudation and ICSI (MI-MII oocytes). Fertilization and early embryonic development were compared between MII and MI-MII oocytes. To investigate the pregnancy potential of MI-MII oocytes, the pregnancy outcome was analyzed in 24 ICSI cycles containing only immature oocytes at retrieval. RESULTS: The fertilization rate of MI-MII oocytes (37.0%) was significantly lower than that of MII oocytes (72.3%). The rates of delayed embryos and damaged embryos did not significantly differ. Eighty-one immature oocytes were retrieved in 24 cycles that retrieved only immature oocytes and 61 (75.3%) of them were in the MI stage. ICSI was performed on 36 oocytes (59.0%) that extruded the first polar body before ICSI and nine MI-MII oocytes (25.0%) were fertilized. Embryo transfers were performed in five cycles. Pregnancy was observed in one cycle, but it ended in biochemical pregnancy. CONCLUSION: In ICSI cycles, oocytes that extruded the first polar body between denudation and ICSI can be used as a source of oocytes for sperm injection. However, their fertilization and pregnancy potential are lower than that of mature oocytes. Therefore, ovarian stimulation should be performed carefully for mature oocytes obtained at retrieval, especially in cycles with a small number of retrieved oocytes.

Oócitos aparentemente maduros injetados em telófase I apresentam piores resultados de reprodução assistida / Apparently matured oocytes injected in telophase I have worse outcomes from assisted reproduction

OBJETIVO: Avaliar o estágio de maturação nuclear de oócitos com o primeiro corpúsculo polar (CP) visível de pacientes inférteis submetidas à estimulação ovariana para injeção intracitoplasmática de espermatozoide (ICSI) e comparar os resultados da injeção intracitoplasmática de espermatozoide entre os oócitos em telófase I (TI) e metáfase II (MII), e entre aqueles em metáfase II com e sem fuso celular visível. MÉTODOS: Estudo prospectivo que incluiu 106 pacientes inférteis submetidas à injeção intracitoplasmática de espermatozoide. Foram incluídas pacientes com idade menor ou igual a 38 anos, hormônio folículo estimulante (FSH) basal menor que 10 mIU/mL e índice de massa corpórea (IMC) menor que 30 kg/m². Foram excluídas pacientes com doenças sistêmicas, com qualquer infecção ativa, tabagistas ou que fizeram uso de medicações hormonais e anti-inflamatórias hormonais e não hormonais nos últimos dois meses, previamente à programação para o procedimento de reprodução assistida. Os oócitos com extrusão do primeiro corpúsculo polar foram avaliados pela microscopia de polarização, imediatamente antes da realização da injeção intracitoplasmática de espermatozoide, e caracterizados quanto ao estágio de maturação nuclear (telófase I ou metáfase II). Os oócitos em metáfase II foram avaliados de acordo com a presença ou não do fuso meiótico. Foram analisadas as taxas de fertilização, clivagem e o número de embriões de boa qualidade no segundo dia (D2) de desenvolvimento. Os dados foram analisados comparativamente através do teste exato de Fisher. Em todas as análises foi considerado o nível de significância de 5% (p<0,05). RESULTADOS: O fuso meiótico de 516 oócitos foi visualizado através da microscopia de polarização. Dezessete dos 516 oócitos avaliados estavam em telófase I (3,3%) e 499 (96,7%) em metáfase II. Os oócitos injetados em telófase I apresentaram taxas de fertilização significativamente menores do que os injetados em metáfase II (53 e 78%, respectivamente) e não produziram nenhum embrião de boa qualidade no segundo dia. Comparando-se os oócitos com e sem fuso celular visível, não foi observada diferença significativa nos resultados de injeção intracitoplasmática de espermatozoide. CONCLUSÕES: Oócitos injetados em telófase I apresentaram menores taxas de fertilização quando comparados aos em metáfase II. É possível que a análise do estágio de maturação nuclear oocitária, por meio da microscopia de polarização, possa ser utilizada como fator de predição das taxas de fertilização pós-injeção intracitoplasmática de espermatozoide.

PURPOSE: To evaluate the nuclear maturation stage and the presence of meiotic spindles of in vivo matured oocytes from infertile women undergoing stimulated cycles for intracytoplasmic sperm injection (ICSI) and compare intracytoplasmic sperm injection outcomes between oocytes in telophase I (TI) and metaphase II (MII), and the ones with and without visible meiotic spindle. METHODS: A prospective and controlled study with 106 infertile patients who underwent ovarian stimulation for intracytoplasmic sperm injection purposes. Patients aged 38 years or less, with basal follicle stimulating hormone (FSH) less than 10 mIU/mL and body mass index (BMI) less than 30 kg/m². Were included patients presenting any systemic diseases, any active infection, smokers or patients who had been using hormonal medications and hormonal and nonhormonal anti-inflammatory drugs for the past two months prior to the assisted reproduction procedure were excluded. The oocytes with the first polar body extruded (in vivo matured oocytes) were imaged by polarization microscopy immediately before intracytoplasmic sperm injection and characterized according to nuclear maturation stage (telophase I and metaphase II) and to the presence of a meiotic spindle. We analyzed the fertilization rates, cleavage, number of good quality embryos on the second day (D2) from oocytes on telophase I versus those in metaphase II, and metaphase II visible spindle versus non-visible ones. Data were analyzed comparatively by Fisher's exact test. The level of significance was set at 5% in all analyses (p<0.05). RESULTS: The meiotic spindles of 516 oocytes were imaged using polarization microscopy. From the 516 oocytes analyzed, seventeen were in telophase I (3.3%) and 499 (96.7%) in metaphase II. The oocytes injected in telophase I had significantly lower fertilization rates than those injected in metaphase II (53 and 78%, respectively) and produced no good quality embryos on day 2. When the oocytes with and without a visible meiotic spindle were compared, there was no significant difference in the intracytoplasmic sperm injection results. CONCLUSIONS: Oocytes injected in telophase I showed lower fertilization rates when compared to those in metaphase II. It is possible that the analysis of oocyte nuclear maturation by polarization microscopy can be used as a predictor of fertilization after intracytoplasmic sperm injection.

In vitro assessment of reproductive toxicity of cigarette smoke and deleterious consequences of maternal exposure to its constituents

Cigarette smoke is known to be a serious health risk factor and considered reproductively toxic. In the current study, we investigated whether constituents of cigarette smoke, pyrazine, 2-ethylpyridine, and 3-ethylpyridine, adversely affect reproductive functioning such as oocyte maturation and sperm capacitation. Our findings indicated that three smoke components were involved in retardation of oocyte maturation in a dose-dependent manner and the lowest-observed-adverse-effect level (LOAEL) was determined to be 10-10M. However, individual smoke components administrated at the LOAEL did not attenuate oocyte maturation, demonstrating that all three toxicants were equally required for the observed growth impairment. When exposed to all three components at 10-10M during in vitro capacitation, murine sperm lost forward progression and were unable to show adequate hyperactivation, which is indicative of the incompletion of the capacitation process. Only sperm administrated with 3-ethylpyridine alone showed significant reduction in capacitation status, suggesting the chemical is the one responsible for disrupting sperm capacitation. Taken together, this is the first report that documents the effect of cigarette smoke components on oocyte maturation and sperm capacitation. The present findings demonstrate the adverse effects of smoke constituents of mammalian reproduction and the differences in sensitivity to smoke components between male and female gametes. Since both processes take place in the female reproductive system, our data provide new insights into deleterious consequences of maternal exposure to cigarette smoke.